PROPOSED
WATER QUALITY
INFORMATION
Volume II
OCTOBER 1973
U.S. ENVIRONMENTAL PROTECTION AGENCY
Washington, D.C. 20460
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FOREWORD
The Federal Water Pollution Control Act Amendments
require the Administrator of the U.S. Environmental
Protection Agency to publish both criteria for water
quality and information for the restoration and
maintenance of aquatic integrity, and the measurement
and classification of water [Section 3Ql*a(a)l and 2,
P.L. 92-500].
Volume I of this two volume series contains the
criteria for water quality for the protection of
human health and for the protection and propagation
of desirable species of aquatic biota. Volume II of
the series contains information on the maintenance
and restoration, measurement, and the classification
of waters. Also those pollutants suitable for maximum
daily load calculations are identified.
Both Volumes I and II are published as proposed
documents with a 180 day period provided for public
comment*
I Russell E. Train
Administrator
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Volume II
Proposed
Information for Water Quality
Environmental Protection Agency
Page
I. Legislative Basis 1
II. Introduction 2
III. Factors for Consideration for the Maintenance and
Restoration of Integrity of Water and for the
Protection and Propagation of Aquatic Life and
Wildlife 304 (a)(2)(A)(P) 3
Aluminum 4
Antimony 4
Arsenic 5
Barium 5
Beryllium 6
Bismuth 6
Boron 6
Bromine 7
Cadmium 7
Chloride 8
Chromium 9
Copper 9
Cobalt 9
Fluoride 10
Iron 11
Lead 12
Lithium 12
Nitrate 13
Nickel 15
Phosphorus 15
Manganese 16
Mercury 16
Molybdenum 16
Selenium 17
Silver 17
Uranium 18
Vanadium 19
Zinc 19
IV. Measurement and Classification of Waters 304(a)(2)(C) 20
A. Measurement Techniques 21
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1. Physical-Chemical Methods 21
Acidity 21
Alkalinity
Electro-metric Titration 22
Automated Methyl Orange 24
Arsenic 25
Biochemical Oxygen Demand (BOD)
Modified Winkler With Full Bottle 26
DO Probe 27
Organic Carbon (Total and Dissolved) 28
Chloride
Titration 30.
Automated Ferricyanide Method 31
Colorinetry or Electrometric Determination 32
Chlorine Requirement 32
Chemical Oxygen Demand (COD) 32
Titration - (High Concentration) 34
Titration - (Low Concentration) 35
Titration - (Saline Waters) 36
Color 37
Cyanide
Colorimetric 38
Titration 40
Dissolved Oxygon (DO)
Modified Winkler - Full Bottle 41
Probe Method 43
Fluoride 45
Hardness 46
Total Hardness 47
Metals (Atomic Absorption) 48
Mercury
Flameless Atomic Absorption 51
Cold Vapor Technique - (Biological Materials) 52
Gas Chromatography - (In Fish) 54
Gas Chromatography - (In Sediment) 56
Atomic Absorption Spectroscopy
for Surface Waters 58
Methylene Blue Active Substances 60
iJitrogen 61
Ammonia 61
Colorimctry fil
Titrinetric G3
Automated Colorimetric 64
Kjeldahl 65
Titrimetric 65
Automated Phenolate 6G
Colorimoteric 68
nitrite , 60
Nitrate and Nitrite 70
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Automated Cadmium Reduction 70
Automated Hydrazine 72
Organic plus Ammonia . 74
Trisodium-tlitrilotriacetic Acid (NTA) 75
Zinc - Zincon 75
Automated Zinc - Zincon 76
Phosphorus 77
Single Reagent .Method 77
Automated Single Reagent Method 79
Automated Stannous Chloride Method 81
Silica ' 82
Oil and Grease 83
pi! 84
Phenolics 85
Solids 87
Dissolved 87
Suspended 88
Total 89
Volatile 90
Sulfate 91
Colorimetry 91
Automated Chloranilate 92
Sulfide 93
Turbidity 94
Temperature 96
Threshold Odor 97
Specific Conductance 99
Identification of Weathered or Unveathered 100
Petroleum Oils 102
Pesticides 103
Electron Capture - Has Liquid Chromatography 103
Organochlorine Pesticides 104
2. Biological Methods 107
Phytoplankton and Periphyton
Cell Counts and Identification 107
Volume of Periphyton 100
Cell Counts and Identification of Phytoplankton 110
Visual Observation 110
Visual Observation (Filter Method) 111
