EPA-430/1-75-003
STREAM SURVEILLANCE AND MONITORING:
FIELD AND LABORATORY PROCEDURES
TRAINING MANUAL
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF WATER PROGRAMS
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EPA-430/1-75-003
March 1975
STREAM SURVEILLANCE AND MONITORING:
FIELD AND LABORATORY PROCEDURES
This course is designed for personnel engaged in programs
concerned with monitoring the quality of surface waters.
Interdisciplinary needs and responsibilities in the accum-
ulation and interpretation of data from field and laboratory
activities are emphasized.
Upon completion of the course the student will be able to
apply suitable methods, techniques, and instrumentation
used in field sampling and measurements, and will be famil-
iar with basic laboratory techniques and procedures in the
chemical, biological, andbacteriological areas. Advantages
and limitations of equipment, methods and techniques will
be considered.
ENVIRONMENTAL PROTECTION AGENCY
Office of Water Program Operations
TRAINING PROGRAM
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CONTENTS
Title or Description Outline Number
Flow Measurement 1
Water Level Recorders ' 2
Tracing Natural Waters 3
Sampling in Water Quality Studies 4
Sample Handling - Field through Laboratory 5
Sample Preservation 6
Bacteriological Sampling in the Field 7
Biological Field Methods 8
The Aquatic Environment (Part 1 through 5) 9
The Identification of Aquatic Organisms 10
Aquatic Organisms of Significance in Pollution Surveys 11
Key to Selected Groups of Freshwater Animals 12
Using Benthic Biota in Water Quality Evaluation 13
Automatic Instruments for Water Quality Measurements 14
DO - Determination by the Winkler lodometric Titratiori 15
and Azide Modification
DO - Determination by Electronic Measurement 16
Dissolved Oxygen: Factors Affecting DO Concentration 17
in Water
COD and COD/BOD Relationships 18
Laboratory Procedures for Routine Level COD 19
Acidity, Alkalinity, pH and Buffers 20
Operating Characteristics and Use of the pH Meter 21
161.3.75
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Contents
Title or Description Outline Number
Specific Conductance • 22
Calibration and Use of a Conductivity Meter 23
Chlorine Determinations and Their Interpretation 24
Phosphorus in the Aqueous Environment 25
Chemical Tests, Observations, and Measurements 26
in the Field
Bacteriological Indicators of Water Pollution 27
Examination of Water for Coliform and Fecal Streptococcus 28
Groups (Multiple Dilution Tube Methods)
Examination of Water for Coliform and Fecal .Streptococcus 29
Groups (Membrane Filter Methods)
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FLOW MEASUREMENT
I INTRODUCTION
Flow measurements are among the more
important data collected during a water
quality survey. Such measurements ar'e
used to interpret data variations, calculate
loadings, and expedite survey planning. If
the analysis of survey data involves estima-
tion of loads, the accurate measurement of
discharge assumes a level of importance equal
to that of laboratory and analytical results.
In the following discussion, procedures for
measurement of stream flow and waste dis-
charge are described. Some of these pro-
cedures are used in long-term, very detailed
water quality and supply studies; others are
more suited to short-term pollution surveys.
II PLANNING
A Station Location
Four factors influence location of gauging
or flow measurement stations:
1 Survey objectives
2 Physical accessibility
3 Characteristics of the stream bed
4 Hydrologic effects
Survey objectives represent the major
-influence on station location; depending
upon objectives, gauging stations may be
located above and/ or below confluences
and outfalls.
Physical accessibility determines the
ease and cost of installation and main-
tenance of the station. The characteristics
of the stream bed may greatly influence
the obtainable accuracy of measurement.
For instance, rocky bottoms greatly
reduce the accuracy of current meters.
Sedimentation in pools behind control
structures may influence stage-discharge
relationships. Hydrologic variations in
stream flow may cause washout or bypass
of the gauging station. In the Southwest,
flash floods have been known to wash out
or bypass gauging stations by assuming
different channels of flow.
B Methodology
Choice of a specific measurement pro-
cedure is dependent upon at least three
considerations:
1 The relation between obtainable and
desired accuracy
2 Overall cost of measurement
3 The quantity of flow to be measured
Ideally, discharge measurements should
be reported to a specific degree of accuracy;
the gauging procedure greatly influences
this accuracy. The influence of overall
cost on the gauging program is readily
apparent. Extensive, detailed studies are
usually characterized by high costs for
automatic instrumentation and low personnel
cost; the opposite is usually true for less
detailed studies. The range of flows to be
measured (within acceptable accuracy) is,
of course, not known prior to the survey.
However, experienced personnel usually
can make reasonable estimates of expected
flows from visual observations and other
data, and may recommend appropriate
gauging procedures. In this regard,
experienced personnel always should be
consulted.
Ill MEASUREMENT
A Streams, Rivers, and Open Channels
1 Current Meter
The current meter is a device for
measuring the velocity of a flowing body .
of water. The stream cross section is
divided into a number of smaller sections,
and the average velocity in each section
is determined. The discharge is then
found by summing the products 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 gauging stations are composed of a
control structure located downstream of
the location of measurement and some
type of water level indicator which iden-
tifies the height of the water surface
above a previously determined datum.
IN. SG. 13a.3.74
1-1
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Flow Measurement
Location of the control structure so
that reliable measurements of flow
will be obtained at all river stages is •
particularly important. The water level
may be continuously recorded by an
automatic recorder located in a wet
well or may be indicated directly on a
staff gauge located at the bank of the
river. Such stations must be calibrated
by measurement of flow by velocity-
area methods (current meter) at all
expected stages of river flow.
3 Weirs
A weir may be defined as a dam or
impediment to flow, over which the
discharge conforms to an equation.
The edge or top surface over which the
liquid flows is called the weir crest.
The sheet of liquid falling over the weir
is called the nappe. The difference in
elevation between the crest and the
liquid surface at a specified location,
usually a point upstream, is called the
weir head. Head-discharge equations
based on precise installation require-
ments have been developed for each
type of weir. Weirs so installed are
called standard weirs. Equations for
non-standard installations or unusual
types may be derived empirically.
Weirs are simple, reliable measure-
ment devices and have been investigated
extensively in controlled experiments.
They are usually installed to obtain
continuous or semi-continuous records
of discharge. Limitations of weirs
include difficulty during installation,
potential siltation in the weir pond,
and a relatively high head requirement,
0. 4 - 2.0 feet. Frequent errors in
weir installation include insufficient
attention to standard installation re-
quirements and failure to assure com-
pletely free discharge of the nappe.
a Standard suppressed rectangular
weir
This type of weir is essentially a dam
placed across a channel. The height
of the crest is so controlled that con-
struction of the nappe in the vertical
direction is fully developed. Since
the ends of the weir are coincident
with the sides of the channel lateral
contraction is impossible. This weir
requires a channel of rectangular
cross section, other special instal-
lation conditions, and is rarely used
in plant survey work. It is more
commonly used to measure the dis-
charge of small streams.
The standard equation for discharge
of a suppressed rectangular weir
(Francis equation) is:
Q = 3.33 LH3/2
where
Q = discharge, cfs
L = length 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 least six forms of the dis-
charge equation are commonly
employed. The standard suppressed
weir is sometimes used when data
must be unusually reliable.
b Standard contracted rectangular weir
The crest of this type of weir is
shaped like a rectangular notch.
The sides and level edge 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 stream discharge.
The standard equation for discharge
of a contracted rectangular weir
(corrected Francis equation) is
Q = 3.33 (L - 0. 2H)H
3/2
where
Q =
L =
H =
0. 2H =
discharge, cfs
length of the level crest
edge, feet
weir head, feet
correction for end contractions
as proposed by Francis
c Cipolletti weir
The Cipolletti weir is similar to the
contracted rectangular weir except
that the sides of the weir notch are
inclined outward at a slope of 1 .
horizontal to 4 vertical. Discharge
through a Cipolletti weir occurs as
though end contractions were absent
and the standard equation does not
include a corresponding factor for
correction.
1-2
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Flow Measurement
Figure 1. STANDARD CONTRACTED
RECTANGULAR WEIR
The standard equation for discharge
through a Cipolletti weir is
Q = 3.367 LH3/2
where
Q =
L =
H =
discharge, cfs
length of the level crest edge, feet
weir head, feet
The discharge of a Cipolletti
weir exceeds that of a suppressed
rectangular weir of equal crest
length by approximately 1 percent.
d 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 especially
useful for measurement of small
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
surveys.
The standard equation for discharge
of a 90° triangular weir (Cone
formula) is
Q = 2.49H2'48
where
Q = discharge, cfs
H = weir head, feet
Crest height and head
are measured to and from the point
of the notch, respectively.
Accuracy and installation
requirements
Quotations of weir accuracy express
the difference in performance between
two purportedly identical weirs and
do not include the effects of random
error in measurement of head. Weirs
installed according to the following
specifications should measure dis-
charge withini 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 crest shall be relieved by a
champfer.
45°
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 weirs
to admit air to the space beneath
the nappe.
1-3
-------
Flow Measurement
4) The approach channel shall be
straight arid of uniform cross
section for a distance above the
weir of 15 to 20 times the maximum
head, or shall be so baffled that a
normal distribution of velocities
exists in the flow approaching the
crest and the water surface at the
point of head measurement is free
of disturbances. The cross-
sectional area of the approach
channel shall be at least 6 times
the maximum area of the nappe at
the crest.
5) The height of the crest above the
bottom of the approach channel
shall be at least twice, and
preferably 3 times, the maximum
head and not less than 1 foot.
For the standard suppressed weir
the crest height shall be 5 times
the maximum head. The height of
triangular weirs shall be measured
from the channel bottom to the
point of the notch.
6) There shall be a clearance of at
least 3 times the maximum head
between the sides of the channel
and the intersection of the maximum
water surface with the sides of the
weir notch.
7) For standard rectangular suppressed,
rectangular contracted, and
Cipolletti weirs the maximum head
shall not exceed 1/3 the length of
the level crest edge.
8) The head on the weir shall be taken
as the difference in elevation
between the crest and the water
surface at a point upstream a
distance of 4 to 10 times the
maximum head or a minimum of
6 feet.
9) The head used to compute dis-
charge shall be the mean of at
least 10 separate measurements
taken at equal intervals. The
head range of the measuring
device shall be 0. 2 - 1.5 feet.
The capacities of weirs which conform
to these specifications are indicated
in Table 1.
4 Parshall flume
The Parshall flume is an open constricted
channel in which the rate of flow is
related to the upstream head or to the
difference between upstream and down-
stream heads. It consists of an
entrance section with converging
vertical walls and level floor, a throat
section with parallel walls and floor
declining downstream, and an exit
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, dependable
accuracy, large capacity range, and
self cleaning capability. Its primary
disadvantage is the high cost of
fabrication; this cost may be avoided
by use of a prefabricated flume. Use
of prefabricated flumes during plant
surveys is becoming increasingly
popular.
a Standard equations
The dimensions of Parshall flumes
are specified to insure agreement
with standard equations. Table of
dimensions are available from
several sources • . For flumes of
6 inch to 8 foot throat width the
following standard equations have
been developed.
1) 6 inch throat width
1.58
Q
2.06 H
2) 9 inch throat width
i
Q = 3.07 H '
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Flow Measurement
TABLE 1 DISCHARGE OF STANDARD WEIRS
Crest Length
(Feet)
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 '
5.0
6.0
7.0
8.0
9.0
10.0
Contracted Rectangular*
Weir
(discharge-cfs)
Max. Min.
.590 .286
1.65 .435
3.34 .584
5.87 .732
9.32 .881
13.8 1.03
19.1 1.18
25.6 1.33
28.8 1.48
34.9 1.78
41.0 2.07
47.1 2.37
53.2 2.67
59.3 2.97
Suppressed Rectangular*
Weir
(discharge-cfs)
Max. Min.
.631 .298
1.77 .447
3.65 .596
6.30 .744
10.0 .893
14.8 1.04
20.4 1.19
27.5 1.34
30.6 1.49
36.7 1.79
42.8 2.08
48.9 2.38
55.0 2.68
61.1 2.98
Cipolletti*
Weir
(discharge-cfs)
Max. Min.
.638 .301
1.79 .452
3.69 .602
6.37 .753
10.1 .903
15.0 1.05
20.6 1.20
27.8 1.35
30.9 1.51
37. .1 ' 1.81
43.3 2.11
49.5 2.41
55.7 2.71
62.0 3.01
90° Triangular*
Weir
(discharge-cfs)
Max. Min.
6.55 .046
' H- 0.2 ft, H- 1.5 ft, H- 1/3 L
Converging
1 section
Diverging
section
Sp^S'^o^^S&g^
FLOW
&Z^-
H~~ —
a
^-y-
~^*!=*'-
^^^5^
*
,_,,-
,. Water surface, s
SECTION
FIGURE 2 PARSHALL FLUME
1-5
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Flow Measurement
3) 1 to 8 foot throat width
1 522W0'026
Q » 4WH 1-ss"w
a
where
Q * free-flow discharge,
defined as that condition
which exists when the
elevation of the down-
stream water surface
above the crest, H,, does
not exceed a prescribed
percentage of the upstreanl
depth above the crest, H .
The prescribed percentage
of submergence is 60
percent for 6 and 9 inch
flumes and 70 percent for
1 to 8 foot flumes
W - throat width, feet
H = upstream head above the
flume crest
b Mead loss
The head required by a Parshall
flume is greater than (H - IL )
because Ha is measured at a point
in the coriverging section where the
water surface has already begun
to decline. Table 2 indicates the
total head requirements of standard
Parshall flumes. These losses
should be added to the normal
channel depth to determine the
elevation of the water surface at
the flume entrance. No head
losses are indicated for discharge-
throat width combinations for which
H& is less than 0.2 ft. or greater
than 2/3 the sidewall depth in the
converging section.
Accuracy and installation require-
ments
A Parshall flume will measure
discharge within ± 5% of the
standard value if the following
conditions are observed.
1) The dimensions of the flume
shall conform to standard
specifications.
*
2) The downstream head, H. , shall
not exceed the recommended
percentage of the upstream head,
H .
TABLE 2 HEAD LOSS IN STANDARD PARSHALL FLUMES
UNDEft FREE DISCHARGE
Discharge
(cfs)
0.6
1.0
2,5
5.0
10.0
30.0
50.0
Head Loss, Feet, in Flume of Indicated Width
1 foot
.08
.14
.26
.42
.70
2 feet
.09
.16
.27
.45
3 feet
.06
.12
.20
.34
.70
4 feet
.10
.16
.27
.56
5 feet
.08
.13
.22
.47
.68
6 feet
.07
.12
.19
.40
.57
7 feet
.06
.10
. 17
.35
.49
8 feet
.05
.09
.15
.30
.41
H > 0.2, H < 2.0
a — a -
1-6
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Flow Measurement
3) The upstream head shall be
measured in a stilling well
connected to the flume by a pipe
approximately 1-1/2 inches in
diameter.
4) The flume shall be installed in a
straight channel with the centerline
of the flume parallel to the direction
of flow.
5) The flume shall be so chosen,
installed, or baffled that a normal
distribution of velocities exists at
the flume entrance.
5 Tracer materials
Techniques, materials, and instruments
are presently being refined to permit
accurate measurement of instantaneous
or steady flow with several tracer
materials. Measurements are made by
one of two methods:
tracer material during its passage past
the sampling point, and At = the total
time of the sampling period.
Disadvantages of tracer methods include
incomplete mixing, natural adsorption
and interference, and high equipment
costs.
6 Floats
Floats may be used to estimate the time
of travel between two points a known
distance apart. The velocity so obtained
may be multiplied by 0. 85 to give the
average velocity in the vertical.
Knowing the mean velocity and the area
of the flowing stream, the discharge
may be estimated. Floats should be
employed only when other methods are
impractical.
B Pipes and Conduits
1 Weirs and Parshall flumes
a Continuous addition of tracer
b Slug injection
With the first method, tracer is injected
into a stream at a continuous and uniform
rate; with the second a single dose of
tracer material is added. Both methods
depend on good transverse mixing and
uniform dispersion throughout a stream.
The concentration of tracer material is
measured downstream from the point
of addition. When continuous addition
is employed, flow rates 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
stream flow rate, and c = the resulting
concentration of the stream flow com-
bined with the tracer. For the slug
injection method
Q '
c At
in which Q = the stream discharge,
S = the quantity of tracer added, c =
the weighted average concentration of
Weirs and Parshall flumes are commonly
installed in manholes and junction boxes '
and at outfalls to measure flow in pipes.
All conditions required for measurement
of open channel flow must 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 especially convenient for
measurement of exfiltration and
infiltration.
3 Depth-slope
If the depth of the flowing stream and
the slope of the sewer invert are known,
the discharge may be computed by
means of any one of several formulas.
a Manning formula
1 4RR 9 /<* 1/9
Q = ii^££ A RZ/V/-i
n
where
Q = discharge, cfs
1-7
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Flow Measurement
n = roughness coefficient
A = area of flow, sq. ft.
R = hydraulic radius, ft.
= area divided by wetted perimeter, ft.
S = slope, ft. per ft.
b Chezy formula
Q «
where
Q == discharge, cfs
C s friction coefficient
A ~ area of flow, sq. ft.
R = hydraulic radius, ft.
= area divided by wetted perimeter, ft.
S = slope, ft. per ft.
4 Open end pipe flow
The following methods can be employed
when other more precise means are not
practical. They can be employed,
however, only when there is free dis-
charge to the air.
a Coordinate method
(Figures 3, 4, and 5)
Discharge may be computed by the
following formula:
._ . .
Q (gpm)
1800 AX
where
A = cross sectional area of liquid
in the pipe (sq. ft. )
X = distance between the end of the
pipe and the vertical gauge in
ft. , measured parallel to the
pipe.
Y = vertical distance from water
surface at the end of the pipe
to the intersection of the water
surface with the vertical gauge,
in ft.
MJmUblc not so tl»t
X ail* Is parsMcl to sevnr
tad T sils Is vertical-
b = ( fro» tattoo of pipe to nrfui of talllnf liquid)
OPEN-PIPE FLOW MEASUREMENT - THIS DEVICE, ADJUSTED TO THE
SLOPE OF A SEWER AND CALIBRATED, CAN THEN BE CLAMPED TO
THE SEWER OUTFALL.
Figure 3
OPEN-PIPE FLOW MEASUREMENT REQUIRES TWO DIMENSIONS THAT
LOCATE THE SURFACE OF STREAM AFTER IT LEAVES THE PIPE.
Figure 4
1-8
-------
Flow Measurement
V = Velocity - fps
A • Cross-Motional --
DlKharce IB CPU
• «SO AV
X ((I) to c«nlvr of sire™
HOW TO MEASURE VELOCITY AND DISCHARGE FROM A PIPE.
Figure 5
b California pipe flow method
(Figure 6)
This method may be used only for
horizontal pipes having free dis-
charge. If the pipe is not horizontal,
a connection must be made to one
that is. The horizontal length must
be not less 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 - ^) '
w = d2'48
where a and d are measured in feet
MEASUREMENTS NEEDED FCC
CALIFORNIA PIPE FLOW METHOD
Inclined pipes should be connected to
e. horizontal lenitb of pipe bj hose.
Figure 6
C Head Measuring Devices
Several of the above gauging methods re-
quire the measurement of water level in
order that discharge may be determined..
Any device used for this purpose must be
referenced to some zero elevation. For;-
example, the zero elevation for weir
measurements is the elevation of the weir
crest. The choice of method is dependent
upon the degree of accuracy and the type
of record desired.
1 Hook gauge
The hook gauge measures water eleva-
tion from a fixed point. The hook is
dropped below the water surface and
then raised until the point of the hook,
just breaks the surface. This method
probably will give the most precise
results when 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
This method involves measurement of
the distance from a fixed reference
point to the water surface, by dropping
a plumb line until it just touches the
water surface.
4 Water level recorder
This 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 float should be placed irt
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 mechanism. When properly in-
stalled and maintained, the water level
recorder will provide an accurate,
continuous record.
1-9
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Flow Measurement
A CKNOWLEDGMENT:
Certain portions of this outline contains
training material from prior outlines by
P.E. Langdon, A.E. Becher, and P.P.
Atkins, Jr.
REFERENCES
1 Planning and Making Industrial Waste
Surveys - Ohio River Valley Water
Sanitation Commission.
2 Stream Gauging Procedure. U. S. Geological
Survey. Water Supply Paper 888. (1943)
3 King, H.W. Handbook of Hydraulics.
4th Edition, McGraw-Hill. (1954)
Water Measurement Manual. United
States Department of the Interior
Bureau of Reclamation. (1967)
This outline was prepared by F.P. Nixon,
formerly Acting Regional Training Officer,
Northeast Regional Training Center, EPA,
WPO, Edison Water Quality Research
Laboratory, Edison, NJ 08817
Descriptors: Chezy Equation, Discharge
Measurement, Flow, Flow Measurement,
Flow Rates, Flumes, Mannings Equation,
Open Channel Flow, Pipe Flow, Streamflow,
Venturi Flumes, Water Flow, Water Level
Recorders, Weirs
1-10
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WATER LEVEL RECORDERS
A Commonly used instruments produce
a graphic record of the rise and fall
of a water surface with respect to
time.
B Advantages over non-continuous staff
gage readings;
1 Continuous record provides most
accurate means of determining
daily average gage height and flow.
2 Maximum and minimum stages are
recorded, and time of occurrence
can be noted.
3 Record can be obtained when ob-
server not there, ot not available.
C Principal elements
1 Time element; clock mechanism
activated by spring, weight, or
electric motor.
2 Gage height element activated by
a float, cable, or tape, and a
counterwieght.
Gear reduction mechanism is pro-
vided in the height element so that
the gage-height reading on the
recorder is changed in proportion
to the rise and fall of the water
surface.
D Common types of recorders
a Drum Recorders
1 Horizontal drum; clock positions
pen along drum axis and gage
height element fotates drum.
2 Vertical drum: clock mechanism
rotates drum and water stage a
function of displacement of pen
along drum axis.
b Strip- chart
1 Totalizer - records flow rates
with respect to time and auto-
matically totalizes volume. Strip-
chart provides graphic record.
E Installation of recorders;
1 Gage tape or cable should be long
enough to accomodate the full range
of water surface changes.
2 Recorder should be installed over
a stilling well and shielded from
the weather.
3 Staff gage needed near stilling well
to check the recorder.
F Stilling Well, Float Well
1 Prime purpose to prevent oscillations
of the float caused by surging water
or wave action.
•
2 Must be anchored to prevent movements
that would introduce oscillations within
the well.
3 Outside surges can be damped out by
restricting area of the water inlet to
about 1/1000 of the inside horizontal
cross-sectional area of the well.
4 Can be made of galvanized iron culvert
pipe, sewer pipe, tongue and groove
creosoted lumber, stove pipe, or other
suitable material.
5 Sometimes built outside the channel in
which the liquid is flowing, and
connected to it by an opening or pipe.
In sewage applications, provision must
be made for frequent cleaning or, water
levels in well will not be representative.
Inlet to float well must be checked fre-
quently for clogging by sludge and
sediment.
WP.SUR. sg. 7. 3.75
2-1
-------
Water Level Recorders
G Errors in Float-Operated Devices
1 Float Lag. If a float performs any
mechanical work, i.e., turns gears,
moves an index hand or a recorder
pen, etc., there is always a lag of
the index behind the true water level.
2 Line Shift. With every change of
stage, a portion of the float line
passes from one side of the float
pulley to the other. This change of
weight changes the depth of flotation
of the float, hence the stylus deviates
from the true water height by a small
amount, depending on the change in
stage since the last .correct setting,
and the weight of the line used be-
tween the float and counterpoise.
3 Submergence of Counterpoise. When
the counterpoise and any portion of
the line becomes submerged, the
pull on the float is reduced and its
depth of flotation is increased.
4 Corrections for errors due to the
above are so small as to be unim-
portant if instruments are correctly
made and properly cared for. All
those errors vary inversely as the
square of the diameter of the float,
and directly as the weight of line
and counterpoise. To minimize them,
therefore, use a sufficiently large
float with light lines and counter-
weights, and keep the instrument
properly oiled and free from dust
to render the force required to move
the index as low as possible.
5 Errors from humidity effects on
charts. All paper- is affected by
humidity changes. It expands with
increasing humidity and contracts as
humidity decreases. The result is
to introduce errors in the graphs
made by recording instruments.
Under ordinary conditions those
errors are quite negligible. In
extreme cases, however, an ex-
pansion from excessive humidity of
as much as 2% has been noted.
The heights, however, may not be
in error by that amount. The actual
error depends on the height itself and
the position of the neutral axis of
expansion.
By far the most practicable procedure
to prevent paper expansion is to reduce
the effect of humidity to a minimum,
by keeping excessively humid air from
the recorder. The use of a desiccator
such as silica gel within the recorder
case is quite effective in keeping the
interior atmosphere reasonably dry.
This turns pink when its capacity is
exhausted. Heating in an oven will
drive off the moisture and restore it
to its normally blue color.
6 The "Human Equation" is the greatest
possible source of error, with the
following among the more conspicuous
reasons for erroneous or incomplete
records:
a Incorrect gage reading
b Inaccurate setting of pen
c Failure to wind clock
d Failure to start clock after winding
e Failure to put stylus on paper
f Wrongly attaching float, so that pen
moves up when it should go down
g Failure to note gage reading and
time on record sheet
h Failure to see that inlet to well is
open
i Failure to oil bearings occasionally
j Use of gummy oil
REFERENCES:
1 Hydrographic Data Book. Leupold &
Stevens, Inc. Portland, Oregon,
8th ed.
2 Water Measurement Manual. U. S. Dept.
of the Interior, Bureau of Reclama-
tion, Denver, CO. 2nd ed.
2-2
-------
Water Level Recorders
3 BUchanari, T.J. & Somers, W.P. This outline was prepared by Charles E.
Stage Measurement at Gaging Sponagle, Sanitary Engineer, National
Stations. Book 3, Chapter A7. Training Center, WPO, EPA, Cincinnati
Techniques of Water Resources OH 45268
Investigations of the U. S\ Geolog-
ical Survey. U. S. Government
Printing Office, Washington, 0. Ci Descriptors; Flow Measurement, Open
1968. Channel Flow, Str'eamflow, Water Level
Recorders
2-3
-------
TRACING NATURAL WATERS •
I INTRODUCTION
The validity of water pollution studies is de-
pendent upon an ability to describe the actual
time and space situation of a pollutant as it
blends with the natural receiving water. This
understanding is important in order to predict
the subsequent effects of the pollutant on the
receiving water and subsequent users or to
detect the sources of pollution.
The nature of this blending is dependent upon
the physical and chemical properties of both
the pollutant and the receiving water as well
as the mixing and flow characteristics of the
receiving channel, basin, or aquifer. Tracing
is an attempt to approximate the actual motion
and mixing of this blend as it moves through
the channel, basin or aquifer.
Although there has been scant reference to
tra'cers in texts there has been rapidly in-
creasing number of technical articles de-
scribing their use in recent years. A com-
prehensive compilation of early radioactive
tracer studies may be found in the report of
the "Time of Flow Studies, Ottawa River,
Lima, Ohio"* ' one of the early attempts to
compare various tracer techniques under
similar field conditions. Several reports
(see Bibliography) have discussed the use Of
dyes during the early 1960's.
More refined and extensive tracer investiga-
tions may be expected as experience is gained
and as more complex studies arise. A water
quality agency should develop competence in
at least one tracer technique and would be
wise to be capable in some others as well.
II PURPOSES
Tracer applications are still in a highly
developmental stage. One finds many words
or phrases to suggest similar field findings.
In broad terms these measurements are con-
cerned with indications of mass movement,
blend mixing, and flow 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 such terms as rate of re-
newal, flushing rate, drift velocity,
mass transfer or net (tidal) drift are
used. The tidal fluctuation imposes a
need for consideration of flow reversal
effects.
3 In subsurface waters such terms as
recharge rate, flow 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
phases which do not necessarily
correspond to slack conditions.
b Obscure current circulation as in'-
fluenced by the interlated factbrs of
tides, winds, Coriolis forces,
topography and density gradients.
2 In ground water
a Aquifer may flow contrary to initial
superficial impression.
b Hydrostatic conditions may change
and reverse normal flow direction.
WP.SUR.tr.lb.3.74
3-1
-------
Tracing Natural Waters
C Mixing Patterns
1 Short circuit effects
a Eddying - primarily the result of
surface channel and basin
configuration.
b Stratification - primarily due to
temperature and density differences.
c Inter-connections - between aquifers
in ground water as well as solution
channels and open fractures.
2 Distribution phenomena
i
. a Dispersion of colloidal, soluble and
suspended substances
b Diffusion of temperatures or gaseous
substances
III TYPES OF STUDIES
Tracers are used in many situations,
including:
A Treatment Plant Units
B Closed Conduits
C Open Channels
D Large Water Bodies
E Hydraulic Models
F Subsurface Aquifers
G Subsurface Basins
IV TYPES OF TRACERS
Materials used for tracers include:
A Floats
1 Surface (wooden and plastic devices as
well as fruit). Influenced by wind
action and debris.
2 Sub-surface "drogues". Apparent di-
rection must be carefully evaluated.
B Salts
1 Common salt - hard to detect when less
than 1 mg/1.
2 Brackish and freshwater mixing in
estuaries and coastal aquifers.
3 Ammonium chloride. (See Table 1)
C Dyes, such as:
1 Rhodamine series - See section VI
2 Pontacyl Brilliant Pink B - most stable
of fluorescent dyes. Rather expensive
dye.
3 Fluorescein - very inexpensive. Fluo-
resces very near natural stream back-
ground level.
4 Uranine - fluoresces near stream
background level.
D Radioactive substances, such as: (see
Table 2) AEC must approve all experi-
ments. AEC has published tables on
permissible concentrations in unrestricted
areas.(13'
1 Rubidium-86
2 Iodine-131
3 Tritium (H3) gives best overall perfor-
mance in subsurface' ' tracing.
4 Krypton-85 - used recently in gas
transfer measurement in laboratory
and stream studies.'12'
E Waste return characteristics; significant
built-in factors such as:
1 Silt - understandably dependent upon
velocity and obstructions.
2 Foam - ABS will foam at levels as low
as 1 - 5 mg/1 depending on hardness.
3-2
-------
Tracing Natural Waters
Table 1. SUBSURFACE NONRADIOISOTOPE FLUID TRACERS
Tracer
Form
Test conditions
Remarks
Chloride NaCl
Dextrose Sugar
Fluorescein
Dye
Chloride NaCl
Dextrose Sugar
Fluorescein
Dye
Dextrose Sugar
Ammonia Chloride
Fluorescein
Dye
Boron I^BO,
Lab: Columns of sand and
sandy loam.
Field: 4 feet thick aquifer of
sand and gravel, 90 feet below
surface.
Field: Added in Cone, of 10,000
ppm, 500 ppm, 50 ppm, and 200
ppm respectively into a 4 foot
thick sand horizon at 2,100 feet
below surface in McKean
County, Pennsylvania.
Boron
Na2B4Cy 10H2O Field: Injection into input wells.
Dilution factor ~5.
H3B°3
Thiocyanate NH4SCN
Alkali-Metal
SCN
Rhodamine B
Field: Water flooding.
Field: Karst topography.
Oxygen-18
Deuterium
H2O
HDD
Chloride most rapid. Fluores-
cein far from satisfactory.
Fluorescein considered best of
dyes. Chloride in the large cone.
required caused density currents.
Adsorption observed: dextrose -
little or none, boron - slight
(cone, dropped 30%) ammonia -
moderate, fluorescein - strong
(moved at rate about 1/10 that of
flood water). Greenburg*33'found
that borax in Hanford fine sandy
loam penetrated only 2 feet.
Limit of detection is reached
after 103 dilution.
Dilution possible 10 .
Dye not detected after 6 X 10
dilution.
Variations resulting from sea-
sonal fluctuations are useful to
study climate. (Also useful in
meteorology and glaciology).
Reproduced with permission of Isotopes, Inc.
3-3
-------
Tracing Natural Waters
Table 2. SUBSURFACE RADIOISOTOPE FLUID TRACERS - FIELD TESTS
(6)
Tracer Half-life
Form
Test conditions
Remarks
H
12. 5 y. HTO
Karst groundwater
Useful results up to 30 km
distance.
Chalk River soil, undistur-
bed, but penetrated with
driven piezometers.
Fluorescein dye traveled only
about 3/4 as fast as tritium.
,35
,131
,131
Co
60
Br
82
Rb
131
86
I
32
89. d
3d. .
8 a.
5.2y.
36 h.
19 d.
8 d.
14 d.
Iodine,
carrier free
KI
EDTA - (Co)
and
K Co(CN)
6 b
NH Br
RbCI
Nal
Na2HP04
Glaciofluvial sand and gravel,
various till soils, and fis-
sures and channels.
Chalk River soil at waste
disposal site.
4 feet thick aquifer of sand
and gravel, 90 feet below
surface.
Tracer compounds used with
carriers KI, EDTA-(Na) and
KgCo(CN)g. Limestone aqui-
fer consisted of some argil-
laceous material and marly
dolomite. Single-well pulse
technique.
Aquifers in alluvium and per-
meable strata in calcareous
marl series.
Groundwater direction at re-
servoir site in Egypt.
Single pulse tracing at dam
site for Jesenitz Reservoir
on Odrava River in
Czechoslovakia.
Chromium complex traveled
as fast as tritium.
Lags behind tritium indicating
some interaction with the soil.
In soil containing appreciable
amounts of gypsum there would .
• be exchange and loss of S .
Should be used with several ppm
stable iodine carrier.
Co complex unsuitable in this
aquifer. K3Co60(CN) appeared
to be most suitable of tracers
tested in limestone aquifer.
Q O
Br satisfactory under.these
conditions.
Tracer detectable 7 miles from
injection after 5 d.
Detected very high subterranean
water velocity necessitating
sealing of porous layer.
I101
82
Br24
Na
8 d.
36 h.
15 h.
Nal
NaBr
NaCI
Single pulse tracing at dam Determined direction and velo-
site on arges River in city of flow of subsurface water.
Roumania.
Reproduced with permission of Isotopes, Inc.
-------
Tracing Natural Waters
3 Acid - severe effects upon total stream
ecology.
4 Temperature - infra-red filming
techniques reveal 1°C temperature
differences.
5 Lignins - "Orzan" is a commercial
product based upon these non-degrading
materials.
6 dyes - prevalent in textile wastes.
F Biota, various techniques possible with:
1 Mammals
2 Fish
3 Shellfish
V DESIRABLE CHARACTERISTICS
Ideally a tracer should be:
A Biologically innocuous to human and
aquatic life, with special reference to
1 Acute toxicity
2 Long-term toxicity
3 Carcinogenic effects
4 Genetic effects
B Stable or persistant despite the effects of
1 Stream chemical constituents
2 Bacteria
3 Sunlight
4 Adsorption
5 Temperature
6 Wind actiori
7 Inherent decay
8 Channel obstructions
9 Stratification
C Readily detectable either
1 Visually and/or
2 Instrumentally despite dilution or
background effects;
D Representative: coincide with the real
waste and stream blend under study: this
involves miscibility and specific gravity
characteristics similar to the blend.
E Economic: this involves careful evaluation
of the costs for
1 Materials
2 Material preparation, and release -
ease of handling
3 Detection equipment
4 Detection technique and recording -
convenience in operation
5 Stream deterioration
F Esthetically agreeable: inoffensive to:
1 Taste
2 Sight - conflict with easy detection
VI ELEMENTS OF A TRACER STUDY -
USING RHODAMINE DYES (Currently
in widespread use for surface studies.)
A Rhodamine Dyes
1 Available frc-m American Cyanamid,
DuPont, Allied Chemical and General
Aniline.
2 Price $4. 95 per pbund except
Rhodamine WT which is $10.00 per
pound.
, 3-5
-------
Trafcihg Natural Waters
3 Specific gravity of 0.99 to 1. 12 depending
on the proportions of Alcohol, Ethylen-e
Glycol, Acetic Acid, and Water used as
solvents in preparing the solution.
f
4 Solutions normally 15%, 20%, 30%, or
40% dye by weight.
5 pH 1.0 to 9.0.
6 Peak wavelength of adsorption at 525-556^
and of fluorescence at 548-585^ are not
normally found in natural waters.
7 Detectable to 0'. 5 ng/1 with continuously
sampling and recording equipment.
8 Visibly red to approximately 1. 0 mg/1.
9 Rapid dispersion when dropped in water.
10 Subject to some destruction by light, but
much less than experienced with
fluorescein.
11 Destroyed by agents such as hypochlorite.
Rhodamine WT is more resistant.
12 Essentially non-toxic.
B Measuring Equipment
1 Available from Turner Associates,
Beckman Instruments, Inc., American
Instrument Co., Inc.
so two or more runs with temperatures '_••'•
covering the expected range of the study . -.
water are desirable. Also it is advisable ,»';•
to make a calibration with the study water..':'
This will check for background and-any
difference in fluorescent characteristics.. ..'
A few helpful hints are: '
1 Pumping a known concentration sample,'.
from a plastic bag immersed in a • • . ; '>"'
constant temperature water bath,
through the meter is a convenient ••• . :;.
calibration procedure. Continuous. ' '. '
dousing with a concentrated solution
into the bag will give various instrument
readings through the detectable ranges.
2 Flush the system with methyl or butyl •
alcohol solution before each calibration •„•'
to remove traces of dye.
3 Bubbles in the flow cuvette will cause
erratic readings. . ' '
4 The fluorometer door is not light tight,. .'
so the instrument should be draped and \
used out of intense light to prevent •
erroneous high readings.. ' ' ;. -. - ' \
5 The sample lines on a continuous'flow
system will transmit light through the .;
instrument door causing erroneous high
readings. Taping these tubes with
black electrical tape for about one foot
will alleviate problem. . . •
<2 Price as low as $1, 000 for photofluorometer D Study Procedures
complete or $500 for unit to modify a
spectrophotometer. ,
3 Available for individual grab sample
analysis or continuous analysis of
sample pumped through instrument.
4 Automatic recorders available.
5 Will measure several dyes (fluorescein,
rhodamine, etc. )
C Fluorometer Calibration
The instrument should be calibrated before
and after a study. The calibration is a
function of temperature (about 2.3%/°C)
1 Select objectives which then dictate:
a Length of time to be studied
b Release point . s' "; '
c Monitoring locations and schedules
2 Determine physical properties of'1";..';
affected area. :'• . ; •
• j Vl /•'•
a Probable net flows
b Total water volumes • ;
c Probable water temperature ' ••.••}•
3-6
-------
Tracing Natural Waters
d Natural fluorescent background
e Salinity
f Suspended solids
3 Estimate required quantities of dye
4 Calibrate fluorometers
5 Conduct mock run to test equipment
6 Public relations
7 Release and monitor dye
8 Process and interpret data
9 Report
2 Using these measurements and a two
dimensional dispersion model com-
pute D and D .
x y
3 Two dimensional model
|£ = D *_| + D
dt xdx2 y
C T"r
ax " vdy
U = velocity in x direction
V = velocity in y direction
Normalized solution:
D_
2inr
where f (t) represents concentration
function and g(t) = decay function
C Coastal Water Dispersion Study, Hilo
Bay, Hawaii*9*
1 From instantaneous release measure
concentration profile during slack
water
D Channel Studies
1 From instantaneous release compute
longitudinal dispersion coefficient
2 Measure time-concentration profile
at given station
3 This C vs. t curve fits equation
C =
M
•n exp -
A(4irDt)2
4 Take log of both sides
(x - ut)'
4 Dt
Ct2 = log
(x - ut)'
4 Dt
log e
5 D can be determined from semi-log
.2
plot of Ct2 vs. (X " Ut)<
t
3-7
-------
Tracing Natural Waters
REFERENCES . 9 O'Connell, R.L., and Walter, C.M. A
Study of Dispersion in Hilo Bay,
1 Straub, C.P., Ludzack, F.J., Hagee, .Hawaii. Report prepared for the
G.R., and Goldin, A.S. Time of U.S. Army Engineer District,
Flow Studies. Ottawa River, Lima, Honolulu, Hawaii. September, 1963.
Ohio. Transactions, Amer. Geo-
physical Union, Vol. 39. pp 420- 10 Tsivoglou, E.G., et al. Tracer Measure-
426. 1958 ments of Atmospheric Reaeration.
I. Laboratory Studies. Presented at
2 Feuerstein, D. L., and Selleck, R.E. the Water Pollution Control Feder-
Flourescent Tracers for Dispersion ation Conference, Bal Harbour,
Measurements. Journal of the Florida. September, 1964.
Sanitary Engineering Division, ASCE,
Vol. 89, No. SA4, Proc. Paper 3586, 11 Cawley, W.A. and Rutledge, W. C.
pp 1-21. August, 1963. Application of Radioactive Tracer
Techniques to Stream Improvement.
3 Buchanan, Thomas J. Time of Travel Journal, San. Eng. Div., ASCE, Vol.
of Soluble Contaminants in Streams. 92, No. SA1, Proc. Paper 4640, p. 1,
Journal of the Sanitary Engineering February, 1966.
Division, ASCE, Vol. 90, No. SA3,
Proc. Paper 3932, ppl-12. June, 1964. 12 Tsivoglou, E.G., O'Connell, R.L.,
Walter, C.M., Godsil, P.J., and
4 Wright, R.R. and Collings, M.R. Appli- Logsdon, G.S. Tracer Measurements
cation of Fluorescent Tracing Tech- of Atmospheric Reaeration-1. Labor-
niques to Hydrologic Studies. Hydraulic atory Studies. Jour. WPCF, Vol, 37,
Engrs., USGS, Atlanta, GA., Jour. p. 1343.
AWWA, Vol. 56:748. June, 1964.
13 U.S. Atomic Energy Commission, Re-
5 Pritchard, D.W. and Carpenter, J.H. print from Federal Register, 17915,
Measurements of Turbulent Diffusion Part 30. 70, Dec. 17, 1964, p. 5.
in Estuarine and Inshore Waters. Ches-
apeake Bay Institute, Johns Hopkins 14 American Cyanamid Company, A Dis-
University. Contribution No. 53. 1960. cussion of Techniques and Tracer
Dyes used in Water Tracing, Dyes
6 Ault, W.U., and Hardeway, J.E. Surface and Textile Chemicals Department-
Tracing with Radioisotopes. Isotopics, Bound Brook, NJ 08805
Isotodes, Inc. Vol. 2 #1. January, 1965.
Diachishin, A.N. Dye Dispersion Studies. This outline was prepared by Dale B. Parke,
Jour. San. Engr. Div., ASCE, Vol. 89, Former Sanitary Engineer, Hudson-Delaware
No. SA1, Proc. Paper 3386, p. 29. Basins Office, EPA, WPO, Edison, NJ
January, 1963.
Descriptors: Fluorescence, Fluorescent Dye,
O'Connell, R.L., and Walter, C.M. Path of Pollutants, Radioisotopes, Tracers
Hydraulic Model Tests of Estuarial
Waste Dispersion. Jour. San. Engr.
Div., ASCE, Vol. 89, No. SA1, Proc. '
Paper 3394, January, 1963. p. 51.
3-8
-------
SAMPLING IN WATER QUALITY STUDIES
I INTRODUCTION
A Objective of Sampling
1 Water quality characteristics are not
uniform from one body of water to
another, from place to place in a
given body of water, or even from time
to time at a fixed location in a given
body of water. A sampling program
should recognize such variations and
provide a basis for interpretation of
their effects.
2 The purpose of collection of samples is
the accumulation 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-
signed that a statistical confidence
limit may be associated with each
element of data.
3 Water quality surveys are undertaken
for a great variety of reasons. The
overall objectives of each survey greatly
influence the location of sampling
stations, sample type, scheduling of
sample collections, and other factors.
This influence should always be kept in
mind during planning of the survey.
4 The sampling and testing program should
be established in accordance with princi-
ples which will permit valid interpre-
tation.
a The collection, handling, and testing
of each sample should be scheduled
and conducted in such a manner as
to assure that the results will be
truly representative of the sou rces
of the individual samples at the time
and place of collection;
b The locations of sampling stations
and the schedule of sample collections
for the total sampling program should
be established in such a manner that
the stated investigational objectives
will be met; and
c Sampling should be sufficiently
repetitive over 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
results from individual samples. While
it is beyond the scope 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 considered in planning the
sampling program.
1 Apparent Variations
a Variations of a statistical nature,
due to collection of samples from
the whole body of water, as con-
trasted with examination of all the
water in the system.
b Variations due to inherent precision
of the analytical procedures.
c Apparent variations are usually
amenable to statistical analysis.
2 True Differences
a Variations of a cyclic nature
Diurnal variations, related to alter-
nating periods of sunlight and
darkness.
Dirunal Variations related to waste
discharges from communities.
WP. SUR. sg. Ib. 3.74
-------
Sampling in Water Quality Studies
Seasonal variations, related to
temperature and its subsequent
effects on chemical and biological
processes and interrelationships.
Variations due to tidal influences,
in coastal and estuarine waters.
b Intermittent variations
Dilution by rainfall and runoff.
Effects of irregular or intermittent
discharges of wastewater, such as
"slugs" of industrial wastes.
Irregular release of water from
impoundments, as from power
plants.
c Continuing changes in water quality
Effects downstream from points of
continuous release of wastewater.
Effects of confluence with other
bodies of water.
Effects of passage of the water
through or over geological forma-
tions of such chemical or physical
nature as to alter the characteristics
of the water.
Continuing interactions of biological,
physical, and chemical factors in
the water, such as in the process of
natural self-purification following
introduction of organic contaminants
in a body of water.
II LOCATION OF SAMPLING STATIONS
A The Influence of Survey Objectives
Much of the sampling design will be
governed by the stated purpose of the
water investigation. As an example of
how different objectives might influence
sampling design, consider a watercourse
with points A and B located as indicated
in Figure 1.
A
•y
flow
Figure 1
B
Point A can be the point of discharge of
wastes from Community A. Point B can
be any of several things, such as an intake
of water treatment plant supplying Com-
munity B, or it might be the place where
the river crosses a political boundary, or
it may be the place where the water is
subject to some legitimate use, such as
for fisheries or for recreational use.
1 Assume that the objective of a water
quality investigation is to determine
whether designated standards of water
quality are met at a water plant 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.
Sampling will be conducted only at
Point B.
2 Alternately, consider that there is a
recognized unsatisfactory water quality
at Point B, and it is alleged that this
is due to discharges of inadequately
treated wastes, originating at Point A.
Assume that the charge is to investigate
this allegation.
In this case the selected sampling sites
will include at least 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 upstream 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 receiving 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 influences
traced from Point A.
-------
Sampling in Water Quality Studies_
B Hydraulic Factors
1 Flow rate and direction
a In a survey of an extended body of
water it is necessary to determine
the rate and direction of water move-
ment influences selection of sam-
pling sites. Many workers plan
sampling stations representing not
less than the distance water flows
in a 24-hour period. Thus, in
Figure 1, intervening sampling
stations would be selected at points
representing the distance water
would flow in about 24 hours.
b In a lake or impoundment direction
of flow is the major problem influenc-
ing selection of sampling stations.
Frequently it is necessary to estab-
lish some sort of grid network of
stations in the vicinity of the sus-
pected sources of pollution."
c In a tidal estuary, the oscillating
nature of water movement will re-
quire establishment of sampling
stations in both directions from
suspected sources of pollution.
2 Introduction of other water
a In situations in which a stream being
studied is joined by another stream
of significant size and character,
sampling 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 of juncture,
b Similar stations will be needed with
respect to other water discharges,
such as from industrial outfalls,
other communities, or other instal-
lations in which water is introduced
into the main stream.
3 Mixing
a Wherever possible, one sampling
point at a sample collection site is
used in stream surveys. This
usually is near the surface of the
water, in the main channel of flow.
b In some streams mixing does not
occur quickly, and introduced water
moves downstream for considerable
distances below the point of con-
fluence with the main streams.
Example: Susquehanna River at
Harrisburg, where 3 such streams
are recognizable in the main river.
Preliminary survey operations
should identify such situations.
When necessary, collect separate
samples at two or more points
across the body of water.
c Similarly, vertical mixing may not
be rapid. This is noted particularly
in tidal estuaries, where it may be
necessary to make collections both
from near the bottom and near the
surface of the water.
d Collection of multiple samples from
a station requires close coordination
with the laboratory, in terms of the
number of samples that can be
examined. Some types of samples
may be composited. The decision
must be reached separately for each
type df sample.
C Types of Analytical Procedure
1 Samples collected for physical, chemi-
cal, and bacteriological tests and
measurements may be collected from
the same series of sampling stations.
2 Sampling stations selected for biological
(ecological) investigation require
selection of a series of similar aquatic
habitats (a series of riffle areas, or a
series of pool areas, or both). The
sites used by the aquatic biologist may
or may not be compatible with those
used for the rest of the survey. Accord-
ingly, in a given stream survey, the
stations used by the aquatic biologist
usually are somewhat different from thp
stations used for other examinations.
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Sampling in Water Quality Studies
D Access to Sampling Stations
For practical reasons, the sampling site
should be easily reached by automobile if
a stream survey, or by boat if the survey
is on a large body of water. Highway
bridges are particularly useful, if the
sample collector can operate in safety.
Ill FACTORS IN SCHEDULING OF SAMPLING
PROGRAMS
A Survey Objectives
B Time of Year
1 In short-term water quality 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 conducted during the
warmer season of the year, at such
times as the water flow rate and
volume is at a minimum and there is
minimum likelihood of extensive
rainfall.
2 In a long-term investigation, sampling •
typically is conducted at all seasons
of the year.
C Daily Schedules
As shown in an introductory paragraph,
water quality is subject to numerous
cyclic or intermittent variations. Sched-
uling of sample collections should be de-
signed to reveal such variations.
1 In short-term surveys it is common
practice to collect samples from each
sampling site at stated intervals through
the 24-hour day, continuing the program
for 1-3 weeks. Sampling at 3-hour
.intervals is preferred by many workers,
though practical considerations may re-
quire extension to 4- or even 6-hour
intervals.
2 In an extended survey there is a ten-
dency to collect samples from each
site at not more than daily intervals,
or even longer. In such cases the
hour of the day should be varied through
the entire program, in order that the
final survey show cyclic or intermittent
variations if they exist.
3 In addition, sampling in tidal waters
requires consideration of tidal flows.
If samples are collected but once daily,
many workers prefer to make the col-
lections at low slack tide.
4 In long-term or any other survey in
which only once-daily samples are
collected, it is desirable to have an
occasional period of around-the-clock
sampling.
IV IDENTIFICATION OF SAMPLING SITES
A River Mile System
The FWPCA method of identifying points
on a water course is by counting river
miles from the mouth (or junction with a
larger stream) back to the source. This
should not be confused with other systems,
such as those in which the river mile is
started at the source of the stream and
proceeds to the mouth of the stream or
confluence with another body of water.
B STORET System
The STORET System is a computer-oriented
data processing system used by FWPCA for
storage, retrieval, and analysis of water
quality data collected by federal, state,
local, and private agencies.
The system includes a complex system -
based on the river mile system - for
identifying sampling locations on all rivers
and streams in the United States. A recent
addition to the system introduces a location
procedure based on geographic coordinates;
this procedure is especially adapted to
location of sampling stations in large bodies
of water such as lakes and impoundments.
k-t*
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Sampling in Water Quality Studies
Not all locations have been coded at this
time, although the coding systems have
been established. The interested worker
should consult Public Health Service Publi-
cation No. 1263, "The Storage and Retrieval
of Data for Water Quality Control. " 1963.
IV SAMPLE COLLECTION
A Types of Samples ,
i
1 "Grab" sample - a grab sample is usually
a manually collected single portion of the
wastewater or stream water. An analysis
of a grab sample shows the concentration
of the constituents in the water at the time
the sample was taken.
2 "Continuous" sample - when several points
are to be sampled at frequent intervals or
when a continuous record of quality at a
given sampling station is required, an
automatic or continuous sampler may be
employed.
a Some automatic samplers collect a
given volume of sample at definite time
intervals; this is satisfactory when the
volume of flow is constant.
b Other automatic samplers take samples
at variable rates in proportion to chang-
ing rates of flow. This type of sampler
requires some type of flow measuring
device.
3 "Composite" sample - a composite
sample is the collection and mixing
together of various individual samples
based upon the ratio of the volume of
flow at the time the individual samples
were taken to the total cumulative
volume of flow. The desired composite
period will dictate the magnitude of the
cumulative volume of flow. The more
frequently the samples are collected,
the more representative will be the
composite sample to the actual situa-
tion. Composite samples may be
obtained by:
a Manual sampling and volume of flow
determination made when each sam-
ple is taken.
b Constant automatic sampling (equal
volumes of sample taken each time)
with flow determinations made as
each sample is taken.
c Automatic sampling which takes
samples at pre-determined time
intervals and 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 is specially designed
for collection of samples from the
bottom muds, at various depths,
or at water surfaces. Special
designs are related to protection of
sample integrity in terms of the
water characteristic or component
being measured.
b For details of typical sampling equip-
ment used in water quality surveys.
see outlines 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 operation is
possible, at lower cost than may be
possible with automatic sampling
equipment.
2 Automatic sampling equipment
Automatic sampling equipment has
several important advantages over
manual methods. Probably the most
important consideration is the reduction
in personnel requirements resulting
from the use of this equipment. It
also allows more frequent sampling
than is practical manually, and elimi-
nates many of the human errors in-
herent in manual sampling.
Automatic sampling equipment has
some disadvantages. Probably the
most important of these is the tendency
of many automatic devices to become
-------
Sampling in Water Quality Studies
clogged when liquids high in solids are
being sampled. Individual portions of
composite samples are usually quite
small which may in some cases be
disadvantageous. In using automatic
samplers, sampling points are fixed,
which results in a certain loss of
mobility as compared to manual
methods.
Automatic sampling equipment should
not be used indiscriminately; some typec
of samples - notably bacteriological,
biological, and DO samples - should
not be composited. In cases of doubt,
the appropriate analyst should be
consulted.
a Compositing samplers
1) Jar and tube sampler - this type-
samples effectively when flow
is nearly constant. As water
drains from the upper carboy,
the vacuum created syphons
waste into the lower one. The
rate-of-flow is regulated by the
pinch clamp to fill the lower
carboy during the sampling
period. (See Figure 2 )
^ WASTE SAMPLE SCREW CLAMP
-WASTE STREAM
C MUST BE GREATER THAN A+B
Figure 2
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Sampling in Water Quality Studies
2) Scoop type
a) Rotating scoop
This device consists of a
power driven scoop mounted
upstream from a weir. The
scoop is so designed and
mounted that the sample vol-
ume grabbed on each rotation
of the scoop is proportional to
the flow, as governed by the
head on the weir. The scoop
may be rotated at a constant
speed or timed to sample
at fixed time intervals.
b) Revolving wheel with cups
(Figure 3)
This device consists of a
power driven wheel or disc
mounted upstream from a
weir. A number of freely
suspended buckets are mount-
ed at varying distances from
the axis so that increased
flow will cause more buckets
to be filled, thereby giving a
sample proportionate to flow.
Both this device and the
rotating scoop sampler can,
of course, be used for non-
proportionate sampling.
c) Bucket elevators
This device may consist of a
single bucket alternately
lowered into and raised out of
the waste stream, or it may
consist of a series of buckets
on an endless chain passing
through the waste stream. In
either case, it will include a
tripping mechanism to cause
the bucket or buckets to spill
into a sampling funnel. Both
types may be operated contin-
uously or timed for intermittent
operation. This method is not
well adapted to proportionate
sampling.
WEIR CREST
Figure 3
WHEEL WITH BUCKETS
3 Pumps
a) Chemical feed pumps have
been found useful for sampling,
because of their ability to
meter out small doses of
liquids. A timing mechanism
may be used to make the pump
run for longer periods during
heavy flow, thereby allowing
collection of the sample in
proportion to flow. These
pumps are usually provided
with adjustable stroke and
variable speed features which
allow variation of the volume
of sample being pumped.
Figure 4 illustrates a battery
operated pump.
b) Automatic shift sampler
(Figure 5 )
Figure 5 shows the automatic
shift sampler. It consists
first of a Randolph or other
"squeegee-" type pump unit.
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Sampling in Water Quality Studies
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-
ment.
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
viaa3/16-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 plastic trough at a
rate equal to one division
every eight hours. With the
10 sample-jar receivers the
timer can be set on Friday,
and the 9 week-end shift
samples can be picked up on
Monday.
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 devices may be employed in
sampling free flowing streams.
(See Figures 6 and 7.)
SOLENOID VU>C
I INSTALL 10 AS NOT
TO ACT AS TRAP)
TM1NO MICH
•OUS* SGKECN TO
MOTCCT
Figure 4
BATTERY OPERATED PUMP
Figure 6
PUMP - SOLENOID VALvE - TIMER
TYPE SAMPLER
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 proportional
sampling is required.
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Sampling in Water Quality Studies
Pump -
Sompli I
Tubing Joint
-SOLENOID PUSHES
-Nomol Position of
Sampling TuM fetimMg
to S«w«r
WASTES
FROM SEWER
7) Drip sampler
Two types of this device are
illustrated in Figures 9 and 10.
Both devices are simple methods
of obtaining a composite sample
at a fairly constant rate.
WIRE ROD SOLDERED
TO FUNNEL AND BENT
TO PASS THROUGH
WATER JET
FUNNEL WITH
NARROW SLOT
CUT IN SIDE
Figure 7
PUMP - SOLENOID-DIVERTER - TIMER
TYPE SAMPLER
6) Air vent control
This type of device is illustrated
in Figure 8. The rate of sample
c collection is controlled by the
bubbling mechanism. It is not
suitable for use when proportion-
ate sampling is required.
VUVt HANOU-
FROM SAMPUH
TO BUBBLES •
mm* INLIT
Figure 9
FUNNEL AND ROD DIVERTER
TUBHM SLOTTED MO
•LID OVCH OLASS TUBE -
PIPC IS* LONG
Figure 8
AIR RELEASE TYPE SAMPLER
SAMPLE COILCCTMO BOTTLES
Figure 10
DRIP TUBE SAMPLERS
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Sampling in Water Quality Studies
VI
b Continuous recording equipment
Instruments have been developed
which provide direct measurement
of temperature, pH, conductivity,
color, and dissolved oxygen. Such
instruments may be equipped for
continuous recording. Instruments
of this type are quite expensive and
their installation is often difficult.
They are best adapted to permanent
installations, although good portable
non-recording instruments are
available for the measurement of
temperature, pH, and conductivity.
SOME CONSIDERATIONS IN SAMPLING
OPERATIONS
All procedures in care and handling of samples
between collection and the performance of
observations and tests are directed toward
maintaining the reliability of the sample as an
indication of the characteristics of the sample
source.
A Sample Quantity
1 Samples for a series of chemical
analyses require determination of the
total sample volume required for all
the tests, and should include enough
sample in addition to provide a safety
factor for laboratory errors or acci-
dents. Many workers collect about
twice the amount of sample actually
required for the chemical tests. As
a rule of thumb, this is on the order
or 2 liters.
2 Bacteriological samples, in general,
are collected in 250 - 300 ml sterile
bottles; approximately 150 - 200 ml of
samples is adequate in practically all
cases.
B Sample Identification
1 Sample identification must be main-
tained throughout any survey. It is
vital, therefore, that adequate records
be made of all information relative to
the source of the sample and conditions
under which the collection was made.
All information must be clearly under-
standable and legible.
2 Every sample should be identified by
means of a tag or bottle marking,
firmly affixed to the sample bottle.
Any written material should be with
indelible marking material.
3 Minimum information on the sample
label should include identification of
the sample site, date and time of col-
lection, and identification of the
individual collecting the sample.
4 Supplemental identification of samples
is strongly recommended, through
maintenance of a sample collection
logbook. If not included on the sample
tag (some prefer to duplicate such infor-
mation) the logbook can show not only
the sample site and date and hour of
collection, but also the results of any
tests made on site (such as temperature,
pH, dissolved oxygen). In addition, the
logbook should provide for notation of
any unusual observations made at the
sampling site, such as rainfall, direc-
tion and strength of unusual winds, or
evidence of disturbance of the collection
site by human or other animal activity.
C Care and Handling of Samples
1 As a general policy, all observations
and tests should be made as soon as
possible after sample collection.
a Some measurements require perfor-
mance at the sampling site, such as
temperature, pH, dissolved oxygen,
chlorine, flow rate, etc.
b Some tests are best made at the
sampling site because the procedures
are simple, rapid, and of acceptable
accuracy. These may include such
determinations as conductivity.
c Some additional determinations, such
as alkalinity, hardness, and turbidity
may be made in the field, provided
-------
Sampling in Water Quality Studies
that ease, convenience, and reliability
of results are acceptable for the pur-
poses of the study.
2 Samples to be analyzed in the laboratory
require special protection to assure that
the quality measured in the sample repre-
sents the condition of the source. Many
samples, especially those subjected to
biological analysis, require special pre-
servation, protection, and handling pro-
cedures. In case of doubt, the appropriate
analyst should be consulted. Most com-
mon procedures for sample protection
include:
a Examination within brief time after
collection.
b Temperature control.
c Protection from light.
d Addition of preservative chemicals.
Applications of these sample protective
procedures are along the following
lines:
3 Early examination of sample
Applicable to all types of samples.
4 Temperature control
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 transport
time of 6 hours. Such samples should
be refrigerated upon receipt in. the
laboratory and processed within 2 hours.
c Chemical samples often require
icing
Preservation by refrigeration at 4°C
is recommended for acidity, alkalinity,
BOD, color, sulfate, threshold odor,
and other samples. Holding times vary.
Quick freezing will permit retention
of many samples for up to several
months prior to laboratory examina-
tion.
5 Protection from light
a Any constituent of water which may
be influenced by physiochemical
reactions involving light should be
protected. DO samples brought to
the iodine stage, for example, should
be protected from light prior to
titration.
b In addition, any water constituent
(such as dissolved oxygen) which
may be influenced by algal activity
should be protected from light.
6 Addition of chemical preservatives
a Bacteriological samples never
should be "protected" by addition
of preservative agents. The only
permissible chemical additive is
sodium thiosulfate, which is used
to neutralize free residual chlorine,
if present.
b Samples for biological examination
should be protected by chemical
additives only under specific
direction of the principal biologist
in a water quality study.
c For chemical tests, preservatives
are useful for a number of water
components. The following examples
are cited:
Nitrogen and phosphorus analyses:
The addition of 40 mg HgCl2 per
liter of sample and refrigeration at
4°C will retard biological activity
which might otherwise alter the
concentration of these constituents
4-11
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Sampling in Water Quality Studies
Metals : The addition of 5 ml of
HNOs acid per liter of sample
prevents precipitation of the metal
in the container.
COD and Organic Carbon: Addition
of 2 ml sulfuric acid per liter of
sample is suggested.
In general, samples requiring re-
tardation of biological activity can
be temporarily preserved by addi-
tion of chloroform; tests should be
run as soon as possible, however.
Cyanide determinations may be
delayed temporarily through addition
of alkali to the sample. Addition of
sodium hydroxide to pH 10 is recom-
mended.
Sulfide analysis may be delayed up
to 7 days by addition of 2 ml/liter of
sample of 2N solution of zinc acetate.
Phenol analysis can be delayed
temporarily by acidification to pH 4. 0
with phosphoric acid and preservation
with 1 gram CuSO4- 5H2O per liter
of sample.
REFERENCES
1 Standard Methods for the Examination
Water, Sewage and Industrial Wastes.
13th Ed. A.P.H.A. 1971
2 Planning and Making Industrial Waste
Surveys. Ohio River Valley Water
Sanitation Commission.
3 Industry's Idea Clinic. Journal of the
Water Pollution Control Federation.
April, 1965.
This outline was prepared by H. L. Jeter,
Director, National Training Center, EPA,
WPO, Cincinnati, OH 45268 and P.P.
Atkins, Jr., formerly Sanitary Engineer,
Training Activities.
Descriptors: Instrumentation, On-Site
Investigations, Preservation, Samplers,
Sampling, Water Sampling, Handling,
Sample, Surface Waters, Wastewater
4-12
-------
SAMPLE HANDLING - FIELD THROUGH LABORATORY
I PLANNING A SAMPLING PROGRAM
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 measures, if any
B Decisive Criteria
1 Nature of the sample source
2 Stability of constituent(s) to be measured
3 Ultimate use of data
H REPRESENTATIVE SAMPLES
If a sample is to provide meaningful and
valid data about the parent population, it
must be representative of the conditions
existing in that parent source at the
sampling location.
A The container should be rinsed two or
three times with the water to be collected.
B Compositjng Samples
1 For some sources, a composite of
samples is made which will represent
the average situation for stable
constituents.
2 The nature of the constituent to be
determined may require a series of
separate samples.
C The equipment used to collect the sample
is an important factor to consider.
ASTM^' 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 benthic deposits. No definite
procedure can be given, but careful
effort should be made to obtain a rep-
resentative sample.
Ill SAMPLE IDENTIFICATION
A Each sample must be unmistakably
identified, preferably with a tag, or label.
The required information should be planned
in advance.
B An information form preprinted on the
tags or labels provides uniformity of
sample records, assists the sampler, and
helps ensure that vital information will
not be omitted.
C Useful Identification Information includes:
1 sample identity code
2 signature of sampler
3 signature of witness
4 description of sampling location de-
tailed enough to accommodate repro-
ducible sampling. (It may be more
convenient to record the details in the .
field record book).
5 sampling equipment used
6 date of collection
7 time of collection
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 field
13 type of analyses to be done in laboratory
WP.SUR.sg.6.3.74
5-1
-------
Sample Handling - Field Through Laboratory
IV SAMPLE CONTAINERS
A Available Materials
1 glass
2 plastic
3 hard rubber
B Considerations
1 Nature of the sample - Organics
attack polyethylene.
2 Nature of constituent('g) to be determined
- Cations can adsorb readily on some
plastics and on certain glassware.
Metal or aluminum foil cap liners can
interfere with metal analyses.
3 Preservatives to be used - Mineral
acids attack some plastics,
4 Mailing Requirements - Containers
should be large enough to allow extra
volume for effects of temperature
changes during transit. All caps
should be securely in place. Glass
containers must be protected against
breakage. Styrofoam linings are
useful for protecting glassware.
C Preliminary Check
Any question of possible interferences
related to the sample container should
be resolved before the study begins. A
preliminary check should be made using
corresponding sample materials, con-
tainers, preservatives and analysis.
D Cleaning
If new containers are to be used, prelim-
inary cleaning is usually not necessary.
If the sample containers have been used-
previously, they should be carefully
cleaned before use.
There are several cleaning methods
available. Choosing the best method in-
volves careful consideration of the nature
of the sample and of the constituents) to
be determined.
1 Phosphate detergents should not be
used to clean containers for phosphorus
samples.
2 Traces of dichromate cleaning solution
will interfere with metal analyses.
E Storage
Sample containers should be stored and
transported in a manner to assure their
readiness for use.
V SAMPLE PRESERVATION
Every effort should be made to achieve
the shortest possible interval between
sample collection and analyses. If there
must be a delay and it is long enough to
produce significant changes in the sample,
preservation measures are required.
At best, however, preservation efforts
can only retard changes that inevitably
continue after the sample is removed
from the parent population.
A Functions
Methods of preservation are relatively
limited. The primary functions of those
employed are:
1 to retard biological action
2 to retard precipitation or the hydrolysis
of chemical compounds and complexes
3 to reduce volatility of constituents
B General Methods
1 pH control - This affects precipitation
of metals, salt formation and can
inhibit bacterial action.
2 Chemical Addition - The choice of
chemical depends on the change to be
controlled.
Mercuric chloride is commonly used
as a bacterial inhibitor. Disposal of
the mercury-containing samples is a
problem and efforts to find a substitute
for this toxicant are underway.
5-2
-------
Sample Handling - Field Through Laboratory
. To dispose of solutions of inorganic
mercury salts, a recommended
procedure is to capture and retain the
mercury salts as the sulfide. at a high
pH. Several firms have tentatively
agreed to accept the mercury sulfide for
re-processing after preliminary con-
ditions are met.*4'
Refrigeration and Freezing - This is
the best preservation technique avail-
able, but it is not applicable to all
types of samples. It is not always a
practical technique for field operations.
C Specific Methods
,(2)
The EPA Methods Manual includes a
table summarizing the holding times and
preservation techniques for several
analytical,,procedures. This information
also can be found in the standard refer-
ences (1. 2, 3) as part of tne presentation
of the individual procedures.
VI METHODS OF ANALYSIS
Standard reference books of analytical
procedures to determine the physical
and chemical characteristics of various
types of water samples are available.
A EPA Methods Manual
The Methods Development and Quality
Assurance Research Laboratory of the
Environmental Protection Agency, has
published a manual of analytical procedures.
to provide methodology for monitoring the
quality of our Nation's Waters and to deter-
mine the impact of waste discharges. The
title of this manual is "Methods for Chem-
cal Analysis of Water and Wastes. "<2)
For some procedures, the analyst is
referred to Standard Methods and/or to
ASTM Standards.
B Standard Methods
The American Public Health Association,
the American Water Works Association
and the Water Pollution Control Federation
prepare and publish a volume describing
methods of water analysis. These include
physical and chemical procedures. The
title of this book is "Standard Methods
for the Examination of Water and Waste-
water. "(3>
C ASTM Standards
The American Society for Testing and
Materials publishes an annual "book"
of specifications and methods for testing
materials. The "book" currently con-
sists of 33 parts. The part applicable
to water is a book titled, "Annual Book of
' ASTM Standards, Part 23. Water;
Atmospheric Analysis". ' '
D Other References
Current literature and other books of
analytical procedures with related in-
formation are available to the analyst.
E NPDES Methodology
When gathering data for National Pollutant
Discharge Elimination System report
purposes, the analyst must consult the
Federal Register for a listing of approved
analytical methodology. There he will be
directed to pages in the above cited
reference books where acceptable pro-
cedures can be found. The Federal
Register also provides information con-
cerning the protocol for obtaining approval
to use analytical procedures other than
those listed.
VII ORDER OF ANALYSES
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.
5-3
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Sample Handling - Field Through Laboratory
A The allowable holding time for samples
depends on the nature of the sample, the •
stability of the constituents) 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 Using preservation techniques, the
holding times lor 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 includes
a table summarizing holding times and
preservation techniques for several
analytical procedures. This information
can also be found in the standard
(123)
references ' ' as part of the
presentation of the individual
procedures.
4 If dissolved concentrations are
sought, filtration should be done in
the field if at all possible. Other-
wise, the sample is filtered as soon
as it is received in the laboratory.
A 0.45 micron membrane filter is
recommended for reproducible
filtration.
B The time interval between collection
and analysis is important and should be
recorded in the laboratory record book.
VIII RECORD KEEPING
The importance of maintaining a bound,
legible record-of pertinent information
on samples cannot be over-emphasized.
A Field Operations
A bound notebook should be used. Informa-
tion that should be recorded includes:
1 Sample identification records (See
Part HI)
2 Any information requested by the
analyst as significant
3 Details of sample preservation
4 A complete record of data on any •
determinations done in the field.
(See B, next)
5 Shipping details and records
B Laboratory Operations
Samples should be logged in as soon as
received and the analyses performed
as soon as possible.
A bound notebook should be used.
Preprinted data forms provide uniformity
of records and help ensure that required
information will be recorded. Such sheets
should be permanently bound.
Items in the laboratory notebook would
include:
1 sample identifying code
2 date and time of collection
3 date and time of analysis
4 the analytical method used
5 any deviations from the analytical
method used and why this was done
6 data obtained during analysis
7 results of quality control checks on the
analysis
8 any information useful to those who
interpret and use the data
9 signature of the analyst
5-4
-------
Sample Handling - Field Through Laboratory
IX SUMMARY
Valid data can be obtained only from a repre-
sentative sample, unmistakably identified,
carefully collected and stored. A skilled
analyst, using approved methods of analyses
and performing the determinations within
the prescribed time limits, can produce data
for the sample. This data will be of value
only if a written record exists to verify sample
history from the field through the laboratory.
REFERENCES
1 ASTM Standards, Part 23, Water;
Atmospheric Analysis.
2 Methods for Chemical Analysis of Water
and Wastes, EPA-MDQARL,
Cincinnati, OH 45268. 1974.
3 Standard Methods for the Examination of
Water and Wastewater, 13th edition,
APHA-AWWA-WPCF, 1971.
Dean, R., Williams. R. and Wise. R.,
Disposal of Mercury Wastes from
Water Laboratories, Environmental
Science and Technology, October, 1971.
This outline was prepared by A. Donahue,
Chemist, National Training,.Center, MPQD
EPA, WPO, Cincinnati, Ohio 45268.
Descriptors: On-Site Data Collections,
On-Site Investigations, Planning, Handling,
Sample, Sampling, Water Sampling,
Surface Waters, Preservation, Wastewater
5-5
-------
SAMPLE PRESERVATION
Complete and unequivocal preservation of
samples, either domestic sewage, industrial
wastes or natural waters, is a practical
impossibility. Regardless of the nature of
the sample, complete stability for every
constituent can never be achieved. At best,
preservation techniques can only retard the
chemical and biological changes that
inevitably continue after the sample is re-
moved from the parent source.
The changes that take place in a sample are
either chemical or biological. In the former
case, certain changes occur in the chemical
structure of the constituents that are a
function of physical conditions. Metal cations
may precipitate as hydroxides or form com-
plexes with other constituents; cations or
anions may change valance states under cer-
tain reducing or oxidizing conditions; other
constituents may dissolve or volatilize with
the passage of time. Metal cations may
also adsorb onto surfaces (glass, plastic,
quartz, etc. ), such as, iron and lead.
Biological changes taking place in a sample
may change the state of an element or a
radical to a different state. Soluble con-
stituents may be converted to organically
bound material in cell structures, or cell
lysis may result in release of cellular
materials into solution. The well known
nitrogen and phosphorus cycles are examples
of biological influence on sample composition.
Methods of preservation are relatively
limited and are intended generally to
(1) retard biological action, (2) retard
hydrolysis of chemical compounds and
complexes and (3) reduce volatility of
constituents.
Preservation methods are generally limited
to pH control, chemical addition, refrigeration,
and freezing. Table 1 shows the various pre-
servatives that may be used to retard changes
in samples.
Preservative
HgCl2
Table 1
Action
Bacterial
Inhibitor
Applicable to:
Nitrogen forms.
Phosphorus forms
Acid (HNO3) Metals sol- Metals
vent.prevents
precipitation
Acid (H2SO4) Bacterial
Inhibitor
Salt forma-
tion with
organic
bases
Alkali (NaOH) Salt forma-
tion with
volatile
compounds
Refrigeration Bacterial
Inhibitor
Organic samples
(COD, oil and
grease, organic
carbon)
Ammonia, amines
Cyanides, organic
acids
Acidity - alkalinity
organic materials,
BOD, color, odor,
organic P, organic
N, carbon, etc.,
biological organisms
(coliform, etc.)
In summary, refrigeration at temperatures
near freezing or below is the best preservation
technique available, but is not applicable to
all types of samples.
The recommended choice of preservatives for
various constituents is given in Table 2.
These choices are based on the accompanying
references and on information supplied by
various Regional Analytical Quality Control
Coordinators.
WP.SUR. sa. 2a. 3.75
6-1
-------
Sample Preservation
Table 2
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT (D
Measurement
Acidity
Alkalinity
Arsenic
BOD
Bromide
COD
Chloride
Chlorine Req.
Color
Cyanides
Dissolved Oxygen
Probe
Winkler
Vol.
Req.
(ml)
100
100
100
1000
100
50
50
50
50
500
300
300
Container
P.G(2)
P.G
P.G
P.G
P.G
P,G
P.G
P.G
P.G
P,G
G only
G only
Preservative
Cool. 4°C
Cool, 4°C
HNOQ to pH< 2
O
Cool, 4°C
Cool, 4°C
H2SO4to pH< 2
None Req.
Cool, 4°C
Cool, 4°C
Cool, 4°C
NaOH to pH 12
Det. on site
Fix on site
Holding
Time(")
24 Hrs.
24 Hrs.
6 Mos.
6Hrs.(3)
24 Hrs.
7 Days
7 Days
24 Hrs.
24 Hrs.
24 Hrs.
No Holding
No Holding
6-2
-------
Sample Preservation
Measurement
Fluoride
Hardness
Iodide
MBAS
Metals
Dissolved
Suspended
Total
Mercury
Dissolved
Total
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT (D
Vol.
Req.
(ml) Container Preservative
300 P, G Cool, 4°C
100 P, G Cool, 4° C
100 P, G Cool,4°C
250 P,G Cool,4°C
200 P, G Filter on site
HNO, to pH< 2
O
Filter on site
100 HNO3topH<2
100 P, G Filter
HNO, topH<2
O
100 P, G HNO3 to PH< 2
Holding
Time (6)
7 Days
7 Days
24 Hrs.
24 Hrs.
6 Mos.
6 Mos.
6 Mos.
38 Days
(Glass)
13 Days
(Hard
Plastic)
38 Days
(Glass)
13 Days
(Hard
Plastic)
6-3
-------
Sample Preservation
Measurement
Nitrogen
Ammonia
Kjeldahl
Nitrate
Nitrite
NTA
Oil and Grease
Organic Carbon
pH
Phenolic s
Phosphorus
Ortho-
phosphate
Dissolved
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT
-------
Sample Preservation
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT^
Measurement
Hydrolyzable
Total
Total.
Dissolved
Residue
Filterable
Non-
Filterable
Total
Volatile
Settleable Matter
Selenium
Silica
Specific
Conductance
Vol.
Req.
(ml)
50
50
50
100
100
100
100
1000
50
50
100
Container
P.G
P.G
P.G
P.G
P.G
P.G
P.G
P,G
P.G
P only
P.G
Preservation
Cool, 4° C
H2SO4 to pH< 2
Cool, 4°C
Filter on site
Cool, 4°C
Cool, 4°C
Cool. 4°C
Cool, 4°C
Cool, 4°C
None Req.
HNO3topH<2
Cool, 4°C
Cool, 4°C
Holding
Time W
24 Hrs. (4)
24Hrs.(4)
24Hrs.<4)
7 Days
7 Days
7 Days
7 Days
24 Hrs.
6 Mos.
7 Days
24Hrs.(5)
Sulfate
50
P.G
Cool. 4°C
7 Days
6-5
-------
Sample Preservation
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT*1)
Measurement
Sulfide
Sulfite
Temperatue
Threshold
Odor
Turbidity
Vol.
Req.
(ml)
50
50
1000
200
100
Container
P.G
P.G
P,G
G only
P,G
Preservation
2 ml zinc
acetate
Cool, 4°C
Det. on site
Cool, 4°C
Cool, 4°C
Holding
Time (6)
24 Hrs.
24 Hrs.
No Holding
24 Hrs.
7 Days
1 More specific instructions for preservation and sampling are found with each procedure
as detailed in this manual. A general discussion on sampling water and industrial
wastewater may be found in ASTM, Part 23, p. 72-91(1973).
2 Plastic or Glass
3 If samples cannot be returned to the laboratory in less than 6 hours and holding time
exceeds this limit, the final reported data should indicate the actual holding time.
4 Mercuric chloride may be used as an alternate preservative at a concentration of 40
mg/1, especially if a longer holding time is required. However, the use of mercuric
chloride is discouraged whenever possible.
5 If the sample is stabilized by cooling, it should be warmed to 25°C for reading, or
temperature correction made and results reported at 25°C.
6 It has been shown that samples properly preserved may be held for extended periods
beyond the recommended holding time.
6-6
-------
Sample Preservation
An excerpt from Manual of Chemical Analy- Descriptors: Preservation, Sampling,
sis of Water and Wastes, 1974, EPA, Wastewater(Pollution), Water.
Methods Development and Quality Assur-
ance Research Laboratory, NERC-Cincinnati,
Ohio 45268.
-------
BACTERIOLOGICAL SAMPLING IN THE FIELD
I INTRODUCTION
The first step in the examination of a water
supply for bacteriological examination is
careful collection and handling of samples.
Selection of sample sites, frequency of
sampling, and numbers of samples are all
directly related to the survey objectives.
II SELECTION OF SAMPLING LOCATIONS
The basis for locating sampling points is
collection of representative samples.
A Grab samples from streams are frequently
collected for control data or application of
regulatory requirements. A grab sample
can be taken in the stream near the surface.
B For intensive stream studies on source
and extent of pollution, representative
samples are taken by considering site,
method, and time of sampling. The
sampling sites may be a compromise
between physical limitations of the labora-
tory, detection of pollution peaks, and
frequency of sample collection in certain
types of surveys. Decisions must be
made as to the total number of samples
that can be processed by laboratory per-
sonnel and the types of samples desired,
as, for instance, those measuring
immediate water quality or those measur-
ing an averaging collection method.
A site designed to measure average con-
ditions should be far enough downstream
to ensure a complete mixing after the
source entry of pollutants. Averaging
does not eliminate variations but tends to
minimize sharp fluctuations. Since these
stations are removed from the point
source, a sampling program should not be
instituted with excessive sampling.
Samples from lakes, ponds, or reservoirs
usually are collected at the same depth
and on frequent occasions may be collected
over the entire surface.
Oceanic or estuarine sample sites can
often cover large areas and may,
depending upon the survey objectives,
necessitate vehicular and/or boat
transporation. Frequently the need for
surface and depth sampling programs are
necessary especially if tidal or current
patterns create a stratified environment.
Ill BOTTLES FOR WATER SAMPLES
A The sample bottles should have capacity
for at least 100 ml of sample, plus an air
space. The bottle and cap must be of
bacteriological inert materials. Resistant
glass or heat resistant plastic are
acceptable. Wide mouth ground-glass
stoppered bottles (Figure 1), plastic
bottles of heat-resistant polyethylene, and
8 ounce glass jars with rubber lined screw
caps are popular types in many laboratories.
Figure 1
W. BA. sa. 2. 3. 75
7-1
-------
Bacteriological Sampling in the Field
All bottles must be properly washed and
sterilized. Protect the top of the bottles.
and cap from contamination by paper or
metal foil hoods. Both glass and heat
resistant plastic bottles may be steri-
lized in an autoclave. Hold plastic at
121°C for at least 10 minutes. Hot air
sterilization, 1 hour at 170°C, may be
used for glass bottles.
B Add sodium thiosulfate to bottles intended
. for halogenated water samples. A quantity
of 0.1 ml of a 10% solution provides 100
mgAiter in a 100 ml sample. This
level shows no effect upon viability or
growth.
C Supply catalogs list wide mouth ground-
glass stoppered bottles of borosilicate
resistance glass, specially for water
samples.
IV TECHNIQUE OF SAMPLE COLLECTION
Follow aseptic technique as nearly as
possible. Nothing but sample water must
touch the inside of the bottle or cap. To
avoid loss of sodium thiosulfate, fill the
bottle directly and do not rinse. Always
remember to leave an 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 flam-
ing the tap before collection. A chlorine
determination is often made on the 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 suggestions 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. A depth of about 6 inches
is satisfactory. 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.
C Samples may be dipped from swimming
pools. Determine residual 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 by wading out
to the two foot depth and dipping the
sample up from about 6 inches below the
surface. Use the procedure described in
III.
E Wells with pumps are similar to distri-
bution systems. With a hand pumped well,
waste water for about five minutes before
taking the sample. Sample a well without
a pump by lowering a sterile bottle
attached to a weight. A device which
opened the bottle underneath the water
would avoid contamination by surface
debris.
F Various types of sampling devices are
available where the sample point is in-
accessible or depth samples are desired.
The general problem is to put a sample
bottle in place, open it, close it, and
return it to the surface. No bacteria but
those in the sample must enter the bottle.
1 The J - Z sampler described by Zobell
in 1941,. was designed for deep sea
sampling but is useful elsewhere
(Figure 2). It has a metal frame,
breaking device for a glass tube, and
sample bottle. The heavy metal
messenger strikes the lever arm
which breaks the glass tubing at a file
mark. A bent rubber tube straightens
and the water is drawn in several inches
from the apparatus. Either glass or
collapsible rubber bottles are sample
containers.
Commercial adaptations are available.
7-2
-------
Bacteriological Sampling in the Field
Figure
Reproduced with permission of the Journal of
Marine Research 4:3, 173-188 (1941) by the
Department of Health, Education and Welfare.
Note the vane and lever mechanism on
the New York State Conservation
Department sampler in Figure 3.
When the apparatus is at proper depth
the suspending line is given a sharp
pull. Water inertia against 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 sample bottle.
A commercial sampler is available
which is an evacuated sealed tube with
a capillary tip. When a lever on the
support rack breaks the tip, the tube
Figure 3
fills. Other samplers exist with a
lever for pulling the stopper, while
another uses an electromagnet.
V DATA RECORDING
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 person to the site.
Reference to bridges, roads, 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
inadvisable.
B While a sanitary survey is an indispensable
part of the evaluation of a water supply, its
discussion is not within the scope of this
lecture. The sample collector could supply
much information if desired.
7-3
-------
Bacteriological Sampling in the Field
VI SHIPPING CONDITIONS
A The examination should commence as soon
as possible, preferably within one hour.
A maximum elapsed time between collec-
tion and examination is 30 hours for
potable water samples and 8 hours for
other water samples (collection 6 hours
and laboratory procedures 2 hours).
Standard Methods (13th Edition) recom-
mends icing of samples between collection
and testing.
VII PHOTOGRAPHS
A photograph is a sample in that it is evidence
representing water quality. Sample collectors
and field engineers may carry cameras to
record what they see. Pictures help the
general public and legal courts to better
understand laboratory data.
REFERENCES
1 APHA, AWWA, WPCF, Standard Methods
for the Examination of Water and
Wastewater. (12 Ed.) 1965.
2 Prescott, S. C., Winslow, C.E.A., and
McCrady, M. H. Water Bacteriology.
6th Ed., 368 pp. John Wiley and Sons,
Inc., New York. 1946.
3 Haney, P. D,, and Schmidt, J.
Representative Sampling and Analytical
Methods in Stream Studies. Oxygen
Relationships in Streams, Technical
Report W58-2 pp. 133-42. U.S.
Department of Health, Education and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering Center,
Cincinnati, Ohio. 1958.
Velz, C. J. Sampling for Effective
Evaluation of Stream Pollution. Sewage
and Industrial Wastes, 22:666-84. 1950.
Bathing Water Quality and Health III
Coastal Water. 134pp. U.S.
Department of Health, Education, and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering,
Cincinnati, Ohio. 1961.
Zobell, C. E. Apparatus for Collecting
Water Samples from Different Depths
for Bacteriological Analysis. Journal
of Marine Research, 4:3:173-88. 1941.
This outline was originally prepared by
A. G. Jose, former Microbiologist, FWPCA
Training Activities, SEC and updated by
the Training Staff, National Training Center,
MPOD, WPO, EPA, Cincinnati, Ohio
45268.
Descriptors: Equipment, Microbiology,
Sampling, Water Sampling
7-4
-------
BIOLOGICAL FIELD METHODS
I INTRODUCTION
A Due to the variability in habitats and
modes of living, methods for the collection
of different types of aquatic organisms
differ. In general we can recognize those
that swim or float and them that crawl,
those that are big and those that are little.
Each comprises a part of "the life" at any
given survey station and consequently a
"complete" collection at any given station
would include all types of organisms that
live there.
B Field collecting methods in this outline
are grouped under 4 general categories.
1 Bottom (or benthic organisms)
2 All types of plankton will be considered.
Benthic forms which break off or loose
their hold to a substrate may become
temporarily a part of the plankton.
3 The periphyton or "aufwuchs" com-
munity, which resembles both the
plankton and the benthics.
4 Fishes represent the fourth major group
and need no further qualification.
C Aquatic mammals and birds, in most
cases, require still other approaches and
are not included here.
D A definite policy should be established
as to the size range of organisms to be
collected and counted, i. e.: microscopic
only, microscopic and macroscopic, those
retained by "30 mesh" screens only or
#10 nets, invertebrates and/or vertebrates,
etc.
II Certain Standard Supplementary Procedures
are a Part of All Collecting Techniques
A In order to be interpreted and used, every
collection must be associated with a record
of environmental conditions at the time of
collection.
1 Data recorded should include the
following as far as practicable:
Location
Station number (particular location of
which a full description should be on
record)
Date
Time
Air temperature
Water temperature (at various depths,
if applicable)
Salinity (at various depths, if applicable)
Tidal flow (ebb or flood)
Turbidity (or light penetration, etc.)
Weather
Wind direction and velocity
Sky or cloud cover
Water color
Depth
Type of bottom
Type of collecting device and
accessories
Method of collecting
Type of sample (quantitative or
qualitative)
Number of samples at each station
Chemical data, e.g., dissolved oxygen,
nutrients, etc.
Collector
Miscellaneous observations (often very
important!)
BI.MET.fm.le. 1. 74
8-1
-------
Biological Field Methods
III
2 All collecting containers should be
identified at least with location, station
number, sample number, and date.
Spares are very handy.
3 Much transcription of data can be
eliminated by using sheets or cards
with a uniform arrangement for includ-
ing the above data. The same field data
sheet may include field or laboratory
analysis.
Compact kits of field collecting equipment
and materials greatly increase collecting
efficiency, especially if collection site is
remote from transportation.
COLLECTION OF BOTTOM OR BENTHIC
ORGANISMS
A Shoreline or Wading Depth Collecting
1 Rakes, shovels, dipnets, or dredges
may be used here depending upon type
of bottom, area to be sampled, or
qualitative or quantitative samples.
2 Ekman dredges are best used on soft
bottoms. This dredge is essentially a
completely closing clamshell type
dredge with spring operated jaws. Size
of dredge is usually 6" X 6" or 9" X 9".
3 For samples of hard packed clay or
sand, or gravelly bottoms, or where
the current is moderately swift a
Petersen dredge may be used.
a This consists of two heavily con-
structed half cylinders hinged
together, and closed by a strong
lever action. The area sampled
is commonly 0. lm^.
b To be effective in cutting into hard
bottoms they are built to weigh 30 -
40 Ibs., and may have additional
weights added td bring the total
weight up to 70 Ibs. or more.
c Due to their weight, especially
when loaded with bottom material
they are best handled by a small
winch or crane. However, they can
be hand operated in shallow waters
using a stout line of large enough
diameter to be handled by wet hands.
4 Ooze suckers and core samplers are
other devices for sampling mud bottom
organisms.
5 After bottom samples to obtain marine
organisms are collected, they are best
placed into some type of container and
then washed and grossly screened and
picked in the field, conditions permitting.
a This removes large stones, etc.,
and gives one a quick idea of what
the sample consists of.
b With many bottom samples, weight
becomes a problem. Washing,
therefore, reduces this weight
making it easier to transport the
samples. Also space is saved with
smaller samples.
6 Hand picking of readily visible plants
and animals is valuable for immediate
qualitative description.
a A frame of known dimensions may
be placed over ah area to be sampled
and the material within cropped out.
This is especially good for larger
plants. This method yields quanti-
tative data.
b Underwater swimming or use of
SCUBA is quite valuable for direct
observation and collecting.
B Deep Water Benthic Collecting
1 When sampling from vessels, a crane
and winch, either hand or power operated,
is used. The Petersen dredge is com-
monly employed. SCUBA diving is used
also. The general ideas described for
shallow waters when applicable, apply
to deeper waters.
2 Since most biological populations are
not normally distributed, but rather
8-2
-------
Biological Field Methods
IV
some form of log normal distribution
occurs, it is most always best to take
2 but preferably 3 or more bottom
samples at a station in order to get a
true estimate of the population.
Besides the types of bottom collecting
gear mentioned here, there are many more
types available, such as the orange-peel
bucket, the Shipdek sampler, the plow
dredge, scallop type dredge, etc. Each
of these has its own advantages and dis-
advantages and it is up to the worker and
his operation to decide what is best for his
particular needs. The Petersen and
Eckman dredges are perhaps the most
commonly used.
THE COLLECTION OR SAMPLING OF
PLANKTON
A Phytoplankton - Planned Program
1 A planned program of analysis should
involve periodic sampling at biweekly,
weekly or even more frequent intervals.
2 A well-planned study and analysis of the
growth pattern of phytoplankton in one
year can provide a basis for predicting
conditions for the following year since
seasonal patterns tend to be repeated
from year to year.
a Since the seasons and years differ,
the more records accumulated, the
more useful they become.
b As the time for an anticipated bloom
approaches, the frequency of collect-
ing and analyses may be increased.
B Field Equipment
1 Two general aspects of plankton
analysis are commonly recognized:
quantitative and qualitative. Either
approach is useful, a combination is
best.
2 Equipment for collecting samples in the
field is varied.
a A Kemmerer type sampler, a
Nansen Bottle, or some similar
vessel of known volume is suggested
for quantitative sampling.
b Plankton nets selectively concentrate
(depending upon mesh size) the
sample in the act of collecting and
capturing certain larger forms
which escape the bottles. Only the
more elaborate nets are quantitative,
however.
c Other instruments such as the Clark-
Bumpus, or the continuous plankton
recorder may be employed for
special purposes.
d For phytoplankton, #20 or #25 size
nets are commonly used. Usually
a net diameter of 5 - 10 inches is
sufficient.
C Zooplankton Collecting
1 Since zooplankton have the ability to
swim away from water bottles, etc.
nets towed at moderately slow speed
are used for their capture.
a Number 12 nets (aperature size
0.119 mm, 125 meshes 1 inch)
or smaller numbered net sizes
are commonly used. A net diameter
greater than 5" is preferred. Fre-
quently half meter nets or larger
are employed. These may be
equipped with flow measuring de-
vices for measuring the amount of
water entering the net.
b Other instruments such as the Clark-
Bumpus, Gulf-Stream, Hardy
continuous plankton recorder, and
high-speed instruments are used for
collecting zooplankton, also..
c The devices used for collecting
plankton capture both the plant and
animal types. The mesh size (net
no.) is a method for selecting which
category of plankton is to be collected.
8-3
-------
Biological Field Methods
2 Plankton is subjected to the force of
the winds and currents. As a result,.
the plankton is often in patches or
"wind rows. " For this reason when
using a net, it is often desirable to
collect at right angles to the wind or
current.
3 Not only do plankton occur horizontally
in patches, but vertically as well.
Zooplankton, especially are very
numerous near the bottom in daylight.
Therefore, a series of tows at different
depths is necessary to obtain a com-
plete sampling. One technique often
employed is to take an oblique tow
from the bottom to the top of the water
column.
D The Location of Sampling Points is
Important
1 Both shallow and deep samples are
suggested. Sometimes, the surface
film itself is significant.
2 The number of sampling stations es-
tablished is limited only by the ability
of the laboratory to analyze the samples.
a There should be at least one sample
station near an outfall.
b Every major natural division of
the bay or estuary should be sampled.
c Additional sampling points should be
established on the basis of experience
and resources.
d Continuous sampling, or the com-
positing of periodic samples over a
24-hour period, for example, is also
very useful.
e For plankton, it is necessary to
sample different periods in the
stage of the tide, otherwise samples
would be biased to a given time, or
type of water carried by the tidal
currents.
Pilot studies to indicate sampling
locations and intervals are often
mandatory.
Some.studies require random sam-
pling points.
V COLLECTING PERIPHYTON, ETC.
A Hand-picking of forms attached to rocks,
pilining, etc. is very valuable.
B Microscopic attached forms or micro-
benthos may be sampled by scraping
or otherwise removing all organisms from
a measured surface area.
C Another technique used widely is to suspend
artificial substrates (slides, cement blocks,
wood, rope, plastic, etc.) with clean sur-
faces in the water. After a designated
time interval organisms become attached
which are subsequently removed and
studied.
VI FISH COLLECTING
A Fish must be sought in the obscure and
unlikely areas as well as the obvious
locations in order for the collection to be
complete. Several techniques should be
employed wherever possible (this is
appropriate for all biota). It is advisable
to check with local authorities to inform
them of the reasons for sampling, because
many of the techniques are not legal for
the layman. In this area, perhaps more
than any other, professionally trained
workers are important. Also, there
must be at least one helper, as a single
individual always has difficulty in pulling
both ends of a 20 foot seine simultaneously.
The more common techniques are listed
below.
B Seines
1 Straight seines range from 4-6 feet
and upwards in length. "Common
8-4
-------
Biological Field Methods
sense" minnow seines with approxi-
mately 1/4 inch mesh are widely used
along shore for collecting the' smaller
fishes.
2 Bag seines have an extra trap or bag
tied in the middle which helps trap
and hold fish when seining in difficult
situations.
C Gill nets are of use in offshore and/or
deep waters. They range in length from
approximately 30 yards upward. A mesh
size is designed to catch a specified size
of fish. The trammel net is a variation
of the gill net.
D Traps range from small wire boxes or
cylinders with inverted cone entrances to
Semi permanent weirs a half mile or more
in length. All tend to induce fish to swim
into an inner chamber protected by an
inverted cone or V-shaped notch to pre-
vent escape. Current operated rotating
fish traps are also very effective (and
equally illegal) in suitable situations.
E Trawls are submarine nets, usually of
considerable size, towed by vessels at
speeds sufficient to overtake and scoop
in 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
vanes or "otter boards" (otter trawl).
1 Beam and otter trawls are usually
fished on the bottom, but otter trawls
when suitably rigged are now being
used to fish mid-depths.
2 The Isaacs-Kidd midwater trawl re-
sembles a plankton net 10 - 15 feet in
diameter. It is proving very effective
for collecting at mid-depths.
F Electric seines and screens are widely
employed by fishery workers in small
and difficult streams. They may also be
used in shallow water areas with certain
reservations.
G Poisoning is much used in fishery studies.
and management. Most widely used and
generally satisfactory is rotenone in
varying formulations, although many
others have been employed from time to
time. Under suitable circumstances,
fish may even be killed selectively
according to species.
H Personal observation by competent
personnel, and also informal inquiries
and discussions with local residents will
often yield information of real use. Many
laymen are keen observers, although they
do not always understand what they are
seeing. The organized creel census
technique yields data on what and how
many fish are being caught.
I Angling remains in its own right a very
good technique in the hands of the skilled
practitioner, for determining what fish
are present. Spear-fishing also is now
being used in some studies.
J Fish are often tagged to trace their move-
ments during migration and at other times.
Miniature radio transmitters can now be
attached or fed to fish (and other organ-
isms) which enable them to be tracked
over considerable distances. Physiological
information is often obtained in this way.
This is known as telemetry.
VII DISPOSITION AND PRESERVATION OF
COLLECTIONS
A The organisms may be picked out on the
spot, or the entire mess taken into the
laboratory for further analysis.
B Organisms may be simply counted,
weighed, or measured volumetrically;
usually a combination is best. They may
be separated and recorded in groups or
species.
C Specimens may be simply observed and
recorded or they may be preserved as a
permanent record.
D Preservation - provisions should be made
for the field stabilization of the sample
until the laboratory examination can be
B-5
-------
Biological Field Methods
made if more than an hour or so (especi-
ally in warm weather) is to elapse.
1 Refrigeration or icing is very helpful.
2 Ninety-five per cent ethanol (ethyl
alcohol) is highly satisfactory. A
final strength of 70% is necessary for
prolonged storage.
3 Formaldehyde is more widely available
and is effective in concentrations of
3 - 10% of the commercial formulation.
However, it shrinks and hardens speci-
mens, collector, and laboratory
analyst without favor! In order to
minimize bad effects from formalin,
commercial neutral formaldehyde is
recommended.
a Phytoplankton is best preserved
with a 3% formalin solution.
b Zooplankton, benthos, and fish are
usually preserved in a 5 - 10%
solution.
4 Lugol's solution is good for
phyloplankton.
5 Other formulations are available.
VIII SOURCE OF COLLECTING EQUIPMENT
Many specialized items of biological collect-
ing equipment are not available from the
usual laboratory supply houses. Conse-
quently, the American Society of Limnology
and Oceanography has compiled a list of
companies handling such items and released
it as: "Special Publication No. 1, Sources
of Limnological Apparatus and Supplies."
This may be obtained from the Secretary of
the Society for $1. 00.
REFERENCES
1 Barnes, H. (ed.). Symposium on New
Advances in Underwater Observations.
Brit. Assoc. Adv. Sci., Liverpool.
pp. 49-64. 1953.
2 Hedgepeth, Joel W. Obtaining Ecological
Data in the Sea Chapter 4 in "Treatise
on Marine Ecology and Paleoecology."
Memoir 67. Geol. Soc. Am. 1963.
3 Isaacs, John D. and Columbus, O. D.
Oceanographic Instrumentation NCR
Div. Phys. Sci. Publ. 309, 1954.
233 pp.
4 Sverdrup, H.U. et al. Observations and
Collections at Sea. Chapter X in: The
Oceans, Their Physics, Chemistry, and
Biology. Prentice-Hall, Inc., N.Y.
1942. 1087 pp.
5 Welsh, Paul S. Limnological Methods.
The Blakiston Co. Pa. 1948.
8-6
-------
THE AQUATIC ENVIRONMENT
Part 1:' The Nature and Behavior of Water
I INTRODUCTION
The earth is physically divisible into the
lithosphere or land masses, and the
hydrosphere which includes the oceans,
lakes, streams, and subterranean waters.
A Upon the hydrospere are based a number
of sciences which represent different
approaches. Hydrology is the general
science of water itself with its various
special fields such as hydrography,
hydraulics, etc. These in turn merge
into physical chemistry and chemistry.
B Limnology and oceanography combine
aspects of all of these, and deal not only
with the physical liquid water and its
various naturally occurring solutions and
forms, but also with living organisms
and the infinite interactions that occur
between them and their environment.
C Water quality management, including
pollution control, thus looks to all
branches of aquatic science in efforts
to coordinate and improve man's
relationship with his aquatic environment.
H SOME FACTS ABOUT WATER
A Water is the only abundant liquid on our
planet. It has many properties most
unusual for liquids, upon which depend
most of the familiar aspects of the world
about us as we know it. (See Table 1)
TABLE 1
UNIQUE PROPERTIES OF WATER
Property
Significance
Highest heat capacity (specific heat) of any
solid or liquid (except NH,)
Stabilizes temperatures of organisms and
geographical regions
Highest latent heat of fusion (except NH,)
Thermostatlc effect at freezing point
Highest heat of evaporation of any substance
Important In heat and water transfer of
atmosphere
The only substance that has Its maximum
density as a liquid <4<>C)
Fresh and brackish waters have maximum
density above freezing point. This is
Important In vertical circulation pattern
In lakes.
Highest surface tension of any liquid
Controls surface and drop phenomena,
important in cellular physiology
Dissolves more substances in greater
quantity than any other liquid
Makes complex biological system possible.
Important for transportation of materials
in solution.
Pure water has the highest dl-electric
constant of any liquid
Leads to high dissociation of inorganic
substances In solution
Very little electrolytic dissociation
Neutral, yet contains both H+ and OH ions
Relatively transparent
Absorbs much energy In Infra red and ultra
violet ranges, but little In visible range.
Hence "colorless"
BI. 2le.l.74
9-1
-------
The Aquatic Environment
B Physical Factors of Significance
1 Water substance
Water is not simply "H-O" but in
reality is a mixture of some 33
different substances involving three
isotopes each of hydrogen and oxygen
(ordinary hydrogen H1, deuterium H2,
and tritium H3; ordinary oxygen O1",
oxygen 17, and oxygen 18) plus 15
known types of ions. The molecules
of a water mass tend to associate
themselves as polymers rather than
to remain as discrete units.
(See Figure 1)
SUBSTANCE OF PURE WATER
TABLE 2
EFFECTS OF TEMPERATURE ON DENSITY
OF PURE WATER AND ICE*
Temperature (°C) Density
Water Ice**
-10
- 8
- 6
- .4
- 2
0
2
4
6
8
10
20
40
60
80
100
.99815
.99869
.99912
.99945
.99970
.99987
.99997
1.00000
.99997
.99988
.99973
.99823
.99225
.98324
.97183
.95838
.9397
.9360
.9020
.9277
.9229
.9168
Figure 1
* -Tabular values for density, etc., represent
estimates by various workers rather than
absolute values, due to the variability of
water.
** Regular ice is known as "ice I". Four or
more other "forms" of ice are known to
exist (ice II, ice III, etc.), having densities
at 1 atm. pressure ranging from 1.1595
to 1.67. These are of extremely restricted
occurrence and may be ignored in most
routine operations.
2 Density
a Temperature and density: Ice.
Water is the only known substance
in which the solid state will float
on the liquid state. (See Table 2)
This ensures that ice usually
forms on top of a body of water
and tends to insulate the remain-
ing water mass from further loss
of heat. Did ice sink, there
could be little or no carryover of
aquatic life from season to season
in the higher latitudes. Frazil or
needle ice forms colloidally at a
few thousandths of a degree
below 0° c. It is adhesive and
may build up on submerged objects
as "anchor ice", but it is still
typical ice (ice I).
9-2
-------
The Aquatic Environment
1) Seasonal increase in solar
radiation annually warms
surface waters in summer
while other factors result in
winter cooling. The density
differences resulting establish
two classic layers: the epilimnion**"
or surface laver, and the
hypolimnion*or lower layer, and
in between is the thermocline *x^
or shear-plane.
2) While for certain theoretical
purposes a "thermocline" is
defined as a zone in which the
temperature changes one
degree centigrade for each
meter of depth, in practice,
any transitional layer between
two relatively stable masses
of water of different temper-
atures may be regarded as a
thermocline.
3) Obviously the greater the
temperature differences
between epilimnion and
hypolimnion and the sharper
the gradient in the thermocline,
the more stable will the
situation be.
4) From information given above,
it should be evident that while
the temperature of the
hypolimnion rarely drops
much below 4° C, the
epilimnion may range from
0° C upward.
5) When epilimnion and hypolimnion
achieve the same temperature,
stratification no longer exists.
The entire body of water behaves
hydrologically as a unit, and
tends to assume 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.
Mineral-rich water from the
hypolimnion, for example,
is mixed with oxygenated
water from the epilimnion.
This usually triggers a
sudden growth or "bloom"
of plankton organisms.
6) When stratification is present,
however, each layer behaves
relatively independently, and
significant quality differences
may develop.
7) Thermal stratification as
described above has no
reference to the size of the
water mass; 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 O16 tends to go off
first in the process of evaporation,
leading to the relative enrichment
of air by O16 and the enrichment
of water by O17 and O18. This
can lead to a measurably higher
OIQ content in warmer climates.
Also, the temperature of water
in past geologic ages can be
closely estimated from the ratio
°f O^g in the carbonate of mollusc
shells.
c Dissolved and/or suspended solids
may also affect the density of
natural water masses (see Table 3)
TABLE 3
EFFECTS OF DISSOLVED SOLIDS
ON DENSITY
Dissolved Solids
(Grams per liter)
0
1
2
3
10
Density
(at 40 c)
1.00000
1.00085
1.00169
1.00251
1.00818
35 (mean for sea water)
1.02822
9-3
-------
The Aquatic Environment
d Types of density stratification
1) Density differences produce
stratification which may be
permanent, transient, or
seasonal.
2) Permanent stratification
exists for example where
there is a heavy mass of
brine in the deeper areas of
a basin which does not respond
to seasonal or other changing
conditions.
3) Transient stratification may
occur with the recurrent
influx of tidal water in an
estuary for example, or the
occasional influx of cold
muddy water into a deep lake
or reservoir.
4) Seasonal stratification is
typically thermal in nature,
and involves the annual
establishment of the epilimnion,
hypolimnion, and the r mo dine
as described above.
5) Density stratification is not
limited to two-layered systems;
three, four, or even more
layers may be encountered in
larger bodies of water.
e A "plunge line" (sometimes called
"thermal line") may develop at
the mouth of a stream. Heavier
water flowing into a lake or
reservoir plunges below the
lighter water mass of the epiliminium
to flow along at a lower level. Such
a line is usually marked by an
accumulation of floating debris.
f Stratification may be modified
or entirely suppressed in some
cases when deemed expedient, by
means of a simple air lift.
The viscosity of water is greater at
lower temperatures (see Table 4).
This is important not only in situations
involving the control of flowing water
as in a sand filter, but also since
overcoming resistance to flow gen-
erates heat, it is significant in the
heating of water by internal friction
from wave and current action.
Living organisms more easily support
themselves in the more viscous
(and also denser) cold waters of the
arctic than in the less viscous warm
waters of the tropics. (See Table 4).
TABLE 4
VISCOSITY OF WATER (In millipoises at 1 atm)
Temp, o C
-in
- 5
0
5
10
30
100
Dissolved solids in g/L
0
oc n
21.4
17.94
15.19
13.10
8.00
2.84
5
18.1
15.3
13.2
8.1
10
18.24
15.5
13.4
8.2
30
18.7
16.0
13.8
8.6
----
4 Surface tension has biological as well
as physical significance. Organisms
whose body surfaces cannot be wet by
water can either ride on the surface
film or in some instances may be
"trapped" on the surface film and be
unable to re-enter the water.
5 Heat or energy
Incident solar radiation is the prime
source of energy for virtually all
organic and most inorganic processes
on earth. For the earth as a whole,
the total amount (of energy) received
annually must exactly balance that
lost by reflection and radiation into
space if climatic and related con-
ditions are to remain relatively
constant over geologic time.
9-4
-------
The Aquatic Environment
a For a given body of water,
immediate sources of energy
include in addition to solar
irradiation: terrestrial heat,
transformation of kinetic energy
(wave and current action) to heat,
chemical and biochemical
reactions, convection from the
atmosphere, and condensation of
water vapor.
b The proportion of light reflected
depends on the angle of incidence,
the temperature, color, and other
qualities of the water; and the
presence or absence of films
of lighter liquids such as oil.
In general, as 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
upper layers.
6 Water movements
a Waves or rhythmic movement
1) The best known are traveling
waves caused by wind. These are
effective only against objects near
the surface. They have little
effect on the movement of large
masses of water.
2) Seiches
Standing waves or seiches occur
in lakes, estuaries, and other
enclosed 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 compensating oscillation in the
overlying water so that no
significant change in surface
level is detected. Shifts in
submerged water masses of
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 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 shores around the world.
2) While part and parcel of the
same phenomenon, it is often
convenient to refer to the rise
and fall of the water level as
"tide, " and to the resulting
currents as "tidal currents. "
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 action are enormously
complicated by local topography,
interaction with seiches, and other
factors. The literature on tides
is very large.
c Currents (except tidal currents)
are steady arythmic water movements
which have had major study only in
oceanography although they are
most often observed in rivers and
streams. They are primarily
concerned with the translocation of
water masses. They may be generated
internally by virtue of density changes,
or externally by wind or terrestrial
topography. 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 laminar flow.
9-5
-------
The Aquatic Environment
d Coriolis force is a result of inter-
action between the rotation of the
earth, .and the movement of masses •
or bodies on the earth. The net
result is a slight tendency for moving
objects to veer to the right in the
northern hemisphere, and to the
left in the southern hemisphere.
While the result in fresh waters is
usually negligible, it may be con-
siderable in marine waters. For
example, other factors permitting,
there is a tendency in estuaries for
fresh waters to move toward the
ocean faster along the right bank,
while salt tidal waters tend to
intrude farther inland along the -
left bank. Effects are even more
dramatic in the open oceans.
e Langmuir circulation (or L. spirals)
is the interlocking rotation of
somewhat cylindrical masses of
surface water under the influence
of wind action. The axes of the
cylinders are parallel to the
direction of the wind.
To somewhat oversimplify the
concept, a series of adjoining cells
might be thought of as chains of
interlocking gears in which at every
other contact the teeth are rising
while at the alternate contacts, they
are sinking (Figure 2).
The result is elongated masses of
water rising or sinking together.
This produces the familiar "wind
rows" of foam, flotsam and jetsam,
or plankton often seen streaking
windblown lakes or oceans. Certain
zoo-plankton struggling to maintain
a position near the surface tend to
collect in the down current between
two Langmuir cells, causing such
an area to be called the "red dance",
while the clear upwelling water
between is the "blue dance".
This phenomenon may be important
in water or plankton sampling on
a windy day.
t)
WATER
SURFACE
\
WATER
RISING
WATER
SINKING
Figure 2. Langmuire Spirals
b. Blue dance, water rising, r. Red
dance, water sinking, floating or
swimming objects concentrated.
9-6
-------
The Aquatic Environment
6 The pH of pure water has been deter-
mined between 5. 7 and 7. 01. by various
workers. The latter value is most
widely accepted at the present time.
Natural waters of course vary widely
according to circumstances.
C The elements of hydrology mentioned
above represent a selection of some of
the more conspicuous physical factors
involved in working with water quality.
Other items not specifically mentioned
include: molecular structure of waters,
interaction of water and radiation,
internal pressure, acoustical charac-
teristics, pressure-volume-temperature
relationships, refractivity, luminescence,
color, dielectrical characteristics and
phenomena,' solubility, action and inter-
actions of gases, liquids and solids,
water vapor, phenomena of hydrostatics
and hydrodynamics in general.
REFERENCES
1 Buswell, A.M. and Rodebush, W. H.
Water. Sci. 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
Physical Tables. Smithsonian
Miscellaneous Collection, 71(1),
7th revised ed., 1929.
4 Hutcheson, George E. A Treatise on
Limnology. John Wiley Company.
1957.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.
Descriptors:
Aquatic Environment, Estuarine Environment,
Lentic Environment, Lotic Environment,
Currents, Marshes, Limnology, Water
Properties
9-7
-------
THE AQUATIC ENVIRONMENT
Part 2: The Aquatic Environment as an Ecosystem
I INTRODUCTION
Part 1 introduced the lithosphere and the
hydrosphere. Part 2 will deal with certain
general aspects of the biosphere, or the
sphere of life on this earth, which photo-
graphs from space have shown is a finite
globe in infinite space.
This is the habitat of man and the other
organisms. His relationships with the
aquatic biosphere are our common concern.
II THE BIOLOGICAL NATURE OF THE
WORLD WE LIVE IN
A We can only imagine what this world
must have been like before there was life.
B The world as we know it is largely shaped
by the forces of life.
1 Primitive forms of life created organic
matter and established soil.
2 Plants cover the lands and enormously
influence the forces of erosion.
3 The nature and rate of erosion affect
the redistribution of materials
(and mass) on the surface of the
earth (topographic changes).
4 Organisms tie up vast quantities of
certain chemicals, such as carbon
and oxygen.
5 Respiration of plants and animals
releases carbon dioxide to the
atmosphere in influential quantities.
CO. affects the heat transmission of
the atmosphere.
C Organisms respond to and in turn affect
their environment. Man is one of the
most influential.
HI ECOLOGY IS THE STUDY OF THE
INTERRELATIONSHIPS BETWEEN
ORGANISMS, AND BETWEEN ORGA-
NISMS AND THEIR ENVIRONMENT.
A The ecosystem is the basic functional
unit of ecology. Any area of nature that
includes living organisms and nonliving
substances interacting to produce an
exchange of materials between the living
and nonliving parts constitutes an
ecosystem. (Odum, 1959)
1 From a structural standpoint, it is
convenient to recognize four
constituents as composing an
ecosystem (Figure 1).
a Abiotic NUTRIENT MINERALS
which are the physical stuff of
which living protoplasm will be
synthesized.
b Autotrophic (self-nourishing) or
PRODUCER organisms. These
are largely the green plants
(holophytes), but other minor
groups must also be included
(See Figure 2). They assimilate
the nutrient minerals, by the use
of considerable energy, and combine
them into living organic substance.
c Heterotrophic (other-nourishing)
CONSUMERS (holozoic). are chiefly
the animals. They ingest (or eat)
and digest organic matter, releasing
considerable energy in the process.
d Heterotrophic REDUCERS are chiefly
bacteria and fungi that return
complex organic compounds back to
the original abiotic mineral condition,
thereby releasing the remaining
chemical energy.
From a functional standpoint, an
ecosystem has two parts (Figure 2)
BI. 21e.l.74
9-9
-------
The Aquatic Environment
CO NSUMERS
PRO DUCERS
REDUCERS
NUTRIENT
MINERALS
FIGURE 1
B
a The autotrophic or producer
organisms, which construct
organic substance.
b The heterotrophic or consumer and
reducer organisms which destroy
organic substance.
3 Unless the autotrophic and hetero-
trophic phases of the cycle approximate
a dynamic equilibrium, the ecosystem
and the environment will change.
Each of these groups includes simple,
single- celled representatives, persisting
at lower levels on the evolutionary stems
of the higher organisms. (Figure 2)
1 These groups span the gaps between the
higher kingdoms with a multitude of
transitional forms. They are collectively
caUed the PROTISTA.
2 Within the protista, two principal sub-
groups can be defined on the basis of
relative complexity of structure.
a The bacteria and blue-green algae,
lacking a nuclear membrane may
be considered as the lower protista
(or Monera).
b The single-celled algae and
protozoa are best referred to as
the Higher Protista.
C Distributed throughout these groups will
be found most of the traditional "phyla"
of classic biology.
IV FUNCTIONING OF THE ECOSYSTEM
A A food chain is the transfer of food energy
from plants through a series of organisms
with repeated eating and being eaten.
Food chains are not isolated sequences but
are interconnected.
9-10
-------
The Aquatic Environment
RELATIONSHIPS BETWEEN FREE LIVING AQUATIC ORGANISMS
Energy Flows from Left to Right, General Evolutionary Sequence is Upward
PRODUCERS |
Organic Material Produced,
Usually by Photosynthesis I
CONSUMERS
Organic Material Ingested or
Consumed
Digested Internally
REDUCERS
Organic Material Reduced
by Extracellular Digestion
and Intracellular Metabolism
to Mineral Condition
ENERGY STORED
ENERGY RELEASED
ENERGY RELEASED
Flowering Plants and
Gymnosperms
Club Mosses, Ferns
Liverworts, Mosses
Multicellular Green
Algae
Red Algae
Brown Algae
Arachnids
Insects
Crustaceans
Mammals
Birds
Reptiles
Segmented Worms Amphibians
Molluscs Fishes
Bryozoa
Rotifers
Roundworms
Flat worms
Sponges
Primitive
Chordates
Echinoderms
Coelenterates
Basidiomycetes
Fungi Imperfect!
Ascomycetes
Higher Phycomycetes
DEVELOPMENT OF MULTICELLULAR OR COENOCYTIC STRUCTURE
H I G" H E R P R 0 T I S T A
Unicellular Green Algae
Diatoms
Pigmented Flagellates
Protozoa
Amoeboid Cilliated
Flagellated,
(non-pigmented)
Suctoria
Lower
Phycomycetes
(Chytridiales, et. al. )
DEVELOPMENT OF A NUCLEAR MEMBRANE
1
LOWER PROTISTA
(or: Monera)
Blue Green Algae
Phototropic Bacteria
Chemotropic Bacteria
Actinomycetes
Spirochaetes
Saprophytic
Bacterial
Types
BI.ECO.pl. 2a. 1. 69
FIGURE 2
9-11
-------
The Aquatic Environment
B A food web is the interlocking pattern of
food chains in an ecosystem. (Figures 3,4)
In complex natural communities, organisms
whose food is obtained by the same number
of steps are said to belong to the same
trophic (feeding) level.