Visual Observation (Counting Chamber Method) 112
Cell Volume Estimates of Plankton and Periphyton 113
Periphyton and Phvtoplankton (Species Connosition) 114
Diatom Species Identification 115
Cell Counts and Identification of Phvtoplankton 116
Chlorophvll a of Phytoplankton 117
In Vitro 117
Spectronhometric 118
Zooplankton Volume and Species Identification 119
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Iri situ Productivity of Phytoplankton 120
Radioactive Hethoc! 120
Oxycren Method 122
Periphyton and Phytoplankton Species Composition 124
Chlorophyll a, h and c of Phytoplankton and Periphyton 125
Cell Surface Area of Phytoplankton 126
Diomass of Hacronhytes 127
Algal r-rowth Potential 128
Chlorophyll a of Phytoplankton (Fluorescence) 130
3. nioassay Procedures 132
B. Classification of Uaters 162
V. Constituents Suitable for Maximum Daily Loading 304(a)(2)(D) 164
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Information for Water Quality
I. Legislative Basis
flection 304 (a) (2) of. the "Federal Water Pollution
Control Act Amendments of 1972" (86 Stat. 816; 33 U.S.C.
1314 1972), hereinafter referred to as the "Act", provides
that the Administrator (EPA) shall, within one year of
enactment, (by Oct. 18, 1973), publish information: (A) on
the factors necessary to restore and maintain the chemical,
physical and biological integrity of the aquatic
environment; (B) on the factors necessary for the protection
and propagation of shellfish, fish and wildlife for all
classes and categories of receiving waters and to allow
recreational activities in and on the water; (C) on the
measurement and classification of water quality; and (D) on
the identification of pollutants suitable for maximum daily
load measurement correlated with the achievement of water
guality objectives. The information shall reflect the
latest scientific knowledge.
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II. Introduction
The national V7ater Quality Standards Program was
initiated with the passage of the Water Quality Act of 1965,
Sec 10 (c). The Water Quality Standards are comprised of use
designations for eeich water body or portion thereof, water
quality criteria to support the use designations, and
implementation plans for scheduling the construction of the
necessary treatment facilities. The designations by water
uses are protection and propagation of fish, aquatic life
and wildlife (fresh water and marine), water for public
water supplies, recreational, agricultural, and industrial
waters. The Water Quality Standards prior to enactment of
FWPCA Amendments of 1972 were applicable to only interstate
waters and their tributaries. The Act extends the coverage
to include all intrastate waters and the state standards
have since been, or are in the process of being revised
accordingly.
The objective of the Act is to restore and maintain the
chemical, physical and biological integrity of the Nation's
waters. The National goal, Sec 101(a)(l), is to eliminate
the discnarge of pollutants into navigable waters by 1985,
with an interim goal, Sec 101(a)(2), being to attain by July
1983, water quality which provides for the protection and
propagation of fish, shellfish and wildlife and for
recreation in and on the Nation's water.
This Information for Water Quality is Volume II of a
two-volume publication. It contains information on what is
found regionally in the Nation's water; on the measurement
and classification of water quality; and on the
identification of pollutants suitable for maximum daily load
measurement. Volume I, under separate cover, is the
Criteria document. The Criteria for Water Quality compiled
within Volume I consists of prescribed limits of
acceptability for pollutants, each followed immediately by
supporting scientific rationale. The Information for Water
Quality is provided to assist the user of Volume I in his
application of the Water Quality Criteria.