C Trophic Levels
1 First - Green plants (producers)
(Figure 5) fix biochemical energy and
synthesize basic organic substances.
This is 'primary production .
2 Second - Plant eating animals (herbivores)
depend on the producer organisms for
food.
3 Third - Primary carnivores, animals
which feed on herbivores.
4 Fourth - Secondary carnivores feed on
primary carnivores.
5 Last - Ultimate carnivores are the last
or ultimate level of consumers.
D Total Assimilation
The amount of energy which flows through
a trophic level is distributed between the
production of biomass (living substance),
and the demands of respiration (internal
energy use by living organisms) in a ratio
of approximately 1:10.
E Trophic Structure of the Ecosystem
The interaction of the food chain
phenomena (with energy loss at each
transfer) results in various communities
having definite trophic structure or energy
levels. Trophic structure may be
measured and described either in terms
of the standing crop per unit area or in
terms of energy fixed per unit area per
unit time at successive trophic levels.
Trophic structure and function can be
shown graphically by means of ecological
pyramids (Figure 5).
Figure 3. Diagram rf the pond ecosystem. Basic units are as follows: I, abiotic substances-basic inorganic and
organic compounds; HA, producers—rooted vegetation; IIB, producers—phytoplankton; III-1A, primary consumers
(herbivores)—bottom forms; Ill-IB, primary consumers (herbivores)— zooplanlcton; III-2, secondary consumers (car-
nivorej); III-3, tertiary consumers (secondary carnivores); IV, decomposers-bacteria and fungi of decay.
9-12
-------
The Aquatic Environment
u-
.-••/; •; Light ..•;-••
Figure 4. A MARINE ECOSYSTEM (After Clark, 1954 and Patten, 1966)
9-13
-------
The Aquatic Environment
(a)
Decomposers 11
r1
1
Carnivores (Secondar
Carnivores (Primary
1 herbivores
Producers
(b)
1
1
(c)
Pn
..wmi
?{]// / / / / /
l///)v / 1 1 1 1 1 / 1
I
I
rf\
/ i i i\
i / i i / iii
Figure 5. HYPOTHETICAL PYRAMIDS of
(a) Numbers of individuals, (b) Biomass, and
(c) Energy (Shading Indicates Energy Loss).
V BIOTIC COMMUNITIES
A Plankton are the macroscopic and
microscopic animals, plants, bacteria,
etc., floating free in the open water.
Many clog filters, cause tastes, odors,
and other troubles in water supplies.
Eggs and larvae of larger forms are
often present.
1 Phytoplankton are plant-like. These
are the dominant producers of the
waters, fresh and salt, "the grass
of the seas".
2 Zooplankton are animal-like.
Includes many different animal types,
range in size from minute protozoa
to gigantic marine jellyfishes.
B Periphyton (or Aufwuchs) - The communities
of microscopic organisms associated with
submerged surfaces of any type or depth.
Includes bacteria, algae, protozoa, and
other microscopic animals, and often the
young or embryonic stages of algae and
other organisms that normally grow up
to become a part of the benthos (see below).
Many planktonic types will also adhere
to surfaces as periphyton, and some
typical periphyton may break off and
be collected as plankters.
C Benthos are the plants and animals living
on, in, or closely associated with the
bottom. They include plants and
invertebrates.
D Nekton are the community of strong
aggressive swimmers of the open waters,
often called pellagic. Certain fishes,
whales, and invertebrates such as
shrimps and squids are included here.
E The marsh community is based on larger
"'higher" plants, floating and emergent.
Both marine and freshwater marshes are
areas of enormous biological production.
Collectively known as "wetlands", they
bridge the gap between the waters and the
dry lands.
VI PRODUCTIVITY
A The biological resultant of all physical
and chemical factors in the quantity of
life that may actually be present. The
ability to produce this "biomass" is
often referred to as the "productivity"
of a body of water. This is neither good
nor bad per se. A water of low pro-
ductivity is a "poor" water biologically,
and also a relatively "pure" or "clean"
water; hence desirable as a water supply
or a bathing beach. A productive water
on the other hand may be a nuisance to
man or highly desirable. It is a nuisance
if foul odors and/or weed-chocked
waterways result, it is desirable if
bumper crops of bass, catfish, or
oysters are produced. Open oceans have
a low level of productivity in general.
9-14
-------
The Aquatic Environment
REFERENCES
1 Clarke, G. L. Elements of Ecology.
John Wiley & Sons, New York. 1954.
2 Cooke, W. B. Trickling Filter Ecology.
Ecology 40(2):273-291. 1959.
3 Hanson, E.D. Animal Diversity.
Prentice-Hall, Inc., New Jersey. 1964.
4 Hedgpeth, J.W. Aspects of the Estuarine
Ecosystem Amer. Fish. Soc., Spec.
Publ. No. 3. 1966.
5 Odum, E.P. Fundamentals of Ecology,
W. B. Saunders Company,
Philadelphia and London. 1959.
6 Patten, B.C. Systems Ecology.
Bio-Science. 16(9). 1966.
7 Whittaker, R.H. New Concepts of
Kingdoms. Science 163:150-160. 1969.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA,
Cincinnati, OH 45268.
Descriptors:
Aquatic Environment, Estuarine Environment,
Lentic Environment, Lotic Environment,
Currents, Marshes, Limnology, Water Properties
9-15
-------
THE AQUATIC ENVIRONMENT
Part 3. The Freshwater Environment
I INTRODUCTION
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 alkaline lakes, springs, etc., are not
treated, as the main objective here in typical
inland water.
All waters have certain basic biological cycles
and types of interactions most of which have
already been presented, hence this outline
will concentrate on aspects essentially
peculiar to fresh inland waters.
n PRESENT WATER QUALITY AS A
FUNCTION OF THE EVOLUTION OF
FRESH WATERS
A The history of a body of water determines
its present condition. Natural waters have
evolved in the course of geologic time
into what we know today.
B Streams
In the course of their evolution, streams
in general pass through four stages of
development which may be called: birth,
youth, maturity, and old age.
These terms or conditions may be
employed or considered in two contexts:
temporal, or spatial. In terms of geologic
time, a given point in a stream may pass
through each of the stages described 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.
1 Establishment or birth. This
might be a "dry run" or headwater
stream-bed, before it had eroded
down to the level of ground water.
During periods of run- off after a
rain or snow-melt, such a gulley
would have a flow of water which
might range from torrential to a
mere trickle. Erosion may proceed
rapidly 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.
Yout hful streams 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 usually 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 are at a peak of biological
productivity. Gradients are moderate,
riffles or rapids are often separated
by long pools.
4 In old age, streams have approached
geologic 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 flat. During normal flow
the channel is refilled and many
shifting bars are developed.
BI.21e. 1.74
9-17
-------
The Aquatic Environment
(Under the influence of man this
pattern may be broken up, or
temporarily interrupted. Thus an '
essentially "youthful" stream might
take on some of the characteristics
of a "mature" stream following soil
erosion, drganic enrichment, and
increased surface runoff. Correction
of these conditions might likewise be
followed by at least a partial reversion
to the "original" condition).
C Lakes and Reservoirs
Geological factors which significantly
affect the nature of either a stream or
lake include the following:
1 The geographical location of the
drainage basin or watershed.
2 The size and shape of the drainage
basin.
3 The general topography, i.e.,
mountainous or plains.
4 The character of the bedrocks and
soils.
5 The character, amount, annual
distribution, and rate of precipitation.
6 The natural vegetative cover of the
land, is, of course, responsive to and
responsible for many of the above
factors and is also severely subject
to the whims of civilization. This
is one of the major factors determining
run-off versus soil absorption, etc.
D Lakes have a developmental history which
somewhat parallels that of streams. This
process is often referred to as natural
eutrophication.
1 The methods of formation vary greatly,
but all influence the character and
subsequent history of the lake.
In glaciated areas, for example, a
huge block of ice may have been covered
with till. The glacier retreated, the
ice melted, ahd the resulting hole
9-18
became a lake. Or, the glacier may
actually scoop out a hole. Landslides
may dam valleys, extinct volcanoes may
collapse, etc., etc.
2 Maturing or natural eutrophication of
lakes.
a If not already present shoal areas
are developed through erosion
and deposition of the shore material
by wave action and undertow.
b Currents produce bars across bays
and thus cut off irregular areas.
c Silt brought in by tributary streams
settles out in the quiet lake water
d Algae grow attached to surfaces,
and floating free as plankton. Dead
organic matter begins to accumulate
on the bottom.
e Rooted aquatic plants grow on
shoals and bars, and in doing so
cut off bays and contribute to the
filling of the lake.
f Dissolved carbonates and other
materials are precipitated in the
deeper portions of the lake in part
through the action of plants.
g When filling is well advanced,
mats of sphagnum moss may extend
outward from the shore. These
mats are followed by sedges and
grasses which finally convert the
lake into a marsh.
3 Extinction of lakes. After lakes reach
maturity, their progress toward
filling up is accelerated. They become
extinct through:
a The downcutting of the outlet.
b Filling with detritus eroded from
the shores or brought in by
tributary streams.
c Filling by the accumulation of the
remains of vegetable materials
growing in the lake itself.
(Often two or three processes may
act concurrently)
-------
The Aquatic Environment
III PRODUCTIVITY IN FRESH WATERS
A Fresh waters in general and under
natural conditions by definition have a
lesser supply of dissolved substances
than marine waters, and thus 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 extraneous materials
(pollutants, nutrients, etc.) The
following notes are directed toward
natural geological and other environ-
mental factors as they affect the
productivity of fresh waters.
B Factors Affecting Stream Productivity
(See Table 1)
TABLE 1
EFFECT OF SUBSTRATE ON STREAM
PRODUCTIVITY*
(The productivity of sand bottoms is
taken as 1)
Bottom Material
Sand
Marl
Fine Gravel
Gravel and silt
Coarse gravel
Moss on fine gravel
Fissidens (moss) on coarse
; gravel
Ranunculus (water buttercup)
Watercress
Anacharis (waterweed)
Relative
Productivity
1
6
9
14
32
89
111
194
301
452
*Selected from Tarzwell 1937
To be 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. Temperatures in moun-
tainous regions are usually low, and
due to the steep gradient, time for
growth is short. Although ample
light is available, growth of true
plankton is thus greatly limited.
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
penetration and consequently the
general plankton production would
diminish.
3 As the stream approaches base level
(old age) and the time available for
plankton growth increases, the
balance between turbidity, nutrient
levels, and temperature and other
seasonal conditions, determines the
overall productivity.
C Factors Affecting the Productivity of
lakes (See Table 2)
1 The size, shape, and depth of the
lake basin. Shallow water is more
productive than deeper water since
more light will reach the bottom to
stimulate rooted plant growth. As
a corollary, lakes with more shore-
line, having more shallow water,
are in general more productive.
Broad shallow lakes and reservoirs
have the greatest production potential
(and hence should be avoided for
water supplies).
TABLE 2
EFFECT OF SUBSTRATE
ON LAKE PRODUCTIVITY *
(The productivity of sand bottoms is taken as 1)
Bottom Material
Sand
Pebbles
Clay
Flat rubble
Block rubble
Shelving rock
Relative Productivity
1
4
8
9
11
77
* Selected from Tarzwell 1937
9-19
-------
The Aquatic Environment
2 Hard waters are generally more
productive than soft waters as there
are more plant nutrient minerals
available. This is often greatly in-
fluenced by the character of the soil
and rocks in the watershed and the
quality an'd quantity of ground water
entering the lake. In general, pH
ranges of 6. 8 to 8.2 appear to be
most productive.
3 Turbidity reduces productivity as
light penetration is reduced.
4 The presence or absence of thermal
stratification with its semi-annual
turnovers affects productivity by
distributing nutrients throughout the
water mass.
5 Climate, temperature, prevalence of
ice and snow, are also of course
important.
Factors Affecting the Productivity of
Reservoirs
1 The productivity of reservoirs is
governed by much the same principles
as that of lakes, with the difference
that the water level is much more
under the control of man. Fluctuations
in water level can be used to de-
liberately increase or decrease
productivity. This can be demonstrated
by a comparison of the TVA reservoirs
which practice a summer drawdown
with some of those in the west where
a winter drawdown is the rule.
2 The level at which water is removed
from a reservoir is important to the
productivity of the stream below.
The hypolimnion may be anaerobic
while the epilimnion is aerobic, for
example, or the epilimnion is poor in
nutrients while the hypolimnion is
relatively rich.
3 Reservoir discharges also profoundly
affect the DO, temperature, and
turbidity in the stream below a dam.
Too much fluctuation in flow may
permit sections of the stream to dry,
or provide inadequate dilution for
toxic waste.
IV CULTURAL EUTROPHICATION
A The general processes of natural
eutrophication, or natural enrichment
and productivity have been briefly out-
lined above.
B When the activities of man speed up
these enrichment processes by intro-
ducing unnatural quantities of nutrients
(sewage, etc.) the result is often called
cultural eutrophication. This term is
often extended beyond its original usage
to include the enrichment (pollution) of
streams, estuaries, and even oceans, as
well as lakes.
V CLASSIFICATION OF LAKES AND
RESERVOIRS
A The productivity of lakes and impound-
ments is such a conspicuous feature that
it is often used as a convenient means of
classification.
1 Oligotrophic lakes are the younger,
less productive lakes, which are deep,
have clear water, and usually support
Salmonoid fishes in their deeper waters.
2 Eutrophic lakes are more mature,
more turbid, and'richer. They are
usually shallower. They are richer
in dissolved solids; N, P, and Ca are
abundant. Plankton is abundant and
there is often a rich bottom fauna.
3 Dystrophic lakes, such as bog lakes,
are low in Ph, water yellow to brown,
dissolved solids, N, P, and Ca scanty
but humic materials abundant, bottom
fauna and plankton poor, and fish
species are limited.
B Reservoirs may also be classified as
storage, and run of the river.
1 Storage reservoirs have a large
volume in relation to their inflow.
2 Run of the river reservoirs have a
large flow-through in relation to their
storage value.
9-20
-------
The Aquatic Environment
According to location, lakes and
reservoirs may be classified as polar,
temperate, or tropical. Differences in
climatic and geographic conditions
result in differences in their biology.
VI SUMMARY
A A body of water such as a lake, stream,
or estuary represents an intricately
balanced system in a state of dynamic
equilibrium. Modification imposed at
one point in the system automatically
results in compensatory adjustments at
associated points.
B The more thorough our knowledge of the
entire system, the better we can judge
where to impose control measures to
achieve a desired result.
6 Tarzwell, Clarence M. Experimental
Evidence on the Value of Trout 1937
Stream Improvement in Michigan.
American Fisheries Society Trans.
66:177-187. 1936.
7 U. S. Dept. of Health, Education, and
Welfare. Public Health Service.
Algae and Metropolitan 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 Whipple. Fresh Water
Biology. (Introduction). John
Wiley Company. 1918.
REFERENCES
1 Chamberlin, Thomas C. and Salisburg,
Rollin P. Geological Processes and
Their Results. Geology 1: pp i-xix,
and. 1-654. Henry Holt 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 Vol. I Geography, Physics
and Chemistry. 1957. Vol. II.
Introduction to Lake Biology and the
Limnoplankton. 1115 pp. 1967.
John Wiley Co.
4 Hynes, H.B.N. The Ecology of Running
Waters. Univ. Toronto Press.
555 pp. 1970.
5 Ruttner, Franz. Fundamentals of
Limnology. University of Toronto
Press, pp. 1-242. 1953.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.
Descriptors:
Aquatic Environment, Estuarine Environment,
Lentic Environment, Lotic Environment,
Currents, Marshes, Limnology, Water
Properties
9-21
-------
THE AQUATIC ENVIRONMENT
Part 4. The Marine Environment and its Role in the Total Aquatic Environment
TABLE 1
I INTRODUCTION
A The marine environment is arbitrarily
defined as the water mass extending
beyond the continental land masses,
including the plants and animals harbored
therein. This water mass is large and
deep, covering about 70 percent of the
earth's surface and being as deep as
7 miles. The salt content averages
about 35 parts per thousand. Life extends
to all depths.
B The general nature of the water cycle on
earth is well known. Because the largest
portion of the surface area of the earth
is covered with water, roughly 70 percent
of the earth's rainfall is on the seas.
(Figure 1)
100* Oceanic
Evaporation
iire 1. THE WATER CICLE
Since roughly one third of the
rain which falls on the land is again
recycled through the atmosphere
(see Figure 1 again), the total amount
of water washing over the earth's surface
is significantly greater than one third of
the total world rainfall. It is thus not
surprising to note that the rivers which
finally empty into the sea carry a
disproportionate burden of dissolved and
suspended solids picked up from the land.
The chemical composition of this burden
depends on the composition of the rocks
and soils through which the river flows,
the proximity of an ocean, the direction
of prevailing winds, and other factors.
This is the substance of geological erosion.
(Table 1)
BI.21e.l.74
PERCENTAGE COMPOSITION OF THE MAJOR IONS
OF TWO STREAMS AND SEA WATER
(Data from Clark, F.W., 1924, "The Composition of River
and Lake Waters of the United States", U. S. Geol. Surv.,
Prof. Paper No. 135; Harvey, H.W., 1957, "The Chemistry
and Fertility of Sea Waters", Cambridge University Press,
Cambridge)
Ion
Na
K
Ca
Mg
Cl
so4
C03
Delaware River
at
Lambertville, N. J.
6.70
1.46
17.49
4.81
4.23
17.49
32.95
Rio Grande
at
Laredo, Texas
14.78
.85
13.73
3.03
21.65
30. 10
11.55
Sea Water
30.4
1.1
1. 16
3.7
55.2
7.7
•t-HCO. 0.35
o
C For this presentation, the marine
environment will be (1) described using
an ecological approach, (2) characterized
ecologically by comparing it with fresh-
water and estuarine environments, and
(3) considered as a functional ecological
system (ecosystem).
H FRESHWATER, ESTUARINE, AND
MARINE ENVIRONMENTS
Distinct differences are found in physical,
chemical, and biotic factors in going from
a freshwater to an oceanic environment.
In general, environmental factors are more
constant in freshwater (rivers) and oceanic
environments than in the highly variable
and harsh environments of estuarine and
coastal waters. (Figure 2)
A Physical and Chemical Factors
Rivers, estuaries, and oceans are
compared in Figure 2 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
seasonal variations) change little, the other
four parameters vary considerably. In
estuaries, they all change.
9-23
-------
The Aquatic Environment
Type of environment
and general direction
of water movement
Salinity
Degree of instability
Temperature
Water
elevation
vertical
strati-
fication
Avail-
ability
of
nutrients
(degree)
Turbidity
Riverine
Oceanic
Figure 2 . RELATIVE VALUES OF VARIOUS PHYSICAL AND CHEMICAL FACTORS
FOR RIVER, ESTUARINE, AND OCEANIC ENVIRONMENTS
B Biotic Factors
1 A complex of physical and chemical
factors determine the biotic composi-
tion of an environment. In general,
the number of species in a rigorous,
highly variable environment tends to be
less than the number in a more stable
environment (Hedgpeth, 1966).
2 The dominant animal species (in
terms of total biomass) which occur
in estuaries are often transient,
spending only a part of their lives in
the estuaries. This results in better
utilization of a rich environment.
C Zones of the Sea
The nearshore environment is often
classified in relation to tide level and
water depth. The nearshore and offshore
oceanic regions together, are often
classified with reference to light penetra-
tion and water depth. (Figure 3)
1 Neritic - Relatively shallow-water
zone which extends from the high-
tide mark to the edge of the
continental shelf.
9-24
-------
The Aquatic Environment
MARINE ECOLOGY
TN^i
BENTHIC (Bottom)
Supro-llltorol
lillorol (Inl.ftiJol)
Subliltorol
Innir
Oulfr
AbtlfOl
BENTHIC
FIGURE 3—Classification of marine environments
a Stability of physical factors is
intermediate between estuarine
and oceanic environments.
b Phytoplankters are the dominant
producers but in some locations
attached algae are also important
as producers.
c The animal consumers are
zooplankton, nekton, and benthic
forms.
Oceanic - The region of the ocean
beyond the continental shelf. Divided
into three parts, all relatively
poorly populated compared to the
neritic zone.
a Euphotic zone - Waters into which
sunlight penetrates (often to the
bottom in the neritic zone). The
zone of primary productivity often
extends to 600 feet below the surface.
1) Physical factors fluctuate
less than in the neritic zone.
2) Producers are the phyto-
plankton and consumers are
the zooplankton and nekton.
b Bathyal zone - From the bottom
of the euphotic zone to about
2000 meters.
1) Physical factors relatively
constant but light is absent.
2) Producers are absent and
consumers are scarce.
c Abyssal zone - All the sea below
the bathyal zone.
1) Physical factors more con-
stant than in bathyal zone.
2) Producers absent and consumers
even less abundant than in the
bathyal zone.
9-25
-------
The Aquatic Environment
in SEA WATER AND THE BODY FLUIDS
A Sea water is a remarkably suitable
environment for living cells, as it
contains all of the chemical elements
essential to the growth and maintenance
of plants and animals. The ratio and
often the concentration of the major
salts of sea water are strikingly similar
in the cytoplasm and body fluids of
,marine organisms. This similarity is
also evident, although modified somewhat
in the body fluids of fresh water and
terrestrial animals. For example,
sterile sea water may be used in
emergencies as a substitute for blood
plasma in man.
B Since marine organisms have an internal
salt content similar to that of their
surrounding medium (isotonic condition)
osmoregulation poses no problem. On the
other hand, fresh water organisms are
hypertonic (osmotic pressure of body
fluids is higher than that of the surround-
ing water). Hence, fresh water animals
must constantly expend more energy to
keep water out (i.e., high osmotic
pressure fluids contain more salts, the
action being then to dilute this concen-
tration with more water).
1 Generally, marine invertebrates are
narrowly poikilosmotic, i.e., the salt
concentration of the body fluids changes
with that of the external medium. This
has special^ significance in estuarine
situations where salt concentrations
of the water often vary considerably
in short periods of time.
2 Marine bony fish (teleosts) have lower
salt content internally than the external
environment (hypotonic). In order to
prevent dehydration, water is ingested
and salts are excreted through special
cells in the gills.
IV FACTORS AFFECTING THE DISTRI-
BUTION OF MARINE AND ESTUARINE
ORGANISMS
A Salinity. Salinity is the single most
constant and controlling factor in the
marine environment, probably followed
by temperature. It ranges around
35, 000 mg. per liter, or "35 parts per
thousand" (symbol: 35%0) in the language
of the oceanographer. While variations
in the open ocean are relatively small,
salinity decreases rapidly as one
approaches shore and proceeds through
the estuary and up into fresh water with
a salinity of "0 %0 (see Figure 2)
B Salinity and temperature as limiting
factors in ecological distribution.
1 Organisms differ in the salinities
and temperatures in which they
prefer to live, and in the variabilities
of these parameters which they can
t olerate. These preferences and
tolerances often change with successive
life history stages, and in turn often
dictate where the organisms live:
their "distribution."
2 These requirements or preferences
often lead to extensive migrations
of various species for breeding,
feeding, and growing stages. One
very important result of this is that
an estuarine environment is an
absolute necessity for over half of
all coastal commercial and sport
related species of fishes and invertebrates,
for either all or certain portions of their
life histories. (Part V, figure 8)
3 The Greek word roots "eury"
(meaning wide) and "steno" (meaning
narrow) are customarily combined
with such words as "haline" for salt,
and "thermal" for temperature, to
give us "euryhaline" as an adjective
to characterize an organism able to
tolerate a wide range of salinity, for
example; or "stenothermal" meaning
one which cannot stand much change
in temperature. "Meso-" is a prefix
indicating an intermediate'capacity.
9-26
-------
The Aquatic Environment
C Marine, estuarine, and fresh water
organisms. (See Figure 4)
Fresh Water
Stenohaline
Marine
Stenohaline
Salinity
ca.35
Figure 4. Salinity Tolerance of Organisms
1 Offshore marine organisms are, in
general, both 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 me so- or euryhaline and
meso- or euryt.hermal.
10
Salinity
25 30 35
Figure 5. DISTRIBUTION OF
ORGANISMS IN AN ESTUARY
a Euryhaline, freshwater
b Indigenous, estuarine, (mesohaline)
c Euryhaline, marine
4 Some well known and interesting
examples of migratory species which
change their environmental preferences
with the life history stage include the
shrimp (mentioned 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 return to the stream where they
hatched to lay their own eggs before
dying.
5 Among euryhaline animals landlocked
(trapped), populations living in lowered
salinities often have 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".
Usually the larvae of aquatic organisms
are more sensitive to changes in
salinity 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
of environmental stress encountered
in any aquatic habitat. Highly specialized
communities have developed in this
zone, some adapted to the rocky surf
zones of the open coast, others to the
muddy inlets of protected estuaries.
Tidal reaches of fresh water rivers,
sandy beaches, coral reefs and
mangrove swamps in the tropics; all
have their own floras and faunas. All
must emerge and flourish when whatever
wateir 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 tide interval. Such a
community is depicted in Figure 6.
9-27
-------
The Aquatic Environment
SNAILS
o Littorina neritoides
C" L. rudis
0 L. obtusata
Q L. littorea
BAK.NACLES
® Chthamalus stellatus
® Balanus balanoides
© B. perforatus
rt «'s i'ir f-j.^v.^-'v.-.;^'"?,-.. • „• • \ .',Y;K.>-:.N /..-.."A ••
«^^fp^ ,• 'W^iV.f^^^jc
ivKf "l/^^^X'-^V^^u: :;^^-:-:ir;'J «
6%> ® « cs ^w&^:m$M&;^
^/OT
* J\ <\S'( 1(\'V • (// v» . 0 \'Y A VA-X'x^AXvv
Figure 6
Zonation of plants, snails, and barnacles on a rocky shore. While
this diagram is based on the situation on the southwest coast of
England, the general idea of zonation may be applied to any temper-
ate rocky ocean shore, though the species will differ. The gray
zone consists largely of lichens. At the left is the zonation of rocks
with exposure too extreme to support algae; at the right, on a less
exposed situation, the animals are mostly obscured by the algae.
Figures at the right hand margin refer to the percent of time that
the zone is exposed to the air, i. e., the time that the tide is out.
Three major1 zones can be recognized: the Littorina zone (above the
gray zone); the Balanoid zone (between the gray zone and the
laminarias); and the Laminaria zone. a. Pelvetia canaliculata;
b. P'ucus spiralis; c. Ascophyllum nodosum; d. Fucus serratus;
e. Laminaria digitata. (Based on Stephenson)
9-28
-------
The Aquatic Environment
V FACTORS AFFECTING THE
PRODUCTIVITY OF THE MARINE
ENVIRONMENT
A The sea is in continuous circulation. With-
out circulation, nutrients of the ocean would
eventually become a part of the bottom and
biological production would cease. Generally,
in all oceans there exists a warm surface
layer which overlies the colder water and
forms a two-layer system of persistent
stability. Nutrient concentration is usually
greatest in the lower zone. Wherever a
mixing or disturbance of these two layers
occurs biological production is greatest.
B The estuaries are also a mixing zone of
enormous importance. Here the fertility
washed off the land is mingled with the
nutrient capacity of seawater, and many
of the would1 s most productive waters
result.
C When man adds his cultural contributions
of sewage, fertilizer, silt or toxic waste,
it is no wonder that the dynamic equilibrium
of the ages is rudely upset, and the
environmentalist cries, "See what man
hath wrought"!
ACKNOWLEDGEMENT:
This outline contains selected material
from other outlines prepared by C. M.
Tarzwell, Charles L. Brown, Jr.,
C. G. Gunnerson, W. Lee Trent, W. B.
Cooke, B. H. Ketchum, J. K. McNulty,
J. L. Taylor, R. M. Sinclair, and others.
REFERENCES
1 Harvey, H. W. The Chemistry and
Fertility of Sea Water (2nd Ed.).
Cambridge Univ. Press, New York.
234pp. 1957.
2 Hedgpeth, J. W. (Ed.). Treatise on
Marine Ecology and Paleoecology.
Vol. I. Ecology Mem. 67 Geol.
Soc. Amer., New York. 1296pp.
1957.
3 Hill, M. N. (Ed.). The Sea. Vol. II.
The Composition of Sea Water
Comparative and Descriptive
Oceanography. Interscience Publs.
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 York. 375 pp. 1961.
6 Sverdrup, Johnson, and Fleming.
The Oceans. Prentice-Hall, Inc.,
New York. 1087 pp. 1942.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.
Descriptors:
Aquatic Environment, Estuarine Environment,
Lentic Environment, Lotic Environment,
Currents, Marshes, Limnology, Water Properties
9-29
-------
THE AQUATIC ENVIRONMENT
Part 5: Wetlands
I INTRODUCTION
A Broadly defined, wetlands are areas
which are "to wet to plough but too
B
saturated with water, salt or fresh,
and numerous channels or ponds of
shallow or open water are common.
Due to ecological features too numerous
and variable to list here, they comprise
in general a rigorous (highly stressed)
habitat, occupied by a small relatively
specialized indigenous (native) flora
and fauna.
They are prodigiously productive
however, and many constitute an
absolutely essential habitat for some
portion of the life history of animal
forms generally 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 of the earth1 s surface,
and the total organic carbon bound in
their mass constitutes an enormous
sink of energy.
D Since our main concern here is with
the "aquatic" environment, 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.
II TIDAL MARSHES AND THE ESTUARY •=--=
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 exist between the bordering
marshland, the moving waters, the tidal
flats, subtidal deposition, and seston
whether of local, oceanic, or riverine
origin.
D The tidal marsh (some inland areas also
have salt marshes) is generally considered
to be the marginal areas of estuaries and
coasts in the intertidal zone, which are
dominated by emergent vegetation. They
generally 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.
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)
Figure 1. Zon«lon In a positive New England eatuarjr. 1. Spring tide level. 2. Mean high tide.
1. Mean low tide. 4. Bog hole. 5. Ice cleavage pool. 6. Chunk ot asjtlna turf depo.lled by Ice.
7. Organic ooie with associated community, B. eelgrass (Zoitero). 8. Bibbed rauinll (modlolu.1-
clom Imml - mud snail (Najja) community. 10. Sea lettuce (Uly»)
BI. 21e.l.74
9-31
-------
The Aquatic Environment
III MARSH ORIGINS AND STRUCTURES
A In general, marsh substrates are high" in
organic content, relatively low in minerals
and trace elements. The upper layers
bound together with living roots called
turf, underlaid by more compacted peat
type material.
1 Rising or eroding coastlines may
expose peat from ancient marsh
growth to wave action which cuts
into the soft peat rapidly (Figure 2).
Such banks are likely to be cliff-like,
and are often undercut. Chunks of
peat are often found lying about on
harder substrate below high tide line.
If face of cliff is well above high water,
overlying vegetation is likely to be
typically terrestrial of the area.
Marsh type vegetation is probably
absent.
Low lying deltaic, or sinking coast-
lines, or those with low energy wave
action are likely to have active marsh
formation in progress. Sand dunes
are also common in such areas
(Figure 3). General coastal
configuration is a factor.
Figure 2. Diagrammatic section of eroding peat cliff
MHW-ig 1300tec
MHW tf 19SO J AO
Figure 3
Development of a Massachusetts Marsh since 1300 BC, involving an
18 foot rise in water level. Shaded area indicates sand dunes. Note
meandering marsh tidal drainage. A: 1300 BC. B: 1950 AD.
9-32
-------
The Aquatic Environment
a Rugged or precipitous coasts or
slowly rising coasts, typically
exhibit narrow shelves, sea cliffs,
fjords, massive beaches, and
relatively less marsh area (Figure 4).
An Alaskan fjord subject to recent
catastrophic subsidence and rapid
deposition of glacial flour shows
evidence of the recent encroachment
of saline waters in the presence of
recently buried trees and other
terrestrial vegetation, exposure
of layers of salt marsh peat along
the edges of channels, and a poorly
compacted young marsh turf developing
at the new high water level (Figure 5).
Figure 4 * River Mouth on a Slowly Rising Coast. Note absence
of deltaic development and relatively little marshland,
although mud flats stippled are extensive.
Figure 5 Some general relationships in a northern fjord with a rising water level. 1. mean low
water, 2. maximum high tide, 3. Bedrock, 4. Glacial flour to depths in excess of
400 meters, 5. Shifting flats and channels. 6. Channel against bedrock, 7. Buried
terrestrial vegetation, 8. Outcroppings of salt marsh peat.
Low lying coastal plains tend to be
fringed by barrier islands, broad
estuaries and deltas, and broad
associated marshlands (Figure 3).
Deep tidal channels fan out through
innumerable branching and often
interconnecting rivulets. The
intervening grassy plains are
essentially at mean high tide level.
9-33
-------
The Aquatic Environment
Tropical and subtropical regions
such as Florida, the Gulf Coast,
and Central America, are frequented •
by mangrove swamps. This unique
type of growth is able to establish
itself in shallow water and move out
into progressively deeper areas
(Figure 6). The strong deeply
embedded roots enable the mangrove
to resist considerable wave action
at times, and the tangle of roots
quickly accumulates a deep layer of
organic sediment. Mangroves
in the south may be considered to
be roughly the equivalent of the
Spartina marsh grass in the north
as a land builder. When fully
developed, a mangrove swamp is an
impenetrable thicket of roots over
the tidal flat affording shelter to an
assortment of semi-? aquatic organisms
such as various molluscs and
crustaceans, and providing access
from the nearby land to predaceous
birds, reptiles, and mammals.
Mangroves are not restricted to
estuaries, but may develop out into
shallow oceanic lagoons, or upstream
into relatively fresh waters.
TOPICAL CONOCARPtrt . AVICCNMA
KWtST Winston ISSOCIU SU.MUKM USOCIU
Figure 6 Diagrammatic transect of a mangrove swamp
showing transition from marine to terrestrial
habitat.
tidal marsh is the marsh grass, but very
little of it is used by man as grass.
(Table 1)
The nutritional analysis of several
marsh grasses as compared to dry land
hay is shown in Table 2.
'TABLE 1. General Orders of Magnitude of Gross Primary Productivity in Terms
of Dry Weight of Organic Matter Fixed Annually
Ecosystem
gms/M /year
(grams/square metere/year) Ibs/acre/year
Land deserts* deep oceans Tens
Grasslands, forests, cutrophic Hundreds
lakes, ordinary agriculture
Estuaries, deltas, coral reefs. Thousands
intensive agriculture (sugar
cane, rice)
Hundreds
Thousands
Ten-thousands
TABLE 2. Analyses of Some Tidal Marsh Grasses
T/A
Dry Wl.
Percentage Composition
Protein Fat Fiber Water
Ash
N-lree Extract
DiVicMis spicara (pure stand, dry)
2.6 5.3 1.7 32.4 8.2 6.7 4S.5
Short Spirtina alterniflora and Saficornia curopaea (in standing water)
1.2 7.7 2.5 31.1 8.8 12.0 37.7
Spartr'na altcrniflora (tall, pure stand m standing water)
3.5 1.f> 2.0 29.0 8.3 15.5 37.3
Sparll'na pa":m 'pijr«: slamj, dry)
3.2 ',.0 '2.2 30.0 8.1 9.0 44.5
Spurtina allcrnitlura and Spjrtinn paroni (mixed stand, wet)
3.4 6.8 1.9 29.0 8.1 10.4 42.8
Spjr/fnj attcrnifltira (short, WIM)
2.2 0.0 2.4 30.4 8.7 13.3 36.3
Comparable Analyses for Hay
1%l«iil li.O 2.0 36.2 6.7 4.2 44.9
ftuJiut Tl.U 3.7 21).5 10.4 5.9 30.5
Analyses performed by Roland W. Gilbert, Department
of Agricultural Chemistry, U. R. I.
IV PRODUCTIVITY OF WETLANDS
A Measuring the productivity of grasslands
is not easy, because today grass is seldom
used directly as such by man. It is thus
usually expressed as production of meat,
milk, or in the case of salt marshes, the
total crop of animals that obtain food per
unit of area. The primary producer in a
9-34
-------
The Aquatic Environment
B The actual utilization of marsh grass is
accomplished primarily by its Decom-
position and ingestion by micro organisms.
(Figure 7) A small quantity of seeds and
solids is consumed directly by birds.
Sto
•PAKTIIU— '- * otTHtTOt
Figure 7 The nutritive composition of
successive stages of decomposition of
Spartina marsh grass, showing increase
in protein and decrease in carbohydrate
with increasing age and decreasing size
of detritus particles.
The quantity of micro invertebrates
which thrive on this wealth of decaying
marsh has not been estimated, nor has
the actual production of small indigenous
fishes and invertebrates such as the
top minnows (Fundulus). or the mud
snails (Nassa), and others.
Many forms of oceanic life migrate
into the estuaries, especially the
marsh areas, for important portions
of their life histories as is mentioned
elsewhere (Figure 8). It has been
estimated that in excess of 60% of the
marine commercial and sport fisheries
are estuarine or marsh dependent in
some way.
O0O-
EGGS
Figure 8 Diagram of the life cycle
of white shrimp (after Anderson and
Lunz 1965).
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 uncountable numbers
of seagulls were counted. One food
requirement estimate for three-
pound poultry in the confined inactivity
of a poultry yard is approximately one
ounce per pound of bird per day.
Oreater yellow legs (left)
and black duck
Great blue heron
Figure 9 Some Common Marsh Birds
9-35
-------
The Aquatic Environment
One-hundred black bellied plovers
at approximately ten ounces each
would weigh on the order of sixty '
pounds. At the same rate of food
consumption, this would indicate
nearly four pounds of food required
for this species alone. The much
greater activity of the wild birds
would obviously greatly increase their
food requirements, as would their
relatively smaller size.
Considering the range of foods con-
sumed, the sizes of the birds, and the
fact that at certain seasons, thousands
of migrating ducks and others pause
to feed here, the enormous productivity
of such a marsh can be better under-
stood.
V INLAND BOGS AND MARSHES
A Much of what has been said of tidal
marshes also applies to inland wetlands.
As was mentioned earlier, not all inland
swamps are salt-free, any more than all
marshes affected by tidal rythms are
saline.
B The specificity of specialized floras to
particular types of wetlands is perhaps
more spectacular in freshwater wetlands
than in the marine, where Juncus,
Spartina, and Mangroves tend to dominate.
1 Sphagnum, or peat moss, is
probably one of the most widespead
and abundant wetland plants on earth.
Deevey (1958) quotes an estimate that
there is probably upwards of 223
billions (dry weight) of tons of peat
in the world today, derived during
recent geologic time from Sphagnum
bogs. Particularly in the northern
regions, peat moss tends to overgrow
ponds and shallow depressions, eventually
forming the vast tundra plains and
moores of the north.
2 Long lists of other bog and marsh plants
might be cited, each with its own
special requirements, topographical,
and geographic distribution, etc.
Included would be the familiar cattails,
spike rushes, cotton grasses, sedges,
trefoils, alders, and many, many
others.
C Types of inland wetlands.
1 As noted above (Cf: Figure 1)
tidal marshes often merge into
freshwater marshes and bayous.
Deltaic tidal swamps and marshes
are often saline in the seaward
portion, and fresh in the landward
areas.
2 River bottom wetlands differ from
those formed from lakes, since wide
flood plains subject to periodic
inundation are the final stages of
the erosion of river valleys, whereas
lakes in general tend to be eliminated
by the geologic processes of natural
eutrophication often involving
Sphagnum and peat formation.
Riverbottom marshes in the southern
United States, with favorable climates,
have luxurient growths such as the
canebrake of the lower Mississippi,
or a characteristic timber growth
such as cypress.
3 Although bird life is the most
conspicuous animal element in the
fauna (Cf: Figure 9), many mammals,
such as muskrats, beavers, otters,
and others are also marsh-oriented.
(Figure 12)
Figure 12
9-36
-------
The Aquatic Environment
VI POLLUTION
A No single statement can summarize the
effects of pollution on marshlands as
distinct from effects noted elsewhere on
other habitats.
B Reduction of Primary Productivity
The primary producers in most wetlands
are the grasses and peat mosses.
Production may be reduced or eliminated
by:
1 Changes in the water level brought
about by flooding or drainage.
a Marshland areas are sometimes
diked .and flooded to produce fresh-
water ponds. This may be for
aesthetic reasons, to suppress the
growth of noxious marsh inhabitating
insects such as mosquitoes or biting
midges, to construct an industrial
waste holding pond, a thermal or a
sewage stabilization pond, a
"convenient" 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 may or may not develop.
b High tidal marshes were often
ditched and drained in former days
to stabilize the sod for salt hay or
"thatch" harvesting which was highly
sought after in colonial days. This
inevitably changed the character
of the marsh, but it remained as
essentially marshland. Conversion
to outright agricultural land has
been less widespread because of the
necessity of diking to exclude the
periodic floods or tidal incursions,
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.
2 Marsh grasses may also be eliminated
by smothering as, for example, by
deposition of dredge spoils, or the
spill or discharge of sewage sludge.
3 Considerable marsh area has been
eliminated by industrial construction
activity such as wharf and dock con-
struction, oil well construction and
operation, and the discharge of toxic
brines and other chemicals.
Consumer production (animal life) has
been drastically reduced by the deliberate
distribution of pesticides. In some cases,
this has been aimed at nearby agricultural
lands for economic crop pest control, in
other cases the marshes have been sprayed
or dusted directly to control noxious
insects.
1 The results have been universally
disastrous for the marshes, and the
benefits to the human community often
questionable.
2 Pesticides designed to kill nuisance
insects, are also toxic to other
arthropods so that in addition to the
target species, such 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,
without seeing a single live crab.
3 DDT and related compounds have been
"eaten up the food chain" (biological
magnification 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.
9-37
-------
The Aquatic Environment
D Most serious of the marsh enemies is
man himself. In his quest for "lebensraum"
near the water, he has all but killed the
water he strives to approach. Thus up to
twenty percent of the marsh—estuarine
area in various parts of the country has
already been utterly destroyed by cut and
fill real estate developments (Figures
10, 11).
E Swimming birds such as ducks, loons,
cormorants, pelicans, and many others
are severely jeopardized by floating
pollutants such as oil.
Figure 10. Diagrammatic representation of cut-and-fill for
real estate development, mlw = mean low water
Figure 11. Tracing of portion of map of a southern
city showing extent of cut-and-fill real
estate development.
9-38
-------
The Aquatic Environment
VII SUMMARY
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 to aquatic life of
all types living in the open waters. They
are essential as habitat for all forms of
wildlife.
B The tidal marsh is the area of emergent
vegetation bordering the ocean or an
estuary.
C Marshes are highly productive areas,
essential to the maintenance of a well
rounded community of aquatic life.-
D Wetlands may be destroyed by:
1 Degradation of the life forms of
which it is composed in the name of
nuisance control.
2 Physical destruction by cut-and-fill
to create more land area.
5 Morgan, J.P. Ephemeral Estuaries of
the Deltaic Environment in: Estuaries,
pp. 115-120, Publ. No. "83, Am.
Assoc. Adv. Sci. 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.
Sci. Washington, DC. 1957.
7 Redfield, A.C. The Ontogeny of a Salt
Marsh Estuary, in: Estuaries, pp.
108-114. Publ. No. 83, Am. Assoc.
Adv. Sci. Washington, DC. 1967.
8 Stuckey, O. H. Measuring the Productivity
of Salt Marshes. Maritimes (Grad
School of Ocean., 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,
Fiscal Year 1968, USDI, BCF, pp. 10-
12.
REFERENCES
1 Anderson, W. W. The Shrimp and the
Shrimp Fishery of the Southern
United States. USDI, FWS, BCF.
Fishery Leaflet 589. 1966.
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 n, Biological
Aspects by J. W. Hedgepeth, pp. 693-
728. in: Treatise on Marine Ecology
and Paleoecology. Geol. Soc. Am.
Mem. 67. Washington, DC. 1957.
4 Hesse, R., W. C. Allee, and K. P.
Schmidt. Ecological Animal
Geography. John Wiley & Sons. 1937.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.
Descriptors: Aquatic Environment, Estuarine
Environment, Lentic Environment Lotic
Environment, Currents, Marshes, Limnology
9-39
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THE IDENTIFICATION OF AQUATIC ORGANISMS
I INTRODUCTION
A There are few major groups of organisms
that are either exclusively terrestrial or
aquatic. The following remarks will
therefore apply in large measure to both,
but primary attention will be directed to
aquatic types.
B One of the first questions usually posed
about an organism is: "What is it, "
usually meaning "What is it's name?"
The naming or classification of biological
organisms is a science in itself (taxonomy).
Some of the principles involved need to be
understood by anyone working with
organisms.
1 Names are the "key number, " "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 biological nomenclature
(very few libraries have a million
books).
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 understanding increase.
b The categories employed are as follows:
Similar species are grouped into
genera (genus) .
Similar genera are grouped into
families
Similar families are grouped into
orders
Similar orders are grouped into
classes
i
Similar classes are grouped into
phyla (phylum)
Similar phyla are grouped into
kingdoms
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 small letter. They
should also be underlined or printed
in italics when used in a technical
sense. For example:
Homo sapiens - modern man
Homo neanderthalis - neanderthal
man
Esox niger - chain pickerel
Esox lucius - northern pike
Esox masquinongy - 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 (an alga), we must simply
use the generic name, and:
Anabaena planctonica,
Anabaena constricta, and
Anabaena flos-aquae
BI.AQ.3f. 1.74
10-1
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The Identification of Aquatic Organisms
are three distinct species which
have different significances to
water treatment plant operations.
that seems most reasonable and either proceed
to the couplet indicated, or else accept the
name of the group as given if the selection
is a terminal statement. "Cf: --" means to
compare with the statement indicated. "POL"
means that members of the group have been
reported to be tolerant of organic pollution.
A complete list of the various cate-
gories to which an organism belongs
is known as its "classification. " This
may be written as follows for Phacus 1& The body Qf the organisms comprising a
Pyrum a green flagellate, for example: single microscopic independent cell, or
many similar and independently functioning
Kingdom Plantae cells associated in a colony, with little
• or no difference between the cells
Phylum Thallophyta (i.e., without forming tissues); or com-
prising masses of multinucleate proto-
Class Algae plasm; mostly microscopic. Single celled
animals Phylum Protozoa
Order Volvocales '
Ib The body of the organism comprising
Family Phacotaceae many cells of different kinds (i. e, form-
ing tissue) may be microscopic or
Genus Phacus macroscopic 2
Species pyrum 2a Body of colony usually forming irregular
masses or layers sometimes cylindrical,
a It should be emphasized that since goblet-shaped, vase-shaped, elongate,
all categories of above species are serrate, or tree-like. Range from
essentially human concepts, there barely visible to large 3
is often divergence of opinion in
regard to how certain organisms 2b Body or colony shows some type of
should be grouped. Changes result. definite symmetry 15
b The most appropriate or correct 3a Colony forming irregular masses, clumps,
name for a given species is also or aggregations of material growing
sometimes disputed, and so species around, over, or clinging to surface of
names too are changed. The species rocks, sticks, pipes, etc 4
itself, as an entity in nature, how-
ever, is relatively timeless and so 3b Irregular, or not obviously symmetrical,
does not change to man's eye. but essentially erect or plant-like individ-
uals or colonies 9
II KEY TO SELECTED LARGER GROUPS
OF AQUATIC ANIMALS
The following key is intended to provide an
introductory acquaintance with some of the
more common aquatic organisms. While
most of the groups mentioned are animals,
a few other 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, without reference to
any other couplet. Select the alternative
4a Freshwater 8
4b Marine 5
5a Surface of colony usually slightly rough
or bristly in appearance under the micro-
scope or hand lens 7
5b Surface smooth 6
5c Colony or organism erect, plant-like,
branched or unbranched 12
6a Mass very thin, usually pink or white
(Coral algae) 13b
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The Identification of Aquatic Organisms
6b Mass gelatinous, either as relatively
thin sheet or clumps of tongue-like club
shaped projections. Transparent or
colored. Star shaped groups of individ-
ual organisms often evident. Colonial
tunicates or ascideans. Phylum Chordata,
Class Tunicata (Cf:12b)
7a Surface webbed or bristly in appearance,
stick-like crystals or "spicules" often
protruding from the general surface.
Pores numerous, one or more usually
large and conspicuous. Sponges.
Phylum Porifera
7b Colonies very thin, surface slightly
roughened. Composed of minute individ-
ual cells or compartments arranged in
various lacy or coral-like patterns. Each
compartment inhabited by a minute bilater-
ally symmetrical animal which protrudes
a crown of delicate tentacles. Moss
animals, bryozoans. ... Phylum Bryozoa
8a Surface of colony rough or bristly in
appearance under the microscope or hand
lens. Grey, green, or brown. Sponges.
(Figure 1) Phylum Porifera
8b Surface colony relatively smooth, general
texture of mass gelatinous, transparent.
Clumps of minute individual organisms
variously distributed. Moss animals,
bryozoans. (Figure 2). .Phylum Bryozoa
9a Freshwater.
10
9b Marine 11
lOa Surface of structures rough or bristly
under the microscope 8a
lOb Individuals or branches tubular. Usually
covered with brownish cuticle to which
much sediment may adhere. Moss
animals, bryozoans. . . . Phylum Bryozoa
(Cf:8b) (Figure 2)
lla Surface of structure appears web-like or
bristly under the microscope 7a
lib Surface of structure an unbroken skin or
cuticle, except for a limited number of
functional apertures 13
12a Colonies minute, plant-like or coral-like.
(Figure 2) 13
12b Solitary, sac-like organism, either stalked
or growing directly on substrate. Trans-
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 several inches in length.
Seasquirts or tunicates.. Phylum Chordata,
Class Tunicata
13a
13b
14a
14b
16a
16b
17a
Compartments or apertures for separate
individual animals evident 14
No evidence of the presence of individual
animals. Structure profusely jointed.
Individual segments hard, smooth, usually
pink or white. May also grow as hard
smooth thin sheet over surface of rocks.
Calcareous algae or coral algae. A
common Genus; . Corallina
Minute organisms protruding from holes
in colony are radially symmetrical. .. 17b
Organisms protruding are bilaterally
symmetrical. (This may be assumed if
they are not obviously radial) Moss
animals, bryozoans. (Figure 2) (Cf:7b)
Phylum Bryozoa
15a Radially symmetrical (body arranged around
an axis) 16
15b Fundamentally bilaterally symmetrical. 13
15c May be superficially spiral as Figure 13 35
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
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
Jelly-like body, massive or ovoid, free
swimming nearly transparent in nature.
Eight rows comb-like paddle plates:
Comb Jellies Phylum Ctenophora
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The Identification of Aquatic Organisms
23b
23c
24a
17b Body tube-like or umbrella-like, tentacles
around mouth, margin of disc, or both.
Jellyfishes, corals, hydroids, sea
anemones, etc... Phylum Coelenterata
18a Microscopic. Action of two ciliated lobes
at anterior end in life often gives appear-
ance of wheels. Body often segmented,
accordion-like. Free swimming or
attached. Rotifers or wheel animalcules
(Figure 3).' Phylum Trochelminthes (Rotifera)
18b Often larger, worm-like or else having
strong skeleton or shell 19 24b
19a Skeleton (or shell) present may be internal
or external 30
19b Body soft and/or worm-like. Skin may
range from soft to parchment-like ... 20
20 a Three or more pairs of well-formed
(though possibly small) jointed legs
present. (Phylum Arthropoda) 36
20b Legs or appendages, if present, limited
to pairs of bumps or hooks. Lobes or
tentacles if present-soft and fleshy, not
jointed 21
21a Body round, oval or flat in cross section
22
21b Inverted U-shaped cross section, with
flat slimy undersurface or foot tapering
off behind. Head with two pairs of
sensitive retractile tentacles. Marine
or terrestrial. Marine forms have fancy
gill structures on back. Slugs.
Class Gastropoda. (Cf: 35a)
(Phylum Mollusca) 35
22a Strongly depressed or flattened 23
226 Oval or round in cross section, or, if
- flat, with single sucker disc at each end.
25
23a Parasitic in_bodies of higher animals.
Extremely long and flat, divided into
sections (like a Roman girdle). Life
history may involve intermediate host.
Tape worms. (Figure 5) Class Cestoda
25 a
26a
Parasitic on bodies of higher animals,
very muscular, strong external sucker
discs (Figure 9) 29b
Body a single unit. Mouth and digestive
system present (but no anus) 24
External or internal parasite on higher
animals. Sucking discs present for attach-
ment. Life history may involve two or
more intermediate hosts or stages.
Flukes Class Trematoda
Free-living. Entire body surface covered
with locomotor cilia. Eyed areas in head
often appear "crossed. " Pol. Free-living
flatworms (Figure 6). Class Turbellaria
Long, slender, with snake-like motion in
life. Covered with glistening cuticle.
Parasitic or free-living. Microscopic to
six feet in length. Round worms
(Figure 7).... Phylum Nemathelminthe s
25b Divided into sections or segments
(Figures 8 and 9) 26
Head a more or less well-formed hard
capsule with jaws, eyes, and antennae.
Pol. (Figure 8) Class Insecta.
(Cf:49a) Order Diptera
26b Head structure soft (except jaws, if
present) (Figure 9) 27
27a Head conical or rounded, lateral appenda^ ,s
not conspicuous or numerous 28
27b Head somewhat broad and blunt. Retractile
jaws usually present. Soft fleshy lobes or
tentacles often present in head region. Tail
usually narrower. Lateral lobes or fleshy
appendages on each segment. (Figure 9A)
Phylum Annelida..... 29
28a Minute dark colored retractile jaws present,
body tapering somewhat at both ends, pairs
or rings of bumps or "legs" often present,
even near tail. Class Insecta. (Cf:49a)
Order Diptera
28b No jaws, sides of body generally parallel
except at ends, (similar to Figure 9).
10-4
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The Identification of Aquatic Organisms
29a
29b
30a
30b
31a
Thickened area or ring usually present
part way back on body. Clumps of
minute bristles on most segments.
(Phylum Annelida) 29
Segments with bristles, and/or fleshy
lobes, or other extensions. Tube builder,
borer, or burrower. Often reddish or
greenish in color. Pol. Earthworms,
sludge worms, clam worms, etc.
Class Chaetopoda
A sucker disc at each end, the larger
one posterior. External bloodsucking
parasites on higher animals, often found
unattached to any host; Pol. Leeches.
(Figure 9B) Class Hirudinea
Skeleton internal, of true bone
(Vertebrates)
58
Body covered with external skeleton or
sheU. (Figure 4) 31
External jointed skeleton or shell covers
legs and other appendages, often leathery
in nature. Jointed legged animals.
(Figures 11, 12, and 14 through 29)
(Phylum Arthropoda) 36
31b Shells limey or calcareous. (Figures 10
and 13) 32
32a Marine, attached, shell conical, or purse-
shaped on fleshy stalk. Crowded individ-
uals may be more or less cylindrical.
Shell composed of several parts, two of
which spread apart to permit extension
of "hand" for feeding. Barnacles.
Phylum Arthropoda, Class Cirripedia
32b Shells one or two, or several arranged
as transverse plates 33
33a Half inch or less in length. Two clam-
like shells. Soft parts inside include
delicate flattened jointed appendages.
Phyllopods or branchipods (Figures 11
and 12) Phylum Arthropoda, Class
Crustacea, Subclass Branchipoda
(Cf:39a)
33b Soft parts covered with thin skin, often
slimy (Phylum Mollusca) 34
34a Marine, oval in shape, eight shells trans-
versely jointed. Chitons.
Class Amphineura
34b Shells one or two in number. Snails,
clams, etc. (Figures 10-13) 35
35a Shell, single may be a spiral, a flattened
cone, degenerate, or missing. Pol.
Snails and slugs Class Gastropoda
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. Leglike appendages or gills
present on abdomens of some larvae. No
marine forms. (Phylum Arthropoda,
Class Insecta) 47
36b More than three pairs of legs apparently
present. (Figures 14-19) 37
37a Body elongated, head broad and flat with
strong jaws. Appendages following first
three pairs of legs are rounded tapering
filaments. Up to 3 inches long. Dobson
fly and fish fly larvae. Class Insecta,
Order Megaloptera
37b Four or more pairs of true legs.
38
38a Four pairs of legs. Body rounded, 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 shrimps, phyllopods, or branchipods.
Subclass Branchipoda
10-5
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The Identification of Aquatic Organisms
39b Less than ten pairs of locomotor
appendages
40 a
40b
41a
41b
42a
42b
43a
43b
44a
44b
45 a
Body and legs enclosed in bivalved
(2 halves) shell which may or may not
completely hide them. (Figures 11-12) 41
Appendages more rounded than leaf-like
in cross section, or if appendages are
flattened for swimming, eyes are on
stalks (pelagic shrimps). May be large
or minute 43
One pair of branched antennae enlarged
for locomotion, extend outside of shell
(carapace). Single eye usually visible.
Cyclops or cladocera. Subclass Cladocera
Locomotion accomplished by body legs
(not antennae). (Figure 16). . . 42
Appendages leaf-like, flattened, more
than ten pairs. (Branchipods) 39a
Animal less than three mm in length.
Appendages more or less slender and
jointed, often used for walking. (Figure 11)
Shells opaque. Ostracodes.
Subclass Ostracoda
Body a series of six or more essentially
similar segments, differing mainly in
size 44
Front part of body enlarged into a some-
what separate body unit (cephalothorax)
often covered with a single piece of shell
(carapace). Back part (abdomen) may be
relatively small, even folded underneath
front part. (Figure 19) 45
Body compressed laterally, i.e., high
and thin. Scuds, amphipods. (Figure 17)
Subclass Aniphipoda
Body depressed, i. e., low and broad.
Flat gills contained in chamber beneath
tail. (Figure 18) Sowbugs.
Subclass Isopoda
Minute in size. Often somewhat elongated
and cylindrical, with abdomen extending
straight out behind ending in two small
projections. Two relatively enormous
masses of eggs are often attached to
40 female. Locomotion by means of two
enlarged, unbranched antennae, the only
large appendages on the body. (Figure 19)
Copepods Subclass Copepoda
45b Larger. Carapace (shell of cephalothorax,
the enlarged front section) usually more
conspicuous. 5 pairs of walking legs
including claws, if present. Appendages
present on abdomen 46
46a Eyes immovable, in broad shell shaped
like a horseshoe, a mud dweller. Long
sharp pointed triangular tail extends out
behind. Flat "book" gills beneath abdomen.
Horseshoe crab. Subclass Arachnoidea,
Genus Limulus
46b Eyes on movable stalks. Abdomen tightly
.. folded back under cephalothorax (crabs)
or continued out behind thorax to end in
an expanded "flipper" or swimming paddle.
Crabs, shrimps, crayfishes, lobsters,
etc Subclass De capo da
47a Two pairs of functional wings (one pair
may be more or less hardened as protection
for the other pair). Adult insects which
normally live on or in the water (Figures 22,
25, 28, 29) 57
47b No functional wings, though "pads" in
which they are developing may be visible.
Some resemble adults very closely, others
differ extremely (Figures 8, 23, 24, 26, 27)
48
48a External pads or cases in which wings
develop clearly visible 53
48b More or less worm-like, no external
evidence of wing development 49
49a No jointed legs (sucker discs, hooks,
prolegs, breathing tubes, and other such
structures may be present. (Figures 8, 9D)
Larvae of two winged flies, midges, etc.
Pol Order Diptera
49b Three pairs of jointed thoracic legs, head
capsule well-formed 50
50a Minute (2 - 4mm), living on surface film.
Tail a strong organ that can be hooked into
10-6
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The Identification of Aquatic Organisms
50b
51a
a "catch" beneath thorax. When released,
animal jumps into the air. Not usually
classed as aquatic. These are adults,
no wings are ever grown. (Figure 20)
Springtails Order Collembola
Larger (rarely collected under 5 mm),
worm-like, living beneath the surface.
51
Construct fixed or movable cases of
bottom materials. (Figure 21) Abdominal
segments often with minute gill filaments,
no long lateral filaments. Generally
cylindric in shape. Caddisfly larvae.
Order Trichoptera
51b Free living, i.e., not case-building. 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 about 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 filaments or other structures
extending out from end of abdomen.
(Figures 24B, 24D, 26, 27) 55
53b Abdomen ending abruptly, unless terminal
segment itself is extended as single
structure. (Figure 24C) 54
54a Head broad, eyes widely spaced. Front
of face covered by extensible folded mouth-
parts often called a "mask. " Larvae of
dragonflies or darning needles. Pol.
(Figure 24E) Order Odonata
(Suborder Anisoptera)
54b Mouthparts for piercing and sucking.
Body form varies extremely. Water bugs,
walking sticks, water boatmen, back-
swimmers, electric light bugs, water
striders, and others. (Figure 25)
Order Hemiptera
55a Tail extensions (caudal filaments) two.
Stonefly larvae. (Figure 26)
Order Plecoptera
55b
56a
56b
57a
57b
58b
Tail extensions usually three. (Figures 24B,
24D, 27) 56
Tail extensions long and slender. Rows
of hairs may give them a feather -like
appearance. Larvae of mayflies.
Order Ephemeroptera
Tail extensions flat, elongated plates.
Head broad with widely spaced eyes,
abdomen relatively long and slender.
Larvae of damselflies. Order Odonata
, (Suborder Zygoptera)
External wings or wing covers form a
hard protective dome over the inner wings
folded beneath, and the abdomen. Beetles.
(Figure 28) Order
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 Hemiptera
58a Appendages present in pairs (fins, legs,
wings) 60
Mouth a round suction disc. No paired
appendages
59
59a Body long and slender. Several holes
alongside of head. Lampreys and hagfishes.
Sub-phylum Vertebrata. 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 appendages 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 short sharp spines. Essentially
marine. Sharks, skates, and rays.
Class Elasmobranchii
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The Identification of Aquatic Organisms
62a Digits with claws, nails, or hoofs. ... 63 63b Body covered with horny scales or plates.
Reptiles Class Reptilia
62b Skin naked, no claws on digits. Frogs,
toads, and salamanders. Class Amphibia 64a Body covered with feathers. Birds.
Class Aves
63a Warm blooded 64 64b Body covered with hair. Mammals.
Class Mammalia
10-8
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The Identification of Aquatic Organisms
APPENDIX A
LIST OF INCLUDED GROUPS
Phyla or
Subphyla
Protozoa
Porifera
Coelenterata
Platyhelminthes
Rotifera
Nemathelminthes
Bryozoa
Annelida
Arthropoda
Classes or
Subphyla
Turbellaria
Trematoda
Cestoda
Chaetopoda
Hirudinea
Crustacea
Classes or
Orders
Other
Arachnoidea
Insecta
Mollusca
Enchinodermata
Chordata
Gastropoda
Pelecypoda
Tunicata
Cyclostomata
Pisces
Amphibia
Reptilia
Aves
Mammalia
Branchiopoda
Cladocera
Ostracoda
Amphipoda
Isopoda
Copepoda
Decapoda
Cirripedia
Acari
Xiphosura
Collembola
Plecoptera
Ephemeroptera
Odonata
Hemiptera
Megaloptera
Trichoptera
Coleoptera
Diptera
Limulus
Algae
Coralllna
10-9
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The Identification of Aquatic Organisms
1. Spongilla spicules
Up to .2 mm. long.
3A. Rotifer, Polyarthra
Up to . 3 mm.
3B. Rotifer, Keratella
Up to . 3 mm.
3C. Rotifer, Philodina
Up to . 4 mm.
4A. Jointed leg
Caddisfly
4B. Jointed leg
Crayfish
2B. Bryozoal mass. Up to
several feet diam.
2A. Bryozoa, Plumatella. Individuals up
to 2 mm. Intertwined masses maybe
very extensive.
4C. Jointed leg
Ostracod
5. Tapeworm head,
Taenia. Up to
25 yds. long
6A. Turbellaria, Mesostoma
• Up to 1 cm.
6B. Turbellaria, Dugesia
Up to 1. 6 cm.
10-10
7. Nematodes. Free living
forms commonly up to
1 mm., occasionally
more.
-------
The Identification of Aquatic Organisms
8B. Diptera, Mosquito
pupa. Up to 5mm.
8A. Diptera, Mosquito larvae
Up to 15 mm. long.
8C. Diptera, Tendiped 8E. Diptera, crane fly
larvae. Up to 2 cm. pupa. Up to 2. 5 cm.
9D. Diptera, Rattailed maggot
Up to 25 mm. without tube.
9A. Annelid,
segmented
worm, up to
1/2 meter
10B. Alasmidonta, end view.
10A. Pelecyopod, Alasmidonta
Side view, up to 18 cm. long.
9B. Annelid, leech up to 20 cm.
11A. Ostracod, Cypericus
Side view, up to 7 mm.
11B. Cypericus, end view.
12A. Branchiopod
Daphnia. Up
to 4 mm.
12B. Branchiopod
Bosmina. Up
to 2 mm.
10-11
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The Identification of Aquatic Organisms
13. Gastropod, Viviparus
Up to 3 inches.
14. Megaloptera,
Alderfly larvae
Up to 25 mm.
16. Fairy Shrimp, Eubranchipus
Up to 5 cm.
15. Water mite,
Up to 3 mm.
17. Amphupod, Pontoporeia
Up to 25 mm.
18. Isopod, Asellus
Up to 25 mm.
20. Collembola, Podura
Up to 2 mm. long
19A. Calanoid copepod, IQB- Copepoid copepod.
Female Female
Up to 3 mm. Up to 25 mm.
10-12
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The Identification of Aquatic Organisms
21A.
21B.
21C.
2 ID.
2 IE.
21. Tricoptera, Larvae cases, mostly 1-2 cm.
22. Megaloptera, alderfly
Up to 2 cm.
23A. Beetle larvae,
Dytisidae,
Usually about | cm.
23B. Beetle larvae,
Hydrophilidae,
Usually about 1 cm.
24A. Odonata, dragonfly
nymph up to 3 or 4
cm.
24B. Odonata, tail of
damselfly nymph
(side view)
is^4 24E- Odonata, front view
of dragonfly nymph
showing "mask"
partially extended
24D. Odonata, damselfly
nymph (top view)
24C. Odonata, tail of
dragonfly nymph
(top view)
10-13
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The Identification of Aquatic Organisms
25A. Hemiptera,
Water Boatman
About 1 cm.
27.Ephemeroptera,
Mayfly nymph
Up to i cm.
25B. Hemiptera,
Water Scorpion
About 4 cm.
26. Plecoptera,
Stonefly nymph
Up to j cm.
28A. Coleoptera,
Water scavenger
beetle. Up to 4 cm.
28B. Coleoptera,
Dytiscid beetle
Usually up to 2^ cm.
29A. Diptera, Crane
fly. Up to 2j cm.
29B. Diptera, Mosquito
Up to 20 mm.
-------
The Identification of Aquatic Organisms
REFERENCES - General
Keeler, C.R. Books on Aquatic Biology.
Freshwater and Marine .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 Edmondson, W. T., Ed. Ward and
Whipple's Freshwater Biology. John
Wiley & Sons. New York. , :
1959.
3 Hedgepeth, J., and Hinton, X. Common
Seashore Life of Southern California.
Naturegraph Company, Heraldsburg,
California. 1961.
4 Jahn, T.L., and Jahn, F. F. How to
Know the Protozoa. Win. C. Brown
Co., Dubuque, Iowa. 1949.
5 Kudo, R. Protozology. Charles C.
Thomas, Publisher, Springfield, Illinois.
1950.
6 Light, S. F., et al. Intertidal Inverte-
brates of the Central California Coast.
University of California Press,
Berkeley. 1961.
7 MacGinitie, G.E., and N. Natural
History of Marine Animals. McGraw-
Hill. 1949.
8 Miner, R.W. Fieldbook 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.
1953.
10 Pratt, H.W. A Manual of the Common
Invertebrate Animals Exclusive of
Insects. The Blakiston Company,
Philadelphia. 1951.
11 Ricketts, E., and Calvin, J. Between
Pacific Tides. Revised by J.
Hedgepeth, Stanford Univ. Press,
Palo Alto, California. 1952.
12 Smith, R.I. Keys to Marine Invertebrates
of the Woods Hole Region, Contribution
No. 11, MBL, Woods Hole, Mass.
1964.
REFERENCES - Fishes
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 $1.00 paper, $2. 00 cloth)
2 Anon. Check List of the Florida Game
and Commercial Marine Fishes with
Common Names. Florida State Board
of Cons. Educational Bull. No. 12
(Free).
3 Benton, A.H., and Stewart. Keys to the
Vertebrates of the Northeastern States.
Burgess 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 Wilby, G.V.
Fishes of the Pacific Coast of Canada.
Fisheries Research 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.
10-15
-------
The Identification of Aquatic Organisms
Zim, H. S., and Shoemaker, H. H.
Fishes--A guide to Fresh and Salt
Water Species. Golden. 1959.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Environmental Protection Agency, OWP.
Cincinnati, OH 45226
Descriptors:
Aquatic Life; Systematics
10-16 '
-------
AQUATIC ORGANISMS OF SIGNIFICANCE IN POLLUTION SURVEYS
I INTRODUCTION
A Any organism encountered in a survey is
of significance. Our problem is thus not
to determine which are of significance
but rather to decide "what is the signifi-
cance of each?"
B The first step in interpretation is
recognition. "The first exercise in
ecology is systematics."
C Recognition implies identification and an
understanding of general relationships
(systematics). The following outline will
thus review the general relationships of
living (as contrasted to fossil) organisms
and briefly describe the various types.
D The species problem
1 Necessity of identifying species
Studies of the ecology 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 unless
we can say what species constitute the
biota. Species vary in their responses
to the impact of the environment.
2 Solutions to the problem
a Evasion
Treat the ecosystem as a "black
box"--a unit—while ignoring the
constitution of the system. This
may produce some broad generalizations
and will certainly yield more questions
than answers.
b Compromise
Work only with those taxonomic
categories with which one has the
competence to deal. 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.
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. *
Lists of expert taxonomists can be
obtained. There will be none for
some groups. Also collaboration
is time consuming.
3 Fungi: extracellular digestion
(enzymes secreted externally.)
Food material then taken in through
cell membrane where it is metab-
olized and reduced to the mineral
condition. Ecologically known as
REDUCERS.
E Each of these groups includes simple,
single-celled representatives, persisting
at lower levels on the evolutionary stems
of the higher organisms.
1 These groups span the gaps between
the higher kingdoms with a multitude
of transitional forms. They are
collectively called the PROTISTA.
2 Within the protista, two principal
sub- groups can be defined on the
basis of relative complexity of
structure.
a The bacteria and blue-green algae,
lacking a nuclear membrane may
be considered as the lower
protista (or Monera).
b The single-celled algae and
protozoa are best referred to as
the higher protista.
F Distributed throughout these groups will
be found most of the traditional ."phyla"
of classic biology.
BI.AQ.22c. 1.74
11-1
-------
Aauatic Organisms of Significance
NSUMERS
PRODUCERS REDUCERS
NUTRIENT
MINERALS
Figure 1. BASIC CYCLES OF LIFE
11-2
-------
Aquatic Organisms of Significance
II PLANTS
A The vascular plants are usually larger
and possess roots, stems, and leaves.
1 Some types emerge above the surface
(emersed).
2 Submersed typed typically do not
extend to the surface.
3 Floating types may be rooted or free-
floating.
B Algae generally smaller, more delicate,
less complex in structure, possess
chlorophyll like other 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
SiC>2 - 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 artificial group.
a Unicellular representatives may be
extremely small.
b Multicellular forms may produce
great floating mats of material.
in FUNGI
Lack chlorophyll and consequently most are
dependent on other organisms. They secrete
extracellular enzymes and reduce complex
organic material to simple compounds which
they can absorb directly through the cell wall.
A Schizomycetes or bacteria are typically
very small and do not have an organized
nucleus.
1 Autotrophic bacteria utilize basic food
materials from inorganic substrates.
They may be photo-synthetic or
chemosynthetic.
2 Heterotrophic bacteria are most
common. The require organic
material on which to feed.
B "True fungi" usually exhibit hyphae as the
basis of structure.
IV ANIMALS
A Lack chlorophyll and consequently feed on
or consume other organisms. Typically
ingest and digest their food.
B The Animal Phyla
1 PROTOZOA are single celled organisms;
many resembling algae but lacking
chlorophyll (cf: illustration in "Oxygen"
lecture).
2 PORIFERA are the sponges; both marine
and freshwater representatives.
3 CNIDARIA (= COELENTERATA)
include corals, marine and fresh-
water jelly fishes, marine and
freshwater hydroids.
4 PLATYHELMINTHES are the flat worms
such as tape worms, flukes and Planeria.
5 NEMATHELMDSTTHES are the round
worms and include both free-living
forms and many dangerous parasites.
6 ROTIFERS are multicellular micro-
scopic predators.
7 BRYOZOA are small colonial sessile
forms, marine or freshwater.
11-3
-------
Aquatic Organisms of Significance
8 MOLLUSCA include snails and slugs, •
clams, mussels and oysters, squids,
and octopi.
9 BRACHIOPODS are bivalved marine
organisms usually observed as fossils.
10 ANNELIDS are the segmented worms
such as earthworms, sludge worms and
many marine species.
11 ECHINODERMS include starfish, sea
urchins and brittle stars. They are
exclusively marine.
12 CTENOPHORES, or comb jellies, are
delicate jelly-like marine organisms.
13 ARTHROPODA, the largest of aU
animal phyla. They have jointed ap-
pendages and a chitinous exoskelton.
a CRUSTACEA are divided into a
cephalothoraz and abdomen, and
have many pairs of appendages,
including paired antennae.
1) CLADOCERA include Daphina
a common freshwater micro-
crustacean; swim by means of
branched antennae.
2) ANOSTRACA (=PHYLLOPODS)
are the fairy shrimps, given
to eruptive appearances in
temporary pools.
3) COPEPODES are marine and
freshwater microcrustacea--
swim by means of unbranced
antennae.
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. Marine
and freshwater.
7) DECAPODA - crabs, shrimp,
crayfish, lobsters, etc.
Marine and freshwater.
b INSECTA - body divided into head,
thorax and abdomen; 3 paris of legs;
adults typically with 2 pairs of
wings and one pair of antennae.
No common marine species. Nine
of the twenty-odd orders include
species with freshwater-inhabiting
stages in their life history as follows:
1) DIPTERA - two-winged flies
2) COLEOPTERA - beetles
3) EPHEMEROPTERA - may flies
4) TRICHOPTERA - caddis flies
5) PLECOPTERA - stone flies
6) ODONATA - dragon flies and
damsel flies
7) NEUROPTERA - alder flies,
Dobson flies and fish flies.
8) HEMIPTERA - true bugs,
sucking insects such as water
striders, electric light bugs
and water boatman
9) LEPIDOPTERA - butterflies
and moths, includes a few
freshwater moths
c ARACHINIDA - body divided into
cephalothorax and abdomen; 4 pairs
of legs - spiders, scorpions, ticks
and mites. Few aquatic repre-
sentatives except for the freshwater
mites and tardigrades.
C CHORDATA
PROTOCHORDATES - primitive marine
forms such as acorn worms, sea
squirts and lancelets
VERTEBRATES - aU animals which
have a backbone
a PICES or fishes: including such
forms as sharks and rays,
lampreys, and higher fishes; both
marine and freshwater
-------
Aquatic Organisms of Significance
b AMPHIBIA - frogs, toads, and
salamanders - marine species
rare.
c REPTILA - snakes, lizards and
turtles.
d MAMMALS - whales and other
warm-blooded vertebrates with
hair.
e AVES - birds - warm-blooded
vertebrates with feathers.
REFERENCE
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 45268, and revised by
Ralph M. Sinclair, Aquatic Biologist,
National Training Center.
Descriptors:
Aquatic Life; Systematics, Streams,
Surveys, Stream Pollution
11-5
-------
Aquatic Organisms of Significance
FUNGI
3/4
Sohizorayoetes - Bacteria, free living representatives
« » A
f I
• ' I A*
' '* *'
+*••
Aerobaoter
aerogenes
Rhizobium
radio ioola
Azotobacter
Phyoonyoetes - Saprolegnia; A,detail of immature reproductive
stages; B, mature oogonium and autheridia, with egp;s and fertili-
zation tubes; C, dead tadpole with growth of S.
Phyoomyoete - Leptomitus.
this genus includes pollution
tollerant species.
H.W.Jaokson
Asoomyoete - Sao char omyoes. a
yeast including poll, tollerant
species. A, single cell; B,budding;
C, ascospore formation.
11-6
PLATE I
-------
Aquatic Organisms of Significance
BLUE GREEN ALGAE
3/4
OBoillatorla spp.. filaments (triohomes) range from .6 to over
60/t in diameter. Ubiquitous, pollution tollerant.
Lypgbia spp.. similar to Osoillatoria "but has a shaath.
A, Lynebla oentorta. reported to be generally intollarant
of pollutione B L. birgei.
B
Aphanizomenjn flos-aquae
A, oolonyi B,filament
Anabaena flos-aquae
A, akinete; B,heterooyst
PLATE II
H.W.Jaokson
11-7
-------
Aquatic Organisms of Significance
1/67
NON-MOTILE GREEN ALGAE: COCCOID
(CHLOROPHYCEAE)
Pediaatruin
Species of the Genus Scenedesmus
3. dlmorphuB
Desmids
Coemarium
PLATE III
11-8
-------
Aquatic Organisms of Significance
NON-MOTILE GREEN ALGAE: FILAMENTOUS
(Chlorophyceae)
PLATE IV
11-9
-------
Aquatic Organisms of Significance
PIGMENTED FLAGELLATES
3/4
Dinobrron
A, form of colony) B,o«ll la lorlea.
I.W. Jaokion
PLATE V
11-10
-------
Aquatic Organisms of Significance
DIATOMS
vaiv«
A discoid or central
diatom suoh as
Stephanodisous
Girdle views,
stylized to show
basic diatom
structure.
A pennate or navioular
diatom suoh as
Praelllarla
A colony of Aeterionella
(girdle views)
A colony of Praglllarla
(girdle views)
Qomnhonema
A,valve view; B,girdle
view.
Diagram showing progressive diminution in the size of certain
frustules tnrough saooesolve cell generations of a diatom.
H.W. Jackson
PLATE VI
11-11
-------
Aquatic Organisms of Significance
FREE LIVING PROTOZOA
I. Flagellated Protozoa, Class llastigophora
Atvthophy sis
Pollution tolerant
6>u
Pollution tolerant
19/t
Colony of Poteriodendron
Pollution tolerant , 35ja
II. Ameboid Protozoa, Class Saroodina
PImastigamoeha
Pollution tolerant
10-50 ,u
Nuelearla.reported
to be intolerant of
pollution, 45/1*
III. Ciliated Protozoa, Class Ciliophora
Colpoda
Pollution tolerant
20-120 ja.
Holophrya.reported
to be intolerant of
pollution, 35 /»
PLATE VII
Difflueta
Pollution tolerant
60-5 00 JB
BplatyllB. pollution
tolerant Colonies often
maorosoopio.
H.W.Jaokson
11-12
-------
Aquatic Organisms of Significance
PLANKTONIC PROTOZOA
Peranema trichophorum
Top
Side
Chaos
Arcella
vulgarls
Actinosphaerium
Vorticella
Codonella
cratera
Tintlnnidium
fluviatle
PLATE VIII
11-13
-------
Aquatic Organisms of Significance
PLANKTONIC ROTIFERS
Various Forms of Keratella cochlearis
Synchaeta
pectinata
Polygarthra
vulgaris~
Brachionus
quadridentata
Rotaria sp
PLATE IX
-------
Aquatic Organisms of Significance
FREE LIVING NEMATHELMINTHES, OR ROUND WORMS
-pharynx
a«rv« ring
-excrBtory pore
•salivary gland
•renette
. Intestine
_te»ti«
-•eminal vesicle
_vaa deferent
-ejaculatory gland
rectal gland
copulatory Bplcule
. gubernaculum
MonhyBtera
Bhabditii
Acaromadora
PLATE X
11-15
-------
Aquatic Organisms of Significance
FRESH WATER ANNELID WORMS
Phylum Annelida
anus
• mouth.
Class? Oligoohaeta, earthworns
Ex: Tub ifex , the sludgeworm
(After Liebman)
I.W.Jackson
mouth
posterior sucker disc
Class Hirudinea, leeches
(After Hegner)
anterior end
Class Polychaeta , polychaet worms
Ex; Manayunkia, a minute, rare, tube
building worm.
(After Leidy)
PLATE XI
11-16
-------
Aquatic Organisms of Significance
SOME MOLLUSCAN TYPES
Class: Cephalopoda*
Squids, octopus,
cuttlefish.
Exclusively marine.
The giant squid shown
was captured In the
Atlantic in the early
ninteenth century.
(After Hegner)
Limax.
a slug
Lymnaea
Campeloma
an air breathing mail a water breathing
snail
Class« Gaetuopodao snails and slugs. (After Buchebanm)
Class: Pelecypodaj clans, mussels, oysters.
Locomotion of a freshwater clam, showing how foot is extended, the tip
expanded, and the animal pulled along to its own anchor. (After Buchs-
baum) H.W.Jackson
PLATE XII
11-17
-------
Aquatic Organisms of Significance
3/4
Class CRUSTACEA
Fairy Shrimp;
Eupranohjpus. Order
Phyllqpoda
20-25 "m
Crayfish, or orawdadj
Cambarus. Order Deoapoda
10-20 em
Sow Bug; Asellus.
Order Jsopoda
10-20 mm
Sbud; Hyalellq
Order Imphipoda
10-15 nm
Pish Louse, Argulus;
a parasitlo Copepod
5-6 HOB
H.W.Jaokson
i "
Copepod; Cyolops. flrder Copepoda
2-5 «a
PLATE XIII
11-18
-------
Aquatic Organisms of Significance
Two Winged Flies
Order DIPTERA
Adult midge
(Chironomid)
Eat-tailed maggot
(IristaUs)
A, adult; B,larva.
Adult sewage fly
(Psychoda)
Sewage fly larva
Midge pupa
Sewage fly pupa
dge larva
H.W.Jackson
After various authors
PLATE XIV
11-19
-------
Aquatic Organisms of Significance
Beetles
Order COLEOPTERA
body
A diving beetle (Dytiseus)
taking air at the surface.
I
Whirligig beetle (Gyrinus) A, Side view
of head-of adult showing divided eye;
B, Larva; C, Adult. Carnivorous.
The riffle beetle (Peephenus);
A. adult; B, dorsal side of larva;
C, ventral side of larva. Predominantly
herbivorous.
A diving beeile (Cybiater). The div-
ing beetles include some of the largest
and most voracious of aquatic insects.
A, larva; B,adult.
PLATE XV
B
Crawling water beetle;
A;adult; B,larva. Predominantly
herbivorous.
H.W.Jackson. After
Needham, Penriak, Morgan,and others.
11-20
-------
Aquatic Organisms of Significance
MINOR PHYLA
Phylum Coelenterata
Hydra with bud;
extended, and contracted^
Medusa of
Crasoedacusta
Phylum Bryozoa
Massive colony on
stick
Creeping colony
on rock
Single zooid, young statoblasts in tube
PLATE XVI
11-21
-------
Aquatic Organisms of Significance
SOME PRIMITIVE FISHES
Class Agnatha, jawless fishes (lampreys and hagfishes) - Family
PETROMYZONTIDAE, the lampreys. Lampetra aepyptera, the
Brook Lamprey A: adult, B: larva (enlarged)
Class Chondrichthyes - cartilagenous fishes (sharks, skates, rays)
Family DASYATIDAE - stingrays. Dasyatis centroura, the Roughtail Stingray
Class Osteichthyes - bony fishes - Family ACIPENSERIDAE, sturgeon.
Acipenser fulvescens, the Lake Sturgeon
Class Osteichthyes - bony fishes - Family POLYODONTIDAE, the
paddlefishes. Polyodon spathula, the Paddlefish. A:side view B:top view
Class Osteichthyes - bony fishes - Family LEP1SOSTEIDAE - gars
Lepisosteus osseus, the Longnose Gar
Class Osteichthyes - bony fishes - Family AMIIDAE, bowfins
Aniia calva, the Bowfin
Reproduced with permission; Trautman, 1957.
BI.AQ.pl. 91. 6. 60
P1ATE XVII
11-22
-------
TYPES OF BONY FISHES
/4
fe^—
male
SKraP2pP£f"
V?'KwWM«$><»WvS»»«?»>.,
lV/>A'£'H#'-i' '*'»'•'''"^T'Trr".-
&'H#!^;§B^B''v: ':•;!': -^ ••••.:f-T~T?
f^j^^S^^vr^-'.'^:'''1--:
'=i^*%^^M:Kife',:--:ari«^' '^^K^
. .... ,iJ^^Tv-,.'•'•.•;•'••';• '•;:
•<^^"-^:,'^;ca^
v:.^^''-f>te::-:r=>^f^vIi;-.';v>'. .:, •','.'.-•'•• -* _J~- - r-
Family CLUPEIDAE - herrings
Dorosoma cepedianum - the eastern gizzard shad
\' /, \
female
H
H
Family POECILIIDAE - livebearers
Gambusia affinis - the mosquitofish
Family ANGUILLEDAE - freshwater eels
Anguilla rostrata - the American eel
Family GADDIDAE - codfishes, hakes, haddock, burbot
Lota lota - the eastern burbot
Family ESOCIDAE - pikes
Esox lucius - the northern pike
Reproduced with permission; Trautman, 1957
BI.AQ.pl. 9m. 6. 60
Family SCIAENIDAE - drums
Aplodinotus grunniens - the freshwater drum
>
iS
il
'o
! 3
! n
i n
-------
KEY TO SELECTED GROUPS OF FRESHWATER ANIMALS
The following key is intended to provide
an introduction to some of the more
common freshwater animals. Technical
language is kept to a minimum.
In using this key, start with the first
couplet (la, Ib), and select the alternative
that seems most reasonable. If you
selected "la" you have identified the
animal as a member of the group, Phylum
PROTOZOA. If you selected "lb'\ proceed
to the couplet indicated. Continue this
process until the selected statement is
terminated with the name of a group.
If you wish more information about the
group, consult references. (See reference
St.)
BI.AQ.21b.5.71
12-1
-------
Key to Selected Groups of Freshwater Animals
la The body of the organism comprising
a single microscopic independent
cell, or many similar and indepen-
dently functioning cells associated
in a colony with little or no differ-
ence between the cells, i. e. , with-
out forming tissues; or body com-
prised of masses of multinucleate
protoplasm. Mostly microscopic,
single celled animals.
Phylum PROTOZOA
Ib The body of the organism com-
prised of many cells of different
kinds, i.e., forming tissues.
May be microscopic or macro-
scopic.
2a Body or colony usually forming
irregular masses or layers some-
times cylindrical, goblet shaped,
-vase shaped, or tree like. Size
range from barely visible to
large.
2b Body or colony shows some type
of definite symmetry.
3a Colony surface rough or bristly
in appearance under microscope
or hand lens. Grey, green, or
brown. Sponges.
Phylum PORIFERA (Fig. 1)
3b Colony surface relatively smooth.
General texture of mass gelatinous,
transparent. Clumps of minute
individual organisms variously
distributed. Moss animals,
bryozoans.
Phylum BRYOZOA (Fig. 2)
4a Microscopic. Action of two
ciliated (fringed) lobes at an-
terior (front) end in life often
gives appearance of wheels.
Body often segmented, accor'dian-
like. Free swimming or attached.
Rotifers or wheel animalcules.
Phylum TROCHELMINTHES
(Rotifera) (Fig. 3)
4b Larger, wormlike, or having
strong skeleton or shell.
5a Skeleton or shell present. Skel- , 15
eton may be external or internal.
5b Body soft and for wormlike. 6
Skin may range from soft to
parchment -like.
6a Three or more pairs of well 19
formed jointed legs present.
Phylum ARTHROPODA (Fig. 4)
6b Legs or appendages, if present, 7
limited to pairs of bumps or hooks.
Lobes or tenacles, if present,
soft and fleshy, not jointed.
7a Body strongly depressed or 8
flattened in cross section.
7b Body oval, round, or shaped like 10
an inverted "U" in cross section.
8a Parasitic inside bodies of higher
animals. Extremely long and flat,
divided into sections like a Roman
girdle. Life history may involve
an intermediate host. Tape worms.
Class CESTODA (Fig. 5)
8b Body a single unit. Mouth and 9
digestive system present, but no
anus.
9a External or internal parasite of
higher animals. Sucking discs
present for attachment. Life his-
tory may involve two or more in-
termediate hosts or stages. Flukes.
Class TREMATODA
9b Free living. Entire body covered
with locomotive cilia. Eye areas
in head often appear "crossed".
Free living flatworms.
Class TURBELLARIA (Fig. 6)
lOa Long, slender, with snake-like
motion in life. Covered with glis-
tening cuticle. Parasitic or free-
living. Microscopic to six feet in
length. Round worms.
Phylum NEMATHELMINTHES
(Fig. 7)
lOb Divided into sections or segments 11
12-2
-------
Key to Selected Groups of Freshwater Animals
lOc Unsegmented, head blunt, one 18
or two retractile tentacles.
Flat pointed, tail.
lla Head a more or less well-formed,
hard, capsule with jaws, eyes,
and antennae.
Class INSECTA order DIPTERA
(Figs. 8A, 8C)
lib Head structure soft, except 12
jaws (if present). Fig. 8E.)
12a Head conical or rounded, lateral 13
appendages not conspicuous or
numerous.
12b Head somewhat broad and blunt. 14
Retractile jaws usually present.
Soft fleshy lobes or tentacles,
often somewhat flattened, may be
present in the head region. Tail
usually narrow. Lateral lobes
or fleshy appendages on each
segment unless there is a large
sucker disc at rear end.
Phylum ANNELIDA (Fig. 9)
13a Minute dark colored retractile
jaws present, body tapering
somewhat at both ends, pairs or
rings of bumps or "legs" often
present, even near tail.
Class INSECTA Order DIPTERA
(Fig. 8)
13b No jaws, sides of body generally 14
parallel except at ends. Thicken-
ed area or ring usually present
if not all the way back on body.
Clumps of minute bristles on most
segments. Earthworms, sludge-
worms .
Order OLIGOCHAETA
14a Segments with bristles and/or fleshy
lobes or other extensions. Tube
builders, borers, or burrowers.
Often reddish or greenish in
color. Brackish or fresh water.
Nereid worms.
Order POLYCHAETA (Fig. 9A)
14b Sucker disc at each end, the large
one posterior. External blood-
sucking parasites on higher animals,
often found unattached to host.
Leaches.
Class HIRUDINEA (Fig. 9B)
15a Skeleton internal, of true bone.
(Vertebrates)
15b Body covered with an external
skeleton or shell.
(Figs. 10, 13, 17, 18, 24,
25, 28)
40
16
19
17
16a External skeleton jointed, shell
covers legs and other appendages,
often leathery in nature.
Phylum ARTHROPODA
16b External shell entire, not jointed,
unless composed of two clam-
like halves.
(Figs. 10, 11, 12)
17a Half inch or less in length. Two
leathery, clam-like shells. Soft
parts inside include delicate,
jointed appendages. Phyllopods
or branchiopods.
Class CRUSTACEA, Subclasses
BRANCHIOPODA (Fig. 12)
and OSTRACODA (Fig. 11)
17b Soft parts covered with thin 18
skin, mucous produced, no jointed legs.
Phylum MOLLUSCA
18a Shell single, may be a spiral cone.
Snails.
Class GASTROPODA (Fig. 13)
18b Shell double, two halves, hinged
at one point. Mussels, clams.
Class BIVALVIA (Fig. 10)
19a Three pairs of regular walking 29
legs, or their rudiments. Wings
present in all adults and rudiments
in some larvae.
Class INSECTA (Figs. 22, 24D,
25, 26, 28, 29)
19b More than three pairs of legs 20
apparently present.
20a Body elongated, head broad and flat
12-3
-------
Key to Selected Groups of Freshwater Animals
with strong jaws. Appendages follow-
ing first three pairs of legs are round-
ded tapering filaments. Up to 3
inches long. Dobson fly and fish fly
larvae.
Class INSECTA Order
MEGALOPTERA (Fig. 14)
20b Four or more pairs of legs. 21
21a Four pairs of legs. Body rounded,
bulbous, head minute. Often
brown or red. Water mites.
Phylum ARTHROPODA, Class
ARACHNIDA, Order ACARI
(Fig. 15)
21b Five or more pairs of walking 22
or swimming legs; gills, two
pairs of antennae. Crustaceans.
Phylum ARTHROPODA,
Class CRUSTACEA
22a Ten or more pairs of flattened,
leaflike swimming and respiratory
appendages. Many species swim
constantly in life; some swim
upside down. Fairy shrimps,
phyllopods, or branchipods.
Subclass BRANCHIOPODA
(Fig. 16)
22b Less than ten pairs of swimming 23
or respiratory appendages.
23a Body and legs inclosed in bi- 24
valved (2 halves) shell which may
or may not completely hide them.
23b Body and legs not enclosed in 26
bivalve shell. May be large or
minute.
(Figs. 17, 18, 19)
24a One pair of branched antennae
enlarged for locomotion, extend
outside of shell (carapace).
Single eye usually visible.
"Water fleas"
Subclass CLADOCERA (Fig. 12)
24b Locomotion accomplished by 25
body legs, not by antennae.
25a Appendages leaflike, flattened,
more than ten pairs.
Subclass BRANCHIOPODA
(See 22 a)
25b Animal less than 3 mm, in length.
Appendages more or less slender
and jointed, often used for walking.
Shells opaque. Ostracods.
(Fig. 11) Subclass OSTRACODA
26a Body a series of six or more 27
similar segments, differing
mainly in size.
26b Front part of body enlarged into 28
a somewhat separate body unit
(cephalothorax) often covered
with a single piece of shell (cara-
pace). Back part (abdomen) may be
relatively small, even folded
underneath front part. (Fig. 19b)
27a Body compressed laterally, i.e.,
organism is tall and thin. Scuds.
amphipods.
Subclass AMPHIPODA (Fig. 17)
27b Body compressed dorsoventrally,
i. e., organism low and broad.
Flat gills contained in chamber
beneath tail. Sowbugs.
Subclass ISOPODA (Fig. 18)
28a Abdomen extending straight out
behind, ending in two small pro-
jections. One or two large masses of
eggs are often attached to female.
Locomotion by means of two enlarged,
unbranched antennae, the only large
appendages on the body. Copepods.
Subclass COPEPODA (Fig. 19)
28b Abdomen extending out behind ending
in an expanded "flipper" or swim-
ming paddle. Crayfish or craw fish.
Eyes on movable stalks. Size range
usually from one to six inches.
Subclass DECAPODA
29a Two pairs of functional wings, 39
one pair may be more or less har-
dened as protection for the other
pair. Adult insects which normally
live on or in the water. (Figs. 25, 28)
12-4
-------
Key to Selected Groups of Freshwater Animals
29b No functional wings, though 30
pads in which wings are develop-
ing may be visible. Some may
resemble adult insects very
closely, others may differ ex-
tremely from adults.
30a External pads or cases in which 35
wings develop clearly visible.(Figs.
24,26,27)
30b More or less wormlike, or at 31
least no external evidence of
wing development.
31a No jointed legs present. Other
structures such as hooks, sucker
discs, breathing tubes may be
present. Larvae of flies,
midges, etc.
Order DIPTERA (Fig. 8)
31b Three pairs of jointed thoracic 32
legs, head capsule well formed.
32a Minute (2-4mm) living on the
water surface film. Tail a
strong organ that can be hooked
into a "catch" beneath the
thorax. When released animal
jumps into the air. No wings
are ever grown. Adult spring-
tails.
Order COLLEMBOLA (Fig. 20)
32b Larger (usually over 5 mm) 33
wormlike, living beneath the
surface.
33a Live in cases or webs in water.
Cases or webs have a silk
foundation to which tiny sticks,
stones, and/or bits of debris
are attached. Abdominal segments
often with minute gill filaments.
Generally cylindric in shape.
CaddisfLy larvae.
Order TRICHOPTERA (Fig. 21)
33b Free living, build no cases. 34
34a Somewhat flattened in cross
section and massive in appear-
ance. Each abdominal segment
with rather stout, tapering, lateral
filaments about as long as body
is wide. Alderflies, fishflies, and
dobsonflies.
Order MEGALOPTERA (Fig. 22, 14)
34b Generally rounded in cross section.
Lateral filaments if present tend
to be long and thin. A few forms
extremely flattened, like a suction
cup. Beetle larvae.
Order COLEOPTERA (Fig. 23)
35a Two or three filaments or other 37
structures extending out from
end of abdomen.
35b Abdomen ending abruptly, unless 36
terminal segment itself is extended •
as single structure.(Figs. 24A, 24C)
36a Mouth parts adopted for chewing.
Front of face covered by extensible
folded mouthparts often called a
"mask". Head broad, eyes widely
spaced. Nymphs of dragonflies
or darning needles.
Order ODONATA (Figs.24A. 24C, 24E)
36b Mouthparts for piercing and sucking.
Legs often adapted for water lo-
comotion. Body forms various.
Water bugs, water scorpions, water
boatmen, backswimmers, electric
light bugs, water striders, water
measurers, etc.
Order HEMIPTERA (Fig. 25)
37a Tail extensions (caudal filaments)
two. Stonefly larvae.
Order PLECOPTERA (Fig. 26)
37b Tail extensions three, at times
greatly reduced in size.
38a Tail extensions long and slender.
Rows of hairs may give extensions
a feather-like appearance.
Mayfly larvae.
Order EPHEMEROPTERA
(Fig. 27)
38b Tail extensions flat, elongated
plates. Head broad with widely
spaced eyes, abdomen relatively
long and slender. Damselfly
nymths.
Order ODONATA (Fig.
38
12-S
-------
Key to Selected Groups of Freshwater Animals
39a External wings or wing covers
form a hard protective dome
over the inner wings folded
beneath, and over the abdomen.
Beetles.
Order COLEOPTERA
(Fig. 28)
39b External wings leathery at base,
Membranaceous at tip. Wings
sometimes very short. Mouth-
parts for piercing and sucking.
Body form various. True bugs.
Order HEMIPTERA (Fig. 25)
40a Appendage present in pairs.
(fins, legs, wings).
40b No paired appendages. Mouth
a round suction disc.
4la Body long and slender. Several
holes along side of head.
Lampreys.
Sub Phylum VERTEBRATA,
Class CYCLOSTOMATA
41b 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
42a Paired appendages are legs 43
42b Paired appendages are fins,
gills covered by a flap
(operculum). True fishes.
Class PISCES
43a Digits with claws, nails, or hoofs 44
43b Skin naked. No claws or digits.
Frogs, toads, and salamanders.
Class AMPHIBIA
42 44a Warm blooded
41 44b Cold blooded. Body covered
with horny scales or plates.
Class REPTILIA
45a Body covered with feathers.
Birds.
Class AVES
45b Body covered with hair.
Mammals.
Class MAMMALIA
45
12-6
-------
Key to Selected Groups of Freshwater Animals
REFERENCES - Invertebrates
1 Eddy, Samuel and Hodson, A.C.
Taxonomic Keys to the Common
Animals of the North Central States.
Burgess Pub. Company, Minneapolis.
162 p. 1961.
2 Edmondson, W. T. (ed.) and Ward and
Whipple's Freshwater Biology. John
Wiley & Sons, New York. pp. 1-1248.
1959.
3 Jahn, T. L. and Jahn, F.F. How to Know
the Protozoa. Wm. C. Brown Company,
Dubuque, Iowa. pp. 1-234. 1949.
4 Klots, Elsie B. The New Field Book of
Freshwater Life. G.P. Putnam's Sons.
398pp. 1966.
5 Kudo, R. Protozoology. Charles C.
Thomas, Publisher, Springfield, Illinois.
pp. 1-778. 1950.
6 Palmer, E. Lawrence. Fieldbook of
Natural History. Whittlesey House,
McGraw-Hill Book Company, Inc.
New York. 1949.
7 Pennak, R.W. Freshwater Invertebrates
of the United States. The Ronald Press
Company, New York. pp. 1-769. 1953.
8 Pimentel, Richard A. Invertebrate
Identification Manual. Reinhold
Publishing Corp. 151 pp. 1967.
9 Pratt, H.W. A Manual of the Common
Invertebrate Animals Exclusive of
Insects. The Blaikston Company,
Philadelphia, Pa. pp. 1-854. 1951.
REFERENCES - Fishes
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. Executive Secretary AFS.
Washington Bid. Suite 1040, 15th &
New York Avenue, N.W. Washington,
DC 20005. (Price $4.00 paper,
$7.00 cloth). 1970.
2 Bailey, Reeve M. A Revised List of
the Fishes of Iowa with Keys for
Identification, IN: Iowa Fish and
Fishing. State of Iowa, Super, of
Printing. 1956. (Excellent color
pictures).
3 Eddy, Samuel. How to Know the
Freshwater Fishes. Wm. C. Brown
Company, Dubuque, Iowa. 1957.
4 Hubbs, C.L. and Lagler, K.F. Fishes
of the Great Lakes Region. Bull.
Cranbrook Inst. Science, Bloomfield
Hills, Michigan. 1949.
5 Lagler, K.F. Freshwater Fishery
Biology. Wm. C. Brown Company,
Dubuque, Iowa. 1952.
6 Trautman, M. B. The Fishes of Ohio.
Ohio State University Press, Columbus.
1957. (An outstanding example of a
State study).
Descriptors: Aquatic Life, Systematics.
12-7
-------
Key to Selected Groups of Freshwater Animals
1. Spongilla spicules
Up to .2 mm. long.
3A. Rotifer, Polyarthra
Up to . 3 mm.
3B. Rotifer, Keratella
Up to . 3 mm.
3C. Rotifer, Philodina
Up to . 4 mm.
4A. Jointed leg
Caddis fly
4B. Jointed leg
Crayfish
2B. Bryozoal mass. Up to
several feet diam.
2A. Bryozoa, Plumatella. Individuals up
to 2 mm. Intertwined masses maybe
very extensive.
4C. Jointed leg
Ostracod
5. Tapeworm head,
Taenia. Up to
25 yds. long
6A. Turbellaria. Mesostoma
Up to 1 cm.
6B. Turbellaria, Dugesia
Up to 1.6 cm.
12-8
7. Nematodes. Free living
forms commonly up to
1 mm., occasionally
more.
-------
Key to Selected Groups of Freshwater Animals
8B. Diptera, Mosquito
pupa. Up to 5mm.
8A. Diptera, Mosquito larvae
Up to 15 mm. long.
8C. Diptera, chironomid 8E'
larvae. Up to 2 cm.
9D. Diptera, Rattailed maggot
Up to 25 mm. without tube.
9A. Annelid,
segmented
worm, up to
1/2 meter
10B. Alasmidonta, end view.
10A. Pelecyopod, Alasmidonta
Side view, up to 18 cm. long.
9B. Annelid, leech up to 20 cm.
i\\\
in
11A. Ostracod, Cypericus
Side view, up to 7 mm.
12A. Branchiopod,
Daphnia. Up
to 4mm.
HB. Cypericus, end view.
12B. Branchiopod,
Bosmina. Up
to 2mm.
12-9
-------
Key to Selected Groups of Freshwater Animals
13. Gastropod, Campeloma
Up to'3 inches.
15. Water mite,
up.to 3 mm.
14. Megaloptera, Sialis
Alderfly larvae
Up to 25 mm.
16. Fairy Shrimp, Eubranchjpus
Up to 5 cm.
17. Amphipod, Pomtoporeia
Up to 25 mm.
18. Isopod, Asellus
Up to 25 mm.
20. Collembola, Podura
Up to 2 mm, long
12-10
\
19A. Calanoid copepod,
Female 19B. C'yclopoid copepod
Up to 3 mm. Female
Up to 25 mm.
-------
Key to Selected Groups of Freshwater Animals
21A.
21B.
21C.
21D. 21E.
21. Trichoptera, l-arval cases,
mostly 1-2 cm.
22. Megaloptera, alderfly
Up to 2 cm.
23A. Beetle larvae, 23B. Beetle larvae, 24A. Odohata, dragonfly
Dytisidae, Hydrophilidae nymph up to 3 or
Usually about 2 cm. Usually about 4 cm
t . 1 cm.
24B. Odonata, tail
of damselfly
nymph
(side view)
Suborder
Zygoptera
(24B, D)
4 24D. Odonata, damselfly
nymph (top view)
24E, Odonata, front view
•(//"„ I of dragonfly nymph
showing "mask"
partially extended
Suborder
Anisoptera
(24A, E, C)
24C. Odonata, tail of
dragonfly nymph
(top view)
12-11
-------
Key to Selected Groups of Freshwater Animals
2SA. Hemiptera,
Water Boatman
About 1 cm.
27 .Epheme ropte ra.
Mayfly nymph
Up to 3cm.
25B. Hemiptera,
Water Scorpion
About 4 cm.
28A. Coleoptera,
Water scavenger
beetle. Up to 4 cm.
26. Plecoptera,
Stone fly nymph
Up to 5cm.
28B. Coleoptera,
Dytiscid beetle
Usually up to 4 cm,
12-12
29A. Diptera. Crane
fly. Up to 2| cm.
29B. Diptera. Mosquito
Up to 20 mm.
-------
USING BENTfflC BIOTA IN WATER QUALITY EVALUATIONS
I BENTHOS ARE ORGANISMS GROWING
ON OR ASSOCIATED PRINCIPALLY
WITH THE BOTTOM OF WATERWAYS
Benthos is the noun.
Benthonic, benthal and benthic are
adjectives.
II THE BENTHIC COMMUNITY
A Composed of 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 locomotion. These
life forms are:
1 Bacteria
A wide variety of decomposers work
on organic materials, breaking them
down to elemental or simple com-
pounds (heterotrophic). Other forms
grow on basic nutrient compounds or
form more complex chemical com-
pounds (autotrophic).
2 Algae
Photo synthetic plants having no true
roots, stems, and leaves. The basic
producers of food that nurtures the
animal components of the community.
3 Flowering Aquatic Plants (Pondweeds)
The largest flora, composed of
complex and differentiated tissues.
Many are rooted.
4 Microfauna
Animals that pass through a U. S.
Standard Series No. 30 sieve, but
are retained on a No. 100 sieve.
Examples are rotifers and micro-
crustaceans. Some forms have
organs for attachment to substrates,
while others burrow into soft
materials or occupy the interstices
between rocks, floral or faunal
materials.
5 Meiofauna
Animals, mostly metazoans, that
can pass a 1.0 mm to 0. 5 mm
screen. Examples are naiad
worms and flatworms.
6 Macrofauna
Animals that are 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
such as sculpins and darters.
B It is a self-contained community, though
there is interchange with other commun-
ities. For example: Plankton settles
to it, fish prey on it and lay their eggs
there, terrestrial detritus leaves are
added to it, and many aquatic insects
migrate from it to the terrestrial en-
vironment for their mating cycles.
C It is a stationary water quality monitor.
The low motility of the biotic. compon-
ents requires that they "live with" the
quality changes of the over-passing
waters. Changes 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.
Ill HISTORY OF BENTHIC OBSERVATIONS
A Ancient literature records the vermin
associated with fouled waters.
B 500-year-old fishing literature refers
to animal forms that are fish food and
used as bait.
BI. MET. fm.Se. 1.74
13-1
-------
Using Benthic Biota in Water Quality Evaluations
C The scientific literature associating
biota to water pollution problems is
over 100 years old (Mackenthun and
Ingram, 1964).
D Early this century, applied biological
investigations were initiated.
1 The entrance of State boards of Health
into water pollution control activities.
2 Creation of state conservation agencies.
3 Industrialization and urbanization.
4 Growth of limnological programs
at universities.
E A decided increase in benthic studies
occurred in the 1950 decade, and much
of today's activities are strongly influenced
by developmental work conducted during
this period. Some of the reasons for this
are:
1 Movement of the universities from
"academic biology" to applied
pollution programs.
2 Entrance of the federal government
into enforcement aspects of water
pollution control.
3 A rising economy and the development
of federal grant systems.
4 Environmental Protection Programs
are a current stimulus.
IV WHY THE BENTHOS?
A It is a natural monitor
B The community contains all of the
components of an ecosystem.
1 Reducers
2 Producers
3 Consumers
a Detritivores and bacterial feeders
b Herbivores
c Predators
C Economy of Survey
1 Manpower
2 Time
3 Equipment
D Extensive Supporting Literature
E Advantages of the Macrobenthos
1 Relatively sessile
2 Life history length
3 Fish food organisms
4 Reliability of Sampling
5 Dollars/information
6 Predictability
7 Universality
V REACTIONS OF THE COMMUNITY TO
POLLUTANTS
A Destruction of Organism Types
1 Beginning with the most sensitive forms,
pollutants kill in order of sensitivity
until the most tolerant form is the last
survivor. This results in a reduction
of variety or diversity of organisms.
2 The usual order of macroinvertebrate
disappearance on a sensitivity scale
below pollution sources is shown in
Figure 2.
Water
Quality
Deteriorati
\
Stoneflies /
Mayflies
ng Caddisffies
Amphipods
Isopods
Midges
^ Oligochaetes
k Water
Quality
Improving
13-2
-------
Using Benthic Biota in Water Quality Evaluations
As water quality improves, these
reappear in the same order.
B The Number of Survivors Increase
1 Competition and predation are reduced
between forms.
2 When the pollutant is a food (plants,
fertilizers, animals, organic materials)
C The Number of Survivors Decrease
1 The material added is toxic or has no
food value.
2 The material added produces toxic
conditions as a byproduct of decom-
position (e.g., large organic loadings
produce an anaerobic environment
resulting in the production of toxic
sulfides, methanes, etc.)
D The Effects May be Manifest in Com-
binations
1 Of pollutants and their effects.
2 Vary with longitudinal distribution
in a stream. (Figure 1)
E Tolerance to Enrichment Grouping
(Figure 2)
Flexibility must be maintained in the
establishment of tolerance lists based
on the response of organisms to the
environment because of complex relation-
ships among varying environmental
conditions. Some general tolerance
patterns can be established. Stonefly
nymphs, mayfly naiads, hellgrammites,
and caddisfly larvae represent a grouping
(sensitive or intolerant) that is general-
ised quite sensitive to environmental
changes. Blackfly larvae, scuds, sow-
bugs, snails, fingernail clams, dragon-
fly nymphs, damselfly nymphs, and most
kinds of midge larve are intermediate
(facultative or intermediate) in tolerance.
Sludge-worms, some kinds of midge
larvae (bloodworms), and some leeches
cc
in
ID
s
I-
DIRECTION OF FLOW •
A.
S /
fe/
i;
8.
Ill
3
5
1
in
in
I
TIME OR DISTANCE
..NUMBER OF KINDS
..NUMBER OF ORGANISMS
.SLUDGE DEPOSITS
Four basic responses of bottom animals to pollution.
A. Organic wastes eliminate the sensitive bottom animals
and provide food in the form of sludges for the surviving toler-
ant forms. B. Large quantities of decomposing organic wastes
eliminate sensitive bottom animals and the excessive quanti-
ties of byproducts of organic decomposition inhibit the tolerant
forms; in time, with natural stream purification, water quality
improves so that the tolerant forms can flourish, utilizing the
sludges as food. C. Toxic materials eliminate the sensitive
bottom rtnimalg; sludge is absent and food is restricted to that
naturally occurring in the stream, which limits the number of
tolerant surviving forms. Very toxic materials may eliminate
all organisms below a waste source. D. Organic sludges with
toxic materials reduce the number of kinds by eliminating
sensitive forms. Tolerant survivors do not utilize the organic
sludges because the toxicity restricts their growth.
Figure 1
are tolerant to comparatively heavy loads
of organic pollutants. Sewage mosquitoes
and rat-tailed maggots are tolerant of
anaerobic environments.
13-3
-------
Using Benthic Biota in Water Quality Evaluations
F Structural Limitations
1 The morphological structure of a
species limits the type of environment
it may occupy.
a Species with complex appendages
and exposed complicated respiratory
structures, such as stonefly
nymphs, mayfly nymphs, and
caddisfly larvae, that are subjected
to a'constant deluge of setteable
particulate matter soon abandon
the polluted area because of the
constant preening required to main-
tain mobility or respirotory func-
tions; otherwise, they are soon
smothered.
b Benthic animals in depositing zones
may also be burdened by "sewage
fungus" growths including stalked
protozoans. Many of these stalked
protozoans are host specific.
2 Species without complicated external
structures, such as bloodworms and
sludgeworms, are not so limited in
adaptability.
a A sludgeworm, for example, can
burrow in a deludge of particulate
organic matter and flourish on the
abundance of "manna."
b Morphology also determines the
species that are found in riffles, on
vegetation, on the bottom of pools,
or in bottom deposits.
VI SAMPLING PROCEDURES
A Fauna
1 Qualitative sampling determines the
variety of species occupying an area.
Samples may be taken by any method
that will capture representatives of .the
species present. Collections from such
samplings indicate changes in the
environment, but generally do not
accurately reflect the degree of
change. Mayflies, for example, may
be reduced from 100 to 1 per square
foot. Qualitative data would indicate
the presence of both species, but might
not necessarily delineate the change in
predominance from mayflies to sludge-
worms . The stop net or kick sampling
technique is often used.
2 Quantitative sampling is performed to
observe changes in predominance. The
most common quantitative sampling
tools are the Petersen and Elkman
REPRESENTATIVE EOT TOM-DWELLING MACROANIMALS
Drawings from Geckler, j., K. M. Mackenthun and W. M. Ingram, 1963.
Glossary of Commonly Used Biological and Related Terms in Water and
Waste Water Control, DHEW. PHS, Cincinnati, Ohio, Pub. No. 999-WP-2.
A
B
C
D
E
F
G
H
(Plecoptera)
(Ephemeroptera)
Stonefly nymph
Mayfly nymph
Hellgrammite or
Dobsonfly larvae (Megaloptera)
Caddisfly larvae (Trichoptera)
Black fly larvae (Simuliidae)
Scud (Amphipoda)
Aquatic sowbug (Isopoda)
Snail (Gastropoda)
I Fingernail clam
J Damselfly nymph
K Dragonfly nymph
L Bloodworm or mi
fly larvae
M Leech
N Sludgeworm
O Sewage fly larvae
P Rat-tailed maggot
(Sphaeriidae)
(Zygoptera)
(Anisoptera)
dge
(Chironomidae)
(Hirudinea)
(Tubificidae)
(Psychodidae)
(Tubifera-Eristalis)
KEY TO FIGURE 2
-------
Using Benthic Biota in Water Quality Evaluations
B X^ C
SENSITIVE
F G
INTERMEDIATE
H
M
TOLERANT
13-5
-------
Using Benthic Biota in Water Quality Evaluations
grabs and the Surber stream bottom
or square-foot sampler. Of these,
the Petersen grab samples the widest
variety of substrates. The Ekman
grab is limited to fine-textured and
soft substrates, such as silt and sludge.
The Surber sampler is designed for
sampling riffle areas; it requires
moving water to transport dislodged
organisms into its net and is limited
to depths of two feet or less.
The collected sample is screened with
a standard sieve to concentrate the
organisms; these are sorted from
the retained material, and the number
of each kind determined. Data are then
adjusted to number per unit area,
usually to number of bottom per
square meter.
Independently, neither qualitative not
quantitative data suffice for thorough
analyses of environmental conditions.
A cursory examination to detect damage
may be made with either method, but
a combination of the two gives a more
precise determination. If a choice must
be made, quantitative sampling would
be best, because it incorporates a
partial qualitative sample.
B Flora
Direct quantitative sampling of natu-
rally growing bottom algae is difficult.
It is basically one of collecting algae
from a standardor uniform area of the
bottom substrates without disturbing
the delicate growths and thereby dis-
tort the sample. Indirect quantitative
sampling is the best available method.
Artificial substrates, such as wood
blocks, glass or plexiglass slides,
bricks, etc., are placed in a stream.
Bottom-attached algae will grow on
these artificial substrates. After two
or more weeks, the artificial sub-
strates are removed for analysis.
Algal growths are scraped from the
substrates and the qunatity measured.
Since the exposed substrate area and
exposure periods are equal at all of
the sampling sites, differences in the
quantity of algae can be related to
changes in the quality of water flowing
over the substrates.
The quantity of algae on artificial sub-
strates can be measured in several
ways. Microscopic counts of algal
cells and dry weight of algal material
are long established methods.
Microscopic counts involve thorough
scraping, mixing, and suspension of
the algal cells. From this mixture
an aliquot of cells is withdrawn for
enumeration under a microscope. Dry
weight is determined by drying and
weighing the algal sample, then ig-
niting the sample to burn off the algal
materials, leaving inert inorganic
materials that are again weighed.
The difference between initial weight
and weight after ignition is attributed
to algae.
Any organic sediments, however, that
settle on the artificial substrate along
with the algae are processed also.
Thus, if organic wastes are present
appreciable errors may enter into
this method.
During the past decade, chlorophyll
analysis has become a popular method
for estimating algal growth. Chloro-
phyll is extracted from the algae and
is used as an index of the quantity of
algae present. The advantages of
chlorophyll analysis are rapidity,
simplicity, and vivid pictorial results.
The algae are scrubbed from the
artificial substrate samples, ground,
then each sample is steeped in equal
volumes, 90% aqueous acetone, which
extracts the chlorophyll from the algal
cells. The chlorophyll extracts may
be compared visually.
13-6
-------
Using Benthic Biota in Water Quality Evaluations
Because the chlorophyll extracts fade
with time, colorimentry should be used
for permanent records. For routine
records, simple colorimeters will
suffice. At very high chlorophyll
densities, interference with colori-
metry occurs, which must be corrected
through serial dilution of the sample
or with a nomograph.
4 Autotrophic Index
The chlorophyll content of the periphyton
is used to estimate the algal biomass and
as 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 largely of
algae, which contain approximately
1 to 2 percent chlorophyll a by dry
weight. If dissolved or participate
organic matter is present in high con-
centrations, large populations of
filamentous bacteria, stalked protozoa,
and other nonchlorophyll 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
result from organic pollution (Weber
and McFarland, 1969; Weber, 1973).