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III. Factors for Consideration for the Haintenance and
Restoration of Integrity of V7ater and for the Protection
and Propagation of Acru.atio Life and Uildlife
Restoration and naintenance of aauatic integrity
reauires the identification and quantification of the
elements or comnonents acting on the environment and an
understanding of the effect each has on the aauatic
environment. Presented, in Table 1 are the ranges and mean
concentrations of inorganic pollutants found in the major
freshv;ater basins in the United States. Data are arranged
by basins, and give the source of pollutants and dates data
vrere collected.
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Agent
Aluminum
Sources
Natural
Weathering of rock
Concent rat i on s
in U.S. Waters
Man-Made
Industrial waste,
mine drainage, and
wash water from water
treatment plants
Basin Areas
Concentrations (tng/1)
New Jersey surface water
1) spring flow
2) summer flovi
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri H. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
0.09-0.
0.08-0.
Mean Positive:
33
31
0.028
0.022
0.117
0.030
0. 141
0.056
0.018
0.017
0.213
0.068
0.050
0.333
0.030
0.063
0.015
0.011
1968
1962-67
Antimony
Industrial effluents,
but is rapidly removed
by precipitation and
adsorption
Principal rivers of U.S.
None detected 1958-59
-------
Sources
Agent
Arsenic
Natural
Weathering of rocks
Man-Made
Waste from industry
mining activity and
residues from
pesticides
Concentrat ions
in U.S. Waters
Basin Areas
Concentrations (mg/1)
Kansas R. at Lawrence
Kansas R. at Topeka
Kansas R. and Lawrence R.
Kansas R.
Northeast Easin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Dasin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
Great Basin
Alaska Basin
0.003
0.00&
0.002-0.010
0.002-0.008
Mean Positive:
0.031
0. 017
0.035
0.050
0.066
0.308
0.069
0.037
0. 123
0.091
0.053
0.022
0.068
0.020
0.034
1970
1970
1970
1962-67
Bariun
Weathering of rock
Brines trom oil well
waste and effluents
from mining areas.
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R, Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
Coosa R. Below Rome, Ga.
Mean Positive:
0.021
0.025
0.026
0.025
0.043
0. 042
0.039
0.015
0.063
0.090
0.060
0.067
0.027
0.042
0. OU1
0.017
0-340
(max. cone.)
1962-67
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Sources
Agent
Beryllium
Natural
Weathering of
mineral beryl
Man-Made
Effluent from atomic
reactors, metallurgy,
aircraft, rocket and
missile fuel indus-
tries
Concentrations
in U.S. Waters
Basin Areas
Concentrations (mg/1)
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee F. Basin
Ohio R. Basin
Lake Erie Basin
Western Great Lakes Basin
Missouri R. Basin
Pacific Northwest Basin
Monongahela R. at
Pittsburgh, Pa.
Mean Positive:
0.002 1962-67
0.00012
0.00005
0.00016
0.00028
0.00017
0.00005
0.00023
0.00002
0.00122 1962-1967
(max. cone.)
Bismuth
Weathering of rock
Metallurgy; medicinal
Principal rivers of U.S.
streams in California
None detected
0.0006-0.0008
1958-59
Boron
Weathering of boron-
bearing rock
Industrial effluents
of weather prooting
wood, tire proofing
fabrics, manufacturing
glass and porcelain,
production of leather
and carpets, cosmetics,
photography, artifi-
cial gems, high
energy fuels, bacteri-
cides and fungicides
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
Colorado R. at Yuma, Ariz.
Mean Positive:
0. 032
0. 012
0. 029
0.02"4
0.067
0.210
0. 105
0.019
0. 15"4
0. 131
0. 179
0.289
0.030
0. 1<»3
0. 08«
0. 028
1. 800
(max. cone.)
1962-67
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Sources
Agent
Natural
Man-Made
Concentrations
in U.S. Waters
Basin Areas
Concentrations (rnq/1)
Bromine
RainfaLl, combustion
of leaded gasoline
containing ethylene
dibroroide, effluents
from industries pro-
ducing salt, chemical
medicinal compounds,
and disinfectants
Lake Superior, Mich.
Lake Superior tributaries
Lake Michiqan
Lake Huron
St. Clair 5.
Lake St. Clair
Detroit R.