... . . T . Ash-free Wgt (mg/m )
Autotrophic Index = -75^ . ,,& .—^T- o(
r Chlorophyll a (mg/m^)
VII FACTORS INVOLVED IN DATA INTER-
PRETATION
Two very important factors in data evalua-
tion are a thorough knowledge of conditions
under which the data were collected and a
critical assessment of the reliability of the
data's representation 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 values usually create acute
conditions in the environment.
B Identification
Precise identification of organisms to
species requires a specialist in limited
taxonomic groups. Many immature
aquatic forms have not been associated
with the adult species. Therefore, one
who is certain of the genus but not the
species should utilize the generic name,
not a potentially incorrect species name.
The method of interpreting biological
data on the basis of numbers of kinds
and numbers of organisms will typically
suffice.
C Lake and Stream Influence
Physical characteristics of a body of
water also affect animal populations.
Lakes or impounded bodies of water
support different faunal 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
naturally supports a higher population
of detritus consumers. For these
reasons, the bottom fauna of a lake
or impoundment cannot be directly
compared with that of a flowing stream.
D Extrapolation
How can bottom-dwelling macrofauna
data be extrapolated to other environ-
mental components? It must be borne
in mind that a component of the total
environment is 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
13-7
-------
Using Benthic Biota in Water Quality Evaluations
would be expected to have a wide vari-
ety of desirable bottom fishes; when
pollution reduces the number of bottom
organisms, a comparable reduction
would be expected in the number of
fishes. Moreover, it would be logical
to conclude that any factor that elim-
inates all bottom organisms would
eliminate most other aquatic forms
of life.
VII IMPORTANT ASSOCIATED ANALYSES
A The Chemical Environment
1 Dissolved oxygen
2 Nutrients
3 Toxic materials
4 Acidity and alkalinity
5 Etc.
B The Physical Environment
1 Suspended solids
2 Temperature
3 Light penetration
4 Sediment composition
5 Etc.
IX AREAS IN WHICH BENTHIC STUDIES
CAN BEST BE APPLIED
A Damage Assessment
If a stream is suffering from pollutants,
the biota will so indicate. A biologist
can determine damages by looking at the
"critter" assemblage in a matter of hours.
Usually, if damages are not found, it will
not be necessary to alert the remainder
of the agency's staff, pack all the equip-
ment, pay travel and per diem, and then
wait five days before enough data can be
assembled to begin evaluation.
B By determining what damages have been
done, the potential cause "list" can be
reduced to a few items for emphasis and
the entire "wonderful worlds" of science
and engineering need not be practiced with
the result that much data are discarded
later because they were not applicable to
the problem being investigated.
C Good benthic data associated with chemical,
physical, and engineering can be data
used to predict the direction of future
changes and to estimate the amount of
pollutants that need to be removed from
the waterways.
REFERENCES
1 Hynes, H. B.N. The Ecology of Running
Waters. Univ. Toronto Press. 1970.
2 Keup, L. E., Ingram, W.M. and
Mackenthun, K. M. The Role of
Bottom Dwelling Macrofauna in Water
Pollution Investigations. USPHS
Environmental Health Series Publ.
No. 999-WP-38, 23 pp. 1966.
3 Keup, L. E., Ingram, W.M. and
Mackenthun, K.M. Biology of Water
Population: A Collection of Selected
Papers on Stream Pollution, Waste
Water, and Water Treatment.
Federal Water Pollution Control
Administration Pub. No. CWA-3,
290pp. 1967.
4 Mackenthun, K. M. The Practice of
Water Pollution Biology. FWQA.
281 pp. 1969.
5 Stewart, R.K., Ingram, W.M. and
Mackenthun, K.M. Water Pollution
Control, Waste Treatment and Water
Treatment: Selected Biological Ref-
erences on Fresh and Marine Waters.
FWPCA Pub. No. WP-23, 126 pp. 1966.
13-8
-------
Using Benthic Biota in Water Quality Evaluations
Weber, Cornelins I., Biological Field
and Laboratory Methods for Measuring This outline was prepared by Lowell E.
the Quality of Surface Waters and Keup, Chief, Technical Studies Branch,
Effluents. U. S. Environmental Pro- Div. of Technical Support, EPA, Wash-
tection Agency, NERC, Cincinnati, ington, D. C. 20242, and revised by
OH . Environmental Monitoring Series Ralph M. Sinclair, Aquatic Biologist,
670/4.73.001 July 1973 National Training Center, EPA, WPO,
Cincinnati, OH 45268.
Descriptors:
Aquatic Life, Benthos, Water Quality,
Degradation, Environmental Effects,
Trophic Level, Biological Communities,
Ecological Distributions
13-9
-------
AUTOMATIC INSTRUMENTS FOR WATER QUALITY MEASUREMENTS
I NEED FOR CONTINUOUS MONITORING
OF WATER QUALITY
Satisfactory evaluation of the quality of water
depends upon the availability of adequate data.
Such data must be not only as accurate as
economically practical but must also be of
sufficient quantity to support reliable con-
clusions. In general, the more information
available, the more reliable will be the
interpretations.
In the past, information on the chemical,
physical, and bacteriological quality of most
surface waters has been obtained by periodic
stream surveys or infrequent "spot" analyses.
Although raw water supplies are sampled
daily, the data obtained are restricted to
those tests of importance in water treatment
and do not include other pollutional param-
eters or water resources not at present being
used for water supply.
It is apparent that in many situations signifi-
cant changes in water quality may occur often
and abruptly. Seasonal changes in flow, the
occurrence of unpredictable industrial dis-
charges or spills, and the changes in flow
from impoundments may alter the concen-
tration of many of the substances of interest
to the water user. In some cases these
changes may occur within a few hours: for
example, the diurnal fluctuations in dissolved
oxygen and the salinity changes in tidal
estuaries. Thus considerable advantage is
gained if continuous monitoring of water
quality can be accomplished. The use of
manual sampling and standard laboratory
analyses would be far too expensive; therefore
a wide variety of automatic instruments have
been developed.
H ELECTRICAL INSTRUMENTS
The diagram below illustrates a typical
system for monitoring instruments which
utilize electrical sensors.
The sensor produces an electrical signal
representative of concentration, the signal
is converted, and amplified, then passed to
the recorder to develop a permanent record
of concentration levels. For a clear
understanding of the operational charac-
teristics, the parts of the system are
discussed separately.
ANALYZER
>
RECORDER
DRAIN
'SENSOR
SAMPLE
A Sensors
Figure 1
The sensor is the part of the system in
contact with the sample. Sensing ele- '
ments may be immersed directly in the
stream, or placed in flow cells through
which the sample is pumped. Both
methods have advantages and limitations.
When the sensor is placed in the stream
the determination is made "in situ. "
Therefore the sample is not affected by
pumping, temperature changes, or time
of travel through the instrument. Such
an installation, however, presents
certain problems. The sensing elements
must be protected from floating debris
and must be mounted so as to remain in
a fixed position in spite of changes in
velocity or direction of current. In bodies
of water which fluctuate in surface level,
such as impoundments, or estuaries, the
relative depth of the sensor may change.
In addition, frequent inspection of the
sensing elements for attached growths or
physical damage is difficult and apt to be
neglected.
CH.MET. 18b.3.74
14-1
-------
Automatic Instruments for Water Quality Measurements
The use of the shore-based system, where
the stream sample is pumped through flow
cells within the instrument housing, is
free from some of the difficulties men-
tioned previously. Inspection of the
sensors is easy, cleaning is simplified,
and replacement of sensing elements is
readily accomplished. It should be
recognized, however, that the precautions
required for satisfactory mounting and
protection of sensor units in the stream
apply equally well to the pump intake.
Further, it is essential that the sample
being tested in the flow cell is truly
representative of the stream water. If
dissolved oxygen is included in the
parameters, a submersible pump is
required, to avoid cavitation and prevent
suction removal of dissolved gases. The
intake screen must be carefully designed,
since a fine screen will quickly clog, while
a coarse screen may permit floating
material to enter the system.
Electrical sensors may be conductimetric,
potentiometric, polarographic, or
coulimetric. The sensor may directly
measure a constituent or property of the
sample or it may be used as an indicating
mechanism in automatic titration.
B Analyzer-Amplifier
The function of the analyzer is to convert
the signal from the sensor to a standard
EMF, usually ranging from 0-50 millivolts.
Often bridge circuits are employed. The
analyzer must be rugged to avoid shock
damage and the electronic circuitry must
be stable over a wide range of environmental
conditions. Considerable advantage is
gained by the use of standard, readily
available components. Provision should
be made for a standard signal to permit
internal checking of the circuits.
In most cases the signal must be amplified
to provide sufficient voltage to drive a
recorder. Amplification is generally
built into the analyzer circuitry.
C Recorder
Although read-out meters can be used, a
permanent record is desirable.. Strip-
chart recorders are the most popular
even though they require line voltage.
A slow chart speed is necessary, since
observation periods of several days
are common. Difficulties with pens are
often encountered due to varying con-
. ditions of humidity and temperature.
The use of pressure-sensitive paper
may offer distinct advantage.
Circular charts have long been used but
they have certain disadvantages. Unless
the chart is inconveniently large the chart
divisions are very close together making
hourly fluctuations difficult to interpret.
In addition, the circular chart does not
lend itself to mechanical handling of data
for computer processing. Chart-drive by
clock mechanism is possible, however,
eliminating the need for power connection.
A preferable system for the recording of
data is the use of digital output. Water
quality instruments can be equipped with
analog-to-digital converters which change
the amplitude of the signal to a digital
value. By the use of a punch-tape readout
the data is recorded on paper tape rather
than on a chart. The punched tape may
then be transferred to a computer for data
processing. Therefore, a monitor equipped
with digital tape readout permits the re-
cording of original signal and the per-
formance of statistical computations without
hand transcription.
When the Surveillance Program includes
several automatic monitors on a river
system , consideration should be given to
a central data logging facility. By
transmitting the data from the monitors
directly to a small computer (by leased
phone lines or radio), the information on
water quality of the entire river basin is
immediately available. The central
facility also provides for the detection of
unusual discharges which may affect
downstream water users.
14-2
-------
Automatic Instruments for Water Quality Measurements
D Parameters Measured Electrically
The following water quality parameters
can be measured by the use of electrodes:
Temperature
Conductivity
PH
Oxygen
Chloride
Residual chlorine
Oxidation-reduc-
tion potential
A prominent instrument manufacturer is
now conducting research into the develop-
ment of a number of glass electrodes
specifically designed for the measurement
of other ions. It appears that the list above
may be considerably expanded in the future.
In most cases precision and accuracy is
not as good as with standard laboratory
tests, but the advantages of continuous
recording outweigh the limitations in
performance.
Ill PHOTOMETRIC INSTRUMENTS
Because of the importance of colorimetric
analysis in the water laboratory, considerable
attention has been given to the development of
continuous monitoring instruments employing
this principle. A typical system is shown
in Figure 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 sample, such as
turbidity or color, is measured directly.
These relatively simple parameters,
however, are rather difficult to determine.
Turbidity measurements are affected by
particle size "and by the true color of the
sample. Conversely, color determinations
are subject to errors caused by turbidity
and by the fact that the wave length of the
color in the sample may vary widely.
Instruments of this type currently
available do not satisfactorily compensate
for these interferences and the data
obtained from them does not correlate
well with standard methods values.
ANALYZER
COLORIMETER
SAMPLE
DRAIN
14-3
-------
Automatic Instruments for Water Quality Measurements
B Colorimetric Analyzers
A second type of photometric instrument
is designed to reproduce laboratory
colorimetric procedures. That is, re-
agents are added to the sample to produce
a color change proportional to the con-
centration of the material being determined.
Since the sample is flowing continuously
the reagents must be metered accurately
and mixed thoroughly before photometric
measurement. In a properly designed
system, almost any colorimetric procedure
can be duplicated and therefore the potential
range of determinations is much wider
than in the electrometric instruments.
A modification of the colorimetric instru-
ment is the continuous titrator. In this
system an indicator is added to the flowing
sample and the reagent is added at a vari-
able rate to maintain a constant 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 metering pump acts as a signal for
the analyzer.
In spite of the apparent advantages of the
photometric systems, certain special
problems are inherent. The color and
turbidity of the sample may interfere, the
accumulation of slime in the cells may
seriously reduce the sensitivity, and the
limitations of the filter photometer (wide
band pass) must be considered. Further,
in most colorimetric procedures the
amount of reagent required is proportional
to the concentration range of the sample,
a factor which would limit the applicability
of the instrument.
C Parameters Measured Photometrically
Continuous analytical procedures have
been developed for:
Turbidity
Color
Hardness
Residual Chlorine
Alkalinity
Fluoride
Silica
Phosphate
Phenols
Chemical Oxygen
Demand
IV PERFORMANCE
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 conducted on a proto-
type instrument supplied by one manufacturer.
The "Acceptable" tolerances were selected as
representative of the usual requirements for
continuous data acquisition and may be too
high or too low, depending upon the accuracy
deemed necessary.
V CALIBRATION AND MAINTENANCE
A Calibration
In the parlance of the instrument manu-
facturer, calibration involves two steps;
(1) the setting of the readout to zero value
in the absence of sensor signal, and
(2) setting of the readout to some standard
value, such as 5mv. when the sensor
signal is replaced by a standard signal.
It is 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 of 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 a true value for
the measured material. 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 standard solutions for calibra-
tion purposes.
The frequency of recalibration depends
upon both the stability of the analyzer and
the sensor. In general, analyzer-amplifiers
are more stable than the sensing systems
now in use. The Basic Data Program,
14-4
-------
Automatic Instruments for Water Quality Measurements
Test
PH
Temperature
Conductance
Dissolved Oxygen
PERFORMANCE DATA
Acceptable deviation Mean deviation found
+ 0.1 unit 0.1 unit
+ 1° 0. 2°
+ 5% 17 (imhos
+ 0.5 mg/1 0.3 mg/1
Acceptable
80%
85%
99%
73%
FWPCA, in performance specifications
for monitoring instruments,has established
a two-week period for unattended per-
formance. While such instrument stability
is desirable, circumstances of site location
and sample characteristics, often
necessitate more frequent checking and
recalib ration.
B Maintenance
It is unwise to assume that any stream
monitoring instrument, no matter how
well designed and built, can function
adequately for extended periods of time
without maintenance. Sensing elements
are subject to physical, chemical, and
biological actions, analyzer components
may fail or perform erratically, and
recorders may stop or fail to print. The
frequency of maintenance depends upon a
large number of factors both controllable
and accidental and can be determined
only by long-term testing and actual field
experience. Ease of maintenance can be
designed into the instrument by the use of
replaceable electronic components and
accessibility for cleaning of flow cells
and sample lines.
The availability of trained personnel for
checking and maintaining the instruments
in the field is a key factor in the successful
stream monitoring program. Such per-
sonnel need not be either electronic
specialists or experienced analysts but
they should have a working knowledge of
the instrument system and a familiarity
with the chemical principles involved in
the use of standard solutions for calibration.
VI APPLICATIONS
Stream monitoring systems are now in use
in a variety of locations throughout the United
States. In most cases the instruments
measure a single parameter or condition of
the stream rather than a number of para-
meters. Some of the instruments employ
well established measurement systems, for
example pH and conductance, while others,
are still largely experimental.
Because of the need for continuous stream
data, the intensive interest on the part of
regulatory agencies, and the development
programs by instrument makers, it is safe
to assume that the use of water quality
monitoring instruments will increase
significantly in the near future.
This outline was prepared by D. G. Ballinger,
Director, EPA, WPO, Methods Development
& Quality Assurance Laboratory, Cincinnati,
OH 45268
Descriptors: Analytical Techniques, Instru-
mentation, Testing Monitoring, Control
Systems, Data Collections, Data Trans-
mission, Remote Sensing
14-.5
-------
DISSOLVED OXYGEN
Determination By The Winkler lodometric Titration and Azide Modification
The basic Winkler procedure (1888) has been
modified many times to improve its work-
ability in polluted waters. None of these
modifications have been completely
successful. The most useful modification
was proposed by Alsterberg and consists of
the addition of sodium azide to control
nitrite interference during the iodometric
titration. The Azide modification of the
iodometric titration is recommended as
official by the EPA- WPO Quality Control
Committee for relatively clean waters.
A Reactions
1 The determination of DO involves
a complex series of interactions
that must be quantitative to provide
a valid DO result. The number of
sequential reactions also compli-
cates interference control. The
reactions will be presented first
followed by discussion of the
functional aspects.
MnSO + 2
2 Mn(OHL
A
MnO(OH)0
£
Mn(SO.)0 H
4 2
I2 + 2 Na2!
KOH ~* Mn(OH)2 + K2SO4
+ O0 -*2 MnO(OH)
^ ^
+ 2 H SO -* Mn(SO ) + 3H
£> rt *± ^ £
h 2 KI -» MnSO + K SO + I
4 ^ 4 Z
3 O ~* Na SO + 2 Na I
(a)
(b)
,0(c)
(d)
(e)
2 Reaction sequence
The series of reactions involves
five different operational steps in
converting dissolved oxygen in the
water into a form in which it can
be measured.
Mn(S04)2
I -+ Thiosulfate (thio) or
£t
phenylarsine oxide (PAO)
titration.
b All added reagents are in excess
to improve contact possibilities
and to force the reaction toward
completion.
The first conversion, O —*•
MnO(OH) (reactions a, B) is an
oxygen transfer operation where
the dissolved oxygen in the water
combines with manganous
hydroxide to form an oxygenated
manganic hydroxide.
a The manganous salt can react
with oxygen only in a highly
alkaline media.
b The manganous salt and alkali
must be added separately with
addition below the surface of
the sample to minimize reaction
with atmospheric oxygen via
air bubbles or surface contact.
Reaction with sample dissolved
oxygen is intended to occur
upon mixing of the reagents and
sample after stoppering the
full bottle (care should be used
to allow entrained air bubbles
to rise to the surface before
adding reagents to prevent
high results due to including
entrained oxygen).
c Transfer of oxygen from the
dissolved state to the pre-
cipitate form involves a two
phase system of solution and
precipitate requiring effective
mixing for quantitative
transfer. Normally a gross
excess of reagents is used
to limit mixing requirements.
Mixing by rapid inversion 25
to 35 times will accomplish
the purpose. Less energy is
required by inversion 5 or 6
CH.O.do.31c.3.74
15-1
-------
Dissolved Oxygen Determination
times, allowing the solids to
settle half way and repeat the
process. Reaction is rapid;
contact is the principal
problem in the two phase
system.
d If the alkaline floe is white,
no oxygen is present.
Acidification (reactions c and d)
changes the oxygenated manganic
hydroxide to manganic sulfate
which in turn reacts with
potassium iodide to form elemental
iodine. Under acid conditions,
oxygen cannot react directly with
the excess manganous sulfate
remaining in solution.
Iodine (reaction e) may be titrated
with sodium thiosulfate standard
solution to indicate the amount of
dissolved oxygen originally
present in the sample.
a The blue color of the starch-
iodine complex commonly is
used as an indicator. This
blue color disappears when
elemental iodine has been
reacted with an equivalent
amount of thiosulfate.
b Phenylarsine oxide solutions are
more expensive to obtain but
have better keeping qualities
than thiosulfate solutions.
Occasional use, field operations
and situations where it is not
feasible to calibrate thio
solutions regularly, usually
encourage use of purchased
PAO reagents.
For practical purposes the DO
determination scheme involves the
following operations.
Fill a 300 ml bottle* under
conditions minimizing DO
changes. This means that the
sample bottle must be flushed
with test solution to displace
the air in the bottle with water
characteristic of the tested
sample*.
*DO test bottle volumes should
be checked - discard those
outside of the limits of 300 ml
+ or - 3 ml.
To the filled bottle:
1) Add MnSO reagent (2 ml)
2) Add KOH, KI, NaN reagent
(2 ml) . d
Stopper, mix by inversion,
allow to settle half way and
repeat the operation.
Highly saline test waters
commonly settle very
slowly at this stage and
may not settle to the half
way point in the time
allotted.
To the alkaline mix (settled
about half way) add 2 ml of
sulfuric acid, stopper and mix
until the precipitate dissolves.
Transfer the contents of the
bottle to a 500 ml Erlenmeyer
flask and titrate with 0.0375
Normal Thiosulfate*. Each
ml of reagent used represents
1 mg of DO/liter of sample.
15-2
-------
Dissolved Oxygen Determination
The same thing applies for
other sample volumes when
using an appropriate titrant
normality such as:
1) For a 200 ml sample, use
0.025 N Thio
2) For a 100 ml sample, use
0.0125 N Thio
*EPA-WPO Method
The addition of the first two DO
reagents, (MnSO4 and the KOH, KI
and NaN3 solutions) displaces an
equal quantity of the sample. This
is not the case when acid is added
because the clear liquid above the
floe does not contain dissolved
oxygen as all of it should be con-
verted to the particulate MnO(OH>2.
Some error is introduced by this
displacement of sample during
dosage of the first two reagents.
The error upon addition of 2 ml of
each reagent to a 300 ml sample
is -i_ X 100 or 1. 33% loss in DO.
300
This may be corrected by an
appropriate factor or by adjust-
ment of reagent normality. It is
generally considered small in
relation to other errors in sampling,
manipulation and interference,
hence this error may be recognized
but not corrected.
Reagent preparation and pro-
cedural details can be found in
reference 1.
IV The sequential reactions for the
Chemical DO determination provides
several situations where significant inter-
ference may occur in application on
polluted water, such as:
A Sampling errors may not be strictly
designated as interference but have the
same effect of changing sample DO.
Inadequate flushing of the bottle con-
tents or exposure to air may raise the
DO of low oxygen samples or lower the
DO of supersaturated samples.
B Entrained air may be trapped in a DO
bottle by:
1 Rapid filling of vigorously mixed
samples without allowing the
entrained air to escape before
closing the bottle and adding DO
reagents.
2 Filling a bottle with low temperature
water holding more DO than that in
equilibrium after the samples warm
to working temperature.
3 Aeration is likely to cool the sample
permitting more DO to be introduced
than can be held at the room or
incubator temperatures.
4 Samples warmer than working
incubator temperatures will be
only partially full at equilibrium
temperatures.
Addition of DO reagents results in
reaction with dissolved or entrained
oxygen. Results for DO are invalid
if there is any evidence of gas
bubbles in the sample bottle.
C The DO reagents respond to any oxidant
or reductant in the sample capable of
reacting within the time allotted. HOC1
or H2O2 may raise the DO titration
while t^S, & SH may react with sample
oxygen to lower the sample titration.
The items mentioned react rapidly and
raise or lower the DO result promptly.
Other items such as Fe or SOl may
or may not react completely within the
time allotted for reaction. Many
organic materials or complexes from
benthic deposits may have an effect upon
DO results that are difficult to predict.
They may have one effect during the
alkaline stage to release iodine from
Kl while favoring irreversible
absorption of iodine during the acid
stage. Degree of effect may increase
with reaction time. It is generally
inadvisable to use the iodometric
titration on samples containing large
amounts of organic contaminants or
15-3
-------
Dissolved Oxygen Determination - II
benthic residues. It would be expected
that benthic residues would tend toward
low results because of the reduced iron
and sulfur content - they commonly
favor high results due to other factors
that react more rapidly, often giving
the same effect as in uncontrolled
nitrite interference during titration.
Nitrite is present to some extent in
natural waters or partially oxidized
treatment plant samples. Nitrite is
associated with a cyclic reaction during
the acid stage of the DO determination
that may lead to erroneous high results.
E
These reactions may be repre-
sented as follows:
2HN02 + 2 HI -J2 + 4H20 + N.O2
.
1/202+
(a)
(b)
These reactions are time, mixing
and concentration dependent and
can be minimized by rapid
processing.
2 Sodium azide (NaNg) reacts with
nitrite under acid conditions to
form a combination of Ng + NgO
which effectively blocks the
cyclic reaction by converting the
HNO to noninterfering compounds
of nitrogen.
3 Sodium azide added to fresh
alkaline KI reagent is adequate to
control interference up to about
20 mg of NO2" N/liter of sample.
The azide is unstable and grad-
ually decomposes. If resuspended
benthic sediments are not detectable
in a sample showing a returning
blue color, it is likely that the
azide has decomposed in the
alkaline KI azide reagent.
Surfactants, color and Fe+++ may
confuse endpoint detection if present
in significant quantities.
F Polluted water commonly contains
significant 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 types of interference.
G The order of reagent addition and prompt
completion of the DO determination is
critical. Stable waters may give valid
DO results after extended delay of
titration during the acidified stage. For .
unstable water, undue delay at any stage
of processing accentuates interference
problems.
A CKNOWLEDGMENTS
This outline contains significant materials
from previous outlines by J. W. Mandia.
Review and comments by C. R. Hirth and
R. L. Booth are greatly appreciated.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, EPA,
WPO, Cincinnati, OH 45268, and revised
by C. R. Feldmann, Chemist, National
Training Center.
Descriptors: Chemical Analysis, Dissolved
Oxygen, Oxygen, Water Analysis
15-4
-------
DISSOLVED OXYGEN
DETERMINATION BY ELECTRONIC MEASUREMENT
I INTRODUCTION
A Electronic measurement of DO is attractive
for several reasons:
1 Electronic methods are more readily
adaptable for automated analysis, con-
tinuous recording, remote sensing or
portability.
2 Application of electronic methods with
membrane protection of sensors affords
a high degree of interference control.
3 Versatility of the electronic system
permits design for a particular measure-
ment, situation or use.
4 Many more determinations per man-
hour are possible with a minor expend-
iture of time for calibration.
B Electronic methods of analysis impose
certain restrictions upon the analyst to
insure that the response does, in fact,
indicate the item sought.
1 The ease of reading the indicator tends
to produce a false sense of security.
Frequent and careful calibrations are
essential to establish workability of the
apparatus and validity of its response.
2 The use of electronic devices requires
a greater degree of competence on the
part of the analyst. Understanding of
the behavior of oxygen must be supple-
mented by an understanding of the
particular instrument and its behavior
during use.
C Definitions
1 Electrochemistry - a branch of chemistry
dealing with relationships between
electrical and chemical changes.
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.
Reduction - any process in which one
or more electrons are added to an atom
or an ion, such as O_ + 2e -»• 2O
The oxygen has been reduced.
Oxidation - any process in which one
or more electrons are removed from
an atom or an ion, such as Zn° - 2e
Zn
+2
The zinc has been oxidized.
Oxidation - reduction reactions - in a
strictly chemical reaction, reduction
cannot occur unless an equivalent
amount of some oxidizable substance
has been oxidized. For example:
2H
+02 -
-4e 3±
°2+4e
2H20
4H hydrogen oxidized
2O" oxygen reduced
Chemical reduction of oxygen may also
be accomplished by electrons supplied
to a noble metal electrode by a battery
or other energizer.
Anode - an electrode at which oxidation
of some reactable substance occurs.
Cathode - an electrode at which
reduction of some reactable substance
occurs. For example in I. C. 3, the
reduction of oxygen occurs at the
cathode.
Electrochemical reaction - a reaction
involving simultaneous conversion of
chemical energy into electrical energy
or the reverse. These conversions are
Note: Mention of Commercial Products and Manufacturers Does
Not Imply Endorsement by the Environmental Protection
Agency.
CH.O. do.32a. 3.74
16-1
-------
Dissolved Oxygen Determination - II
equivalent in terms of chemical and
electrical energy and generally are
reversible.
9 Electrolyte a solution, gel, or mixture
capable of conducting electrical energy
and serving as a reacting media for
chemical changes. The electrolyte
commonly contains an appropriate
concentration of selected mobile ions
to promote the desired reactions.
10 Electrochemical cell - a device con-
sisting of an electrolyte in which 2
electrodes are immersed and connected
via an external metallic conductor.
The electrodes may be in separate
compartments connected by tube con-
taining electrolyte to complete the
internal circuit.
a Galvanic (or voltaic) cell - an
electrochemical cell operated in
such a way as to produce electrical
energy from a chemical change,
such as a battery (See Figure 1).
Polarographic (electrolytic) cell -
an electrochemical cell operated in
such a way as to produce a chemical
change from electrical energy
(See Figure 2).
Cathode
Anode
POLAROGRAPHIC CELL
Figure 2
Anode
Cathode
GALVANIC-CELL
Figure 1
D As indicated in I. C. 10 the sign of an
electrode may change as a result of the
operating mode. The conversion by the
reactant of primary interest at a given
electrode therefore designates terminology
for that electrode and operating mode.
In electronic oxygen analyzers, the
electrode at which oxygen reduction occurs
is designated the cathode.
E Each cell type has characteristic advantages
and limitations. Both may be used
effectively.
1 The galvanic cell depends upon
measurement of electrical energy
produced as a result of oxygen
16-2
-------
Dissolved Oxygen Determination
reduction. If the oxygen content of the
sample is negligible, the measured
current is very low and indicator driving
force is negligible, 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 change 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.
H ELECTRONIC MEASUREMENT OF DO
A Reduction of oxygen takes place in two
steps as shown in the following equations:
1 O0 + 2H0O + 2e -* HO + 2OH
ft ci 2i &
2 H2O2 + 2e -+ 2OH
Both equations require electron input to
activate reduction of oxygen. The first
reaction is more important for electronic
DO measurement because it occurs at a
potential (voltage) which is below that
required to activate reduction of most
interfering components (0.3 to 0.8 volts
relative to the saturated calomel electrode •
SCE). Interferences that may be reduced
at or below that required for oxygen
usually are present at lower concentrations
in water or may be minimized by the use
of a selective membrane or other means.
When reduction occurs, a definite quantity
of electrical energy is produced that is
proportional to the quantity of reductant
entering the reaction. Resulting current
measurements thus are more specific for
oxygen reduction.
B Most electronic measurements of oxygen
are based upon one of two techniques for
evaluating oxygen reduction in line with
equation II.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
supplying the activating potential; one
employs a source of outside energy, the
other uses spontaneous energy produced
by the electrode pair.
1 The polarographic 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 DO
using the DME but the difficulty of
maintaining a constant mercury drop
rate, temperature 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 sufficiently 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 availability.
Interrupting the outside circuit may
produce erratic behavior for a time
after reconnection. The resulting
16-3
-------
Dissolved Oxygen Determination - II
current produced by oxygen reduction
may be converted to oxygen concen-
tration by use of a sensitivity coefficient'
obtained during calibration. Provision
of a pulsed or interrupted signal makes
it possible to amplify or control the
signal and adjust it for direct reading
in terms of oxygen concentration or to
compensate for temperature effects.
Ill E LE CTRONIC DO A NA LY ZER
APPLICATION FACTORS
A Polarographic or galvanic DO instruments
operate as a result of oxygen partial
pressure at the sensor surface to produce
a signal characteristic of oxygen reduced
at the cathode of some electrode pair.
This signal is conveyed to an indicating
device with or without modification for
sensitivity and temperature or other
influences depending upon the instrument
capabilities and intended use.
1 Many approaches and refinements have
been used to improve workability,
applicability, validity, stability and
control of variables. Developments
are continuing. It is possible to produce
a device capable of meeting any reasonable
situation, but situations differ.
2 Most commercial DO instruments are
designed for use under specified con-
ditions. Some are more versatile than
others. Benefits are commonly reflected
in the price. It is essential to deter-
mine the requirements of the measure-
ment situation and objectives for use.
Evaluation of a given instrument in
terms of sensitivity, response time,
portability, stability, service
characteristics, degree of automation,
and consistency are used for judgment
on a cost/benefit basis to select the
most acceptable unit.
B Variables Affecting Electronic DO
Measurement
1 Temperature affects the solubility of
oxygen, the magnitude of the resulting
signal and the permeability of the
protective membrane. A curve of
oxygen solubility in water versus
increasing temperature may be concave
downward while a similar curve of
sensor response versus temperature
is concave upward. Increasing
temperature decreases oxygen solubility
and increases probe sensitivity and
membrane permeability. Thermistor
actuated compensation of probe
response based upon a linear relation-
ship or average of oxygen solubility
and electrode sensitivity is not precisely
correct as the maximum spread in
curvature occurs at about 17° C with
lower deviations from linearity above
or below that temperature. If the
instrument is calibrated at a temperature
within + or - 5° C of working temperature,
the compensated readout is likely to be
within 2% of the real value. Depending
upon probe geometry, the laboratory
sensor may require 4 to 6% correction
of signal per o C change in liquid
temperature.
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
negligible.
Inclusion of entrained gases results in
erratic response that increases with
depth of immersion.
Electrode sensitivity changes occur as
a result of the nature and concentration
of contaminants at the electrode sur-
faces and possible physical chemical or
electronic side reactions produced.
These may take the form of a physical
barrier, internal short, high residual
current, or chemical changes in the
metal surface. The membrane is
intended to allow dissolved gas pene-
tration but to exclude passage of ions
or particulates. Apparently some ions
or materials producing extraneous ions
within the electrode vicinity are able
to pass in limited amounts which
16-4
-------
Dissolved Oxygen Determination
become significant in time. Dissolved
gases include 1) oxygen, 2) nitrogen,
3) carbon dioxide, 4) hydrogen, sulfide,
and certain others. Item 4 is likely 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 H.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 exposed 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 DO; continuous use in the mixed
liquor required electrode cleanup in 2
to 4 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
advisable.
4 Electrolytes may consist of solutions
or gels of ionizable materials such as
acids, alkalies or salts. Bicarbonates,
KC1 and KI are frequently used. The
electrolyte is 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. Generally 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 of contaminating sub-
stances.
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
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
signal.
6 Test media characteristics control the
interval of usable life between cleaning
and rejuvenation for any type of
electrode. More frequent cleanup is
essential 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 mg/1.
7 Agitation of the sample in the vicinity
of the electrode is important because
DO 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 is 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
16-5
-------
Dissolved Oxygen Determination
mixing may create a vortex and expose
the sensing surface to air rather than.
sample liquid. This should be avoided.
A linear liquid velocity of about 1 ft/sec
at the sensing surface is usually
adequate.
8 DO sensor response represents a
potential or current signal in the
milli-volt or milli-amp range in a
high resistance system. A high quality
electronic instrument is essential to
maintain a usable signal-to-noise ratio.
Some of the more common difficulties
include:
a Variable line voltage or low batteries
in amplifier power circuits.
b Substandard or unsteady amplifier
or resistor components.
c Undependable contacts or junctions
in the sensor, connecting cables, or
instrument control circuits.
d Inadequately shielded electronic
components.
e Excessive exposure to moisture,
fumes or chemicals in the wrong
places lead to stray currents,
internal shorts or other malfunction.
C Desirable Features in a Portable DO
Analyzer
1 The unit should include steady state
performance electronic and indicating
components in a convenient but sturdy
package that is small enough to carry.
2 There should be provisions for addition
of special accessories such as bottle
or field sensors, agitators, recorders,
line extensions, if needed for specific
requirements. Such additions should
be readily attachable and detachable
and maintain good working characteristics.
3 The instrument should include a
sensitivity adjustment which upon
calibration will provide for direct
reading in terms of mg of DO/liter.
4 Temperature compensation and temp-
erature readout should be incorporated.
5 Plug in contacts should be positive,
sturdy, readily cleanable and situated
to minimize contamination. Water
seals should be provided where
necessary.
6 The sensor should be suitably designed
for the purpose intended in terms of
sensitivity, response, stability, and
protection during use. It should be
easy to clean, and reassemble for use
with a minimum loss of service time.
7 Switches, connecting plugs, and con-
tacts preferably should be located on
or in the- instrument box rather than
at the "wet" end of the line near the
sensor. Connecting cables should be
multiple strand to minimize separate
lines. Calibration controls should be
convenient but designed so that it is
not likely that they will be inadvertently
shifted during use.
8 Agitator accessories for bottle use
impose special problems because they
should be small, self contained, and
readily detachable but sturdy enough
to give positive agitation and electrical
continuity in a wet zone.
9 Major load batteries should be
rechargeable or readily replaceable.
Line operation should be feasible
wherever possible.
10 Service and replacement parts avail-
ability are a primary consideration.
Drawings, parts identification and
trouble shooting memos should be
incorporated with applicable operating
instructions in the instrument manual
in an informative organized form.
D Sensor and Instrument Calibration
The instrument box is likely to have some
form of check to verify electronics,
battery or other power supply conditions
for use. The sensor commonly is not
included in this check. A known reference
16-6
-------
Dissolved Oxygen Determination
sample used with the instrument in an
operating mode is the best available
method to compensate for sensor variables
under use conditions. It is advisable to
calibrate before and after daily use under
test conditions. Severe conditions,changes
in conditions, or possible damage call for
calibrations during the use period. The
readout scale is likely to be labeled -
calibration is the basis for this label.
The following procedure is recommended:
1 Turn the instrument on and allow it to
reach a stable condition. Perform the
recommended instrument check as
outlined in the operating manual.
2 The instrument check usually includes
an electronic zero correction. Check
each instrument against the readout
scale with the sensor immersed in an
agitated solution of sodium sulfite
containing sufficient 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 result of residual or stray
currents, internal shorting in the
electrode, or membrane rupture.
Minor adjustments may be made using
the indicator rather than the electronic
controls. Serious imbalance requires
electrode reconditioning if the electronic
check is O.K. Sulfite must be carefully
rinsed from the sensor until the readout
stabilizes to prevent carry over to the
next sample.
3 Fill two DO bottles with replicate
samples of clarified water similar to
that to be tested. This water should
not contain significant test interferences.
4 Determine the DO in one by the azide
modification of the iodometric titration.
5 Insert a magnetic stirrer in the other
bottle or use a probe agitator. Start
agitation after insertion of the sensor
assembly and note the point of
stabilization.
a Adjust the instrument calibration
control if necessary to compare
with the titrated DO.
b If sensitivity adjustment is not
possible, note the instrument
stabilization point and designate
it as ua. A sensitivity coefficient,
<)> is equal to trrr where DO is the
titrated value for the sample on
which ua was obtained. An unknown
ua
DO then becomes DO =
This
factor is applicable as long as the
sensitivity does not change.
Objectives of the test program and the
type of instrument influence calibration
requirements. Precise work may
require calibration at 3 points in the
DO range of interest instead of at zero
and high range DO. 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 difference in
oxygen content of air and of water is
too large for air calibration to be
satisfactory for precise calibration
for use in water.
IV This section reviews characteristics of
several sample laboratory instruments.
Mention of a specific 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 or may not
represent the current available model.
A The electrode described by Carrit 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 reference cell with
a platinum disc cathode (1-3 cm diameter).
The salt bridge consisted of N/2 KC1 and
16-7
-------
Dissolved Oxygen Determination - II
KOH. The polyethylene membrane was
held in place by a retaining ring. An
applied current was used in a polarographic
mode. Temperature effects were relatively
large. Thermistor correction was studied
but not integrated with early models.
— Silver Ring
'latinum Disk t Electrolyte Layer
Figure 3
The Beckman oxygen electrode is another
illustration of a polarographic DO sensor
(Figure 4). It consists of a gold cathode,
a silver anode, an electrolytic gel con-
taining KC1, covered by a teflon membrane.
The instrument has a temperature readout
and compensating thermistor, a source
polarizing current, amplifier with signal
adjustment and a readout DO scale with
recorder contacts.
SENSOR
ELECTRONICS
AMPUFIK
-GOIO CATMOOf
The YSI Model 51 (3) is illustrated in
Figure 5. This is another form of
polarographic DO analyzer. The cell
consists of a silver anode coil, a gold
ring cathode and a KC1 electrolyte with
a teflon membrane. The instrument has
a sensitivity adjustment, temperature and
DO readout. The model 51 A has temp-
erature compensation via manual preset
dial. A field probe and bottle probe are
available.
YSI Model 51 DO Sen»or
'O' Ring
Membrane
KCL Solution
^^
Anode Coil
Cathode Ring—-tt
Figure 5
D The Model 54 YSI DO analyzer (4) is based
upon the same electrode configuration but
modified to include automatic temperature
compensation, DO readout, and recorder
jacks. A motorized agitator bottle probe
is available for the Model 54 (Figure 6).
Figure 4, THE BECKMAN OXYGEN
SENSOR
.Mimbran* ••teln*r
Ring
Agitator Ring
16-8
-------
Dissolved Oxygen Determination -
E The Galvanic Cell Oxygen Analyzer (7, 8)
employs an indicator for proportional DO
signal but does not include thermistor
compensation or signal adjustment.
Temperature readout is provided. The
sensor includes a lead anode ring, and
a silver cathode with KOH electrolyte
(4 molar) covered by a membrane film
(Figure 7).
Precision Galvanic Call Oxygen Probe
adl
Thermistor Cable
Retainer
Tapered Section
to fit BOD Bottle!
Plaitic Membrane
Retainer Ring
Lead Anode Ring
Silver Cathode
Polyethylene Membrane
F The Weston and Stack Model 300 DO
Analyzer (8) has a galvanic type sensor
with a pulsed current amplifier adjustment
to provide for signal and temperature
compensation. DO and temperature
readout is provided. The main power
supply is a rechargeable battery. The
sensor (Figure 8) consists of a lead anode
coil recessed in the electrolyte cavity
(50% KI) with a platinum cathode in the tip.
The sensor 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
sampler respectively. The thermistor
and agitator are mounted in a sleeve that
also provides protection for the membrane.
G The EIL Model 15 A sensor is illustrated
in Figure 9. This is a galvanic cell with
thermistor activated temperature com-
pensation and readout. Signal adjustment
is provided. The illustration shows an
expanded scheme of the electrode which
when assembled compresses into a sensor
approximating 5/8 inch diameter and 4 inch
length exclusive of the enlargement at the
upper end. The anode consists of com-
pressed lead shot in a replaceable capsule
(later models used fine lead wire coils),
a perforated silver cathode sleeve around
the lead is covered by a membrane film.
The electrolyte is saturated potassium
bicarbonate. The large area of lead
surface, silver and membrane provides
a current response of 200 to 300 micro-
amperes in oxygen saturated water at
200 C for periods of up to 100 days use (8).
The larger electrode displacement favors
a scheme described by Eden (9) for
successive DO readings for BOD purposes.
V Table 1 summarizes major characteristics
of the sample DO analyzers described in
Section IV. It must be noted that an ingenious
analyst may adapt any one of these for special
purposes on a do-it-yourself program. The
sample instruments are mainly designed for
laboratory or portable field use. Those
designed for field monitoring purposes may
include similar designs or alternate designs
generally employing larger anode, cathode,
and electrolyte capacity to approach better
response stability with some sacrifice in
response time and sensitivity. The electronic
controls, recording, telemetering, and
accessory apparatus generally are semi-
permanent installations of a complex nature.
ACKNOWLEDGMENTS:
This outline contains certain materials from
previous outlines by D. G. Ballinger,
N. C. Malof, and J.W. Mandia. Additional
information was provided by C.R. Hirth,
C.N. Shadix, D.F. Krawczyk, J. Woods,
and others.
16-9
-------
Dissolved Oxygen Determination
WESTON & STACK
DO PROBE
CORD
CORD RESTRAINER
SERVICE CAP
PROBE SERVICE CAP
ELECTROLYTE FILL SCREW
•
PROBE BODY
PLATINUM CATHODE
CONNECTOR PINS
PIN HOUSING
LEAD ANODE
REMOVABLE PROBE SHIELD
AND THERMISTOR HOUSING
Figure 8
16-10
-------
Cable Sealing
Nut
A 15017
Cable
Connection
Cover
A15016A
'O'JRmg
R524
Model A15A ELECTRODE COMPONENT PARTS
Lead Anode
Complete
'O' Ring
R389
(A15024A)
Membrane Securing
' Rin "
R317
'O' Ring
R385
Membrane— Securing
'O' Ring
R317
Silver Cathode
A15013A
Filler Screw
Z471
mimnirmT
Anode
Contact
A -15 0140
(With Sleeve S24)
'O' Ring
R612
'O' Ring
R622
Anode
Contact
Holder
A15015A
End Cap
AlSOllA
'O' Ring
R622
Note: Red wire of cable connects to Anode Contact Holder
Black wire of cable connects to Anode Contact
Membrane not shown E. I. L. part number T22.
OJ
i
Figure 9
-------
Dissolved Oxygen Determination
TABLE 1
CHARACTERISTICS OF VARIOUS LABORATORY DO INSTRUMENTS
Anode
Cathode Elec
DO
Sig.
Type Membr Adj.
Temp.
Comp. Accessories for
Temp. Rdg. which designed
Carrit &
Kanwisher
Beckman
Yellow Springs
51
Yellow Springs
54
Precision
Sci
Weston &
Stack
300
EIL
Delta
75
Delta
85
silver- Pt
silver ox. disc
ring
Aq
ring
Ag
coil
II
Pb
ring
Pb
coil
Pb
Lead
Lead
Au
disc
Au
ring
11
silver
disc
Pt
disc
Ag
Silver
disc
Silver
disc
KC1
KOH
N/2
KC1
gel
KC1
soln
sat.
II
KOH
4N
KI
40%
KHCOg
KOH
IN
KOH
IN
pol* polyeth
pol tenon
pol teflon
galv**polyeth
galv tenon
galv tenon
galv teflon
galv teflon
no
yes
yes
yes
no
yes
yes
yes
yes
no
yes
yes
no*
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
no
yes
yes
Recording temp.
& signal adj. self
assembled
recording
field and bottle
probe
recording field
bottle & agitator
probes
agit. probe
depth sampler
recording
field bottle &
agitator probe
field bottle &
agitator probe
*Pol - Polarographic (or amperometric)
**Galv - Galvanic (or voltametric)
REFERENCES
1 Carrit, D. E. and Kanwisher, J.W.
Anal. Chem. 31:5. 1959.
2 Beckman Instrument Company. Bulletin
7015, A Dissolved Oxygen Primer,
Fuller-ton, CA. 1962.
3 Instructions for the YSI Model 51 Oxygen
Meter, Yellow Springs Instrument
Company, Yellow Springs, OH 45387.
4 Instructions for the YSI Model 54 Oxygen
Meter, Yellow Springs Instrument
Company, Yellow Springs, OH 45387.
5 Technical Bulletin TS-68850 Precision
Scientific Company, Chicago, IL 60647.
6 Mancy, K. H., Okun, D.A. and Reilley,
C.N. J. Electroanal. Chem. 4:65.
1962.
7 Instruction Bulletin, Weston and Stack
Model 300 Oxygen Analyzer. Roy F.
Weston. West Chester, PA 19380.
8 Briggs, R. and Viney, M. Design and
Performance of Temperature Com-
pensated Electrodes for Oxygen
Measurements. Jour, of Sci.
Instruments 41:78-83. 1964.
16-12
-------
Dissolved Oxygen Determination
9 Eden, R. E. BOD Determination Using 11 Methods for Chemical Analysis of Water
a Dissolved Oxygen Meter. Water and Wastes. EPA-AQCL, Cincinnati,
Pollution Control, pp. 537-539. 1967. OH, July. 1971.
10. Skoog, D. A. and West, D. M. Fundamentals
of Analytical Chemistry. Holt,
Rinehart & Winston, Inc. 1966.
This outline was prepared by F. J. Ludzack
Chemist, EPA, WPO, National Training
Center, Cincinnati, OH 45268 and Nate
Malof, Chemist, EPA, WPO, National
Field Investigations Center, Cincinnati, OH
Descriptors ; Chemical Analysis, Dissolved
Oxygen, Dissolved Oxygen Analyzers,
Instrumentation, On-Site Tests, Water Analysis,
Analysis, Wastewater
16-13
-------
DISSOLVED OXYGEN
Factors Affecting DO Concentration in Water
I The Dissolved Oxygen determination is
a very important water quality criteria for
many reasons:
A Oxygen is an essential nutrient for all
living organisms. Dissolved oxygen is
essential for survival of aerobic
organisms and permits facultative
organisms to metabolize more effectively.
Many desirable varieties of macro or
micro organisms cannot survive at
dissolved oxygen concentrations below
certain minimum values. These values
vary with the type of organisms, stage
in their life history, activity, and other
factors.
B Dissolved oxygen levels may be used as
an indicator of pollution by oxygen
demanding wastes. Low DO concen-
trations are likely to be associated with
low quality waters.
C The presence of dissolved oxygen
prevents or minimizes the onset of
putrefactive decomposition and the
production of objectionable amounts of
malodorous sulfides, mercaptans,
amines, etc.
D Dissolved oxygen is essential for
terminal stabilization wastewaters.
High DO concentrations are normally
associated with good quality water.
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.
F The BOD or other respirometric test
methods for water quality are commonly
based upon the difference between an
initial and final DO determination for a
given sample time interval and con-
dition. These measurements are
useful to indicate:
II
1 The rate of biochemical activity in
terms of oxygen demand for a
given sample and conditions.
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.
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.
5 Oxygenation requirements
necessary to meet the oxygen
demand in treatment units or
surface water situations.
The DO test is the only chemical test
included in all Water Quality Criteria,
Federal, State, Regional or local.
FACTORS AFFECTING THE DO
CONCENTRATION IN WATER
A Physical Factors:
DO solubility in water for an
air/water system is limited to
about 9 mg DO/liter of water at
20°C. This amounts to about
0. 0009% as compared to 21% by
weight of oxygen in air.
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
17-1
-------
Dissolved Oxygen Determination
interface area, mixing phenomena
and other items.
Certain factors tend to confuse
reoxygenation mechanisms of
water aeration:
a The transfer of oxygen in air
to dissolved molecular oxygen
in water has two principal
variables:
1) Area of the air-water
interface.
2) Dispersion of the oxygen-
saturated water at the
interface into the body liquid.
The first depends upon the surface
area of the air bubbles in the water
or water drops in the air; the
second depends upon the mixing
energy in the liquid. If diffusors
are placed in a line along the wall,
dead spots may develop in the core.
Different diffusor placement or
mixing energy may improve oxygen
transfer to the liquid two or threefold.
b Other variables in oxygen transfer
include:
3) Oxygen deficit in the liquid.
4) Oxygen content of the gas phase.
5) Time.
If the first four variables are
favorable, the process of water
oxygenation is rapid until the liquid
approaches saturation. Much more
energy and time are required to
increase oxygen saturation from
about 95 to 100% than to increase
oxygen saturation from 0 to about
95%. For example: ^n oxygen-
depleted sample often will pick up
significant DO during DO testing;
changes are unlikely with a sample
containing equilibrium amounts
of DO.
The limited solubility of oxygen
in water compared to the oxygen
content of air does not require
the interchange of a large mass of
oxygen per unit volume of water
to change DO saturation. DO
increases from zero to 50%
saturation are common in passage
over a weir.
Aeration of dirty water is practiced
for cleanup. Aeration of clean
water results in washing the air and
transferring fine particulates and
gaseous contaminants to the liquid.
One liter of air at room temperature
contains about 230 mg of oxygen.
A 5 gal carboy of water with 2 liters
of gas space above the liquid has
ample oxygen supply for equilibration
of DO after storage for 2 or 3 days
or shaking for 30 sec.
Aeration tends toward evaporative
cooling. Oxygen content becomes
higher than saturation values at
the test temperature, thus
contributing to high blanks.
Oxygen solubility varies with the
temperature of the water.
Solubility at IQOC is about two
times that at 30o C. Temperature
often contributes to DO variations
much greater than anticipated by
17-2
-------
Dissolved Oxygen Determination
solubility. A cold water often has
much more DO 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 partially 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 30° C will show a water
level about 1/2 inch below the mark
when the water temperature is
reduced to 20° C. BOD dilutions
should be'adjusted to 20° C + or -
1 1/20 before filling and testing.
Water density varies with tem-
perature with maximum water
density at 4°C. Colder or warmer
waters tend to promote stratification
of water that interferes with
distribution of DO 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
decreases 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
controls the oxygen solubility
limits in the water. For example,
the equilibrium concentration of
oxygen in water is about 9 mg DO/1
under one atmospheric pressure of
air, about 42 mg DO/liter in
contact with pure oxygen and 0 mg
DO/liter in contact with pure
nitrogen (@ 20° C).
B Biological or Bio-Chemical Factors
1 Aquatic life requires oxygen for
respiration to meet energv
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
availability of food and favor-
ability of conditions.
2 Algae, 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.
a Photosynthesis occurs only
under the influence of adequate
light intensity.
b Respiration of alga is
continuous.
c The dominant effect in terms
of oxygen assets or
liabilities of alga depends upon
algal activity, numbers and
light intensity. Gross algal
productivity contributes to
significant diurnal DO
variations.
3 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
17-3
-------
Dissolved Oxygen Determination
deoxygenation rates commonly are
associated with significant growth
or regrowth of organisms.
Micro-organisms tend to flocculate
or agglomerate to form settleable
masses particularly at limiting
nutrient levels (after available
nutrients have been assimilated or
the number of organisms are large
in proportion to available food).
a Resulting benthlc deposits
continue to respire as bed
loads.
b Oxygen availability is limited
because the deposit is physically
removed from the source of
surface oxygenation and algal
activity usually is more
favorable near the surface.
Stratification is likely to limit
oxygen transfer to the bed load
vicinity.
c The bed load commonly is
oxygen deficient and decomposes
by anaerobic action.
d Anaerobic action commonly is
characterized by a dominant
hydrolytic or solubilizing action
with relatively low rate growth
of organisms.
e The net effect is to produce low
molecular weight products
from cell mass with a corre-
spondingly large fraction of
feedback of nutrients to the
overlaying waters. These
lysis products have the effect
of a-high rate or immediate
oxygen demand upon mixture
with oxygen containing waters.
f Turbulence favoring mixing of
surface waters and benthic
sediments commonly are
associated with extremely
rapid depletion of DO.
Recurrent resuspension of
thin benthic deposits may
contribute to highly erratic
DO patterns.
g Long term deposition areas
commonly act like point
sources of new pollution as
a result of the feedback of
nutrients from the deposit.
Rate of reaction may be low
for old materials but a low
percentage of a large mass of
unstable material may produce
excessive oxygen demands.
C Tremendous DO variations are likely
in a polluted water in reference to
depth, cross section or time of day.
More stabilized waters tend to show
decreased DO variations although it is
likely that natural deposits such as leaf
mold will produce differences related
to depth in stratified deep waters.
A CKNOWLEDGMENTS
This outline contains significant materials
from previous outlines by J. W. Mandia.
Review and comments by C. R. Hirth and
R. L. Booth are greatly appreciated.
REFERENCE
1 Methods for Chemical Analysis of
Water and Wastes, EPA-AQCL,
Cincinnati, OH, July 1971.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, MDS,
WPO, EPA, Cincinnati, OH 45268 and
revised by Charles R. Feldmann, Chemist,
National Training Center.
Descriptors : Aeration, Aerobic Conditions,
Air-Water Interfaces, Anaerobic Conditions,
Benthos, Biolcg ical Oxygen Demand, Dissolved
Oxygen, Water Pollution, Water Quality
17-4
-------
CHEMICAL OXYGEN DEMAND AND COD/BOD RELATIONSHIPS
DEFINITION
The Chemical Oxygen Demand (COD) test
is a measure of the oxygen equivalent of
that portion of the organic matter in a
sample that is susceptible to oxidation
under specific conditions of oxidizing
agent, temperature 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 (OC) preferred by
some, but unpopular.
3 Chemical oxygen demand (COD) current
preference.
4 Complete oxygen demand (COD)
misnomer.
5 Dichromate oxygen demand (DOC)
earlier distinction of the current pre-
ference for COD by dichromate or a
specified analysis such as Standard
Methods.
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,
but none have been completely
satisfactory.
1 Ceric sulfate has been investigated,
but in general it is not a strong
oxidant.
2 Potassium permanganate was one of
the earliest oxidants 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-
ganate 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. '•*)
5 Effective determination of elemental
carbon in wastewater was sought by
Buswell as a water quality criteria.
a Van Slyke* ' 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 Hall, et al., '^) used a heated
combustion tube with infrared
detection to determine carbon quickly
and effectively by wet sample
injection.
6 Current development shows a trend to
instrumental methods automating
CH.O. oc. 10e.3.75
18-1
-------
Chemical Oxygen Demand and COD/BOD Relationships
II
conventional procedures or to seek
elemental or more specific group
determination.
RELATIONSHIP OF THE COD TEST WITH
OTHER OXIDATION CRITERIA IS
INDICATED IN TABLE 1.
A Table 1
Test
BOD
COD
IDOD
Van Slyke
Carbon detn.
Carbon by
combustion
+IR
Chlorine
Demand
Test
Temp. °C
20
145
20
400+
950
20
Reaction
time
days
2 hrs.
15'
1 hr.
minutes
20 min.
Oxidation
system
Biol. prod.
Enz. Oxidn.
50% H2SO4
K2Cr207
May be cata-
lyzed
Diss. oxyg.
H3P°4
HI03*
H2S04
K2Cr2°7
Anhydrous
Oxygen atm.
catalyzed
HOC1 soln.
Variables
Compound, environ-
ment, biota, time,
numbers. Metabolic
acceptability, etc.
Susceptibility of
the test sample to
the specified
oxidation
Includes materials
rapidly oxidized by
direct action,
Fe , SH.
Excellent approach
to theoretical oxi-
dation for most
compounds (N-nil)
Comparable to
theoretical for
carbon only.
Good NH3 oxidn.
Variable for other
compounds.
B From Table 1 it is apparent that oxidation
is the only common item of this series of
separate tests.
1 Any relationships among COD & BOD
or any other tests are fortuitous be-
cause the tests measure
the oxidizability of a given sample
under specified conditions, which are
different for each test.
If the sample is primarily composed
of compounds that are oxidized by
both procedures (BOD and COD) a
relationship may be established.
18-2
-------
Chemical Oxygen Demand and COD/BOD Relationships
The COD procedure may be sub-
stituted (with proper qualifications)
for BOD or the COD maybe used
as an indication of the dilution
required for setting up BOD
analysis.
If the sample is characterized by a
predominance of material that can
be chemically, but not biochemi-
cally oxidized, the COD will be
greater than the BOD. Textile
wastes, paper mill wastes, and
other wastes containing high con-
centrations of cellulose have a
high COD, low BOD.
If the situation in item b is reversed
the BOD will be higher than the
COD. Distillery wastes or refinery
wastes may have a high BOD, low
COD, unless catalyzed by silver
sulfate.
Any relationship established as in
2a will change in response to
sample history 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.
Ill ADVANTAGES AND LIMITATIONS OF
THE COD TEST*2) AS RELATED TO BOD
A Advantages
1 Time, manipulation, and equipment
costs are lower for the COD test.
2 COD oxidation conditions are effective
for a wider spectrum of chemical
compounds.
3 COD test conditions can be standardized
more readily to give more precise
results.
4 COD results are available while the
waste is in the plant, not several
days later, hence, plant control is
facilitated.
5 COD results are useful to indicate
downstream damage 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 flask 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
test.
3 Dichromate in hot 50% sulfuric acid
requires close control to maintain
safety during manipulation.
4 Oxidation of chloride to chlorine is not
closely related to BOD but may affect
COD results.
5 It is not advisable to expect precise
COD results on saline water.
IV BACKGROUND OF THE STANDARD
METHODS COD PROCEDURE
A The COD procedure considered dichro-
mate oxidation in 33 and 50 percent sul-
furic acid. Results indicated preference
of the 50 percent acid concentration for
oxidation of sample components. This is
the basis for the present standard
procedure.
18-3
-------
Chemical Oxygen Demand and COD/BOD Relationships
B Muers*' suggested addition of silver
sulfate to catalyze oxidation of certain
low molecular weight aliphatic acids and
alcohols. The catalyst also improves
oxidation of most other organic components
to some extent but does not make the COD
test universally applicable for all chemical
pollutants.
C The unmodified COD test result (A) includes
oxidation of chloride to chlorine. Each mg
of chloride will have a COD equivalent of
0. 23 mg. Chlorides must be determined
in the sample and the COD result corrected
accordingly.
1 For example, if a sample shows 300
mg of COD per liter and 200 mg Cl~
per liter the corrected COD result will
be 300 -(200 x 0.23)or 300 - 46 = 254
mg COD/1 on a chloride corrected basis.
2 Silver sulfate addition as a catalyst
tends to cause partial precipitation of
silver chloride .even in the hot acid- solu-
: tion. Chloride corrections are ques-
tionable unless the chloride is oxidized
before addition of silver sulfate, i.e.,
.reflux for 15 minutes for chloride ox-
idation, add Ag.SO., and continue the
reflux or use of HgSO4(D).
tn\
D Dobbs and Williams V" proposed prior
complexation of chlorides with HgSO4 to
prevent chloride oxidation during the test.
A ratio of about 10 of Hg++ to 1 of Cl~ (wt.
basis) appears essential. The Cl~ must
be complexed in acid solution before addi-
tion of dichromate and silver sulfate.
1 For unexplained reasons the HgSO4
complexation does not completely
prevent chloride oxidation in the
presence of high chloride concentrations.
2 Factors have been developed to provide
some estimate of error in the result
due to incomplete control of chloride
behavior. These tend to vary with the
sample and technique employed.
E It is not likely that COD results will be
precise for samples containing high
chlorides. Sea water contains 18000 to
21000 mgCr/1 normally. Equivalent
chloride correction for COD exceeds
4000 mg/ 1. The error in chloride
determination may give negative COD
results upon application of the correction.
Incomplete control of chloride oxidation
with HgSO4 may give equally confusing
results.
HgSO4 appears to give precise results
for COD when chlorides do not exceed
about 2000 mg/1. Interference in-
creases with increasing chlorides at
higher levels.
F The 12th edition of Standard Methods re-
duced the amount of sample and reagents
to 40% of amounts utilized in previous
editions. There has been no change in
the relative proportions in the test. This
step was taken to reduce the cost of pro-
viding expensive mercury and silver sul-
fates required. Results are comparable
as long as the proportions are identical.
Smaller aliquots of sample and reagents
require more care during manipulation
to promote precision.
G The EPA Methods for COD
1 For routine level COD (samples having
an organic carbon concentration
greater than 15 mg/liter and a chloride
concentration less than 2000 mg/liter),
the EPA specifies the procedures found
in Standard Methods ^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 ^)_ The difference from
the routine procedure primarily in-
volves a greater sample volume and
more dilute solutions of dichromate
and ferrous ammonium sulfate.
3 For saline samples (chloride level
exceeds 2000 mg/liter), EPA provides
an analytical procedure* ' involving
preparation of a standard curve of COD
versus mg/liter chloride to correct
the calculations. Volumes and concen-
trations for the sample and reagents
are adjusted for this type of determination.
-------
Chemical Oxygen Demand and COD/BOD Relationships
/Q\
V PRECISION AND ACCURACYV
Eighty six analysts in 58 laboratories
analyzed a distilled water solution contain-
ing oxidizable organic material equivalent
to 270 mg/1 COD. The standard deviation
was ± 17. 76 mg/1 COD with an accuracy
as percent relative error (bias) of —4. 7%.
For a solution equivalent to 12. 3 mg/1
COD (low level), the standard deviation
was i 4. 15 mg/1 with an accuracy as percent
relative error (bias) of 0. 3%. (EPA Method
Research Study 3)
VI
REMARKS PERTINENT TO EFFECTIVE
COD DETERMINATIONS INCLUDE:
Sample size and COD limits for 0. 250 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 mg/1, (low level).
Sample Size
20 ml
10 ml
5 ml
mg COD/1
2000
4000
8000
D
Most organic materials oxidize rela-
tively rapidly under COD test condi-
tions. A significant fraction of
oxidation occurs during the heating upon
addition of acid but the orange color of
dichromate should remain. If the
sample color changes from orange 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 reaction conditions.
Chloride concentrations should be known
for all test samples so appropriate
analytical techniques can be used.
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.
Plug the condenser breather tube
with glass wool to minimze dust
entrance.
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 sulfuric acid
must be of very high quality to
maintain low blanks on the refluxed
samples for the 0. 025 N oxidant.
VII NPDES METHODOLOGY
Under the National Pollutant Discharge
Elimination System, the accepted method
(Federal Register, Tuesday, October 16,
1973, Volume 38, number 199, Part II)
for doing the Chemical Oxygen Demand
Test is given in Standard Methods, ^2^ p. 495;
ASTM,(8) p. 219 and the EPA Manual, <9>
p. 20.
ACKNOWLEDGEMENT:
Certain portions of this outline contain
training material from prior outlines by
R. C. Kroner, R. J. Lishka, and
J. W. Mandia.
REFERENCES:
1 Moore, W. A., Kroner, R. C. and
Ruchhoft, C. C. Anal. Chem.
21:953. 1949.
18-5
-------
Chemical Oxygen Demand and COD/ BOD Relationships
2 Standard Methods, 13th Edition, APHA-
AWWA-WPCF. 1971.
3 Moore, W. A., Ludzack, F. J. and
Ruchhoft, C. C. Anal. Chem.
23:1297, 1951.
4 Van Slyke, D. D. and Folch, J. J.
Biol. Chem. 136:509, 1940.
5 Van Hall, C. E., Safranko, J. and
Stenger, V. A., Anal. Chem.
35:315, 1963.
6 Muers, M. M. J. Soc. Chem.
Ind. (London) 55:711, 1936.
7 Dobbs, R. A. and Williams, R. T.,
Anal. Chem. 35:1064. 1963.
ASTM Standards, Part 23, Water:
Atmospheric Analysis, 1972.
Methods for Chemical Analysis
of Water and Wastes, EPA-
MDQARL, Cincinnati, Ohio
45268, 1974.
This outline was prepared by F. J.
Ludzack, Chemist, EPA, WPO, National
Training Center, Cincinnati, OH 45268.
Descriptors: Analysis, Biochemical
Oxygen Demand, Chemical Analysis,
Chemical Oxygen Demand, Chlorides,
Oxygen Demand, Wastewater, Water
Analysis
18-6
-------
LABORATORY PROCEDURE FOR ROUTINE
LEVEL CHEMICAL OXYGEN DEMAND
I REAGENTS
A Standard Potassium Dichromate (0.250 N):
Dissolve 12. 259 g of primary standard
grade K Cr O^, which had been dried at
103° C for two nours, in distilled water and
dilute to one liter.
B Ferrous Ammonium Sulfate (0. IN):
Dissolve 39 g of Fe (NH4>2
in distilled water.
6H20
Carefully add 20 ml of concentrated H SO .
Cool and dilute to one liter.
C Ferroin Indicator:
Dissolve 1.485 g 1, 10-phenanthroline
monohydrate and 0.695 g FeSO • 7H O in
water and dilute to 100 ml. This indicator
may be purchased already prepared.
D Concentrated Sulfuric Acid (36 N):
E Mercuric Sulfate: Analytical Grade
F Silver Sulfate: Analytical Grade
G Concentrated Sulfuric Acid - Silver Sulfate:
Dissolve 22 g of silver sulfate in a 9 Ib
bottle of concentrated sulfuric acid.
(1-2 days required for dissolution)
H EQUIPMENT PREPARATION
Before use, the Erlenmeyer flask (500 ml,
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 K Cr2O?, 50 ml of distilled water,
and several bumping stones to the flask.
Carefully add 20 ml of concentrated HgSO
and mix thoroughly. Connect the flask
and condenser, but do not turn on the water
to the condenser. Boil the mixture so that
steam emerges from the top of the con-
denser for several minutes. Cool the
mixture, carefully discard the acid, and
rinse the condenser 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.
IH STANDARDIZATION OF FERROUS
AMMONIUM SULFATE
A Dilute 10. 0 ml of the standard
potassium dichromate to about 100 ml
with distilled water.
Add 30 ml of concentrated H SO and
allow to cool.
C Add 2-3 drops of ferroin indicator and
titrate to a reddish-brown end point
with the ferrous ammonium sulfate.
Calculate the normality, N, of the
ferrous ammonium sulfate.
D Calculation
N of Fe (NH4)2
ml K2Cr20? XN of
mlFe(NH4)2
IV PROCEDURE
A Place 0. 4 g HgSO in a 500 ml 24/40
standard taper Erlenmeyer flask.
Add 20 ml of sample, or an aliquot
diluted to 20 ml with distilled water.
B Add 3 ml of concentrated H SO and
swirl to dissolve the HgSO..
CH.O.oc.lab.3b.3.75
19-1
-------
Laboratory Procedure for Routine Level Chemical Oxygen Demand
C Add 10. 0 ml of the 0. 250 N
and mix.
D Carefully add 27 ml of the sulfuric acid
silver sulfate reagent.
E Add several pumice granules or glass
beads to prevent bumping, and then
swirl the mixture to insure complete
mixing.
F Reflux the mixture for two hours.
J Calculation
mg COD/1
(A-B> NX 8X 1000
ml of sample
COD = chemical oxygen demand
A • ml Fe(NH,), (SO.L used for blank
4 £t 4 6t
B » ml Fe(NH ) (SO.), used for sample
4 £t TC £
N -Nof Fe(NH.L (SO.L
4 Cl 4 &
8 B equivalent weight of oxygen
G Allow the solution to cool, wash down
the condenser with distilled water, and
add about 75 ml water to bring the
volume to about 150 ml.
H Add 2-3 drops of the ferroin indicator
and titrate the solution to a reddish -
brown end point with the ferrous
ammonium sulfate.
I A blank consisting of 20 ml of distilled
water and containing all reagents is
refluxed and titrated in the same manner
as the sample.
A CKNOWLEDGEMENT:
Portions of this outline were taken from
an outline prepared by R. J. Liska.
REFERENCES
1 Methods for Chemical Analysis of
Water and Wastes, EPA-MDQARL,
Cincinnati, Ohio, 1974.
2 Standard Methods, APHA-AWWA-
WPCF, 13th Edition, 1971.
This outline was prepared by Charles R.
Feldmann, Chemist, EPA, WPO, National
Training Center, Cincinnati, OH 45268.
Descriptors ; Chemical Analysis, Chemical
Oxygen Demand, Organic Compounds,
Oxidation, Oxygen, Oxygen Demand, Oxygen
Requirements, Water Analysis
19-2
-------
ACIDITY. ALKALINITY. pH AND BUFFERS
I DEFINITIONS OF ACIDS AND BASES
A Arrhenius Theory of Acids and Bases
(Developed about 1887)
1 Acid: A substance which produces,
in aqueous solution, a hydrogen ion
(proton), IT1".
2 Base: A substance which produces,
in aqueous solution, a hydroxide
ion, OH".
3 The Arrhenius theory was confined
to the use of water as a solvent.
B Bronsted and Lowry Theory of Acids
and Bases (Developed about 1923).
1 Acid: A substance which donates,
in chemical reaction, a hydrogen
ion (proton).
2 Base: A substance which accepts,
in chemical reaction, a hydrogen
ion (proton).
3 Bronsted and Lowry had expanded
the acid-base concept into non-
aqueous media; i.e., the solvent
could, but did not have to be .water.
C There are other acid-base theories. The
two above are probably the most commonly
used ones when discussing wastewater
topics however.
II DEFINITIONS OF ACIDITY,
ALKALINITY AND 'NEUTRALITY
A A cidity
A condition in which there is a prepon-
derance of acid materials present in
the water.
B Alkalinity
A condition in which there is a prepon-
derance of alkaline (or basic) materials
present in the water.
C Neutrality
D
III
It is possible to have present in
the water chemically equivalent
amounts of acids and bases. The
water would then be described as
being neutral; i.e., there is a
preponderance of neither acid nor
basic materials. The occurrence
of such a condition would be rare.
The term "neutralization" refers to
the combining of chemically equiv-
alent amounts of acids and bases.
The two products of neutralization
are a salt and water.
HC1
NaOH
NaCl
Hydro- Sodium
chloric Hydroxide
acid
Sodium
Chloride
(a salt)
The key word in the above definitions
is "preponderance. " It is possible to
have a bas ion of acidity while there
are basic materials present in the
water, as well as conversely.
HOW ARE DEGREES OF ACIDITY AND
ALKALINITY EXPRESSED?
The pH scale is used to express various
degrees of acidity and alkalinity. Values
can range from Oto 14. These two ex-
tremes are of theoretical interest and would
never be encountered in a natural water or
in a wastewater. pH readings from 0 to
just under 7 indicate an acidic condition;
from just over 7 to 14, an alkaline condition.
Neutrality exists if the pH value is exactly
7. pH paper, or a pH meter, provides the
most convenient method of obtaining pH
readings. It should be noted, that under NPDES
Methodology, pH measurements are to be made
using a pH meter. Some common liquids and pH
values are listed in Table I.
CH.ALK. 3.3.75
20-1
-------
Acidity, Alkalinity. pH and Buffers
TABLE 1. pH Values of
Household lye
Bleach
Ammonia
Milk of magnesia
Borax
Baking soda
Sea water
Blood
Distilled water
Milk
Corn
Boric acid
Orange juice
Vinegar
Lemon juice
Battery acid
5.9 - 8.4 is the common
natural waters.
Common Liquids
13.7
12.7
11.3
10.2
9.2
8.3
8.0
7.3
7.0
6.8
6.2
5.0
4.2
2.8
2.2
0.2
pH range for most
IV HARD AND SOFT WATERS
In addition to being acidic or basic, water
can also be described as being hard or soft.
A Hard water contains large amounts of
calcium, magnesium, strontium, man-
ganese and iron ions, relative to the
amount of sodium and potassium ions
present. Hard water is objectionable
because it forms insoluble compounds
with ordinary soap.
B Soft water contains small amounts of
calcium, magnesium, strontium, man-
ganese and iron ions, relative to the
amount of sodium and potassium ions
present. Soft water does not form in-
soluble compounds with ordinary soap.
V TITRATIONS
A The conversion of pH readings into such
quantities as milligrams (mg) of acidity,
alkalinity, or hardness, is not easily
carried out. These values are more
easily obtained by means of a titration.
B In a titration, an accurately measured
volume of sample (of unknown strength)
is combined with an accurately measured
20-2
volume of standard solution (of known
strength) in the presence of a suitable
indicator.
C The strength (called normality) of the
sample is then found using the following
expression:
milliliters (ml) of sample X normality
(N) of sample ° ml of standard solu-i
tion X N of standard solution.
Three of the four quantities are known,
and
N of sample = ml of standard solution
X N of-standard solution/ml of sample.
D In modified form, and a more specific
application of the above equation, alka -
linity is calculated in the following
manner (13th ed. Standard Methods).
mg of alkalinity as mg CaCO3/liter (1)
= ml of standard HgSO^ X N of standard
H2SO4 X 50 X 1000/ml sample.
VI INDICATORS
The term "suitable indicator" was used
above. At the end of a titration, the pH of
the solution will not necessarily be 7. It
may be above or below 7. A suitable indi-
cator, therefore, is one which undergoes
its characteristic color change at the appro-
priate pH. Below are a few examples of
indicators and the pH range in which they
undergo their characteristic color changes.
In some cases, mixed indicators may be
used in order to obtain a sharper and more
definite color change. Again it should be noted
that under NPDES, pH meters are to be used
for the measurement of pH.
Indicator
Methyl Yellow
Methyl Orange
Methyl Red
Cresol Purple
Phenolphthalein
Alizarine Yellow
TABLE 2. pH
Operational
pH Range
2.8 - 4.0
3. 1 - 4.4
4.4 - 6.2
7.4 - 9.0
8.0 - 9.6
10.0 - 12.0
Range of Indicators
-------
Acidity, Alkalinity, pH and Buffers
VII BUFFERS
A A buffer is a combination of substances
which, when dissolved in water, resists
a pH change in the water, as might be
caused by the addition of acid or alkali.
Listed below are a few chemicals
which, when combined in the proper
proportions, will tend to maintain the
pH in the indicated range.
Chemicals pH Range
AceticAcid + SodiumAcetate 3.7- 5.6
Sodium Dihydrogen Phosphate +
Disodium Hydrogen Phosphate 5.8 - 8.0
6.8 - 9.2
9.2 - 11.0
Boric Acid + Borax
Borax + Sodium Hydroxide
TABLE 3. pH Range of Buffers
B
A buffer functions by supplying ions
which will react with hydrogen ions
(acid "spill"), or with hydroxide ions
(alkali "spill").
In many instances, the buffer is composed
of a weak acid and a salt of the weak acid;
e.g., acetic acid and sodium acetate.
1 In water, acetic acid ionizes or
"breaks down" into hydrogen ions
and acetate ions.
HC2H302 =
H+
(acetic acid) (hydrogen ion) (acetate ion)
(proton)
This ionization occurs to only a
slight extent, however, most of the
acetic acid remains in the form of
HC2H3C>2; only a small amount of
hydrogen and acetate ions is formed.
2 Thus, acetic acid is said to be a
weak acid.
3 In the case of other acids, ionization
into the component ions occurs to a
large degree, and the term strong acid
is applied; e.g., hydrochloric acid.
HC1
(hydrochloric
acid)
(hydrogen ion)
(proton)
cr
(chloride
ion)
D
4 The terms "strong" and "weak" are
also applied to bases. In water
solutions, those which break down
into their component ions to a large
extent are termed "strong", and
those which do not are "weak".
Sodium hydroxide is a relatively
strong base, while ammonium
hydroxide is realtively weak.
5 Sodium acetate (a salt of acetic acid)
dissociates or "breaks down" into
sodium ions and acetate ions when
placed in water.
NaC2H302 « Na+ + C2H3O2-
(sodiumacetate) (sodium ion) (acetate ion)
This dissociation occurs to a large
extent, and practically all of the .
sodium acetate is in the form of
sodium ions and acetate ions.
It would be difficult and expensive to
prepare large quantities of buffers for
use in a treatment plant. However,
certain naturally occurring buffers may
be available (carbon dioxide is an ex-
ample). It dissolves in water to form
the species indicated below.
C02 + H20
(carbon dioxide) (Water)
H2C03 - H+
3 H2C03
(carbonic acid)
H+ + HCO3~
(hydrogenion)(hydrogen car-
proton) bonate ion)
(bicarbonate)
The hydrogen ions react with hydroxide
ions which might appear in the water
as the result of an alkali "spill".
H+
(in the
buffer)
(hydroxide ion
"spilled")
H20
The hydrogen carbonate ions react with
hydrogen ions which might appear in
the water as the result of an acid "spill".
H+ + HCO3- = H2CO3
(hydrogen ion) (in the
(proton) buffer)
"spilled"
This buffering action will be in effect as
long as there is carbonic acid present.
20-3
-------
Acidity. Alkalinity, pH and Buffers
E Buffering action is not identical with
a process in which acid wastes are This outline was prepared by C. R. Feldmann,
"neutralized" with alkali wastes, or Chemist, National Training Center, MDS,
conversely. The desired effect is WPO, EPA, Cincinnati, OH 45268
achieved in both cases, however (i. e.,
the pH is maintained within a desired
range.) Descriptors: Acids, Acidity, Alkalis,
Alkalinity, Analytical Techniques, Buffers,
Buffering Capacity, Chemical Analysis,
Hydrogen Ion Concentration, Indicators,
Neutralization, Water Analysis.
20-4
-------
OPERATING CHARACTERISTICS AND USE
OF THE pH METER
I INTRODUCTION
pH is a term used to describe the intensity
of the acid or alkaline condition of a
solution. The concept of pH evolved from
a series of developments that led to a
fuller understanding of acids and alkaline
solutions (bases). Acids and bases were
originally distinguished by their difference
in physical characteristics (acids -sour,
bases-soapy feel). In the 18th century it
was recognized that acids have a sour taste
(vinegar-acetic acid), that they react with
limestone with the liberation of a gaseous
substance (carbon dioxide) and that neutral
substances result from their interaction
with alkaline solutions.
Acids are also described as compounds
that yield hydrogen ions when dissolved
in water. And that bases yield hydroxide
ions when dissolved in water. The process
of neutralization is then considered to be
the union of hydrogen (H ) ions and hydroxyl
(OH~) ions to form neutral water
(H+ + OH
H20).
It has been determined that there are
1/10, 000, 000 grams of hydrogen ions and
17/ 10, 000,000 grams 'of hydroxyl ions in
one liter of pure water. The product of the
H+ and OH" ions equal a constant value.
Therefore, if the concentration of the H+
ions is increased there is a corresponding
decrease in OH" ions. The acidity or
alkalinity, hydrogen ion concentration of a
solution is given in terms of pH. The pH
scale extends from 0 to 14 with the neutral
point at 7. 0.
II INSTRUMENTATION
A General
Because of the differences between the
many makes and model of pH meters
which are available commercially, it is
impossible to provide detailed instruc-
tions for the correct operation of every
instrument. In each case, follow the
manufacturer's instructions. Thoroughly
wet the glass electrode and the calomel
electrode and prepare for use in accor-
dance with the instructions given.
Standardize the instrument against a
buffer solution with a pH approaching
that of the sample, and then check the
linearity of electrode response against
at least one additional buffer of a
different pH. The readings with the
additional buffers will afford a rough
idea of the limits of accuracy to be
expected of the instrument and the technic
of operation.
B Electrode Design
About 1925 it was discovered that an
electrode could be constructed of glass
which would develop a potential related
to the hydrogen-ion concentration with-
out interference from most other ions.
The glass pH electrode is the nearest
approach to a universal pH indicator
known at present. It works on the
principle of establishing a potential
across a pH-sensitive, glass membrane
whose magnitude is proportional to the
difference in pH of the solution
separated by this membrane.
All glass pH indicating electrodes have
a similar basic design. Contained on
one side of an appropriate glass membrane
is a solution of constant pH. In contact
with the other side of this pH sensitive
glass is the solution of unknown pH.
Between the surfaces of the glass mem-
brane, a potential is established which
is proportional to the pH difference of
these solutions. As the pH of one
solution is constant, this developed
potential is a measure of the pH of the
other.
CH.pH. 2.3.75
21-1
-------
Operating Characteristics and Use of the pH Meter
To measure this potential, a half-cell
is introduced into both the constant,
internal solution and into the unknown,
external solution. These half-cells are
in turn connected to your pH meter. The
internal reversible half-cell sealed
within the chamber of constant pH is
almost exclusively a wire of silver-
silver chloride. The external
reversible half-cell is often silver-
silver chloride. If both the internal and
external electrodes are combined in a
common pH measuring device, the
electrode is a combination pH electrode.
As the function of these half-cells is to
provide a steady reference voltage
against which voltage changes at the
glass pH sensitive membrane can be
referred, they must be protected from
contamination and dilution by the unknown
solutions. This is accomplished by
permanently sealing the internal half-
cell in a separate chamber which makes
electrical contact to the unknown
solution through a porous ceramic plug.
This ceramic plug allows current to flow,
but does not permit exchange of solution
to this chamber. Graduallythe KC1
solution is slowly lost, therefore a filling
port is placed in this electrode so that
additional saturated potassium chloride
can be added.
C Instrument Calibration
The pH balance control, by adding a
voltage in series with the pH electrode
system, allows the operator to adjust
the meter readout to conform to the pH
of the calibrating buffer. In general,
calibrate the meter in the general range
of the unknown solution. Appropriate
buffers can be selected (pH 4. 0, 6. 8,
7.4 and 10. 0). Always set the tem-
perature compensator on the instrument
to the temperature of the standard buffer
solution.
For most accurate analysis the pH of the
sample should be determined, and then
buffered solutions of a pH above and
below the determined pH should be
selected to re-calibrate the instrument
and the determination of the pH of the
sample repeated for a final reading.
D Precision and Accuracy
The precision and accuracy attainable
with a given pH meter will depend upon
the type and condition of the instrument
employed and the technique of standard-
ization and operation. With the proper
care, a precision of t 0.02 pH unit and
an accuracy of t 0. 05 pH unit can be
achieved with many of the new and
improved models. However, 1 0. 1 pH
unit represents the limit of accuracy
under normal conditions. For this
reason, pH values generally should be
reported to the nearest 0. 1 pH unit.
E Maintenance Practices
The reference chamber of the pH .
electrode system should always be kept
nearly full of saturated KC1 solution.
Routinely check the level and saturation
of potassium chloride in this reference
chamber and add saturated KC1 if
necessary.
The pH sensitive glass membrane
dehydrates when removed from water,
and thus it is imperative that dry
electrodes be soaked in buffer or water
for several hours before use. To
avoid this break-in period always keep
the glass pH sensitive membrane wet
between periods of use.
The buffers are pH standards; do not
contaminate them.
If the meter is a battery operated
instrument. To conserve the battery
life, the instrument should be turned
off when not in use.
Ill PROCEDURE
In the measurement of pH values of industrial
wastes, effluents, sludges and similar
21-2
-------
Operating Characteristics and Use of the pH Meter
samples, the electrodes must be thoroughly
rinsed with buffer solution between samples
and after calibrating. Buffer solutions can
be prepared by use of the formulations shown
in Table I.
The electrodes should be kept free of oil
and grease and stored in water when not in
use. In testing samples containing gaseous
or volatile components which affect the pH
value, any handling technic such as stirring
or heating may cause loss of such components
and thereby introduce error. For example,
the loss of carbon dioxide from an anaerobic
sludge digester sample due to stirring will
result in an observed pH value which is too
Table I
Preparation of pH Standard Solutions
Standard Solution (Molality) pH at 25 C
Primary standards
Potassium hydrogen tartrate
(saturated at 25°C) 3. 557
0. 05 potassium dihydrogen citrate 3. 776
0. 05 potassium hydrogen phthalate 4.008
0. 025 potassium dihydrogen
phosphate + 0. 025 disodium
hydrogen phosphate 6. 865
0.008695 potassium dihydrogen
phosphate + 0. 03043 disodium
hydrogen phosphate 7.413
0.01 sodium borate decahydrate
(borax) 9.180
0. 025 sodium bicarbonate + 0. 025
sodium carbonate 10.012
Secondary Standards
0. 05 potassium tetroxalate dihydrate 1. 679
Calcium hydroxide (saturated at 25°C) 12. 454
Weight of Chemicals Needed per 1, 000
ml of Aqueous Solution at 25 C
11.41gKH2C6H507
10. 12gKHC8H404
3. 388gKH2PO4t + 3.533gNa2HPO4tt
179gKH2P04t + 4. 302gNa2HPO4tt
2. 092gNaHCO3 + 2.
12.61gKH3C408-2H20
1.5gCa(OH)2*
*Approximate solubility
tDry chemical at 110-130 C for 2 hr.
tPrepare with freshly boiled and cooled distilled water (carbon dioxide-free)
21-3
-------
Operating Characteristics and Use of the pH Meter
high. If a sample of sludge or mud is highly
buffered, a small amount of water may be
added but the result cannot be considered
valid unless further dilutions yield the same
pH value. All dilutions should be reported
along with the result.
3 Instruction - Manual IL 175 Porto-matte
pH meter, Instrumentation Laboratory,
Inc. Lexington, Massachusetts,
Standard Methods, APHA-AWWA-WPCF.,
13th Ed., 1971.
REFERENCES
1 Nebergall, W. H., Schmidt, F. C. and
Holtzclaw, Jr., H. F. College Chem.,
2nd Ed. Heath and Co., Boston, 1963.
2 Sawyer, C. N., and McCarty. P. L.
Chem. for San. Eng. 2nd Ed.
McGraw-Hill, New York 1967
This outline was prepared by P. F. Hallbach,
Chemist, National Training Center, DTTB,
MDS, WPO.EPA, Cincinnati, Ohio 45268
Descriptors: Hydrogen Ion Concentration,
Instrumentation, Chemical Analysis
21-4
-------
SPECIFIC CONDUCTANCE
I INTRODUCTION
An electrical conductivity measurement of a
solution determines the ability of the solution
to conduct an electrical current. Very
concentrated solutions have a large population
of ions and transmit current easily or with
small resistance. Since resistance is ^
inversely related to conductivity K = —,
a very concentrated solution has a very
high electrical conductivity.
Electrical conductivity is determined by
transmitting an electrical current through
a given solution, using two electrodes. The
resistance measured is dependent principally
upon the ionic concentration, ionic charge,
and temperature of the solution although
electrode characteristics (surface area and
spacing of electrodes) is also critical. Early
experiments in standardizing the measurement
led to construction of a "standard cell" in
which the electrodes were spaced exactly 1 cm
and each had a surface area of 1 cm . Using
this cell, electrical conductivity is expressed
as "Specific Conductance". Modern specific
conductance cells do not have the same
electrode dimensions as the early standard
cell but have a characteristic electrode spacing/
area ratio known as the "cell constant".
K
sp
1
7^
R
distance (cm)
~T — 9 T — ,
(cm'')
area
sp
1
— A
R
k = cell constant
Specific conductance units are Mhos/cm or
reciprocal ohms/cm. Most natural, fresh
waters in the United States have specific
conductances ranging from 10 to 1, 000
micromhos/cm. (1 micromho = 10 mho).
II CONDUCTIVITY INSTRUMENTS
Nearly all of the commercial specific con-
ductance instruments are of a bridge circuit
design, similar to a Wheatstone Bridge.
Null or balance is detected either by meter
movement, electron "ray eye" tubes, or
headphones. Since resistance is directly
related to temperature, some instruments
have automatic temperature compensators,
although inexpensive models generally have
manual temperature compensation.
Conductivity instruments offer direct specific
conductance readout when used with a cell
matched to that particular instrument.
Electrodes within the cell may become
damaged or dirty and accuracy may be
affected; therefore, it is advisable to
frequently check the instrument readings
with a standard KC1 solution having a known
specific conductance.
Ill CONDUCTIVITY CELLS
Several types of conductivity cells are
available, each having general applications.
Dip cells are generally used for field
measurement, flow cells for measurement
within a closed system, and pipet cells for
laboratory use. Many modifications of the
above types are available for specialized
laboratory applications; the Jones cells and
inductive capacitance cells are perhaps the
most common.
Examples of various cell ranges for the RB3
- Industrial Instruments model (0-50
micromhos/cm scale range) are in Table 1.
Relative
Cell Conductivity
Number Value
Maximum range
micromhos / cm
Most accurate range
micromhos / cm
Cell VSO2
Cell VS2
Cell VS20
1
10
100
0 - 50
0 - 500
0 - 5000
Table 1
2 - 30
20 - 300
200 - 3000
CH. COND. 2d.3.75
22-1
-------
Specific Conductance
IV Computation of Calibration Constant
A calibration constant is a factor to which
scale readings must be multiplied by com-
pute specific conductance.
V
K = cM
sp
where K
sp
= actual specific conductance
= calibration constant
M = meter reading
For example, a 0. 001 N KC1 solution
(147 micromhos/cm standard) may show
a scale reading of 147.
147 = c!47, C=TT£
147
147
1.00
In this case the cell is perfectly "matched"
to the instrument, the calibration constant
is 1.00, and the scale reading represents
actual specific conductance. A variety of
cells, each covering a specific range,
may be used with any one instrument.
However, a calibration constant for each
cell must be computed before solutions of
unknown specific conductance can be
determined.
RELATIONSHIP OF SPECIFIC CON-
DUCTANCE TO IONIC CONCENTRATION
Natural water consists of many chemical
constituents, each of which may differ
widely in ionic size, mobility, and solubility.
Also, total constituent concentration and
proportions of certain ions in various natural
waters range considerably. However, it
is surprising that for most natural waters
having less than 2, 000 mg/1. dissolved
solids, dissolved solids values are closely
related to specific conductance values,
ranging in a ratio of . 62 to . 70. Of course
this does not hold true for certain waters
having considerable amounts of nonionized
soluble materials, such as organic com-
pounds and nonionized, colloidal inorganics.
Properties of some inorganic ions in regard
to electrical conductivity are shown below:
Ion
Calcium
Magnesium
Sodium
Potassium
Bicarbonate
Carbonate
Chloride
Micromhos/cm
per meq/1 cone.
,0
,6
52.
46.
48.9
72.0
43.6
84.6
75.9
VI ESTIMATION OF CONSTITUENT
CONCENTRATIONS
Generally speaking, for waters having a
dissolved solids concentration of less than
1, 000 mg/1, calcium and magnesium (total
hardness), sodium, bicarbonate and
carbonate (total alkalinity), and sulfate are
the principal or most abundant ions,
representing perhaps 90-99% of the total
ionic concentration of the water. Specific
conductance, total hardness and total
alkalinity are all simple and expedient
measurements which can be performed in
the field. Therefore, the remaining principal
ions are sodium and sulfate, and concentrations
of these can be estimated by empirical
methods. For example, we find that a certain
water has:
KSp = 500 micromhos/cm
Total Hardness • 160 mg/1 or 3. 20 meq/1
Total Alkalinity = 200 mg/1 or 3.28 meq/1,
as bicarbonate.
Next we multiply the specific conductance by
*0.011 (500X0.011 = 5.50) to estimate the
total ionic concentration in meq/1.
This factor may vary slightly for
different waters
Cations (meq/1)
3.20
Calcium
Magnesium
Sodium 5.50-3.20
2.30
Total Cations 5. 50
Anions (meq/1)
Carbonate 0. 00
Bicarbonate 3.28
Sulfate 5.50-3.28 »
2.22
Total Anions 5.50
22-2
-------
Specific Conductance
Realizing that several variables are involved
in empirical analysis, application rests
entirely upon testing the formula with previous
complete laboratory analyses for that
particular water. If correlation is within
acceptable limits, analytical costs may be
substantially reduced. Empirical analysis
can also be used in determination of proper
aliquots (dilution factor) necessary for
laboratory analysis.
Records of laboratory chemical analysis may
indicate that a particular stream or lake
shows a characteristic response to various
streamflow rates or lake water levels. If
the water's environment has not been altered
and water composition responds solely to
natural causes, a specific conductivity
measurement may be occasionally used in
substitution for laboratory analyses to
determine water quality. Concentration of
individual constituents can thus be estimated
from a specific conductance value.
VII APPLICATIONS FOR SPECIFIC
CONDUCTANCE MEASUREMENTS
(2)
A Laboratory Operations
1 Checking purity of distilled and de-
ionized water
2 Estimation of dilution factors for
samples
3 Quality control check on analytical
accuracy
4 An electrical indicator
B Agriculture
1 Evaluating salinity
2 Estimating Sodium Adsorption Ratio
(1)
C Industry* '
1 Estimating corrosiveness of water in
steam boilers
2 Efficiency check of boiler operation
D Geology
1 Stratigraphic identification and
characterization
a geological mapping
b oil explorations
E Oceanography
1 Mapping ocean currents
2 Estuary studies
F Hydrology
1 Locating new water supplies
a buried stream channels (See Fig. 1)
b springs in lakes and
streams (See Fig. 2)
2 Detection and regulation of sea water
encroachment on shore wells
G Water Quality Studies
/o)
1 Estimation of dissolved solids
(See Section V, also Fig. 3)
2 Empirical analysis of constituent
concentrations (See Section VI, also
reference 2)
3 Quality control check for salt water
conversion studies
4 Determination of mixing efficiency
of streams (See Fig. 4)
5 Determination of flow pattern of
polluted currents (See Fig. 4)
6 Identification of significant fluctuations
in industrial wastewater effluents
7 Signal of significant changes in the
composition of influents to waste
treatment plants
22-3
-------
Specific Conductance
FIGURE 1
DETECTION OF BURIED STREAM CHANNELS
TEST WELLS
X-SECT
FIGURE 2
DETECTION OF SPRINGS IN LAKES AND STREAMS
MAN LOWERING
CONDUCTIVITY CELL
LAND SURFACE
-------
Specific Conductance
POLLUTION STUOffS
FIGURE 3
INDUSTIIAL
WASTi [FFLUENT
VIII EPA METHODOLOGY
A The current EPA Methods Manual
specifies that specific conductance be
measured with a self-contained con-
ductivity meter, Wheatstone bridge-'
type or equivalent.
Samples should preferably be analyzed
at 25 C. If not, temperature corrections
should be made and results reported
at 25°C.
1 The instrument should be standard-
ized using KC1 solutions
2 It is essential to keep the conductivity
cell clean
The EPA manual specifies using the
procedure as described in Standard
Methods*2) or in ASTM Standards*3*.
These are approved in 40 CFR 136
for NPDES Report purposes.
Precision and Accuracy
Forty-one analysts in 17 laboratories
analyzed 6 synthetic water samples
containing the following Kgp increments
of inorganic salts: 100, 1Q6, 808, 848,
1640 and 1710 micromhos/cm.
The standard deviation of the reported
values was 7.55, 8.14, 66.1, 79.6, 106
and 119/n mhos/cm respectively.
The accuracy of the reported values was
-2.0, -0.8, -29.3, -38.5, -87.9 and
— 86. 9 n mhos /cm bias respectively.
REFERENCES
1 Methods for Chemical Analysis of Water
and Wastes, MDQARL, Cincinnati,
OH 45268. 1974.
2 Standard Methods for the Examination
of Water and Wastewater, APHA-AWWA-
WPCF, 13th edition, 1971.
3 ASTM Standards, Part 23, 1973.
This outline was prepared by John R.
Tilstra, Chemist, National Eutrophication
Research Program, Corvallis. Oregon
with additions by Audrey E. Donahue, Chemist,
MPOD, WPO, EPA, Cincinnati,
OH 45268.
22-5
-------
CALIBRATION AND USE OF A CONDUCTIVITY METER
I EQUIPMENT AND REAGENTS
A Equipment
1 Solu Bridge conductivity meters
2 Probes
a Cell VSO2
b Cell VS2
c Cell VS2O
3 Thermometers
4 400 ml beakers
B Reagents
1 Standard KC1 solutions
Normality of Specific Conductance
KC1 Solution micromhos/cm.
0.0001 14.9
0.001 147.0
0.01 1413.0
0.1 12900.0
2 Distilled water
II CHECKING THE INSTRUMENT
A The measurement of specific conductivity
as presented in sections II and m is
written for one type of conductivity meter
and probe.
B A battery check is made by depressing
the Battery Check switch, and at the
same time pressing the on-off button.
The meter needle should deflect to the
right (positive) and come to rest in the
green zone.
C Place a 10, 000 ohm resistor in the holes
of the electrical contacts on the meter.
Turn the temperature knob to read 25°C.
Depress the on-off button and bring the
meter needle to a reading of 0 by
turning the specific conductance switch.
The specific conductance reading should
be approximately 200 micromhos/cm.
Ill DETERMINATION OF THE CALIBRATION
CONSTANT
A Determine the temperature of the
standard KC1 solutions and move the
temperature knob to that value.
B Connect probe Cell VS02 to the con-
ductivity meter.
C Rinse the probe in the beaker of
distilled water, wipe the excess water
with a kimwipe and place probe in the
first beaker of KC1 solution (0.0001 N).
D Make certain the cell is submerged to
a point at least 1/2 inch above the air
hole and that no entrapped air remains.
The cell should also be at least 1/2
inch from the inside walls of the flask.
E Press and hold down the ON-OFF
button, simultaneously rotating the main
scale knob until the meter reads zero.
Release the button. (If the meter needle
remains off scale or cannot be nulled,
discontinue testing in that solution.)
F Record the scale reading in Table 1 and
proceed to KC1 solutions 0. 001N,
0. 01N, 0. 1 N using Steps C, D and E.
G Repeat steps C through F using the VS2,
then the VS20 probe.
H Compute the cell calibration constant-a
factor by which scale readings must be
multiplied to compute specific conductance:
K = cM
sp
where K = actual specific conductance,
c = calibration constant
M= meter reading
(continued next page)
CH. COND.lab.3c. 3.75
23-1
-------
Calibration and Use of a Conductivity Meter
TABLE 1 DATA FOR CALIBRATION CONSTANTS
Probe
KC1
Solutions
Test #
1
Cell
Constant
Cell VSO2
0.0001N
0.001N
0.01N
0. IN
Cell VS2
0.0001N
0. COIN
0.01N
0. IN
: Cell VS20
0.0001N
0. 001N
0.0 IN
0. IN
For each cell, calculate the cell constant
by using the meter reading closest to the
400 - 600 range. The known specific
conductance for the corresponding KC1
solution can be found in IB Reagents. Record
the cell constants on Table I.
IV DETERMINATION OF Ksp FOR SAMPLES
Obtain meter readings, M, for samples
A, B and C using Section III C, D and E.
Record M in Table 2. See Table I for
the appropriate cell constant, c, to
calculate Kgp for each sample where
K0 = cM. Record results in Table 2.
sp
V EPA METHODOLOGY
The current EPA Manual^1' specifies
using the procedures found in References
2 and 3. These procedures have been
adapted for this laboratory session and
all are approved in 40CFR136 for NPDES
report purposes.
A CKNOWLEDGMENT
This outline contains certain portions of
previous outlines by Messrs. J. W. Mandia,
and J. R. Tilstra.
REFERENCES
1 Methods for Chemical Analysis of
Water and Wastes, EPA-MDQARL
Cincinnati, OH 45268, 1974.
2 Standard Methods for the Examination
of Water and Wastewater, 13th
Edition. 1971.
3 Book of ASTM Standards, Part 23, 1973
This outline was prepared by C. R. Feldmann,
Chemist, National Training Center, and
revised by Audrey E. Donahue, Chemist,
National Training Center, MPOD, WPO, .•
USEPA, Cincinnati, OH 45268.
Descriptors: Analytical Techniques,
Conductivity, Electrical Conductance,
Specific Conductivity, Water Analysis
TABLE 2. SPECIFIC CONDUCTIVITY TESTS
Sample
Probe
1
Cell
Constant
Sp. Cond.
u mhos /cm
A
Cell
VSO2
Cell
VS2
Cell
VS2O
B
Cell
VSO2
Cell
VS2
Cell
VS2O
C
Cell
VSO2
Cell
VS2
Cell
VS2O
23-2
-------
CHLORINE DETERMINATIONS AND THEIR INTERPRETATION
INTRODUCTION
Chlorine normally is applied to water as a
bactericidal agent; it reacts with water con-
taminants to form, a variety of products
containing chlorine. The difference between
applied and residual chlorine represents the
chlorine demand of the water under conditions
specified. Wastewater chlorination is parti-
cularly difficult because the concentration of
organisms and components susceptible to
interaction with chlorine are high and variable.
Interferences with the chlorine determination
in wastewater confuse interpretation with
respect to the chlorine residual at a given
time and condition, its bactericidal potency,
or its future behavior.
II CHEMISTRY OF CHLORINATION
A Chlorine compounds (C12) dissolve in water,
and hydrolyze immediately according to the
reaction.
C1
H2O
HOC1 + H1" + Cl
The products of this reaction are hypo-
chlorous and hydrochloric acid. The
reaction is reversible, but at pH values
above 3. 0 and concentrations of chlorine
below 1000 mg/1 the shift is predominantly
to the right leading to hypochlorous acid
(HOC1).
Hypochlorous acid is a weak acid and con-
sequently ionizes in water according to the
equation:
HOC1
H + OCl
This reaction is reversible. At a pH value
of 5. 0 or below almost all of the chlorine
is present as hypochlorous acid (HOCl)
whereas above pH 10. 0 nearly all of it
exists as hypochlorite ion (OCl"). The pH
value that will control is the pH value
reached after the addition of chlorine.
Chlorine addition tends to lower the pH
and the addition of alkali hypochlorites
tends to raise the pH.
The initial reactions on adding chlorine to
wastewaters may be assumed to be funda-
mentally the same as when chlorine is
added to water except for the additional
complications due to contaminants and
their concentration.
Hypochlorous acid (HOCl) reacts with
ammonia and with many other complex
derivatives of ammonia to produce com-
pounds known as chloramines. Formation
of the simple ammonia chloramines includes:
1 NH3 + HOCl
NH2C1 +
monochloramine
H20
2 NH2C1 + HOCl— *NHC12 + H
dichloramine
NHC1 -
3 NH2C1
N2 + 3 HC1
The distribution of the ammonia chloramines
is dependent on pH, as illustrated below:
Ph
5
6
7
8
9
Percentage of Chlorine Present as
Monochloramine Dichloramine
16
38
65
85
94
84
62
35
15
6
PC. lib. 9.74
24-1
-------
Chlorine Determinations and Their Interpretation
The formation of the ammonia chloramines
are dependent on pH, temperature, and
chlorine-ammonia ratio. Chlorine re-
actions with amino acids are likely;
product disinfecting powers are lower
than those of chlorine or of ammonia
chloramines.
Ill TERMINOLOGY
A Terms used with Respect to Application
Site
1 Pre-chlorination - chlorine added
prior to any other treatment.
2 Post-chlorination - chlorine added
after other treatment.
3 Split chlorination - chlorine added at
different points in the plant - may
include pre- and post-chlorination.
B Terms Used in Designating Chlorine
Fractions
1 Free available residual chlorine - the
residual chlorine present as hypo-
chlorous acid and hypochlorite ion.
2 Combined available residual chlorine -
the residual chlorine present as
chloramines and organic chlorine con-
taining compounds.
3 Total available residual chlorine - the
free available residual chlorine + the
combined available residual chlorine -
may represent total amount of chlorine
residual present without regard to type.
In ordinary usage these terms are
shortened to free residual chlorine,
combined residual chlorine and total
residual chlorine. In the chlorination
of wastewaters only combined residual
chlorine is ordinarily present and is
often improperly termed chlorine
residual.
C Breakpoint chlorination specifically refers
to the ammonia-chlorine reaction where
applied chlorine hydrolyzes and reacts to
form chloramines and HC1 with the
chloramines eventually forming N2 + HC1
as in II. B. 3. Assuming no other chlorine
demand, the total chlorine residual will
rise, decrease to zero and rise again with
increasing increments of applied chlorine.
Other substances may produce humps in
the applied chlorine vs residual chlorine
plot due to oxidation of materials other
than ammonia. Sometimes these are
erroneously considered as a breakpoint.
IV ANALYTICAL METHODS
lodometric and amperometric titration
methods are the basis for deter-
mining chlorine residuals in water.
The relative advantages of a specific
determination depend upon the form in which
the reactable chlorine exists and the amount
and nature of interferences in the water.
A Amperometric Titration - Direct Method
1 Scope and application
This method is applicable to the
determination of free, combined or
total residual chlorine in all types of
water and wastewaters that do not
contain substantial amounts of organic
matter.
2 Summary of Method
When the cell of the titrator is immersed
in a sample at pH 7. 0, the cell unit pro-
duces a small direct current if free
chlorine (an oxidizing agent) is present.
As phenylarsine oxide (PAO) solution
is added, it reduces the free chlorine.
When all the chlorine is neutralized, the
generation of current ceases. At this
end point, the microammeter pointer on
the apparatus no longer deflects
down-scale.
To determine combined chlorine, pH 4. 0
buffer and potassium iodide are then
added to the sample. Free iodine
released by the combined chlorine also
causes the cell to produce a small direct
current. Addition of PAO reduces the
free iodine and the generation of current
ceases. Again, the end point occurs
24-2
-------
Chlorine Determinations and Their Interpretation
when the microammeter pointer no
longer deflects down-scale.
In either titration, the amount of PAO
reducing agent used to reach the end
point is ultimately stoichiometrically
proportional to chlorine present in the
sample. The sum of the free and
combined chlorine is the total residual
chlorine in the sample.
Total residual chlorine can be deter-
mined directly by adding pH 4. 0 buffer
and potassium iodide to the sample
before beginning the titration. Any
free or combined chlorine present will
stoichiometrically liberate free iodine
which is then reduced with PAO
titrant. The amount of PAO titrant
used measures the total amount of free
and combined chlorine originally
present in the sample.
3 Interferences
a Organic matter reacts with liberated
iodine.
b Cupric ions may cause erratic
behavior of the apparatus.
c Cuprous and silver ions tend to
poison the electrode by plating out
on it.
B Amperometric Titration - Indirect Method
1 Scope and Application
This method is applicable to the
determination of total chlorine residual
in all types of water and wastewaters.
In contrast to the direct amperometric
titration, this back-titration procedure
minimizes interferences in waters
containing substantial amounts of
organic matter.
2 Summary of Method
A sample is treated with a measured
excess of standard phenylarsine oxide
(PAO) solution followed by addition
of potassium iodide and a buffer to
maintain the pH between 3. 5 and 4. 2.
Any form of chlorine present will
stoichiometrically liberate iodine
which immediately reacts with the
PAO before significant amounts are
lost to reactions with organic matter
in the sample.
When the cell of the amperometric
titrator is immersed in a sample so
treated, no current is generated since
neither free chlorine nor free iodine
is present.
The amount of PAO used to reduce the
liberated iodine is then determined by
titrating the excess with a standard
iodine solution. No current is generated
until all the excess PAO has been
oxidized by the iodine. At this end
point the next small addition of iodine
causes current to be generated and the
microammeter pointer permanently
deflects up-scale.
The excess PAO thus measured is
subtracted from the original amount
of PAO added. The difference is the
PAO used to reduce the liberated iodine
and is ultimately a measure of the total
chlorine originally present in the sample.
NOTE: Sodium thiosulfate solution may
be used instead of PAO, but PAO is
more stable and is to be preferred.
3 Interferences
a Cupric ions may cause erratic
behavior of the apparatus.
b Cuprous and silver ions tend to
poison the electrode by plating out
on it.
Colorimetric Starch-Iodide Titration -
Indirect Method
1 Scope and Application
This method is applicable to the
determination of total chlorine residual
in all types of water and wastewater.
A back-titration procedure is used to
minimize interferences in waters con-
taining substantial amounts of organic
matter.
24-3
-------
Chlorine Determinations and Their Interpretation
2 Summary of Method
A sample is treated with a measured
excess of standard phenylarsine oxide
(PAO) solution followed by addition
of potassium iodide and a buffer to
maintain the pH between 3. 5 and 4. 2.
Any form of chlorine present will
stoichiometrically liberate iodine
which immediately reacts with PAO
before significant amounts are lost to
reactions with organic matter in the
sample.
The amount of PAO used to reduce the
liberated iodine is then determined by
titrating the excess with a standard
iodine solution in the presence of starch
until the PAO is completely oxidized.
At this end point, the next small addition
of iodine causes a faint blue color to
persist in the sample.
The excess PAO thus measured is
subtracted from the original amount
of PAO added. The difference is the
PAO used to reduce the liberated
iodine and is ultimately a measure of
the total chlorine originally present
in the sample.
NOTE: Sodium thiosulfate solution may
be used instead of PAO, but PAO is
more stable and is to be preferred.
3 Interferences
a An unusually high content of organic
matter may cause some uncertainty
in the end point. If manganese,
iron and nitrite are definitely absent,
this uncertainty can be reduced by
acidification to pH 1.0.
b Color and turbidity in the sample
cause difficulty with end-point
detection.
V EVALUATION OF METHODS
lodometric titration using amperometric
end point detection appears to be the most
accurate residual chlorine method
available.
1,2
Besides being inherently
more accurate than color detection methods,
this electrical end point is free of interfer-
ence from color and turbidity.
The accuracy of the colorimetric starch-
iodide end point is improved by employing
the indirect titration method described
above. By adding an excess of the standard
reducing agent and back-titrating, contact
between the liberated iodine and organic
matter in the sample is minimized.
1 Standard Methods for the Examination of
Water and Wastewaters, 13th Ed.,
APHA, AWWA, WPCF. 1971.
2 Book of ASTM Standards, Part 23 -
Industrial Water; Atmospheric
Analysis. American Society for
Testing and Materials.
Philadelphia, PA. 1972.
3 Sawyer, C. N. Chemistry for Sanitary
Engineers. McGraw-Hill Book
Company. New York. 1960.
4 Moore, E. W. Fundamentals of
Chlorination of Sewage and. Wastes.
Water and Sewage Works. Vol. 98.
No. 3. March 1951.
5 Day, R. V., Horchler, D. H., and
Marks, H. C. Residual Chlorine
Methods and Disinfection of Sewage.
Industrial and Engineering Chemistry.
May 1953.
24-4
-------
Chlorine Determinations and Their Interpretation
Marks, H. C., Joiner, R. R., and
Strandskov, F. B. Amperometric
Titration of Residual Chlorine in
Sewage. Water and Sewage Works.
May 1948.
This outline was prepared by J. L. Holdaway,
Chemist, Technical Program, EPA, Region III,
Charlottesville, VA 22901 (1971); and revised
by Audrey E. Donahue, Chemist, National
Training Center, MPOD, WPO, USEPA,
Cincinnati, OH 45268.
Descriptors: Chemical Analysis, Chlorination,
Chlorine, Disinfection, Sewage Treatment,
Wastewater Treatment, Water Analysis.
24-5
-------
PHOSPHORUS IN THE AQUEOUS ENVIRONMENT
I Phosphorus is closely associated with
water quality because of (a) its role in
aquatic productivity such as algal blooms,
(b) its sequestering action, which causes
interference in coagulation, (c) 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
significant 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 phosphorylation.
b The release of energy for meta-
bolic purposes is likely to
include a triphosphate exchange
mechanism.
B Most natural waters contain relatively low
levels of P (0. 01 to 0. 05 mg/1) in the
soluble state during periods of significant
productivity.
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.
The concentrations of P in hydrosoils,
sludges, treatment plant samples and
soils may range from 10 to 106 times
that in stabilized surface water. Both
concentration and interfering compo-
nents affect applicability of analytical
techniques.
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
dusts, fumes, stack gases, or other
discharges
III Phosphorus terminology is commonly
confused because of the interrelations among
biological, chemical, engineering, physical,
and analytical factors.
CH.PHOS.4c. 3.75
25-1
-------
Phosphorus in the Aqueous Environment
A Biologically, phosphorus may be available
as a nutrient, synthesized into living mass,
stored in living or dead cells, agglomerates,
or mineral complexes, or converted to
degraded materials.
B Chemically, P exists in several mineral
and organic forms that may be converted
from one to another under favorable
conditions. Analytical estimates are
based upon physical or chemical techniques
necessary to convert various forms of P
into ortho phosphates 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 systems to favor
acceptable water quality objectives.
Phosphorus removal is associated with
solids removal.
D Solubility and temperature are major
physical factors in phosphorus behavior.
Soluble P is much more available than
insoluble P for chemical or biological
transformations and the rate of conversion
from one to another is strongly influenced
by temperature.
E Table 1 includes a classification of the
four main types of chemical P and some
of the relationships controlling solubility
of each group. It is apparent that no
clear-cut separation can be made on a
solubility basis as molecular weight,
substituent and other factors affect
solubility.
Table 2 includes a scheme of analytical
differentiation of various forms of P
based upon:
1 The technique required to convert an
unknown variety of phosphorus into
ortho P which is the only one quanti-
tated by the colorimetric test.
Solubility characteristics of the sample
P or more precisely the means required
to clarify the sample.
a Any clarification method is subject
to incomplete separation. Therefore,
it is essential to specify the method
used to interpret the yield factor of
the separation technique. The
degree of separation of solubles
and insolubles will be significantly
different for:
1 Membrane filter separation
(0. 5 micron pore size)
2 Centrifugation (at some specified
rpm and time)
3 Paper filtration (specify paper
identification)
4 Subsidence (specify time and
conditions)
Analytical separations (Table 2) like those
in Table 1, do not give a precise separa-
tion of the various forms of P which may
be included quantitatively with ortho or
poly P. Conversely some of the poly and
organic P will be included with ortho P if
they have been partially hydrolyzed
during storage or analysis. Insolubles
may likewise be included as a result of
poor separation and analytical conditions.
The separation methods provide an
operational type of definition adequate
in most situations if the "operation"
is known. Table 2 indicates the nature
of incidental P that may appear along
with the type sought.
25-2
-------
Phosphorus in the Aqueous Environment
Table 1
PHOSPHORUS COMPOUNDS CLASSIFIED BY
CHEMICAL AND SOLUBILITY RELATIONS
Form
1. Ortho phosphates
' "4(P3010)"5(P3°9)
and others depending upon
the degree of dehydration.
as in 1 above
Increasing dehydration
decreases solubility
(a) as in 1 above
(b) multi P polyphosphates
(high mol. wt.) in-
cluding the "glassy"
phosphates
3. Organic phosphorus
R-P, R-P-R (2)
(unusually 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-
erated
(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
phosphosilicates
(d) certain mineral com-
plexes.
(1) Used in reference to predominance under common conditions.
-3
(2) R represents an organic radical, Prepresents P, PO , or its derivatives.
Total P in Table 2 includes liquid and
separated residue P that may exist in
the whole sample including silt, organic
sludge, or hydrosoils. This recognizes
that the feedback of soluble P from
deposited or suspended material has a
real effect upon the kinetics of the
aqueous environment.
25-3
-------
Phosphorus in the Aqueous Environment
Table 2
PHOSPHORUS COMPOUNDS CLASSIFIED BY
ANALYTICAL METHODOLOGY
1.
2.
Desired P Components
Ortho phosphates
Polyphosphates
(2)-(l) = poly P
(hydrolyzable)
Technique
No treatment on clear
samples
acid hydrolysis on clear
samples, dilute
(a) H2S04
(b) HC1
heated
(2)
Incidental P Included '
Easily hydrolyzed
(a) poly phosphates -
(b) organic -P, -
(c) Mineral -P, + or -
(a) ortho-P +
(b) organic -P + or -
(c) mineral -P + or'-
3. Organic phosphorus
(3) - (2) + orgP
(hydrolyzable)
acid + oxidizing hydrolysis
on whole sample, dilute
(a) H2S04
(b) HS0
(a) ortho P +
(b) poly P +
(c) mineral P + or -
heated
4. Soluble phosphorus
(preferably classified
by clarification method)
clarified liquid following
filtration, centrifugation
or subsidence
generally includes
(a) 1, 2. or 3
(b) particulates not
completely separated
5. Insoluble phosphorus
(residue from clari-
fication)
Retained residues separated
during clarification
See (6)
(a) generally includes
sorbed or complexed
solubles.
6. Total phosphorus
Strong acid + oxidant
digestion
(a)
^ + HNO,
4 3
(b) H.SO . + HNO, + HCK)
^ 4 3 4
(c)
+ 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) Coding: + quantitative yield
- a small fraction of the amount present
+ or - depends upon the individual chemical and sample history
-------
Phosphorus in the Aqueous Environment
IV Polyphosphates are of major interest 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 follows:
NaO
= O
P = O
H2°
7
mono sodium ortho
phosphate (2)
disodium dihydrogen
polyphosphate
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.
1 The major factors affecting the rate of
reversion of poly to orthophosphates
include:
a) Temperature, increased T increases
rate
b) pH, lower pH increases rate
The general form for producing
polyphosphates from mono substituted
orthophosphates is:
heat
200-4000C
(NaPO ) + n HO
o n £
Di-substituted ortho phosphates or
mixtures of substituted ortho phosphates
lead to other polyphosphates:
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
disodium hydrogen
ortho phosphate
heat ,
mono sodium
di hydrogen
ortho phosphate
Na5P3°10
• pentasodium
tri-phosphatc
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
Items a, b and c have a large effect
upon reversion rate compared with
other factors listed. The actual
reversion rate is a combination of
listed items and other conditions or
characteristics.
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.
25-5
-------
Phosphorus in the Aqueous Environment
V SAMPLING AND PRESERVATION
TECHNIQUES
A Sampling
1 Great care should be exercised to
exclude any benthic deposits from
water samples.
2 Glass containers should be acid rinsed
before use.
3 Certain plastic containers may be
used. Possible adsorption of low con-
centrations of phosphorus should be
checked.