Lake Erie
Lake Ontario
0.007-
0.005-
0. 01 1-
0.013-
0.019-
O.OU5-
0.020-
0.020-
0.038-
0.033
0.260
0.021
0.029
0.039
0.055
0.028
0.05U
0.077
1969
Cadmium
Weatherinq of rock
Effluents from indus-
tries using cadmium
such as metallurqy,
electroplating,
Northeast Dasin
North Atlantic Basin
Southeast Basin
Ohio R. Basin
Mean Positive:
ceramics, piqmentation. Lake Erie Easin
photography, nuclear
reactors, insecticides
and antihelminthics
Upper Mississippi R. Basin
Western Great Lakes Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
Great Basin
Cuyahoqa R. at Cleveland,
Ohio
0.005
0.003
0.005
0.007
C.050
0.006
0.005
0,002
0.010
0.005
0.001
0. 120
(max. cone.)
1962-67
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Sources
Agent
Natural
Concentrations
in U.S. Waters
Man-Made
Basin Areas
Concentrations (nxj/1)
Chloride weathering of rock,
aniir.
-------
Sources
Agent
Chromium
Natural
Man-Made
Concentrat ions
in U.S. Maters
Basin Areas
Concentrations (mq/1)
Industrial effluents
using chromium such
as metal pickling anJ
plating operations,
anodizing aluminum,
leather processing,
manufacture of paints,
dyes, explosives,
ceramics, paper, glass,
corrosion inhibitm-ii
and many others
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Fasin
Alaska Basin
Mean Positive:
0.006
0.007
0.012
0.007
0.006
0.017
0.016
0.016
0.025
0.006
0.015
0.0014
0.009
1962-67
St. Lawrence R. at Massena,
N.Y.
0.112 1962-67
(max. cone.)
Copper
Weathering of rock
Corrosive action ot
water in copper pipes,
industrial effluents
such as discharged
from textile mills,
pigmentation, tanning,
photography, engraving,
electroplating, in-
secticides, fungicides
and in many other in-
dustrial processes
Northeast Basin
North Atlantic Basin
southeast Basin
Tennessee R- Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R, Basin
western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado K. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
Mean Positive:
0.015
0.017
0.01U
0.011
0.023
0.011
0.01U
0.007
0.017
0.019
0.010
0.011
0.009
0.012
0.012
0.009
1962-67
Cobalt
Weathering of rock
Industrial effluents
from plants usinq
cobalt such as metal-
lurgical firms, cer-
amics and glass manu-
Northeast Basin
North Atlantic Basin
southeast Basin
Ohio R. Basin
Lake Erie Basin
facturintj, the tungsten Upper Mississippi R. Basin
carbide tool industry Western Great Lakes Basin
and others Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Pacific Northwest Basin
Allegheny R. at Pittsburqh, Pa.
Mean Positive:
0.01t
0.009
0.001
0.019
0.033
0.018
0.011
0.008
C.036
0.011
0.008
O.OU8
(max. cone.)
1962-67
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10
Agent Natural
Man-Made
in U.:;. .-.a'-ers
hasin Areas
Concent r if i or.:-
Fluoride Weathering of rock
containing fluoride
Industrial effluents
from plants usinq
fluorides in the
production ot steel,
disinfectants, pro-
served wood arid
muci]a<.jes, cjldSE and
enamels, chemicals
It is aiiued i-o many
public water supplies
to reduce incidoi.ce
of cavitios in t.et-th.
0. 1
o. u
surface waters of
Drinkinq water of 1t>3
atfas in
HocKy Creek an^i Ejst
Gallatin R. (various
locations alnnq rivers
f rciii sources ot pol I'.jric
Colorado R. Basin (;nax at
K. iielow Littlef ield , Ariz.)
Pacific Slope Basins in Calif.
(max. at colusa Trough riear
Colusu, cilit.)
Great Basin excluding c;redt
SalV Lake (max. at !!umbolii-
V. near Kye patch, "Jev. )
Paciric Slope Basiro in
w.isnincjton and Upne.r
Coluirhia R. Basin (max.
at OruL Creek near Smyrna,
kashi nqton)
Pacific Slope Basins ir. Ore.
anri Lower Columbia 'A. Basin
(max at V>dllii Walla K.
near Touchet, Wash.)