4 If a differentiation of phosphorus forms
is to be made, filtration should be
carried out immediately upon sample
collection. A membrane filter of
0.45M pore size is recommended for
reproducible separations.
B Preservation
1 If at all possible, samples should be
analyzed on the day of collection. At
best, preservation measures only
retard possible changes in the sample.
a Possible physical changes include
solubilization, precipitation,
absorption on or desorption from
suspended matter.
b Possible chemical changes include
reversion of poly to ortho P and
decomposition of organic or min-
eral P.
c Possible biological changes
include microbial decomposition
of organic P and algal or
bacterial growth forming organic
P.
2 Refrigeration at 4 C is recommended
if samples are to be stored up to
24 hours. This decreases hydrolysis
and reaction rates and also losses due
to volatility.
Addition of 40 mg HgCl2/liter is
recommended for longer storage
periods. This chemical limits
biological changes.
a HgCL is an interference in the
analytical procedure if the
chloride level is low (less than
50 mg. Cl /I). See Part VI
B 3 below.
VI THE EPA ANALYTICAL PROCEDURE*6*
A This is a colorimetric determination,
specific for orthophosphate. Depending
on the nature of the sample and on the
type of data sought, the procedure in-
volves two general operations:
1 Conversion of phosphorus forms to
soluble orthophosphate (See Pig. 1);
a sulfuric acid-hydrolysis for
polyphosphates, and some
organic P compounds,-
b persulfate digestion for organic
P compounds.
2 The color determination involves
reacting dilute solutions of phosphorus
with ammonium molybdate and
potassium antimonyl tartrate in an
acid medium to form an antimony-
phosphomolybdate complex. This
complex is reduced to an intensely
blue-colored complex by ascorbic
acid. The color is proportional to
the orthophosphate concentration.
Color absorbance is measured at
880 nm or 650 nm and a concentration
value obtained using a standard curve.
Reagent preparation and the detailed
procedure can be found in the EPA
manual.
The methods described there are
usable in the 0. 01 to 0. 5 mg/liter
phosphorus range.
25-6
-------
Phosphorus in the Aqueous Environment
SAMPLE i Totnl Sjirnolo CAe, VJ If i-.nrlfln) ,
1
1
Direct
Coloriaetry
Total
Orthophosphate
1
H2SOi 1 Persulfato
liydrolystj & 1 Digestion
Colorinetry 1 4 Colorioetry
Total Hydrolyzeble Totnl
& Orthophosphate Phosphorus
Filter 0.45 Micron Membrane Filter
v'- A
! Residue Filtrate j
*
il
Direct | H2SO^
Colo rime try I Hydrolysis
J, & Colorimetry
Ortho?
.
hosphote & Orthophosphate
inorganic polyphosphate
phosphorus + some organic
c expounds phosphorus
Persulfata
Digestion
6 Colorimetry
Phosphorus
inorganic +
organic
phosphorus
Compounds compounds
FIGURE 1
ANALYTICAL SCHEME FOR DIFFERENTIATION OF PHOSPHORUS FORMS
B Interferences
1 Erroneous results from contaminated
glassware is avoided by cleaning it
with hot 1:1 HC1, treating it with
procedure reagents and rinsings
with distilled water. Preferably
this glassware should be used only
for the determination of phosphorus
and protected from dust during
storage. Commercial detergents
should never be used.
2 High iron concentrations in samples
can precipitate phosphorus.
3 If HgCl is used as a preservative,
it interferes if the chloride level of
the sample is less than 50 mg
Cl/liter. Spiking with NaCl is then
recommended.
4 Others have reported interference
from chlorine, chromium, sulfides,
nitrite, tannins, lignin and other
minerals and organics at high con-
centrations.
I C\
C Precision and Accuracy
1 Organic phosphate - 33 analysts in
19 laboratories analyzed natural
water samples containing exact in-
crements of organic phosphate of
0.110, 0.132, 0.772, and 0.882 mg
P/liter.
Standard deviations obtained were
0.033, 0.051, 0.130 and 0.128
respectively.
Accuracy results as bias, mg P/liter
were: +0.003, +0.016, +0.023 and
- 0.008, 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/liter.
Standard deviations obtained were
0.010, 0.008, 0.018 and 0.023
respectively.
25-7
-------
Ph
in the Aqueous Environment
Accuracy results as bias, mg P/liter
were-0.001, -0.002, -0.009 and
-0.007 respectively.
D Automated Methods
The EPA Manual also contains a
procedure for an automated colorimetry
method using the ascorbic acid reduction
method.
VII VARIABLES IN THE COLORIMETRIC
PROCEDURE
Several important variables affect
formation of the yellow heteropoly
acid and its reduced form, molybdenum
blue, in the colorimetric test for P.
A Acid Concentration during color develop-
ment is critical. Figure 2 shows that
color will appear in a sample containing
no phosphate if the acid concentration
is low. Interfering color is negligible
when the normality with respect to
H2SO approaches 0.4.
1 Acid normality during color develop-
ment of 0.3 to slightly more than
0.4 is feasible for use. It is prefer-
able to control acidity carefully and
to seek a normality closer to the
higher limits of the acceptable range.
2 It is essential to add the acid and
molybdate as one solution.
3 The aliquot of sample must be
neutralized prior to adding the
color reagent.
1.0
u
8
on
D
0.05 mg P
BLANK
0.1 0.2 0.3 0.4 0.5 0.6
H2SO NORMALITY
Figure 2
0-PHOSPHATE COLOR
VS ACIDITY
Choice of Reductant - Reagent stability,
effective reduction and freedom from
deleterious side effects are the bases
for reductant selection. Several re-
ductants have been used effectively.
Ascorbic acid reduction is highly
effective in both marine and fresh water.
It is the reductant specified in the
EPA method.
Temperature affects the rate of color
formation. Blank, standards, and
samples must be adjusted to the same
temperature (^ 1°C), (preferably room
temperature), before addition of the
acid molybdate reagent.
Time for Color Development must be
specified and consistent. After addition
of reductant, the blue color develops
rapidly for 10 minutes then fades grad-
ually after 12 minutes.
25-8
-------
Phosphorus in the Aqueous Environment
VIII DETERMINATION OF TOTAL
PHOSPHORUS
A Determination of total phosphorus
content involves omission of any
filtration procedure and using the acid-
hydrolysis and persulfate treatments
to convert all phosphorus forms to the
test-sensitive orthophosphate form.
B Determining total phosphorus content
yields the most meaningful data since
the various forms of phosphorus may
change from one form to another in a
short period of time. (See part V, Bl)
IX DEVELOPMENT OF A STANDARD
PROCEDURE
Phosphorus analysis received intensive
investigation; coordination and validation of
methods is more difficult than changing
technique.
A Part of the problem in methods arose
because of changes in analytical objectives
such as:
1 Methods suitable to gather "survey"
information may not be adequate for
"standards".
2 Methods acceptable for water are not
necessarily effective in the presence
of significant mineral and organic
interference characteristic of hydro-
soils, marine samples, organic
sludges and benthic deposits.
3 Interest has been centered on "fresh"
water. It was essential to extend them
for marine waters.
4 Instrumentation and automation have
required adaptation of methodology.
B Analysts have tended to work on their own
special problems. If the method
apparently served their situations, it was
used. Eacn 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
acidity, Mo content, reductant and
separation techniques are the major
variables.
C At the present time there is not sufficient
data to warrant EPA endorsement of the
P procedure for sediment-type samples,
sludges, algal blooms, etc. Following
is a procedure (not included in the EPA
manual) which is useful when solids
are present in samples:
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 H2SO4, 5 ml
concentrated HNO~o, 2 berl saddles
and digest on hot plate.
4 Digest until the disappearance of nitric
acid fumes and the appearance of white
803 fumes. Continue digestion for
approximately 5 minutes. Cool before
proceeding with Step 5.
5 Add 2 ml of HNO3-HC1O4 mixture
and 5 ml concentrated HNOg. 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 most organics for safety.
25-9
-------
Phosphorus in the Aqueous Environment
8
Cool. Add approximately 40 ml
distilled water and transfer to 100 ml
volumetric flask.
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
samples to the calibration standards to
equalize salt content during color
development.
Determine orthophosphate according
to the usual color procedure.
ACKNOWLEDGMENT:
Materials in this outline include significant
portions of previous outlines by J. M. Cohen,
L. J. Kamphake, andR. J. Lishka. Important
contributions and assistance were made by
B. C. Kroner, E. F. Earth, W. Allen Moore,
Lloyd Kahn, Clifford Risley, Lee Scarce,
John Winter, and Charles Feldmann.
REFERENCES
2 Gales, Morris E., Jr., Julian. Elmo C..
and Kroner, Robert C., Method for
Quantitative Determination of Total
Phosphorus in Water. JAWWA 58:
(10) 1363. October 1966.
3 Lee, G. Fred, Clesceri, Nicholas L. and
Fitzgerald, George P., Studies on the
Analysis of Phosphates in Algal Cultures.
Int. J. Air & Water Poll. 9:715. 1965.
4 Earth, E. F. and Salotto, V. V.,
Procedure for Total .Phosphorus
in Sewage and Sludge, Unpublished
Memo, Cincinnati Water Research
Laboratory, FWQA. April 1966.
5 Moss, H. V., (Chairman, AASGP
Committee) Determination of Ortho
Phosphate, Hydrolyzable Phosphate
and Total Phosphate in Surface Water,
JAWWA 56:1563. December 1958.'
6 Methods for Chemical Analysis of Water
& Wastes, EPA, MDQARL. Cincinnati.Ohio
45268, 1974.
This outline was prepared by Ferdinand J.
Ludzack, Chemist, Audrey E. Donahue,
Chemist, both with National Training Center,
MPOD, WPO, EPA, Cincinnati, Ohio
45268.
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.
Descriptors :
Chemical Analysis, Nutrients, Phosphates,
Phosphorus, Phosphorus Compounds,
Pollutant Identification, Sampling, Water
Analysis, Water Pollution Sources.
25-10
-------
CHEMICAL TESTS,OBSERVATIONS,AND MEASUREMENTS IN THE FIELD
I INTRODUCTION
A Laboratory determinations with approved
equipment and convenient facilities by
experienced specialists generally are
easier, faster, and more reliable. It is
advisable to transport the samples to the
laboratory whenever it is feasible to do so.
B Field tests are essential because:
1 Certain sample components are
inherently unstable with respect to
biological, chemical, or physical
changes. 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 field investigation
to identify and evaluate problems, locate
critical areas, and minimize surprises.
II FIELD TEST CONDITIONAL
CONSIDERATIONS
A Moving the laboratory into the field means
improvisation and adaptation to more
"primitive" conditions.
1 Rugged construction of field equipment
is 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
fragile glass.
2 Portability is essential, particularly
when the site is not accessible by boat
or car. Ideally, the field kit should
be small enough and light enough to be
carried by one man for extended periods.
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.
Instrumentation increases objectivity
of the measurements but the instruments
must be adapted to the items outlined in
II. A. 1, 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 calibration on site with simple
adjustment or repair for emergencies.
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:
a The individuals must be good
observers arid recognize what may
be significant later.
b They must be good interpreters
of a variety of observed and
derived test information.
c They must be good technicians to
follow prescribed procedures
diligently and recognize anomalous
behavior when it occurs.
d They must be good reporters to
describe what happened, where
it happened, and when. Failure
to report anomalous behavior
CH.4b.3.74
26-1
-------
Chemical Tests Observations and Measurements in the Field
and related events in an understandable
and consistent fashion may render the
entire effort meaningless because of
some doubt or inconsistency.
Subsequent operations also may be
mislead by the early observer.
B Use of field data imposes certain con-
straints relatable to the item, the methods,
and to the personnel involved:
1 How valid is it?
2 How consistent is it in line with other
observed or recorded information?
3 What backup information is required
to verify suggested information?
C Decisions must be made on all field
data applications:
1 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
laboratory?
3 What use is to be made of the
preliminary field estimates?
4 What do you "need" to know?
5 What would be "nice" to know?
6 In line with facilities, time, cost,
manpower and objectives, what
can you determine?
Ill COMPONENT CHARACTERISTICS
FAVORING FIELD TESTING:
\ Any item that changes rapidly during a
holding period as a result of temperature
or pressure variations, or is highly
reactive from a biological, chemical,
or physical standpoint, usually means
that it must be determined on site and in
place with minimum time lapse and
manipulation.
26-2
1 Dissolved gases, such as O_, CO9,
H2S, C12, are sensitive to pressure
or temperature changes and may
react with other sample components
readily. These require in place 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 promptly. Examples
of this include the relative ratios of
oxidized or reduced substances, such
as Cu+| and Cu+2, Fe and Fe+3, Cr
and Cr ; hydrolysis is the principal
factor in the ratios of organic and
NHg-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/insoluble ratios commonly
change with time and conditions due
to complexation, hydrolysis
precipitation, agglomeration, or
other factors. Particulate size may
increase or decrease. Solubles may
besorbed on or desorbed from solid
surfaces. Settleability or turbidity
are transient phenomena subject 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 change in response
to nutrition and conditions.
IV SITUATIONS FAVORING FIELD TESTING
A 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.
-------
Chemical Tests Observations and Measurements in the Field
1 It may be necessary to locate suspected
inflows, channels, or sources of items
affecting water quality.
2 Definition of mixing zones often is
required.
3 Laboratory time may be reduced if
approximate concentrations of items
sought are known.
4 Field tests or observations may
suggest other tests that are more
critical in evaluation of the given
situation.
5 Stratification may be evaluated for
guidance 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 testing.
1 An established 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
apparent.
4 It may be necessary to "track" some
hazardous discharge down stream
to guide subsequent users on the
choice of intake or storage water.
5 A complaint or inquiry may be
evaluated by field tests.
In-plant field type tests are essential
to guide operations toward the production
of continuous high quality effluents.
Record tests commonly are provided too
late to do anything about the situation;
quick test results are emphasized for
process control because time, manpower,
and change are critical.
1 Surprises in the form of wastewater
changes in flow, concentration, or
significant components are common
for treatment process operators. They
need quick numbers rather than
impressions to distinguish real from
apparent problems.
2 Process upsets are minimized or
prevented by regular and meaningful
tests 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.
V TYPICAL FIELD TESTS - INSTRUMENTAL
A 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 CO_ 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 CC"
and alter pH response. Electronic pH
instruments are produced for field use
by many apparatus supply firms. With
care, they will function effectively.
B 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.
26-3
-------
Chemical Tests,Observations, and Measurements in the Field
VI
DO analyzers are available for field
use application from several manufacturers.
They differ in portability accessory
equipment and in versatility so that it is
possible to obtain one or more fitting almost
any requirement. These units are based
upon reduction of oxygen at the cathode
surface to convert chemical to electrical
energy. Direct measurement of electrical
energy produced or the change in some
carrier current or voltage may be used
for readout. The signal may be amplified
in line with sensitivity to give direct
concentration readout. Temperature
compensation is useful and available.
Membrane covered sensors selective
for dissolved gases protect sensor
surfaces from most interfering components.
The analyst must learn to use his
particular instrument effectively to enable
him to obtain valid results under conditions
in which a wet chemical procedure would
be misleading.
TYPICAL FIELD TESTS -
WET CHEMICAL
DO titrations for clean water samples
may be adapted for field use by shifting
to dry reagents encased in plastic
pillows, substituting phenyl arsene
oxide for -thio and using plastic burets,
sample containers, etc. Powdered
starch substitutes also are available.
Sample sizes 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 packaged for field
use. "Home made" kits are common.
Alkalinity and hardness titrations are
commonly sought under field conditions
using standard acid and EDTA solutions.
Methyl purple or mixed indicators
commonly are used in place of methyl
orange indicator because of better
visibility in the field. Powder pillows
for buffer and indicator are available
for the hardness test.
Chlorine tests using ortho tolidine in a
comparator, such as those by Hilger
or Taylor and others, frequently are
used for rapid on site measurements.
The comparators, reagents and comparator
tubes commonly are available from
apparatus, equipment, or chlorine
suppliers.
VII SUMMARY
Simple and rapid field test kits are
available from several manufacturers,
suppliers and scientific apparatus
houses for individual tests, as well
as combination test kits for the items
mentioned and others.
It is possible for the analyst to devise
one suited to his particular requirements
with available or improvised materials.
Effectiveness of the field operation
depends on the knowledge and care
exercised in using the equipment and
procedure.
CAUTION: Field test results are
useful, but not always valid
enough for the record.
ACKNOWLEDGEMENT:
This outline contains significant
material from a previous outline by
D. G. Ballinger.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, WPO,
Cincinnati, OH 45268.
Descriptors : Chemical Analysis, On-Site
Tests, Water Analysis
26-4
-------
BACTERIOLOGICAL INDICATORS OF WATER POLLUTION
Part 1. General Concepts
I INTRODUCTION
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
often 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
Origin
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,
Staphylo coccus, Proteus, Pseudomonas,
certain spore-forming bacteria, and
others.
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 Vibrio comma.
b Viruses: A wide variety, including
that of infectious hepatitis, Po}io-
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
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-
groupings
b Fecal streptococci and certain
sub groupings
c Miscellaneous indicators of pollution
Evidence of water contamination by
intestinal wastes of warm-blooded
animals is regarded as evidence of
health hazard in the water being tested.
II PROPERTIES OF AN IDEAL INDICATOR
OF POLLUTION
A An "ideal" bacterial indicator of pollution
should:
1 Be applicable in all types of water
W.BA.48g. 2. 75
27-1
-------
Bacteriological Indicators of Water Pollution
in
Always be present in water when
pathogenic bacterial constituents of
fecal contamination are present.
Ramifications of this include —
a Its density should have some direct
relationship to the degree of fecal
pollution.
b It should have greater survival time
in water than enteric pathogens,
throughout its course of natural
disappearance from the water body.
c It should disappear rapidly from
water following the disappearance
of pathogens, either through natural
or man-made processes.
d It always should be absent in a
bacteriologically safe water.
3 Lend itself to routine quantitative
testing procedures without interference
or confusion of results due to extra-
neous bacteria
4 Be harmless to man and other animals
In all probability, an "ideal" bacterial
indicator does not exist. The discussion
of bacterial indicators of pollution in the
following parts of this outline include
consideration of the merits and limitations
of each group, with their applications in
evaluating bacterial quality of water.
APPLICATIONS OF TESTS FOR
POLLUTION INDICATORS
A Tests for Compliance with Bacterial
Water Quality Standards
1 Potability tests on drinking water to
meet Interstate Quarantine or other
standards of regulatory agencies.
2 Determination of bacterial quality of
environmental water for which quality
standards may exist, such as shellfish
waters, recreational waters, water
resources for municipal or other
supplies.
3 Tests for compliance with established
standards in cases involving the pro-
tection or prosecution of municipalities,
industries, etc.
B Treatment Plant Process Control
1 Water treatment plants
2 Wastewater treatment plants
C Water Quality and Pollutant Source Monitoring
1 Determination of intestinal pollution
in surface water to determine type and
extent of treatment required for com-
pliance with standards.
2 Tracing sources of pollution
3 Determination of effects on bacterial
flora, due to addition of organic or
other wastes
D Special Studies, such as
1 Tracing sources of intestinal pathogens
in epidemiological investigations
2 Investigations of problems due to the
Sphaerotilus group
3 Investigations of bacterial interference
to certain industrial processes, with
respect to such organisms as Pseudo-
monas, A chromobacter, or others
IV SANITARY SURVEY
The laboratory bacteriologist is not alone in
evaluation of indication of water pollution of
intestinal origin. On-site study (Sanitary
Survey) of the aquatic environment and
adjacent areas, by a qualified person, is a
necessary collateral study with the laboratory
work and frequently will reveal information
regarding potential bacteriological hazard
which may or may not be demonstrated
through laboratory findings from a single
sample or short series of samples.
27-2
-------
Bacteriological Indicators of Water Pollution
Part 2. The Coliform Group and Its Constituents
I ORIGINS AND DEFINITION
A Background
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
coli- commune and _B. lactis aerogenes.
In 1895, another bacteriologist,
Migula, rename dE5. coli commune as
Escherichia coli. which today is the
official 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 fact 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 subdivided 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"
(13th 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 35°C. "
n
! 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,
JE. freundii, E. intermedia
b Aerobacter aerogenes, _A. cloacae
c Biochemical intermediates between
the genera Escherichia and Aero-
bacter
The above terminology is in accordance
with the current editions of Standard
Methods and Bergey's Manual of Deter-
minative Bacteriology and will be
consistent throughout this manual until
these sources are modified.
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.
SUBDIVISION OF COLIFORMS INTO
"FECAL" AND "NONFECAL"
CATEGORIES
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 of fecal
origin while _A. 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.
27-3
-------
Bacteriological Indicators of Water Pollution
Without discussion of their relative merits,
several may be cited here:
B Types of Single-Test Differentiation
1 Determination of gas ratio
Fermentation of glucose by E3. coli
results in gas production, with
hydrogen and carbon dioxide being
produced in equal amounts.
Fermentation of glucose by A.
aerogenes results in generation of
twice as much carbon dioxide as
hydrogen.
Further studies suggested absolute
correlation between H2/CO_ ratios
and the terminal pH resulting from
glucose fermentation. This led to the
substitution of the methyl red test.
2 Methyl red test
Glucose fermentation by E_. coli
typically results in a culture medium
having terminal pH in the range 4.2-
4. 6 (red color a positive test with the
addition of methyl red indicator).
A_. aerogenes typically results in a
culture medium having pH 5. 6 or
greater (yellow color, a negative test).
3 Indole
When tryptophane, an amino acid, is
incorporated in a nutrient broth,
typical E_. coli strains are capable of
producing indole (positive test) among
the end products, whereas A_. aerogenes
does not (negative test).
In reviewing technical literature, the
worker should be alert to the method
used to detect indole formation, as the
results may be greatly influenced by
the analytical procedure.
4 Voges-Proskauer test (acetylmethyl
carbinol test)
The test is for detection of acetylmethyl
carbinol, a derivative of 2, 3, butylene-
glycol, as a result of glucose
fermentation in the presence of
peptone. A. aerogenes produces
this end product (positive test)
whereas E. coli gives a negative test.
a Experience with coliform cultures
giving a positive test has shown a
loss of this ability with storage on
laboratory media for 6 months to
2iyears, in 20 - 25% of cultures
(105 out of 458 cultures).
b Some workers consider that all
coliform bacteria produce acetyl-
methyl carbinol in glucose metab-
olism. These workers regard
acetylmethyl carbinol-negative
cultures as those which have
enzyme systems capable of further
degradation of acetylmethyl
carbinol to other end products
which do not give a positive test
with the analytical procedure.
Cultures giving a positive test for
acetylmethyl carbinol lack this
enzyme system.
c This reasoning leads to a hypothesis
(not experimentally proven) that the
change of reaction noted in certain
cultures in 4. a above is due to the
activation of a latent enzyme system.
5 Citrate utilization
Cultures of E_. coli are unable to use
the carbon of citrates (negative test)
in their metabolism, whereas cultures
of _A_. aerogenes are capable of using
the carbon of citrates in their metab-
olism (positive test).
Some workers (using Simmons Citrate
Agar) incorporate a pH indicator
(brom thymol blue) in the culture
medium in order to demonstrate the
typical alkaline reaction (pH 8.4 - 9.0)
resulting with citrate utilization.
6 Elevated temperature (Eijkman) test
a The test is based on evidence that
E. coli and other coliforms of fecal
27-4
-------
Bacteriological Indicators of Water Pollution
origin are capable of growing and
fermenting carbohydrates- (glucose
or lactose) at temperatures signif-
icantly 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 ±
0.2oc are the current standard
medium and method.
While the "EC" terminology of the
medium suggests "_E. coli" the
worker should not regard this as a
specific procedure for isolation of
E. coli.
c A similar medium, Boric Acid
Lactose Broth, has developed
by Levine and his associates. This
medium gives results virtually
identical with those obtained from
EC Broth, but requires 48 hours of
incubation.
d Elevated temperature tests require
incubation in a water bath. Standard
Methods 13th Ed. requires this
temperature to be 44. 5+0. 2°C.
Various workers have urged use of
temperatures ranging between
43. 00C and 46.00C. Most of these
recommendations have provided a
tolerance of + 0. 5° C from the rec-
ommended levels. However, some
workers, notably in the Shellfish
Program of the Public Health Service,
stipulate a temperature of 44. 5 +
0. 2°C. This requires use of a water
bath with forced circulation to main-
tain this close tolerance. This
tolerance range was 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,
after 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. nonfecal origin. A summary
follows:
Test
No. of times
used for dif-
ferentiation
Voges-Proskauer 22
reaction
Methyl red test 20
Citrate utilization 20
Indole test 15
Uric acid test 6
Cellobiose fermentation 4
Gelatin liquefaction 3
Eijkman test 2
Hydrogen sulfide 1
production
Sucrose fermentation 1
a-Methyl-d-glucoside 1
fermentation
27-5
-------
Bacteriological Indicators of Water Pollution
2 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 each
culture. Thus, a typical culture of
E. coli would have a code ++—, and a
typical A. aerogenes culture would
have a code --++.
3 Groupings 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 this page.
D Need for Study of Multiple Cultures
All the systems used for differentiation
between coliforms of fecal vs. those of
nonfecal origin require isolation and study
of numerous pure cultures. Many workers
prefer to study at least 100 cultures from
any environmental source before attempting
to categorize the probable source of the
coliforms.
NATURAL DISTRIBUTION OF COLIFORM
BACTERIA
A Sources of Background Information
Details of the voluminous background of
technical information on coliform bacteria
recovered from one or more environ-
mental media are beyond the scope of
this discussion. References of this
outline are suggested routes of entry
for workers seeking to explore this
topic.
B Studies on Coliform Distribution
1 Since 1960 numerous workers
have engaged in a continuing study of
the natural distribution of coliform
bacteria and an evaluation of pro-
cedures for differentiation between
coliforms of fecal vs. probable non-
fecal origin. Results of this work
have special significance because:
a Rigid uniformity of laboratory
methods have been applied through-
out the series of studies
b Studies are based on massive
numbers of cultures, far beyond
any similar studies heretofore
reported
Groupings of Coliforms into Fecal, Nonfecal and Intermediate Groups
Organism
Indole
Methyl
red
Voges-
Proskauer
Citrate
E. coli. Variety I
Variety n
E_. freundii
(Intermediates)
Variety I
Variety n
A_. aerogenes
Variety I
Variety n
±
+
+
+
27-6
-------
Bacteriological Indicators of Water Pollution
c A wider variety of environmental and
biological sources is being gtudied
than in any previous series of reports.
d All studies are based on freshly
recovered pure culture isolates
from the designated sources.
e All studies are based on cultures
recovered from the widest feasible
geographic range, collected at all
seasons of the year. It is believed
that no more representative series
of studies has been made or is in
progress.
2 Distribution of coliform types
Table 1 shows the consolidated results
of coliform distributions from various
biological and environmental sources.
a The results of these studies show a
high order of correlation between
known or probable fecal origin and
the typical J3. coli IMViC code
(++--). On the other hand,
human feces also includes
numbers of_A. 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 codes than fecal
sources, without clear dominance of
any one 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 those 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_. coli are
recovered in relatively smaller
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-
ments .
e The distribution of coliform types
from human sources should be
regarded as a representative value
for large numbers of sources.
Investigations have shown that there
can be large differences in the
distribution of IMViC types from
person to person, or even from an
individual.
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
sources.
a IMViC type ++- - is a measurement
of E. 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 large 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.
27-7
-------
to
-J
I
CO
Table 1. COLIFORM DISTRIBUTION BY IMViC TYPES AND ELEVATED TEMPERATURE
TEST FROM ENVIRONMENTAL AND BIOLOGICAL SOURCES
*120 of these *129 of these
were -H—, were ++--,
15 --++, 27 -+-+,
11 -+-- 5 ++-+
IMViC
type
Vegetation
No.
strains
4-+--
--++
-+--
+++-
-+- +
++- +
-+++
++++
+-++
h
--H--
--+-
+-+-
+ — +
+
Total
No. EC +
%EC +
128
237
23
2
168
116
32
291
88
87
5
19
2
5
0
1203
169*
14. 1*
% of
total
10.6
19. 7
1. 9
0. 2
14.0
9. 6
2. 7
24.2
7. 3
7. 2
0.4
1. 6
0. 2
0.4
<0. 1
Insects
No.
strains
.
134
113
0
0
332
118
28
254
46
42
0
0
0
8
9
1084
162*
14.9*
% of
total
12.4
10.4
<0. 1
<0. 1
30. 6
Soil
Undisturbed
No.
strains
131
443
78
7
1131
10.9 | 87
2.6
23.4
4.2
3.9
<0. 1
<0. 1
<0. 1
0. 7
0.8
181
159
67
4
1
53
6
0
0
2348
216
9.2
% of
total
5.6
18. 8
3.3
0.3
48. 1
3. 7
7. 7
6. 8
2.9
0.2
<0. 1
2.3
0. 3
<0. 1
<0. 1
Polluted
No.
strains
536
13
1
0
87
22
5
0
0
1
0
0
0
0
0
665
551
82.9
% of
total
80. 6
2. 0
0.2
<0. 1
13.0
3. 3
0. 7
<0. 1
<0. 1
0.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
Fecal sources
Human
No.
strains
3932
245
99
106
50
35
21
6
14
2
0
0
0
0
2
4512
4349
96.4
% of
total
87.2
5.4
2. 2
2.4
1. 1
0. 8
0.5
0. 1
0.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
Livestock
No.
strains
2237
0
14
59
1
27
0
0
0
0
0
0
0
0
0
2339
2309
98.7
%of
total
95.6
<0. 1
0.6
2.5
<0. 1
1.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
Poultry
No.
strains
1857
1
20
0
5
% of
total
97.9
0. 1
1. 1
<0. 1
0. 3
i
11 i 0.6
0
0 '
0
0
0
0
0
0
2
1896
1765
93.0
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
bd
P
o
o
ra
o
I
GO
O
t^>
8.
0)
o
I—•
rf
H>>
O
-------
Bacteriological Indicators of Water Pollution
HUMAN
EC ® 96.4
BALB® 94.7
SUM/MARY
Type
n- —
-+ —
-------
Bacteriological Indicators of Water Pollution
Table 2. COMPARISON OF COLIFORM STRAINS ISOLATED FROM WARM-BLOODED ANIMAL
FECES, FROM UNPOLLUTED SOILS AND POLLUTED SOILS .WITH USE OF THE
IMViC REACTIONS AND THE ELEVATED TEMPERATURE TEST IN EC MEDIUM
AT 44. 50 C ( +0. 50) (12th ed. 1965; Standard Methods for the Examination of Water
and Wastewater)
Test
+ + - -
+ + - -,
+ and
- + - -
Indole positive
Methyl red positive
Voges-Proskauer positive
Citrate utilizers
Elevated temperature (EC).
positive .
Number of cultures
studied
Warm-blooded
animal feces
91.8%
93. 3%
94. 0%
96. 9%
5. 1%
3.6%
96. 4%
8, 747
Soil:
Unpolluted
5.6%
8.9%
19.4%
75. 6%
40. 7%
88. 2%
9.2%
2,348
Soil:
Polluted
80. 6%
80. 7%
82. 7%
97. 9%
97. 3%
19. 2%
82. 9%
665
Vege-
tation
10. 6%
12. 5%
52. 5%
63. 6%
56. 3%
85. 1%
14. 1%
1,203
Insects
12. 4%
13. 2%
52.4%
79. 9%
40. 6%
86. 7%
14. 9%
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 smaller,
but demonstrable, percentages of
such organisms in environmental
sources not interpreted as being
polluted could be attributed largely
to the warm-blooded wildlife in the
area, including birds, rodents, and
other small 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. Therefore, it is
the official standard method for
differentiation coliforms of
probable direct fecal origin from those
which may have become established
in the bacterial flora of the aquatic
or terrestrial habitat.
IV EVALUATION OF COLIFORMS AS
POLLUTION INDICATORS
A The Coliform Group as a Whole
1 Merits
a The absence of coliform bacteria is
evidence of a bacteriologically safe
water.
b The density of coliforms is roughly
proportional to the amount of
excretal pollution present.
c If pathogenic bacteria of intestinal
origin are present, coliform
bacteria also are present, in much
greater numbers.
d Coliforms are always present in the
intestines of humans and other warm-
blooded animals, and are eliminated
in large numbers in fecal wastes.
27-10
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Bacteriological Indicators of Water Pollution
e Coliforms are more persistent in
the aquatic environment than are
pathogenic bacteria of intestinal
origin.
f Coliforms are generally harmless
to humans and can be determined
quantitatively by routine laboratory
procedures.
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.
b Some strains of the coliform group
may multiply in certain polluted
waters ("aftergrowth"), of high
nutritive values thereby adding to
the difficulty of evaluating a pollution
situation in the aquatic environment.
Members of the _A. aerogenes 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
some circumstances.
d Tests for coliforms are subject to
interferences due to other kinds of
bacteria. False negative results
sometimes occur when species of
Pseudomonas are present. False
positive results sometimes occur
when two or more kinds of non-
coliforms produce gas from lactose,
when neither can do so alone
(synergism).
The Fecal Coliform Component of the
Coliform Group (as determined by elevated
temperature test)
1 Merits
a The majority (over 95% of the coli-
form bacteria from intestines of
warm-blooded animals grow at the
elevated temperature.
b These organisms are of relatively
infrequent occurrence except in
association with fecal pollution.
c Survival of the fecal coliform group
is shorter in environmental waters
than for the coliform group as
whole. It follows, then, that high
densities of fecal coliforms is
indicative of relatively recent
pollution.
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.
e In some wastes, notably those from
pulp and paper mills, Klebsiella has
been found in large numbers
utilizing the elevated temperature
testi There has been much contro-
versy about whether the occurrence
of Klebsiella is due to aftergrowth
due to soluble carbohydrates in such
wastes. The significance of
Klebsiella as an indicator of direct
discharge of intestinal wastes thus
is under challenge. The issue is
still further complicated by questions
over whether Klebsiella is in and of
itself a pathogenic organism or is
potentially pathogenic. This is a
serious problem which is the subject
of current intensive research by this
Agency.
2 Limitations
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 criterion of
differentiation. These organisms
are E. coli varieties by present
taxonomic classification.
b There is at present no established
and consistent correlation between
27-11
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Bacteriological Indicators of Water Pollution
ratios of total coliforms/fecal
coliforms in interpreting sanitary-
quality of environmental waters.
In domestic sewage, the fecal
coliform density commonly is
greater than 90% of the total
coliform density. In environmental
waters relatively free from recent
pollution, the fecal coliform density
may range from 10-30% 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.
c Studies have been made
regarding the survival of fecal
coliforms in polluted waters
compared with that of enteric
pathogenic bacteria. In recent
pollution studies, species of
Salmonella have been found in the
presence of 220 fecal coliforms per
100 ml (Spino), and 110 fecal
coliforms per 100 ml (Brezenski,
Raritan Bay Project).
The issue of the Klebsiella problem
described in an earlier paragraph
may ultimately be resolved as a
merit or as a limitation of the value
of the fecal coliform test.
V APPLICATIONS OF COLIFORM TESTS
A Current Status in Official Tests
1 The coliform group is designated, in
"Standard Methods for the Examination
of Water and Wastewater" (13th ed.,
1971), through the Completed Test
MPN procedure as 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
Completed Test. The membrane filter
method also is accepted for examination
of waters subject to interstate regulation.
2 The 12th edition of Standard Methods
introduced a 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 continued in
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 supplies, or
industrial supplies. 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 surveys, as an adjunct
to determination 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.
27-12
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Bacteriological Indicators of Water Pollution
Part 3. The Fecal Streptococci
I INTRODUCTION
Investigations regarding streptococci
progressed from the streptococci of medical
concern to those which were distributed in
differing environmental conditions which,
again, related to the welfare of man. The
streptococci were originally reported by Laws
and Andrews (1894), and Houston (1899, 1900)
considered those streptococci, which we now
call "fecal streptococci, "as ... "indicative
of dangerous pollution, since they are readily
demonstrable in waters recently polluted and
seemingly altogether absent from waters above
suspicion of contamination.
From their discovery to the present time the
fecal streptococci appear characteristic of
fecal pollution, being consistently present in
both the feces of all warm-blooded animals
and in the environment associated with animal
discharges. As early 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 methods of
detection and enumeration appeared after
World War II.
Renewed interest in the group as indicators
began with the introduction of azide dextrose
broth in 1950, (Mallmann & Seligmann, 1950).
The method which is in the current edition
of Standard Methods appeared soon after.
(Litsky, et al. 1955).
With the advent of improved methods for
detection and enumeration of fecal strep-
tococci, significant body of technical
literature has appeared.
This outline will consider the findings of
various investigators regarding the fecal
streptococci and the significance of discharges
of these organisms into the aquatic environment.
H FECAL MATERIALS
A Definition
The terms "enterococci, " "fecal
streptococci, " "Group D streptococci, "
"Streptococcus fecalis, " 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 differentiate the Group D
streptococci from the various groups.
Although they overlap, the three groups,
fecal streptococcus, 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, usually capable of
development at 45° 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. "
However, 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 fecal
excreta of warm-blooded animals,
which includes all of the enterococcus
group in addition to other groups of
streptococci."
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Bacteriological Indicators of Water Pollution
B Species Isolated
1 Findings
a Human feces
Examination of human fecal specimens
yields a high percentage of the
enterococcus group and usually
demonstration of the S. salivarius
which is generally considered a
member of the human throat flora
and to be surviving in human fecal
materials rather than actively
multiplying in the enteric tract.
Also 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. bovis and/or S. equinus
organisms with a concomitantly
reduced percentage of the
enterococcus group.
2) Fowl excreta
Excrement from fowl characteris-
tically yields a large percentage
of enterococcal biotypes as well
as a significant percentage of
enterococcus group.
2 Significance
Species associations with particular
animal hosts is an established fact and
leads to the important laboratory
technique of partition counting of colonies
from the membrane filter or agar
pour plates in order to establish or
confirm the source of excretal
pollution in certain aquatic investi-
gations .
It is important to realize that a suitable
medium is necessary in order to
allow all of the streptococci which
we consider to be fecal streptococci
HI
to grow in order to give credence to
the derived opinions. Use of liquid
growth media into which direct
inoculations from the sample are
made have not proven to be successful
for partition counting due to the differing
growth rates of the various species of
streptococci altering the original
percentage relationships. Due to the
limited survival capabilities of some
of the fecal streptococci it is necessary
to sample fresh fecal material 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 range of 4. 0-9. 0.
FECAL STREPTOCOCCI IN THE
AQUATIC ENVIRONMENT
A General
From the foregoing it is appears that
the preponderant human fecal streptococci
are composed of the enterococcus group
and, as this is the case, several media are
presently available which will detect only
the enterococcal group will be suitable
for use with aquatic samples which are
known to be contaminated or potentially
contaminated with purely domestic
(human) wastes. On the other hand,
when it is known or suspected that other-
than-human wastes have potential egress
to the aquatic environment under investi-
gation, it is necessary to utilize those
media which are capable of quantitating
the whole of the fecal streptococci group.
B Stormwaters and Combined Sewers
1 General
Storm sewers are a series of pipes
and conduits which receive surface
runoffs from the action of rainstorms
and do not include sewage which are
borne by a system of sanitary sewers.
Combined sewers receive both the storm
runoff and the water-borne
wastes of the sanitary system.
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Bacteriological Indicators of Water Pollution
Both storm water and combined
sewer flows have been found to
usually contain large quantities of
fecal streptococci in numbers which
generally are larger than those of
the fecal coliform indicator organisms.
2 Bacteriological Findings
Table 1 represents, in 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 1
DISTRIBUTION OF FECAL STREPTOCOCCI
IN DOMESTIC SEWAGES AND STORMWATER
RUNOFFS
Fecal Streptococci
per 100 ml Ratio
Water Source median values FC/FS
5.3
4.5
4.9
4.4
4.0
27.9
16.9
Domestic Sewage
Preston, ID
Fargo, ND
Moorehead, MN
Cincinnati, OH 2,
Lawrence, MA 4,
Monroe, MI
Denver, CO 2,
Stormwater
Business District
Residential
Rural
64, 000
290, 000
330, 000
470, 000
500, 000
700, 000
900, 000
51,000
150, 000
58, 000
0.26
0.04
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 is less than 0. 7 for stormwaters.
The use of this ratio is useful to
identify the source of pollution as
Table 2. ESTIMATED PER CAPITA CONTRIBUTION OF INDICATOR MICROORGANISMS
FROM SOME ANIMALS*
Average indicator
density per gram
of feces
Average contribution
per capita per 24 hr
Animals
Man
Duck
Sheep
Chicken
Cow
Turkey
Pig
Avg wt of
Feces/24 hr,
wet wt, g
150
336
1, 130
182
23,600
448
2,700
Fecal
coliform,
million
13.0
33.0
16.0
1.3
0.23
0.29
3.3
Fecal
streptococci,
million
3.0
54.0
38.0
3.4
1.3
2.8
84.0
Fecal
coliform,
million
2,000
11,000
18, 000
240
5,400
130
8,900
Fecal
streptococci,
million
450
18,000
43, 000
620
31,000
1,300
230, 000
Ratio
FC/FS
4.4
0.6
0.4
0.4
0.2
0.1
0.04
*Publication WP-20-3, P. 102
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Bacteriological Indicators of Water Pollution
being human or nonhuman warm-
blooded animal polluted. When the ratio
is greater than 4. 0 it is considered to be
human waste contaminated while a ratio
of less than 0.7 is considered to be
nonhuman. • It is evident that the storm-
waters have been primarily polluted by
excreta of rats and other rodents and
possibly domestic and/or farm animals.
Species differences are the main cause
of different fecal coliform-fecal
streptococci ratios. Table 2 compares
fecal streptococcus and fecal coliform
counts for different species. Even
though individuals vary widely, masses
of individuals in a species have charac-
teristic proportion of indicators.
C Surface Waters
In general, the occurrence of fecal
streptococci indicates 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 isolated and
occurrences of small numbers can be
attributed to wild life and/or snow melts
and resultant drainage flows.
Various examples of fecal streptococcal
occurrences are shown in Table 3 in
relation to surface waters of widely varying
quality. (Geldreich and Kenner 1969)
IV FECAL STREPTOCOCCI:
AND LIMITATIONS
ADVANTAGES
A General
Serious studies concerning the streptococci
were instituted when it became apparent
that they were the agents 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 microbiological techniques
have been brought to bear on 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
INDICATOR ORGANISMS IN SURFACE
WATERS
Densities/100 ml
Fecal Fecal
Water Source coliform streptococci
Prairie Watersheds
Cherry Creek, WY 90 83
Saline River, KS 95 180
Cub River, ID 110 160
Clear Creek, CO 170 110
Recreational Waters
Lake Mead 2 444
Lake Moovalaya 9 170
Colorado River 4 256
Whitman River 32 88
Merrimack River 100 96
Public Water Intakes
Missouri River (1959)
Mile 470.5 11,500 39,500
Mile 434.5 22,000 79,000
Mile 408.8 14,000 59, 000
Kabler (1962) discussed the slow acceptance
of the fecal streptococci as indicators of
pollution resulting from:
1 Multiplicity and difficulty of laboratory
procedures
2 Poor agreement between methods of
quantitative enumeration
3 Lack of systematic studies of ....
a sources
b survival, and
c interpretations, and
4 Undue attention to the S. faecalis group.
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Bacteriological Indicators of Water Pollution
Increased attention to the fecal streptococci,
especially during the last decade, have
clarified many of the earlier cloudy issues
and have elevated the stature of these
organisms as indicators of pollution.
Court precedents establishing legal status
and recommendations of various technical
advisory boards have placed the fecal
coliform group in a position of primacy
in many water quality applications. The
fecal streptococci have evolved from a
position of a theoretically useful indicator
to one which was ancillary to the coliforms
to one which was useful when discrepancies
or questions evolved as to the validity of
the coliform data to one where an equality
status was achieved in certain applications.
In the future it is anticipated that, for
certain applications, the fecal streptococci
will achieve a position of primacy for
useful 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. "
B Advantages and Limitations
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 coliforms. 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 coliforms.
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 oxygen 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 totally eliminated sooner.
2 Resistance to Disinfection
In artificial pools the source of
contamination 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. salivarius,
can be a more reliable indicator
when detected along with the other
fecal streptococci especially 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 dieoff curve
differences are insignificant when
the curves of the fecal coliforms
are compared to various Gram
negative pathogenic bacteria which
reduces their effectiveness as
indicators.
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. faecalis
is characterized by its ability to
hydrolyze starch while the varietal
form, liquefaciens, is nonbeta
haemolytic and capable 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 strains constitute the majority
or all of the isolations when analyzed
biochemically.
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Bacteriological Indicators of Water Pollution
V STANDARDS AND CRITERIA
Acceptance and utilization of Total Coliform
criteria, which must now be considered a
pioneering effort, has largely been supplanted
in concept and in fact by the fecal coliforms
in establishing standards for recreational
waters.
.The first significant approach to the utiliza-
tion of the fecal streptococci as a criterium
for recreational water standards occurred in
1966 when a technical committee recommended
the utilization of the fecal streptococci with the
total coliforms as criteria for standards
pertaining to the Calumet River and lower
Lake Michigan waters. Several sets of
criteria were established to fit the intended
uses for this area. The use of the fecal
streptococci as a criterium is indicated to
be tentative pending the accumulation of
existing densities and could be modified in
future standards.
With the existing state-of-the-art knowledge
of the presence of the fecal streptococci in
waters containing low numbers of fecal
coliforms it is difficult to establish a specific
fecal streptococcus density limit of below
100 organisms/100 ml when used alone or
in con junction .with the total coliforms.
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Bacteriological Indicators of Water Pollution
Part 4. Other Bacterial Indicators of Pollution
I TOTAL BACTERIAL COUNTS
A Historical
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 gelatin plating medium
incubated 3 days at 2QQ C.
2 Later developments led to inoculation
of samples on duplicate plating media,
with one set incubated at 37° C and the
other at 20° C.
a Results were used to develop a ratio
between the 37° C counts and the
20°C counts.
b Waters having a predominant
count at 37° C were regarded as
being of probable sanitary signifi-
cance, while those giving
predominant counts at 20° C were
considered to be of probable soil
origin, or natural inhabitants of
the water being examined.
B Groups Tested
There is no such thing as "total" bacterial
count in terms of a laboratory determination.
1 Direct microscopic counts do not
differentiate between living and dead
cells.
2 Plate counting methods enumerate only
the bacteria which are capable of using
the culture medium provided, under the
temperature and other growth conditions
used as a standard procedure. No one
culture medium and set of growth
conditions can provide, simultaneously,
an acceptable environment for all the
heterogeneous, often conflicting,
requirements of the total range of
bacteria which may be recovered from
waters.
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 control procedures in
treatment plant operations
c Determination of sanitary conditions
in plant equipment or distributional
systems
2 Serious limitations in total bacterial
counts exist because:
a No information is given regarding
possible or probable fecal origin
of bacterial changes. Large numbers
of bacteria can sometimes be
cultivated from waters known to be
free of fecal pollution.
b No information of any kind is given
about the species of bacteria
cultivated.
c There is no differentiation between
harmless or potentially dangerous
forms.
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
27-19
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Bacteriological Indicators 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 used until the 1940's.
B Spore-Forming Bacteria (Clostridium
perfringens, or C. welchii)
1 Distribution
This is one of the most widely distributed
species of bacteria. It is regularly
present in the intestinal tract of warm-
blooded animals.
2 Nature of organism
C. perfringens is a Gram-positive,
spore-forming rod. The spores cause
a distinct swelling of the cell when
formed. The organism is extremely
active in fermentation of carbohydrates,
and produces the well-known "stormy
fermentation" of milk.
3 Status
The organism, when present, indicates
that pollution has occurred at some
time. However, because of the ex-
tremely extended viability of the spores,
it is impossible to obtain even an
approximation of the recency of pollution
based only on the presence of
C!. perfringens.
The presence of the organism does not
necessarily indicate an unsafe water.
C Tests for Pathogenic Bacteria of Intestinal
Origin
1 Groups considered include Salmonella
sp, Shigella sp, Vibrio comma,
Mycobacterium sp, Pasteurella sp,
Leptospira sp, and others.
2 Merits of direct tests:
Demonstration of any pathogenic
species would demonstrate an
unsatisfactory water quality, hazardous
to persons consuming or coming into
contact with that water.
3 Limitations
a There is no available routine pro-
cedure for detection of the full
range of pathogenic bacteria cited
above.
b Quantitative methods are not avail-
able for routine application to any
of the above.
c The intermittent release of these
pathogens makes it impossible to
regard water as safe, even in the
absence of pathogens.
d After detection, the public already
would have been exposed to the
organism; thus, there is no built-in
margin of safety, as exists with
tests for the coliform group.
4 Applications
a In tracing the source of pathogenic
bacteria in epidemiological investi-
gations
b In special research projects
c In water quality studies concerned
with enforcement actions against
pollution, increasing attention is
being given to the demonstration of
enteric pathogenic bacteria in the
presence of the bacterial indicators
of pollution.
D Miscellaneous Indicators
It is beyond this discussion to explore the
total range of microbiological indicators
of pollution that have been proposed and
27-20
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Bacteriological Indicators of Water Pollution
investigated to some 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
certain 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.
REFERENCES
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. 1971.
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.
1938.
4 Clark, H. F. and Kabler, P.W. The
Physiology of the Coliform Group.
Proceedings of the Rudolfs 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 Geldreich et al. The Fecal Coli-Aerogenes
Flora of Soils from Various Geographic
Areas. Journal of Applied Bacteriology
25:87-93. 1962.
7 Geldreich, E.E., Kenner, B.A., and
Kabler, P.W. Occurrence of
Coliforms, Fecal Coliforms, and
Streptococci on Vegetation and Insects.
Applied Microbiology. 12:63-69. 1964.
8 Kabler, P.W., Clark, H.F., and
Geldreich, E.E. Sanitary Significance
of Coliform and Fecal Coliform
Organisms in Surface Water. Public
Health Reports. 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.
EL. Quantification in Feces. Am. J.
Public Health. 50:1553-59. 1960.
11 Litsky, W., Mailman, W.L., and Fifield,
C.W. Comparison of MPN of
Escherichia coli and Enterococci in
River Water. Am. Jour. Public Health.
45:1949. 1955.
12 Medrek, T. F. and Litsky, 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.
27-21
-------
Bacteriological Indicators of Water Pollution
16 Morris. W. and Weaver, R.H.
Streptococci as Indices of Pollution
in Well Water. Applied Microbiology,
2:282-285. 1954.
17 Mundt, J.O., Goggin, J.H., Jr., and
Johnson, L. F. Growth of
Streptococcus fecalis var. liquefaciens
on Plants. AppliedMicrobiology.
10:552-555. 1962.
18 Geldreich, E.E. Sanitary Significance
of Fecal Coliforms 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.
1969.
20 Kabler, P.W. Purification and Sanitary
Control of Water (Potable and Waste)
Ann. Rev. of Microbiol. 16:127. 1962.
21 Litsky, W., Mailman, W. L., and Fifield,
C. W. Comparison of the Most Probable
Numbers of Escherichia coli and
Enterococci in River Waters. A. J, P. H.
45:1049. 1955.
22 Geldreich, E.E. Applying Bacteriological
Parameters to Recreational Water
Quality. J. AWWA. 62:113. 1970.
23 Geldreich, E.E., Best, L. C., Kenner, B.A.
and Van Donsel, D. J. The Bacteriolog-
ical Aspects of Stormwater Pollution.
J. WPCF. 40:1860. 1968.
24 FWPCA Report of Water Quality Criteria
Calumet Area - Lower Lake Michigan,
Chicago, IL. Jan. 1966.
This outline was prepared by H. L. Jeter,
Director, National Training Center and
revised by R. Russomanno, Microbiologist.
National Training Center, WPO, EPA,
Cincinnati, OH 45268.
Descriptors; Coliforms, Escherichia coli
Fecal Goliforms, Fecal Streptococci, Indicator
Bacteria, Microbiology, Sewage Bacteria,
Water Pollution
27-22
-------
EXAMINATION OF WATER FOR COLIFORM AND
FECAL STREPTOCOCCUS GROUPS
(Multiple Dilution Tube [MPN] Methods)
I INTRODUCTION
The subject matter of this outline is contained
in three parts, as follows:
A Part 1
1 Fundamental aspects of multiple dilution
tube ("most probable numbers") tests,
both from a qualitative and a quantitative
viewpoint.
2 • Laboratory bench records.
3 Useful techniques in multiple dilution
tube methods.
4 Standard supplies, equipment, and
media in multiple dilution tube tests.
B Part 2
Detailed, day-by-day, procedures in tests
for the coliform group and subgroups
within the coliform group.
C Part 3
Detailed, day-by-day, procedures in tests
for members of the fecal streptococci.
D Application of Tests to Routine Examinations
The following 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 level is 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.
TABLE 1
Examination Terminated at -
Type of Receiving
Water
Sewage Receiving
Treatment Plant - Raw
Chlorinated
Bathing
Drinking
Other Information
Presumptive
Test
Applicable
Applicable
Not Done
Not Done
Not Done
Confirmed Test
Applicable
Applicable
Applicable
Applicable
Applicable
Applicable in all
cases where Pre-
sumptive Test alone
is unreliable.
Completed Test
Important where results
are to be used for control
of raw or finished water .
Application to a statis-
tically valid number of
samples from the
Confirmed Test to estab-
lish its validity in
determining the sanitary
quality.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Environmental Protection Agency.
W. BA. 3L. 2.75
28-1
-------
MPN Methods
H BASIS OF MULTIPLE TUBE TESTS
A Qualitative Aspects
1 For purely qualitative aspects of testing
for indicator organisms, it is convenient
to consider the tests applied to one
sample portion, inoculated into a tube
of culture medium, and the follow-up
examinations and tests on results of the
original inoculation. Results of testing
procedures are definite: positive
(presence of the organism-group is
demonstrated) or negative (presence of
the organism-group is not demonstrated.)
2 Test procedures are based on certain
fundamental assumptions:
a First, even if only one living cell of
the test organism is present in the
sample, it will be able to grow when
introduced into the primary inoculation
medium;
b Second, growth of the test organism
in the culture medium will produce
a result which indicates presence of
the test organism; and,
c Third, extraneous organisms will
not grow, or if they do grow, they
will not limit growth of the test
organism; nor will they produce
growth effects that will be confused
with those of the bacterial group for
which the test is designed.
3 Meeting these assumptions usually
makes it necessary to conduct the tests
in a series of stages (for example, the
Presumptive, Confirmed, and Completed
Test stages, respectively, of standard
tests for the coliform group).
4 Features of a full, multi-stage test
a First stage: The culture medium
usually serves primarily as an
enrichment medium for the group
tested. A good first-stage growth
medium should support growth of all
the living cells of the group tested,
and it should include provision for
indicating the presence of the test
organism being studied. A first-
stage medium may include some
component which inhibits growth
of extraneous bacteria, but this
feature never should be included
if it also inhibits growth of any
cells of the group for which the
test is designed. The Presumptive
Test for the coliform group is a
good example. The medium
supports growth, presumably, of
all living cells of the coliform
group; the culture container has a
fermentation vial for demonstration
of gas production resulting from
lactose fermentation by coliform
bacteria, if present; and sodium
lauryl sulfate may be included in
one of the approved media for
suppression of growth of certain
noncoliform bacteria. This
additive apparently has no adverse
effect on growth of members of the
coliform group in the concentration
used. If the result of the first-stage
test is negative, the study of the
culture is terminated, and the result
is recorded as a negative test. No
further study is made of negative
tests. If the result of the first-
stage test is positive, the culture
may be subjected to further study
to verify the findings of the first
stage.
b Second stage: A transfer is made
from positive cultures of the first-
stage test to a second culture medium.
This test stage emphasizes provision
to reduce confusion of results due to
growth effects of extraneous bacteria,
commonly achieved by addition of
selective inhibitory agents. (The
Confirmed Test for coliforms meets
these requirements. Lactose and
fermentation vials are provided for
demonstration of coliforms in the
medium. Brilliant green dye and
bile salts are included as inhibitory
agents which tend to suppress growth
of practically all kinds of noncoliform
bacteria, but do not suppress growth
of coliform bacteria when used as
directed).
28-2
-------
MFN Methods
If result of the second- stange test is
negative, the study of the culture is
terminated, and the result is recorded
as a negative test. A negative test here
means that the positive results of the
first-stage test were "false positive, "
due to one or more kinds of extraneous
bacteria. A positive second-stage test
is partial confirmation of the positive
results obtained in the first-stage test;
the culture may be subjected to final
identification through application of still
further testing procedures. In routine
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 stage. This practice should be
followed only if adequate testing is done
to demonstrate that the assumption is
valid. Some workers recommend contin-
uing at least 5% of all sample examina-
tions to the third stage to demonstrate
the reliability of the second-stage results.
B Quantitative Aspects of Tests
1 These methods for determining bacterial
numbers are based on the assumption
that the bacteria can be separated from
one another (by shaking or other means)
resulting in a suspension of individual
bacterial cells, uniformly distributed
through the original 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 each of a stipulated series of de-
creasing sample volumes is inoculated
into the first-stage culture medium.
Through decreasing the sample incre-
ments, eventually a volume is reached
where only one cell is introduced into
some tubes, and no cells are introduced
into other tubes. Each of the several
tubes of sample-inoculated first-stage
medium is tested independently,
according to the principles previously
described, in the qualitative aspects
of testing procedures.
3 The combination of positive and
negative results is 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 ^s 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 present.
5 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, 0.01,0.001, and 0.0001ml,
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 ml.
28-3
-------
MPN Methods
IH LABORATORY BENCH RECORDS
A Features of a Good Bench Record Sheet
1 Provides complete identification of the
sample.
2 Provides for full, day-by-day informa-
tion about all tests performed on the
sample.
3 Provides easy step-by-step record
applicable to any portion of the sample.
4 Provides for recording of the quantitative
result which will be transcribed to sub-
sequent reports.
5 Minimizes the amount of writing by the
analyst.
6 Identifies the analyst(s).
There is no such thing as "standard"
bench sheet for multiple tube tests; there
are many versions of bench sheets. Some
are prescribed by administrative authority
(such as the Office of a State Sanitary
Engineer); others are devised by laboratory
or project personnel to meet specific needs.
It is not the purpose of this discussion to
recommend an "ideal" bench form; however,
the form used in this training course
manual is essentially similar to that used
in certain research laboratories of this
organization. The student enrolled in the
course for which this manual is written
should make himself thoroughly familiar
with the bench sheet and its proper use.
See Figure 1.
IV NOTES ABOUT WORKING PROCEDURES
IN THE LABORATORY
A Each bacteriological examination of water
by multiple dilution tube methods requires
a considerable amount of manipulation;
much is quite repetitious. Laboratory
workers must develop and maintain good
routine working habits, with constant
alertness to guard against lapses into
careless, slip-shod laboratory procedures
and "short cuts" which only can lead to
lowered quality of laboratory work.
The student reader is urged to review the
form for laboratory surveys (PHS-875,
Rev. 1966) used by Public Health Service
personnel charged with responsibility for
accreditation of laboratories for examination
of water under Interstate Quarantine
regulations.
B Specific attention is brought to the following
by no means exhaustive, critical aspects of
laboratory procedures in multiple dilution
tube tests:
1 Original sample
a Follow prescribed care and handling
procedures before testing.
b Maintain absolute identification of
sample at all stages in testing.
c Vigorously shake samples (and
sample dilutions) before planting
in culture media.