Snake R. Basin (max. at J5nake
P. at Kinq Hill, Idaho)
Alaska (max. at Tonsina K. at
Tonsina, Alaska)
North Atlantic Slope Oasins
(max. at Wissachickon cre°k
at Fort Washington, Pa.)
South Atlantic Slope* Basins
(max. at Alafia R. at Lithia,
Kla.)
Ohio R. Basin (max. at Tus-
carawas R. at Newcorrerstcwn,
Ohio)
-St. Lawrence K. Bacin (max
at Black R. =it Elyria,
Onio)
Cct. TJf. 1
U.a-U.9
J.1-0.1
). 1-0.7
o.o-i. a
0. 0-17-0
C.O-U. 2 1964-t>S
0.0-1.7
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11
Sources
Agent
Natural
Concentrat ions
in U.S. Waters
BdSin Areas
Concentrations (mq/1)
Missouri R. Basin (nax at
Cedar Creek near Columbia,
Mo.
Hudson P. and Upper Missis-
sippi R. Basins (max at
Souris H. near Verendrye,
N. Dak.)
Lower Mississipfi R. Basin
(max at Center Creek at
Oronooo, Mo.)
Western Gulf of Mexico Basins
(max at Double Mountain Fork
Brazos R. at Justiceburq,
Tex.)
Illinois surtace waters
at Illinois R. at
Peoria-1961)
(max
0.0-3.U 1964-65
0.0-1.2
0.0-15.0
0.0-2.1
0.0-2.U 1956-66
Iron
ot iron
salts from soil and
rock into both
qround and surface
waters
Aciu mine dr.iinaqe,
the effluents from
the many industries
usinq iron in their
processing, and the
oxidation ot iron used
in such items as
ships, automobiles,
refriqerators and
many others
Northeast Easin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
Sabine R. near Ruliff, Tex.
Mean Positive:
0.051
0. 019
0. 120
.037
.028
.035
.035
.022
,037
.069
0.010
0. 173
0.032
O.OU6
0.070
0.025
0.952
(max. cone.)
1962-67
-------
sources
Concentrat ions
in U.S. Waters
Agent
Lead
Natural
Weathering ot lime-
stone and galena
Man-Made
Corrosion ot leau
in pipes and near
industrial outf
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13
Sources
Concentrat ions
in U.S. Waters
Agent
Nitrate
Natural
tecal nidtoridl
Effluents trcm plants
producing cl;emical
fertilizer. Applica-
tion or chemical
fertilizers on agri-
cultural l.inds that
are then loached into
the surface ami perco-
latinq ground waters.
Application of ferti-
lizers on residential
lawns and gardens.
Municipal treatment
nlant effluent
Easin Areas
Concentrations (roq/1)
nurtace waters of 98 U.S. rivers
Surface waters of U.S.
Range: 0.1-10.1
0.5
3.2
1.1
Missouri R. water
in Omaha, Neb.
in Kansas City, Mo.
in St. touis County, Mo.
in St. Louis, Ho.
Hudson R.
Iowa
Ohio, 10» of municipal water supply
Mahoninq R.
Ohio rural water supplies
Drinkinq water in 163 metropolitan
areas in U.S.
Missouri wells
Lake Erie
Colorado R. basin (max. at Gila F.
below Gillespie Dam, Ariz.)
Pacific Slope Basins in Calif, (max.
at Salinas R. near Sprechles, Cal.)
Great Basin excluding Great Salt take
(max. at Jordan R. at Salt Lake
City, Utah)
Pacific Slope Basins in Washington
and Upper Columbia R. Basin (max.
at Flett Creek at Tacoma, Hash.)
Pacific Slope Basins in Oregon and
Lower Columbia K. Basin (max. at
V»alla walla R. near Touchet, Vlash.)