2 Sample measurement into primary
culture medium
a Sample portions must be measured
accurately into the culture medium
for reliable quantitative tests to be
made. Standard Methods prescribes
that calibration errors should not
exceed + 2. 5%.
28-4
-------
Project
Sample Station
Collection Data
Date £./(f/G, 7 Time
TempeVature___^°C
Other Observations
Analytical Record
By
Bench Number of Sample _,
Analyst ^&^g^
Test started at
^Coliform MPN/100 ml
Confirmed:
Completed:
Fecal Coliform MPN:
Figure 1. SAMPLE BENCH SHEET
Fecal Streptococcus MPN/100^ml
A-D - EVA:
28-5
-------
Methods
Suggested sample measuring practices
are as follows: Mohr measuring
.pipets are recommended. 10 ml
samples are delivered at the top of
the culture tube, using 10 ml pipets.
1. 0 ml samples 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
desired amount of sample has been
delivered. 1. 0 ml or 2. 0 ml pipets
are used for measurement of this
volume. 0.1 ml samples are
delivered in the same manner as 1. 0
ml samples, using, great care that
the sample actually gets into the
culture medium. Only 1. 0 ml pipets
are used for this sample volume.
After delivery of all sample incre-
ments into the culture tubes, the
entire rack of culture tubes may be
shaken gently to carry down any of
the sample adhering to the wall of
the tube above the medium.
Workers should demonstrate by actual
tests that the pipets and the technique
in use actually delivers the rated volumes
within the prescribed limits of error.
Volumes as small as 0.1 ml routinely
can be delivered directly from the
sample with suitable pipets. Lesser
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 Figure 2.
b Gas in any quantity is a positive test.
It is necessary to work in conditions
of suitable lighting for easy recog-
nition of the extremely small amounts
of gas inside the tops of some
fermentation vials.
Reading of liquid culture tubes for
growth as indication of a positive test
requires good lighting. Growth is
shown by any amount of increased
turbidity or opalescence in the culture
medium, with or without deposit of
sediment at the bottom of the tube.
Transfer of cultures with inoculation
loops 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 any non-
sterile object before making the
transfer.
b After sterilization, allow sufficient
time for cooling, in the air, to avoid
heat-killing bacterial cells on the
hot wire.
c Loops should be 3 mm in inside
diameter, with a capability of holding
a drop of water or culture.
For routine standard transfers
requiring transfer of 3 loopsful of
culture, many workers form three
3-mm loops on the same length of
wire.
Reading of culture tubes for gas
production
a On removal from the incubator.
shake culture rack gently, to
encourage release of gas which
may be supersaturated in the culture
medium.
As an alternative to use of standard
inoculation loops, the use of
"applicator sticks" have now been
sanctioned by the 13th Edition of
Standard Methods.
28-6
-------
MPN Methods
Figure 2. PREPARATION OF DILUTIONS
Dilution Ratios:
Delivery volume
1:10000
1ml
Tubes «.
Petri Dishes or Culture Tubes
Actual volume
of sample in tube
1ml O.lml 0.01 ml 0.001ml
0. 0001 ml o. 00001 ml
The applicator sticks are dry heat
sterilized (autoclave sterilization is
not acceptable because of possible
release of phenols if the wood is
steamed) and are used on a single-
service basis. Thus, for every culture
tube transferred, a new applicator
stick is used.
This use of applicator sticks is
particularly 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
gas burners.
7 Streaking cultures on agar surfaces
a All streak-inoculations should be
made without breaking the surface
of ifhe agar. Learn to use a light
touch with the needle; however,
many inoculation needles are so
sharp that they are virtually useless
in this respect. When the needle is
platinum or platinum- iridium wire,
it sometimes is beneficial to fuse
the working tip into a small sphere.
This can be done by momentary
insertion of a well-insulated (against
electricity) wire 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 entirely too heat-retentive
to be useful.
28-7
-------
MPN Methods
When the needle is nichrome
resistance wire, it cannot be heat-
fused; the writer prefers to bend
the terminal 1/16 - 1/8" of the wire
at a slight angle to the overall axis
of the needle. The side of the
terminal bent portion of the needle
then is used for inoculation of agar
surfaces.
b When streaking for colony isolation,
avoid using too much inoculum. The
streaking pattern is somewhat
variable according to individual
preference. The procedure favored
by the writer is shown in the
accompanying figures. Note
particularly that when going from
any one stage of the streaking to the
next, the inoculation needle is heat-
sterilized.
Preparation of cultures for Gram
stain
a The Gram stain always should be
made from a culture grown on a
nutrient agar surface (nutrient agar
slants are used here) or from nutrient
broth.
The culture should be young, and
should be actively growing. Many
workers doubt the validity of the
Gram stain made on a culture more
than 24 hours old.
Prepare a thin smear for the staining
procedure. Most beginning workers
tend to use too much bacterial sus-
pension in preparing the dried smear
for staining. The amount of bacteria
should be so small that the dried film
is barely visible to the naked eye.
V EQUIPMENT AND SUPPLIES
Consolidated lists of equipment, supplies,
and culture media required for all multiple
dilution tube tests described in this outline
are shown in Table 2.;. Quantitative infor-
mation is not presented; this is variable-
according to the extent of the testing pro-
cedure, the number of dilutions used, and
the number of replicate tubes per dilution.
It is noted that requirements for alternate
procedures are fully listed and choices are
made in accordance to laboratory preference.
28-8
-------
MPN Methods
a Flame-sterilize an inoculation needle and air-cool.
b Dip the tip 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 agar surface.
d Flame-sterilize the inoculation needle and air-cool.
a Turn the Petri dish one-quarter-turn and streak 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-sterilize the inoculation needle and air-cool.
a Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (2) into
area (3), the remaining unstreaked area.
b Flame-sterilize the inoculation needle and set it aside.
c Close the culture container and incubate as prescribed.
Figure 3. A SUGGESTED PROCEDURE FOR COLONY ISOLATION BY A
STREAK-PLATE TECHNIQUE
AREA 1 (Heavy inoculum)
AREA 3 (Isolated colonies]
AREA 2
(Moderate growth)
APPEARANCE OF STREAK - PLATE
AFTER INCUBATION INTERVAL
28-9
-------
MPN Methods
TABLE 2. APPARATUS AND SUPPLIES FOR STANDARD
FERMENTATION TUBE TESTS
Description of Item
Lauryl tryptose broth or Lactose
broth. 20 ml amounts of 1. 5 X
concentration medium, in 25 X 150 mm
culture tubes with inverted fermen-
tation vials, suitable caps.
Lauryl tryptose broth or Lactose
broth. 10 ml amounts of single
strength medium in 20 X 150 mm
culture tubes with inverted fermen-
tation vials, suitable caps.
Brilliant green lactose bile broth, 2%
in 10 ml amounts, single strength,
in 20 X 150 mm culture tubes with
inverted fermentation vials,
suitable caps.
Eosin methylene blue agar, poured
in 100 X 15 mm Petri dishes
Endo Agar. poured in 100 X 15 mm
dishes
Nutrient agar slant, screw cap tube
Boric actd lactose broth, 10 ml
amounts of single strength medium
in fermentation tubes.
EC Broth, 10 ml amounts of single
strength medium in fermentation
tubes:
Formate ricinoleate broth
(provisional)
Culture tube racks, 10 X 5 openings;
each opening to accept 25 mm dia-
meter tubes.
Pipettes, 10 ml, Mohr type, sterile,
in suitable cans.
Pipettes, 2 ml (optional), Morh type,
sterile, in suitable cans
Pipettes, 1 ml, Mohr type, sterile
in metal suitable cans
Standard buffered dilution water,
sterile, 99-ml amounts in screw-
capped bottles.
Gas burner, Bunsen type
Inoculation loop, loop 3mm dia-
meter, of nichrome or platinum-
iridium wire, 26 B & S gauge, in
suitable holder, (or sterile applicator.
stick)
Inoculation needle, nichrome, or
platinum -iridium wire, 26 B & S
gauge, in suitable holder.
Incubator, adjusted to 35 + 0. 50 C
Waterbath incubator, adjusted to
43+0.2°C
Waterbath incubator, adjusted to
44.5+ 0.2°C.
Glass microscopic slides, . 1 " X 3 "
Slide racks (optional)
Gram-stain solutions, complete set
Compound microscope, oil immer-
sion lens. Abbe' condenser
Basket for discarded cultures
Container for discarded pipettes
Total Coliform Group
Presumptive
Test
X
X
X
X
X
X
X
X
X
X
Confirmed
Test
X
X
X
X
X
X
X
X
X
Completed
Test
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Fecal Coliforms
(BALE)
X
X
X
X
X
X
(EC broth)
X
X
X
X
X
X
28-10
-------
Part 2
DETAILED TESTING PROCEDURES FOR MEMBERS OF THE
COLIFORM GROUP BY MULTIPLE DILUTION TUBE METHODS
I SCOPE
A Tests Described
1 Presumptive Test
2 Confirmed Test
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 is somewhat
repetitious, inasmuch as the Presumptive
Test 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 is to be the
stage at which the test is to be ended, and
the details of the procedures throughout,
in order to prevent the possibility of
discarding gas-positive tubes before
proper transfer procedures have been
followed.
Thus, if the worker knows that the test will
be ended at the Confirmed Test, he will
turn at once to Section HI, TESTING TO
THE CONFIRMED TEST STAGE, and will
ignore Sections II and IV.
The Fecal Coliform Test is described
separately, in Section V, as an
adjunct to the Confirmed Test and to the
Completed Test.
H TESTING TO PRESUMPTIVE TEST
STAGE
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 of lauryl tryptose broth
required for .the initial planting of the
sample (Table 3). 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, corresponding 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: Be sure to use tubes 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
Wastewater).
28-11
-------
MPN Methods
Tabla 3. SUGGESTED LABELING SCHEME FOR ORIGINAL CULTURES AND
' SUBCULTURES IN MULTIPLE DILUTION TUBE TESTS
Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Tube
1
312
A
312
a
312
a
312
la
312
2a
Tube
2
312
B
312
b
312
t±
312
Ib
312
2b
Tube
3
312
C
312
c
312
c
312
Ic
312
2c
Tube
4
312
D
312
d
312
d
312
Id
312
2d
Tube
5
312
E
312
e
312
e
312
le
312
2e
Sample volume
represented
Tubes with 10 ml
of sample
Tubes with 1 ml
of sample
Tubes with 0. 1 ml
of sample
Tubes with 0.01 ml
of sample
Tubes with 0.001 ml
of sample
Typical Example
RB
312-
A ,
^x
Lab. Worker
Side ntif ication
-Bench Number
"Sample Volume
Tube of Culture Medium
The labeling of cultures can be reduced by labeling only the first tube of
each series of identical sample volumes in the initial planting of the sample.
All subcultures from initial plantings should be labeled completely.
Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
Shake the sample vigorously, approxi-
mately 25 times, in an arc of one foot
within seven seconds and withdraw the
sample portion at once.
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.
a Use a 10 ml pipet for 10 ml sample
portions, and 1 ml pipets for portions
of 1 ml or less. Handle sterile 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 sample.
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently 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 35° + 0.50C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake gently. If gas is about to appear
in the fermentation vials, the shaking
will speed the process.
28-12
-------
MPN Methods
2 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 negative (-) test. GAS IN ANY 2
QUANTITY IS A POSITIVE TEST.
3 Discard all gas-positive tubes of lauryl
tryptose broth, and return all the gas-
negative tubes to the 35°C incubator for
an additional 24 i 2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 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 elsewhere in this manual.
Ill TESTING TO CONFIRMED TEST STAGE
Note that the description starts with the
sample inoculation and includes the
Presumptive Test stage. The Confirmed
Test preferred in Laboratories of this agency
is accomplished by means of the brilliant
green lactose bile broth (BGLB) and the
acceptable alternate tests are mentioned in
HI F. In addition, the Fecal Coliform Test is
included as an optional adjunct to the procedure.
A First-Day Procedures
1 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 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) 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 identification of the worker(s)
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 medium,- 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 tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
Shake the sample vigorously, approxi-
mately 25 times, 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.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-ml pipets for portions
of 1 ml or less. Handle sterile 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
sample.
28-13
-------
MPN Methods
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver sample
portions of 1 ml or less down into
the culture tube near the surface of
the medium. Do not deliver small
sample volumes at the top of the tube
and allow them to run down inside
the tube; too much of the sample
will fail to reach the culture medium.
d Prepare preliminary dilutions of
samples for portions of 0. 01 ml or
less before delivery into the culture
medium. See Table 1 for preparation
of dilutions. NOTE: Always deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much
as 30 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently 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 35° + 0. 5° C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake gently. If gas 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
gas in the fermentation vial as a
positive (+) test and each tube not
showing gas as a negative (-) test.
GAS IN ANY QUANTITY IS A POSITIVE
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 tubes of lauryl
tryptose broth culture for Confirmed
Test procedures. Confirmed Test
procedures may not be required for all
gas-positive cultures. If, after 24-hours
of incubation, all five replicate cultures
are gas-positive for two or more con-
secutive sample volumes, then select
the set 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 gas-positive
cultures representing smaller volumes
of sample, in which some tubes were
gas-positive and some were gas-negative.
5 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of lauryl tryptose broth
selected for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop 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 gas-positive
tube.
7 After making the transfers, the rack
should contain some 24-hour gas-
negative tubes of lauryl tryptose broth
and the newly inoculated BGLB.
8 If the Fecal Coliform Test is included
in the testing procedures, consult
Section V of this part of the outline of
testing procedures.
28-14
-------
MPN Methods
9 Incubate the 24-hour gas-negative
BGLB tubes and any newly-in.oculated
tubes of BGLB an additional 24 ft- 2
hours at 35°+ 0.5°C. "~
C 48-hour Procedures
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
results from LTB under the 48-hour
LTB column and the BGLB results under
the 24-hour column of the data sheet.
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
positive at 48 hours are to be transferred.
The option described above for 24-hour
cultures does not apply 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
BGLB tubes and any newly-inoculated
tubes of BGLB 24 + 2 hours at 35° +
0.5QC.
6 Discard all tubes of LTB and all 24-hour
gas-positive BGLB cultures.
D 72-hour Procedures
1 If any cultures remain to be examined,
all will be BGLB. 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-hour gas-negative cultures
for incubation 24 + 2 hours at 35 +
0.5°c.
4 Discard all gas-positive BGLB cultures
and all 48-hour gas-negative cultures
from BGLB.
5 It is possible that all cultural work and
results for the Confirmed Test have
been finished at this point. If so, codify
results and determine Confirmed Test
coliforms per 100 ml as described in
the outline on use of MPN Tables.
E 96-hour Procedures
At most only a few 48-hour cultures in
BGLB may be present. Read and record
gas production of such cultures in the "48"
column under BGLB on the data sheet.
Codify results and determine Confirmed
Test coliforms per 100 ml.
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 of these media in
preference to BGLB (also approved in
Standard Methods) is advised to refer to
the current edition of "Standard Methods-
for the Examination of Water and Waste-
water" for procedures.
28-15
-------
MPN Methods
IV TESTING TO COMPLETED TEST STAGE
(Note that this description starts with the
sample inoculation and proceeds through the
Presumptive and the Confirmed Test stages.
In addition, the Fecal Coliform Test is
referred to as an optional adjunct to the
procedure.)
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) performing the laboratory
tests, and the sample volumes planted.
2 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 identification of the worker(s)
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. Guidance on labeling for
laboratory data'number and identification
of individual tubes is described else-
where ;in this outline.
NOTE: If 10-ml samples are being
plated, it is necessary to use tubes
containing the correct concentration
of culture medium. This has previously
been noted elsewhere in this outline"
and referral is made to tables.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
5 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.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-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 any sample.
When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
When delivering the sample into the
culture medium, deliver sample
portions of 1 ml or less down into
28-16
-------
MPN Methods
the culture tube near the surface of
the medium. Do not deliver small
sample volumes at the top of the
tube and allow them to run down
inside the tube; too much of the
sample will fail to reach the culture
medium.
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 deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much as
3 0 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently 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 35O + 0. 5OC for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose broth
cultures from the incubator, and shake
gently. If gas 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
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY
QUANTITY IS A POSITIVE 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 tubes of lauryl
tryptose broth culture for the Confirmed
Test procedures. Confirmed Test
procedures may not be required for
all gas-positive cultures. If, after
2 4-hours of incubation, all five
replicate cultures are gas-positive for
two or more consecutive sample
volumes, then select the set 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 gas-positive cultures
representing smaller volumes of
sample, in which some tubes were
gas-positive and some were gas-
negative .
5 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of lauryl tryptose broth
selected for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop 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
gas-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 some 24-hour gas-
negative tubes of lauryl tryptose borth
and the newly inoculated BGLB.
Incubate the rack of cultures at 35° C
+ O.SOCfor 24 + 2 hours.
C 48-hour Procedures
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
28-17
-------
MPN Methods
bile broth (BGLB). Be sure to record
results from LTB under the 48-hour .
LTB column and the BGLB results
under the 2 4-hour column of the data
sheet.
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 positive at 48 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 35° + 0. 5° C.
Retain all~24-hour gas-positive cultures
in BGLB for further test procedures.
5 Label a Petri dish preparation of eosin
methylene blue agar (EMB agar) to
correspond with each gas-positive
culture in BGLB.
6 Prepare a streak plate for colony
isolation from each gas-positive culture
in BGLB on the correspondingly-labeled
EMB agar plate.
Incubate the EMB agar plates 24 + 2
hours at 35°+ 0.5° C.
D 72-hour Procedures
1 Remove the cultures from the incubator.
Some may be on BGLB; several EMB
agar plates also can be expected.
2 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 agar plates 24 +
2 hours at 35 + 0.5° C. Discard the
gas-positive BGLB cultures after
transfer.
4 Reincubate any gas-negative BGLB
cultures 24 + 2 hours at 35° + 0.50 C.
5 Discard all 48-hour gas-negative BGLB
cultures.
6 Examine the EMB agar plates for the
type of colonies developed thereon.
Well-isolated 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 may or 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-
labeled 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 least two well-isolated
colonies representative of those
appearing on the EMB plate, and trans-
fer them to lactose broth and to agar
slants.
8 Incubate all cultures transfered from
EMB agar plates 24+2 hours at 35 +
0.50C.
E 96-hour Procedures
1 Subcultures from the samples being
studied may include: 48-hour tubes
of BGLB, EMB agar plates, lactose
broth tubes, and agar slant cultures.
28-18
-------
MPN Methods
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. 5O C.
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
plate.
Examine and record results from the
lactose broth cultures.
Prepare a Gram-stained smear from
each of the agar slant cultures, as
follows:
NOTE: Always prepare Gram stain
from an actively growing culture,
preferably 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 oily film.
b Place one drop of distilled water on
the slide.
c Use the inoculation needle to suspend
a tiny amount of growth from the
nutrient agar slant culture in the
drop of water.
d Mix the thin suspension of cells with
the tip of the inoculation needle, and
allow the water to evaporate.
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
paper.
h Flood the smear with Lugol's
iodine for 1 minute.
i Wash the slide in gently running
water and blot dry with filter paper.
j Decolorize the smear with 95%
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.
1 Examine the slide under the
microscope, using the oil
immersion lens. Goliform
bacteria are Gram-negative,
nonspore-forming, rod-shaped
cells, occurring 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 ml.
28-19
-------
MPN Methods
F 120-hour Procedures and following:
1 Any procedures to be undertaken from
this point are "straggler" cultures on
media already described, and requiring
step-by-step methodology already given
in detail. Such cultures may be on:
EMB plates, agar slants, or lactose
broth. The same time-and-temperature
of incubation required for earlier studies
applies to the "stragglers" as do the
observations, staining reactions, and
interpretation of results. On con-
clusion of all cultural procedures,
codify results and determine Completed
Test coliforms per 100 ml.
V FECAL COLIFORM TEST
A General Information
1 The procedure described is an elevated
temperature test for fecal coliform
bacteria.
2 Equipment required for the tests are
those required for the Presumptive
Test of Standard Methods, a water-bath
incubator, and the appropriate culture
media.
B Fecal Coliform Test with EC Broth
1 Sample: The test is applied to gas-
positive tubes from the Standard
Methods Presumptive Test (lauryl
tryptose broth), in parallel with
Confirmed Test procedures.
2 24-hour Operations. Initial procedures
are the planting procedures described
for the Standard Methods Presumptive
Coliform test.
a After reading and recording gas-
production on lauryl tryptose broth,
temporarily retain all gas-positive
tubes.
b Label a tube of EC broth to corre-
spond with each gas-positive tube
of lauryl tryptose broth. The option
regarding transfer of only a limited
number..of tubes to the Confirmed
Test 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 correspondingly
labeled tube of EC broth.
d Incubate EC broth tubes 24 i 2 hours
at 44. 5 t 0. 2°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 possible
after inoculation and always within
30 minutes after inoculation.
3 48-hour operations
a Remove the rack of EC cultures
from the waterbath, shake gently,
and record gas production for each
tube. Gas in any quantity is a
positive test.
b As soon as results are recorded,
discard all tubes. (This is a 24-
hour test for EC broth inoculations
and not a 48-hour test.)
c Transfer any additional 48-hour
gas-positive tubes of lauryl tryptose
broth to correspondingly labeled
tubes of EC broth. Incubate 24 +
2 hours at 44. 5 ± 0.2°C.
4 72-hour operations
a Read and record gas production for
each tube. Discard all cultures.
b Codify results and determine fecal
coliform count per 100 ml of sample.
28-20
-------
MPN Methods
TESTS FOR COUfOKM GROUP
/
l-
SUMPTIVE TES
Of
0.
V
'
vt
Q
UJ
%
£
S
b
>
1
COMPLETED TEST
^
(
\
i
*
»
LACTOSE OR LAURYl TRYPTOSE BROTH AMD ARE MCUBATCD AT 35 DEC Ct
FERMENTATION TUBES (SERIAL DILUTION) OJ DEC C.
yX" 'V GAS POsrnvK TUBES (ANY AMOUNT
/ ^\ Of GAS) CONSTITUTE A POSIT ATE
1 1 PRESUMPTIVE TEST
,. GAS POSITIVE GAS NEGATIVE
| (24 HR.t 2 HR.) | TOTAL INC UB A TON TIME FOR LACTOSE
! GAS POSITIVE GAS NEGATIVE
! COIIFORM GROUP ABSENT
1
1 '
i ^ k n
! CONFIRMATORY BROTH
! BRILLIANT GREEN LACTOSE BILE
1 / V INfllBATF BfilB TIIBFS FOR 18 HffS
•i EMB PLATES -AilS^Mm / \ ± 3 HRS. AT 35 DEC. C± OJ DEG. C.
^ COWrllW^tO f x. *
tNDO AGAR / \
"ATES GAsrosmvr GASNtGATnrf INCUBATE EMB OR ENDO AGAR
T PLATES FOR 24 HRS. t 2 HRS AT
COIFORM GROUP COIIFORM GROUP or nrG r+ OJ DEG C
CONFIRMED NOTCONFKMED ~
_> /
~r /
^^JEMB PLATES 1
^s^ ^"^^"^T^~""^^
^^^ \
^ 1 1
^NUTRIENT AGAR SLANT LACTOSE BROTH TUBE
GRAM + AND GRAM NEGATIVE GAS POSITIVE GAS NEGATIVE
RODS AND/O* RODS ^X'
SPOREFORMERS NO SPORES ^X^ COUFORM GROUP ABSENT
ifOJH COMflcFED TEST
GAS POSITIVE GAS NEGATIVE
•^ COIIFORM GROUP ABSENT
TRANSFER TO EMB PLATE
AND REPEAT PROCESS
28-21
-------
Part 3
LABORATORY METHODS FOR FECAL STREPTOCOCCUS
(Day-By-Day Procedures)
I GENERAL INFORMATION
A The same sampling and holding procedures
apply as for the coliform test.
B The number of fecal streptococci 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
test. For example: If a sample planting
series of 1.0, 0.1, 0.01, and 0.001 ml
is planned for the coliform test, it is
suggested 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
Presumptive and Confirmed Tests, except
for the differences in culture media.
H STANDARD METHODS (Tentative)
PROCEDURES
A First-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
portions.
2 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
3 Measure the predetermined sample
volumes into the labeled tubes of azide
dextrose broth, using the sample
measurement and delivery techniques
used for the Presumptive Test.
4 Shake the rack of tubes of inoculated
culture media, to insure good mixing
of sample with medium.
5 Place the rack of inoculated tubes in
the incubator at 35° + 0. 5° C for 24 +
2 hours.
B 2 4-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. Evidence of growth
consists either of turbidity of the
medium, a "button" of sediment at the
bottom of the culture tube, or both.
2 Label 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
tubes.
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 hours at 35° + 0. 5°C.
28-23
-------
MPN Methods
C 48-hour 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, is a
positive test. Be sure to report the
results of the azide dextrose broth
medium under the "48" column for that
medium and the results of the ethyl
violet azide broth cultures under the
"24" column for that medium.
2 Any 48-hour growth-positive cultures
of azide dextrose broth are to be
transferred (three loopfulls) to ethyl
violet azide broth. Discard all 48-hour
growth-negative tubes of azide dextrose
broth and all 24-hour growth-positive
tubes of ethyl violet azide broth.
3 Incubate the 24-hour growth-negative
and the newly-inoculated tubes of ethyl
violet azide broth 24 + 2 hours at 35°
+ 0.5QC.
D 72-hour Operations
1 Read and report growth results of all
tubes of ethyl violet azide broth.
2 Discard all growth-positive cultures
and all 48-hour growth-negative
cultures.
3 Reincubate any 24-hour growth-negative
cultures in ethyl violet azide broth 24
+ 2 hours at 35O + 0.5QC.
E 96-hour Operations
1 Read and report growth results of any
remaining tubes of ethyl violet azide
broth.
Codify results and determine fecal
streptococci per 100 ml.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater (13th Ed)".
Prepared and published jointly by
American Public Health Association,
American Water Works Association,
and Water Pollution Control
Federation. 1971.
2 Geldreich, E.E., Clark, H.F., Kabler,
P.W., Huff, C.B. andBordner, R.H.
The Coliform Group. II. Reactions
in EC Medium at 45° C. 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 the Bacteriological
Examination of Sea Water and Shellfish.
3rd Edition, American Public Health
Association. 1962.
This outline was prepared by H. L. Jeter,
Director, National Training Center, Water
Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
Descriptors: Coliforms, Fecal Coliforms,
Fecal Streptococci, Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Microbiology, Most Probable Number, MPN,
Sewage Bacteria, Water Analysis
28-24
-------
EXAMINATION OF WATER FOR COLIFORM
AND FECAL STREPTOCOCCUS GROUPS
(Membrane Filter Methods)
Part 1 - Overview
I INTRODUCTION
A Scope
1 The membrane filter consists of a
porous disk of cellulose esters 47 mm
in diameter and approximately 0. 15 mm
thick. Filters used in water bacter-
iology have pores 0. 45 microns in
diameter. The basic principles of use
in water bacteriology are as follows:
Currently, active research is under-
way to reevaluate the pore size in an
effort thereby to improve colony
productivity.
a A sterile membrane 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 conditions 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.
Basic Principles and Origins of
Membrane Filter Techniques
The purpose of this discussion is to
provide orientation in the types and
amount of equipment and supplies
needed in membrane filter analyses
in water quality surveys, 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.
II REQUIREMENTS FOR EQUIPMENT AND
SUPPLIES
A Laboratory Supplies and Equipment
1 Membrane filters
a Membrane filters are available
from several different commercial
sources, representing the United
States, Britain, and Germany.
Each source applies a distinctive
tradename to its MF product; such
as:
"Millipore Filters" - Millipore
Filter Corp., U. S. A.
"Bac-T-Flex Filters" - Schleicher
and Schuell, U.S.A. representatives
for Sartorius - Werke, Germany.
"Oxoid Filter" - Oxo Limited,
Britain
b Membrane filters are available in
a wide variety of controlled pore
W.BA. mem. 86.2.75
29-1
-------
Examination of Water
sizes. Each commercial source
will designate the filter most suitable
for water bacteriological tests upon •
request.
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
filters, ordinarily are sold with the
membrane filters. Additional supplies
are obtainable from filter manufacturers.
These pads may be the source of cult-
ural problems due to chemical
impurities.
3 Filter-holding units
a The filter-holding unit is a device
for supporting the membrane filter
and for holding the sample until it
passes through the filter. During
the filtration, the sample passes
through a circular area, usually
about 35 mm in diameter, in the
center of the filter. 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 receptable,
supports the membrane filter on
a plate about 50 mm in diameter.
The central part of this plate is
porous, to allow 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 flat ring that
rests firectly over the nonporous
part of the filter support plate.
3) When assembled, the two
elements of the filter-holding
unit are joined by a locking ring
or by one or more clamps or
springs.
b Filter-holding units are either
metal or glass. Metal units are
available from at least six commer-
cial sources, and glass units are
available from at least two sources.
Most workers prefer to use metal
filter-holding units, due to their
greater durability. Most of the use
of glass units seems to be dictated
by economic considerations, as
these are much lower in initial cost.
4 Culture media
a Culture media are commercially
available for use with membrane
filters in the determination of
bacterial plate counts, for coliform
counts, fecal coliform counts, fecal
streptococcus counts and, in addition,
for several special applications.
b These media can be obtained in
several forms, including:
1) Dehydrated media, requiring
weighing, addition of a suitable
amount of distilled water, and
some form of sterilization prior
to use. This form of media is
preferred 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.
5 Culture containers
Several forms of culture containers are
suitable for membrane filter works.
These include small glass Petri dishes,
and plastic Petri dishes. The plastic
Petri dishes may be preferred, as they
are sufficiently cheap to encourage
single-service use.
29-2
-------
Examination of Water
6 Vacuum sources
In the fixed laboratory, central vacuum
service, laboratory-scale electric
Vacuum pumps, or water pumps
("aspirators") are suitable for vacuum
filtration of samples.
7 Glassware
Conventional laboratory glassware is
used in fixed laboratories for measure-
ment and delivery of samples for filtra-
tion, sample dilutions, and the like.
Under field conditions it sometimes is
necessary to use a limited amount of
laboratory glassware, with some form
of field application of sterilization
methods.
8 General laboratory facilities
Such facilities as autoclave, incubators,
weighing equipment, and the like are
required in conventional bacteriology
laboratories, and are needed equally
when membrane filter methods are
used.
B Field Equipment
Field operations cannot be carried on
indefinitely without support from a central
laboratory. Functions such as preparation
of distilled water, pre-weighing of culture
media, sterilization of membrane filters
and culture containers, and other general
preparations are best performed in a
central laboratory facility.
1 With the introduction of membrane
filters to the bacteriological analysis
of water, a number of investigators
gave 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
usefulness:
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.
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 transporation of
samples.
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 developed field equipment
is currently available from only one
source.
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 equipment 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.
29-3
-------
Examination of Water
III MEMBRANE FILTER PROCEDURES IN
WATER QUALITY AND POLLUTANT
SOURCE MONITORING
A Standard Coliform Test
1 Basic procedures
a Sample filtration volumes are
selected (usually 3 volumes) to give
at least one volume which will con-
tain 20-80 typical coliform colonies.
b The sample volumes are filtered
through membrane filters, and
incubated at 35°C for 20-22 hours
in an atmosphere at or near
saturation.
c Following incubation, the typical
coliform colonies are counted and
reported as coliforms per 100 ml
of sample.
2 Applications
The total coliform count has been used
most widely of all procedures when
bacteriological data are collected in a
survey. Applications of membrane
filter methods in surveys necessarily
have a short history. The method has
been used in certain studies of the
Potomac River System, in the Illinois -
Great Lakes studies, and in certain
surveys 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" Test with Membrane
Filters
1 Procedures
a Sample filtration procedures are the
same as for the Standard Membrane
Filter coliform test. Sample
filtration volumes should be
selected so that at least one filter
will produce 20-60 fecal coliform
colonies.
b The culture medium is M-FC
Broth. The medium requires
addition of a rosolic acid solution
prior to use.
c Within 20 minutes after sample
filtration, the membrane filters
are placed in small plastic bags,
which are sealed, and immersed
in a forced circulation water bath
at 44.5°C ± 0. 2°C for a 24 hour
incubation time.
d Fecal coliform colonies are blue or
blue-hues, generally 1-3 mm in
diameter. The fecal coliform
density is reported as fecal coliforms
per 100 ml of sample.
2 Application
The fecal coliform test is being used
increasingly in water quality studies.
Fecal coliform criteria are being
established for compliance with
effluent limits for NPDES Permit
Compliance.
C Fecal Streptococcus Tests
1 Basic procedures
a The filtration procedures are the
same as with the coliform tests.
The sample volumes should be such
that at least one membrane filter
will produce 20 - 100 fecal strepto-
coccus colonies.
b The culture medium can be the
M-Enterococcus Agar developed by
Slanetz and his associates, or it can
be KF-Broth, developed by Kenner
and his associates. In either case,
incubation is 48 hours at 35 C.
29-4
-------
Examination of Water
IV
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.
LIMITATIONS OF MEMBRANE FILTER
METHODS
Most of the recognized limitations of the
membrane filter method 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
Volume
1 In tests for coliform bacteria, the
sample filtration volumes 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
differentiated and sample volume be-
gins to fail. This is especially trouble-
some in waters containing large numbers
of noncoliform bacteria capable of
growing on the filters.
2 In tests 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
as clay, and has relatively low counts of
the indicator organisms being studied,
cultural difficulties often result. The
particulate matter suspended in the water
is deposited on the surface of the membrane
filter in sample filtration, with any
bacteria contained in the sample. Culture
medium diffusing through the membrane
filter forms a capillary layer around all
the particles of matter deposited on the
filter. This results in development of
spreading, poorly defined bacterial
colonies.
The solutions to problems of this type are:
1) to filter the sample in several smaller
increments so that the combined sample
filtration volume is spread over several
membrane filters, each 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.
29-5
-------
Examination of Water
1 In colif orm testing, this ^can-he A
serious problem. The difficulty arises
when appreciable numbers of non-
coliform bacteria are present and are
capable of growing on the Endo-type
culture medium used. When large
numbers of noncoliform bacteria grow
on the medium, and few coliform
bacteria are present, the sheen-
producing capacity of many of the
coliform colonies is impaired, and
the result indicates the presence of
fewer coliform bacteria than actually
are present.
A solution for this type of problem
is to filter several smaller sample
increments, or else to use the MPN
method.
2 In tests for fecal streptococci, the
presence of large numbers of bacteria
of other groups has not proven trouble-
some. The 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.
Increasingly, problems in sensitivity
of the test are being recognized in
tests on chlorinated effluents from
wastewater treatment facilities.
OFFICIAL STATUS OF MEMBRANE
FILTER METHODS
American Public Health Association,
American Water Works Association,
and Water Pollution Control Federation,
in "Standard Methods for the Exam-
ination of Water and Wastewater. "
(13th Edition, 1971)
1 A single-stage procedure, using
M-Endo Broth MF for tests for
members of the coliform group, is
described as an official testing
procedure.
2 A new two-stage procedure has been
added.
3 A delayed incubation procedure, using
benzoated M-Endo Broth MF, is
designated a Tentative Method.
4 A test for enterococci (fecal strepto-
cocci), using M Enterococcus Agar
or KF broth, is designated a
Tentative Method.
B The Technical Committee on Methods for
the Bacteriological Examination of
Boundary Waters Recommended use of
membrane filter methods for the
examination of boundary waters. The
Advisory Committee adopted the recom-
mendation in January, 1957.
C The U. S. Government, in Interstate
Quarantine Drinking Water Standards
1 (Federal Register, October 23, 1956,
pp. 8110-11 and March 1, 1957,
Vol. 22, p. 1271). Preliminary and
final authorization was granted to use
membrane filter coliform tests for
the examination of potable waters at
interstate carrier water points and
other waters under water quality
control regulations of the Public
Health Service or other agencies of
the U. S. Government.
D The U. S. Government, in test procedures
for compliance with water quality criteria.
Federal Register, October jj>, 1973,
pp. 28758-60, Volume 38, No. 199. The
same information also appears in the
Code of Federal Regulations, Title 40,
Part 136, pp. Ill ff, state July 1, 1974.
29-6
-------
DETAILED MEMBRANE FILTER METHODS
Part 2
I BASIC PROCEDURES
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 membrane
filter procedures described here are not
repeated elsewhere in this text 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.
C "Sterilizing" Media
Set tubes of freshly prepared medium in a
boiling waterbath for 10 minutes. This
method suffices for medium in tubes up to
25X150 mm. Frequent agitation is needed
with media containing agar.
Alternately, coliform media can be
directly heated on a hotplate to the first
bubble of boiling. Stir the medium
frequently if direct heat is used, to avoid
charring the medium.
Do not sterilize in the autoclave.
D General Laboratory Procedures with
Membrane Filters
1 Prepare data sheet
Minimum data required are: sample
identification, test performed including
media and methods, sample filtration
volumes, and the bench numbers
assigned to individual membrane filters.
2 Disinfect the laboratory bench surface.
Use a suitable disinfectant solution and
allow the surface to dry before
proceeding.
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.
Forceps sterility 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 chars the filter.
*When an agar medium is used, absorbent pads are not 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.
29-7
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT. SUPPLIES AND MEDIA
Item
Funnel unit assemblies
Ring stand, with about a 3" split ring, to
support the filtration funnel
Forceps, smooth tips, type for
MF work
Methanol, in small wide-mouthed bottles.
about 20 ml for sterilizing forceps
Suction flasks, glass, 1 liter, mouth to
fit No. 8 stopper
Rubber tubing, 2-3 feet, to connect
suction flask to vacuum services, latex
rubber 3/16" I.D. by 3/32" wall
Pinch clamps strong enough for tight
compression of rubber tubing above
Pipettes, 10 ml, graduated, Mohr type.
sterile, dispense 10 per can per working
space per day. (Resterilize daily to
meet need).
Pipettes, 1 ml, graduated, Mohr type.
sterile, dispense 24 per can per working
space per day. (Resterilize daily to
meet need).
Pipette boxes, sterile, for 1 ml and
10 ml pipettes (sterilize above pipettes
in these boxes).
Cylinders, 100 ml graduated, sterile.
(resterllize daily to meet need).
Jars, to receive used pipettes
Gas burner, Bunsen or similar
laboratory type
Wax pencils, red, suitable for writing
on glass
Sponge in dilute iodine, to disinfect the
desk tops
Membrane filters (white, grid marked.
sterile, and suitable pore size for
microbiological analysts of water)
Absorbent pads for nutrient, (47 mm in
diameter), sterile, in units of 10 pads
per package. Not required if medium
•contains agar.
Fetri dishes,' disposable, plastic.
50 X 12 mm, sterile
Waterbath incubator 44.5 + 0.2°C
Vegetable crispers, or cake boxes.
plastic, with tight fitting covers, for
membrane filter incubations
Fluorescent lamp, with extension cord.
Ring stand, with clamps, utility type
Tot
M-Endo
Broth
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
••
X
X
X
X
1 Conforms
L.E.S.
Coliform
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Delayed
Coliform
X
X
X
X
X
X
X
X
X
X
X
X
X
x
X
X
X
X
X
X
X
Fecal
Coliform
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Fecal
Streptococcus
X
X
X
X
X
X
X
X
X
X
X
X
x.
X
X
X
X
X
X
X
X
Verified
Tests
X
X
29-8
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)
Item
Half-round glass paper weights for
colony counting, with lower half of a
2-oz metal ointment box
Hand tally, single unit acceptable,
hand or desk type
Stereoscopic (dissection) microscope,
magnification of 10X or 15X, prefer-
able binocular wide field type
Bacteriological inoculating needle
Wire racks for culture tubes,
10 openings by five openings pre-
ferred, dimensions overall approxi-
mately 6" X 12"
Phenol Red Lactose Broth in 16 X
150 mm fermentation tubes with
metal caps, 10 ml per tube
Eosin Methylene Blue Agar
(Levine) in petri plates, prepared
ready for use
Nutrient agar slants, in screw
capped tubes, 16 X 126 mm
Gram stain solutions, 4 solutions
per complete set
Microscope, compound, binocular,
with oil immersion lens, micro-
scope lamp and immersion oil
Microscope slides, new, clean,
l"X3"size
Water proof plastic bags
for fecal coliform culture
dish incubation
M-Endo medium, MF dehydrated
medium in 25 X 95 mm flat bottomed
screw- capped glass vials, 1.44g
per tube, sufficient for 30 ml of
medium
Ethanol, 95% in small bottles or
screw-capped tubes, about 20 ml
per tube
Sodium benzoate solution, 12%
aqueous, in 25 X 150 mm screw-
capped tubes, about 10 ml per tube
L. E. S. Endo Agar MF, dehydrated
M-Endo medium, 0. 36 g per 25 X
95 mm flat bottomed screw-capped
glass vial, plus 0.45 g agar, for 30 ml
Lactose Lauryl Sulfate Tryptose Broth
in 25 X 150 mm test tube without
included gas tube, about 25 ml, for
enrichment in L. E. S. method
Total Coliform s
M-Endo
Broth
X
X
X
X
X
L.E.S.
Coliform
X
X
X
X
X
X
Delayed
Coliform
X
X
X
X
X
X
Fecal
Coliform
X
X
X
X
Fecal
Streptococcus
X
X
X
Verified
Tests
X
X
X
X
X
X
X
X
X
29-9
-------
Detailed Membrane Filter Methods
Table 1. EQUIPMENT. SUPPUES AND MEDIA (Cont'd)
Item
M-FC Broth for fecal collform.
dehydrated medium in 25 X 95 mm
flat bottomed screw-capped glass
vials, 1. 11 g per tube, sufficient
for 30 ml of culture medium
Rosolic acid, 1% solution, in
0. 2N NaOH, in 25 X 150 mm flat
bottomed screw-capped tubes.
about 5 ml per tube, freshly
prepared
M-Enterococcus Agar. dehydrated
medium In 25 X 150 mm screw-
capped tubes, sufficient for 30 ml,
1.26 g per tube
Dilution bottles, 6-oz, preferable
boro- silicate glass, with screw-
cap (or rubber stopper protected
by paper) . each containing 99 ml
of sterile phosphate buffered
distilled water
Electric hot plate surface
Beakers, 400 - 600 ml (for water-
bath in preparation of membrane
filter culture media)
Crucible tongs, to be used at
electric hot plates, for removal
of hot tubes of culture media for
boiling waterbath
Total Coliforms
M-Endo
Broth
X
X
X
X
L. E.S.
Collform
X
X
X
X
Delayed
Collform
X
X
X
X
Fecal
Collform
X
X
X
X
X
X
Fecal
Streptococcus
X
X
X
X
X
Verified
Test
29-10
-------
Detailed Membrane Filter Methods
6 Deliver enough culture medium to
saturate each absorbent pad, using
a sterile pipette.
Exact quantities cannot be stated
because pads and culture containers vary.
Sufficient medium should be applied so
that when the culture container is tipped,
a good-sized drop of culture medium
freely drains cut of the absorbent pad.
7 Organize supplies and equipment for
convenient sample filtration. In
training courses, laboratory instructors
will suggest useful arrangements;
eventually the individual will select a
system 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 minimize 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 outer part
of the filter disk, outside the part
of the filter through which the sample
passes.
9 Attach the funnel element to the base
of the filtration unit.
To avoid damage to the membrane
filter, locking forces should only be
applied at the locking arrangement.
The funnel element never should be
turned or twisted while 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 colonies
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 ml,
measure the sample with a sterile
10 ml or 20 ml pipette, and pipette
on a dry membrane in the filtration
assembly.
c With samples of 2 ml to 10 ml, pour
about 20 ml of sterile dilution water
into the filtration assembly, then
measure the sample into the sterile
buffered dilution water with a 10 ml
sterile pipette.
d With samples of 0. 5 to 2 ml, 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 0. 5 ml is to
be filtered, prepare appropriate
dilutions in sterile dilution water,
and proceed as applicable in item c
or d above.
When dilutions 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.
29-11
-------
Detailed Membrane Filter Methods
30 minutes. Always shake sample •
dilutions thoroughly before delivering
measured volumes.
12 Turn on the vacuum.
Open the appropriate spring 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 source of contamination
of later samples. (In laboratory
practice the funnel unit is not routinely
sterilized between successive filtrations
of a series). The purpose of the funnel
rinse is to flush all droplets of a sample
from the funnel walls to the membrane
filter. Extensive tests have shown that
with proper rinsing technique, bacterial
retention on the funnel walls is negligible.
13 Rinse the sample through the filter.
After all the sample has passed through
the membrane filter, rinse down the
sides of the funnel walls with at least
20 ml of sterile dilution water. Repeat
the rinse twice after all the first rinse
has passed through the filter. Cut off
suction on the filtration assembly.
14 Remove the funnel element of the filter
holding unit.
If a ring stand with split ring is used,
hang the funnel element on the ring;
otherwise, place the inverted funnel
element on the inner surface of the
wrapping material. This requires
care in opening the sterilized package,
but it is effective as a protection of the
funnel ring from contamination.
15 Take the membrane filter from the
filter holder and carefully place it.
grid-side up on the medium.
Check that no air bubbles have been
trapped between the membrane filter
and the underlying absorbent pad or
agar. Relay the membrane if necessary.
16 Place in incubator after
filtration series.
Invert the containers. The immediate
atmosphere of the incubating membrane
filter must be at or very near 100%
relative humidity.
17 Count colonies which have appeared
after incubating for the prescribed
time.
A stereoscopic microscope magnifying
10- 15 times and careful illumination
give best counts.
For reporting results, the computation
is:
bacteria/ 100 ml =
No. colonies counted X 100
Sample volume filtered in ml
Example:
A total of 36 colonies grew after
filtering a 10 ml sample. The
number reported is:
36lCOm"ieS X 10° = 36° Per 10° ml
Report results to two significant figures.
Example:
A total of 40 colonies grew after
filtering a 3 ml sample.
This calculation gives:
40
10° • 1333. 33 per 100 ml
But the number reported should be
1300 per 100 ml.
29-12
-------
Detailed Membrane Filter Methods
H MF LABORATORY TESTS FOR
COLIFORM GROUP
A Standard Coliform Test (Based on M-Endo
Broth MF)
1 Culture medium
a M-Endo Broth MF Difco 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
Tryptose
Lactose
Sodium desoxycholate
Dipotassium phosphate
Monopotassium phosphate
Sodium chloride
.Sodium lauryl sulf ate
Basic fuchsin (bacteriological)
Sodium sulfite
Distilled water (containing
20.0 ml ethanol)
1.5
5.0
5.0
10.0
12.5
0.1
4.375 g
1.375g
5.0 g
g
g
g
0.05
1 . 05
2.1
g
g
g
g
g
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 that the laboratory
worker preweigh the dehydrated
medium in closed tubes for several
days, or even weeks, at one operation.
b
With this system, a large number
of increments of dehydrated medium
(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
maintained.
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.
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.
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 light source perpendicular to the
plane of the membrane filter. A
small fluorescent lamp is ideal for
the purpose.
b 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.
c Record the colony counts on the
data sheet, and compute the coliform
count per 100 ml of sample.
29-13
-------
Detailed Membrane Filter Methods
B Standard Coliform Tests (Based on L. E. S.
Endo Agar)
The distinction of the L. E. S. count is a
two hour enrichment incubation on LST
broth. M-Endo L.E.S. medium 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 &)
Bacto-Yeast Extract
Bacto- Casitone
Bacto-Thiopeptone
Bacto-Tryptose
Bacto-Lactose
Dipotassium phosphate
Monopotassium phosphate
Sodium chloride
Sodium desoxycholate
Sodium lauryl sulfate
Sodium sulfite
Bacto-Basic fuchsin
Agar
Distilled water (containing
20 ml ethyl alcohol)
1.
3.
3.
7.
9.
3.
1.
3.
0.
2
7
7
5
4
3
0
7
1
g
g
g
g
g
g
g
g
g
0.05 g
1.6 g
0.8 g
15 g
1000 ml
b To rehydrate the medium, suspend
51 grams in the water-ethyl alcohol
solution. ;
c Medium is "sterilized" as directed
in I, C.
d Pour 4-6 ml of freshly prepared Agar
into the smaller half of the container.
Allow the medium to cool and solidify.
2 Procedures for filtration and incubation
a Lay out the culture dishes in a row
or series of rows as usual. 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 all
29-14
manipulations of the pads.
(Agar occupies smaller half or
bottom).
c Using a sterile pipette, deliver
enough single strength lauryl
sulfate tryptose broth to saturate
the pad only. Avoid excess medium.
d Follow general procedures for
filtering in I, D. Place filters on
pad with lauryl sulfate tryptose
broth.
e Upon completion of the filtrations,
invert the culture containers and
incubate at 350 C for 1 1/2 to 2
hours.
3 2-hour procedures
a Transfer the membrane filter from
the enrichment pad in the upper half
to the agar medium in the lower
half 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
is optional.
c The container is inverted and
incubated 22 hours + 2 hours + 0.5 C.
4 Counting procedures are as in I, D.
5 L.E.S. Endo Agar may be used as a
single-stage medium (no enrichment
step) in the same manner as 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
is unnecessary when the interval be-
tween sample collection and plating is
within acceptable limits.
Preparation of culture media for
delayed incubation coliform test
a Preservative media M-Endo Broth
base
-------
Detailed Membrane Filter Methods
To 30 ml of M-Endo Broth MF
prepared in accordance with
.directions in n, A, 1 of this
' outline, add 1. 0 ml of a sterile
12% aqueous solution of sodium
benzoate.
L. E. S. MF Holding Medium-
Coliform: Dissolve 12.7 grams in
1 liter of distilled water. No
heating is necessary. Final pH
7.1 + 0.1. .This medium contains
.sodium benzoate.
b Growth media
M-Endo Broth MF is used, prepared
as described in II, 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
Broth.
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 lay out
the culture containers on the labora-
tory bench.
d Remove the tops from the culture
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 culture container.
f Place a sterile absorbent pad in
each culture container, and deliver
enough freshly prepared M-Endo
Broth to saturate each pad.
g Using 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 underlying
absorbent pad. Discard the
absorbent pad containing pre-
servative medium.
h After incubation of 20 + 2 hours
at 35° C, count colonies as in the
above section A, 2.
i If L. E.S. Endo Agar is 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 applies to identification
of colonies growing on Endo-type media
used for determination of total coliform
counts. Isolates from these colonies are
studied for gas production from lactose
and typical coliform morphology. In
effect, the procedure corresponds with
the Completed Test stage of the multiple
fermentation tube test for coliforms.
Procedure:
1 Select a membrane filter bearing
several well-isolated coliform-type
colonies.
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
29-15
-------
Detailed Membrane Filter Methods
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 coliforms; "NCr
for colonies not conforming to
coliform colony appearance on
Endotype media..
3 Incubate the broth tubes at 35° C+ 0.5°C,
4 At 24 hours:
a Read and record the results from
the lactose broth fermentation tubes.
The following code is suggested:
Code
O
No indication of acid or gas
production, either with or
without evidence of growth.
Evidence of acid but not gas
(applies only 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.
Tubes not showing gas production are
returned to the 35° C incubator.
c Gas-positive tubes are transferred
as follows:
1) Prepare a streak inoculation 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 35° C' + o. 5°C.
5 At 48 hours:
a Read and record results of lactose
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-negative cultures are discarded
without further study; they are
coliform- negative.
c Examine the cultures transferred
to EMB agar plates and to nutrient
agar slants, as follows:
1) Examine the EMB agar 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.
ft 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 stain should
be made on a culture not more
than 24 hours old. Examine under
oil immersion for typical coliform
morphology, and record results.
6 At 72 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 lactose at 35° 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+0. 2°C.
1 Preparation of Culture Medium
(M-FC Broth Base) for Fecal
Coliform Count
29-16
-------
Detailed Membrane Filter Methods
a Composition
Tryptose 10. 0 g
Proteose Peptone No. 3 5.0g
Yeast extract 3.0 g
Sodium chloride 5. 0 g
Lactose 12.5 g
Bile salts No. 3 1.5 g
Rosolic acid* (Allied 10. 0 ml
Chemical)
Aniline blue (Allied Chemical) 0.1 g
Distilled water
1000 ml
Filter membranes for fecal coliform
counts consecutively and immediately
place them in their culture containers.
Insert as many as six culture containers
all oriented in the same way (i. 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 submerge
the sacks with culture containers in-
verted beneath the surface of a 44. 5
+ 0. 2 C waterbath.
b To prepare the medium dissolve
37.1 grams in a liter of distilled
water which contains 10 ml of 1%
rosolic acid (prepared in 0.2 N
NaOH).
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 2-8° C.
2 Special supplies
Small water proof plastic 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 + 0. 2°C as rapidly
as possible.
Ill
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 - 10X magnification.
b Low angle lighting from the side is
advantageous.
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)
TESTS FOR FECAL STREPTOCOCCAL
GROUP-MEMBRANE FILTER 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.
^Prepare 1% solution of rosolic acid in 0. 2 N NaOH. This dye is practically insoluble in water.
29-17
-------
Detailed Membrane Filter Methods
Test for Members of Fecal Streptococcal
Group (Tentative, Standard Methods) M-
Enterococcus Agar Medium
1 Preparation of the culture medium
a Formula (The Difco formula is shown,
but equivalent constituents from
other sources are equally acceptable).
Bacto tryptose 20. 0 g
Bacto yeast extract 5. 0 g
Bacto dextrose 2. 0 g
Dipotassium phosphate 4. 0 g
Sodium Azide 0.4 g
Bacto agar 10.0 g
2, 3, 5. Triphenyl 0.1 g
tetrazolium chloride
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 containers
(about 25 X 150 mm) in quantities
sufficient for preparation of 30 ml
of culture medium (1. 26 g per tube).
c Follow I, C, for "sterilizing" medium
and dispense while hot into culture
containers. Allow plates to harden
before use.
2 List of apparatus, materials, as given
in Table I.
3 Procedure, in general, as given in I.
Special instructions
a Incubate for 48 hours, inverted,
with 100% relative humidity, after
filtrations are completed. If the
entire incubator does not have
saturated humidity, acceptable
conditions can be secured by placing
the cultures in a tightly closed
container with wet paper, towels,
or other moist material.
After incubation, remove the
cultures from the incubator, and
count all colonies under wide field
binocular dissecting microscope
with magnification set at 10X 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 count per
100 ml of sample. This is con-
veniently computed:
No. fecal streptococci per 100 ml =
No. fecal streptococcus colonies counted
Sample filtration volume in ml
X100
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 liter of reconstituted
medium.
Bacto proteose peptone #3 10. 0
Bacto yeast extract 10. 0
Sodium chloride (reagent grade) 5. 0
g
g
g
g
g
g
g
Sodium glycerphosphate 10.0
Maltose (CP) 20.0
Lactose (CP) 1. 0
Sodium azide (Eastman) 0.4
Sodium carbonate 0.636 g
(Na2CO reagent grade)
Brom cresol purple 0. 015 g
(water soluble)
Bacto agar 20.0 g
b Reagent
2, 3, 5-Triphenyl tetrazolium
chloride reagent (TPTC)
This reagent is prepared by making
a 1% aqueous solution of the above
chemical passing it through a Seitz
filter or membrane filter. It can
29-18
-------
Detailed Membrane Filter Methods
be kept in the refrigerator in a
screw-capped tube until used.
The dehydrated medium described
above is prepared for laboratory
use as follows:
Suspend 7. 64 grams of the dehydrated
medium in 100 ml of distilled water
in a flask with an aluminum foil
cover.
Place the flask in a boiling water-
bath, melt the dehydrated medium,
and leave in the boiling waterbath
an addional 5 minutes.
Cool the medium to 50°-60OC, add
1. 0 ml of the TPTC reagent, and
mix.
For membrane filter studies, pour
5-8 ml in each 50 mm glass or
plastic culture dish or enough to
make a layer approximately 1/8"
thick. Be sure to pour plates before
agar cools and solidifies.
For plate counts, pour as for standard
agar plate counts.
NOTE: Plastic dishes containing
media may be stored in a dark, cool
place up to 30 days without change
in productivity of the medium, pro-
vided that no dehydration occurs.
Plastic dishes may be incubated in
an ordinary air incubator. Glass
dishes must be incubated in an
atmosphere with saturated humidity.
Apparatus,
Table 1.
and materials as given in
3 General procedure is as given in I.
Special instructions
a Incubate 48 hours, inverted with
100% relative humidity after
filtration.
b After incubation, remove the
cultures from the incubator, and
count colonies under wide field
binocular dissecting microscope,
with magnification set at 10X or
2 OX. Fecal streptococcus colonies
are pale pink to dark wine- color.
In size 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 ml =
No. fecal streptococcus colonies
Sample filtration volume in ml
C Verification of Streptococcus Colonies
1 Verification of colony identification
may be required in waters containing
large numbers of Micrococcus 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 use
this reference for the step-by-
step procedure.
IV PROCEDURES FOR USE OF MEMBRANE
FILTER FIELD UNITS
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 be sent to
the field, pre weighed, in vials or
capped culture tubes. The medium
then requires only the addition of a
suitable volume of distilled water-
ethanol prior to sterilization.
29-19
-------
Detailed Membrane Filter Methods
3 Sterilization procedures in the field
are the same as for laboratory methods.
4 Laboratory preparation of the media,
ready for use, would be permissible
provided that the required limitations
on time and conditions of storage are
met.
B Operation of Millipore Water Testing Kit,
Bacteriological
1 Supporting supplies and equipment are
the same as for the laboratory
procedures.
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+ 0.5°C.
3 Sterilize the funnel unit assembly by
exposure to formaldehyde or by
immersion in boiling water. If a
laboratory autoclave is available, this
is preferred.
Formaldehyde is produced by soaking
an asbestos ring (in the funnel base)
with methanol, igniting, and after a
few seconds of burning, closing the
unit by placing the stainless steel
flask over the funnel and base. This
results in incomplete combustion of
the methanol, whereby formaldehyde
is produced. Leave the unit closed
for 15 minutes to allow adequate
exposure to formaldehyde.
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
application.
C Other commercially available field kits
should be used according to manu-
facturer's instructions. It is emphasized
that the standards of performance are
required for field devices as for laboratory
equipment.
D .Counting of Colonies on Membrane Filters
1 Equipment and materials
Membrane filter cultures to be
examined
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 coliform
and noncoliform colonies. Coliform
and noncoliform colonies. Coliform
colonies have a "metallic" surface
sheen .under reflected light, which
may cover the entire colony or may
appear only on the center.
29-20
-------
Detailed Membrane Filter Methods
Noncoliform colonies range from
colorless to pink or red, but do not
have the characteristic "metallic""
sheen.
Enter the colony counts in the data
sheets.
Enter the coliform count per 100 ml
of sample for each membrane having
a countable number of coliform
colonies. Computation is as follows:
No. coliform per 100 ml =
No. coliform colonies on MF
No. milliliters sample filtered
X100
McCarthy, J.A., Delaney, J.E. and
Grasso, R.J. Measuring Coliforms
in Water. Water and Sewage Works.
1961: R-426-31. 1961.
This outline was prepared by H. L. Jeter,
Director, National Training Center,
EPA, WPO, Cincinnati, OH 45268.
Descriptors: Biological Membranes,
Coliforms, Fecal Coliforms, Fecal
Streptococci, Filters, Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Membranes, Microbiology, Water Analysis
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater. APHA,
AWWA, WPCF. 12th Edition. 1965.
29-21
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