1960-
Aug. 19
Sept 19
Oct 19
19
1951-
0.2-8,6 monthly
1.8
1.C-9.0 "
2.1-11.3 "
0.2-1.0
10.0-100.0
1.0
10
10.0-100.0
Average: 2.3
•5-300
1.0
0.00-60.0
0.0-38.0
0.0-17.0
0.0-12.0
0.0-4.2
19
19
July 19
March 19
19
1950-
-------
14
Sources
Agent
Natural
Man-Made
Concentrations
in U.S. Waters
Basin Areas
Concentrations (mq/1)
Snake R. Basin (max. at Palouse R. 0.0-11*.0
at Hooper, Wash.)
Alaska (max. at Trail R. near Lowing, 0.0-2.8
Alaska
North Atlantic Slope Basins (max. at 0-0-62.0
Rockaway R. at Pine Brook, N.J.)
South Atlantic Slope and Eastern Gulf 0.0-620.0
of Mexico Basins (max. at Planta-
tion Rd. Canal near Fort
Lauderdale, Fla.)
Ohio R. Basin (max. at Great Miami 0.0-23.0
R. at Elizabetbtown, Ohio)
St.. Lawrence R. Basin (max. at 0.2-113.0
Black R. at Elyria, Ohio)
Missouri R. Basin (max. at Horse 0.0-77.0
Creek near Vale, S- Dak.)
Hudson R. and Upper Mississippi R. 0.0-302.0
Basins (max. at Blue Earth R. near
Rapidan, Minn.)
Lower Mississippi R. Basin (max. at 0.0-302.0
Fountain Creek at Pueblc, Colo.)
Western Gulf of Mexico Basins (max. 0-0-5U.O
at Yegua Creek near Somerville,
Tex.)
Illinois surface waters (max. at 0.0-«8.i»
Illinois R. Meredosia-1962)
Lehiqh R. Easin (max. at Black Creek 0.0-23.0
near Weatherly-1963)
196U-
1964-
1956-6
1945-6
-------
15
Sources
Aqent
Nickel
Natural
Man-Made
Concentrat ions
in U.S. Waters
Basin Areas
New Jersey surface waters
1) spring flow
2) summer flow
New Jersey surface waters
Big Flat Brook
Calcareous - site 1
Acidic - Site 2
Lower Mississippi R. Basin:
Californiaa Gulch and
Arkansas H. at Malta, Colo.
(max. at California Gulch)
Concentrations (mq/1)
0.002-0.009
0.001-0.017
0.006
0.001
0.0-0.069
1968
196«-65
Phosphorus
The decomposition of
orqanic matter
Siltation. Applied
fertilizer. Effluents
of plants makinq
fertilizers, rr.atciies
armaments, orqanic
chemicals, ortho-
phosphoric acid, foo.i
supplement in animal
foods, water soften-
ers, anu in metalurqy
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R, Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missour R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California
Great Basin
Alaska Basin
Mean Positive:
O.OU4
O.OU8
O.OU3
0.042
0. 130
0. 153
0.2U3
0.031
0. 353
0.081
0. 121
0, 173
O.OU7
0.083
0.037
O.OUO
1962-67
-------
16
Sources
Agent
Natural
Man-Made
Concentrations
in U.S. Waters
Basin Areas
Concentration:.
Manganese
Leached from rock
into groundwaters
Mining effluents from
from industries using
manganese in the
production of steel
alloys, dry cell bat-
teries, qlass and
ceramics, paints
and varnish, inks,
matches, fireworks
and in agriculture
to nourish manganese
deficient soils
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska
Mean Positive:
0.0035 1962-67
0.0027
0.0028
0.0037
0. 232
0. 138
0.0098
0.0023
0. 0138
0. 009
0.012
0.010
0.0028
0.0028
0.078
0.0018
Mercury
Leaching of rock
and volatilization
Effluents to air
emissions from chlor-
alkali plants, ap-
plications of pesti-
cides, fuel burning,
catalytic processes,
ore refining, sewage
treatment, waste
incineration, phosphate
rock, processing, paint
manufacture, and use and
breakage of mercury-
containing devices, and
many others
Ranqe of Hg found in all of
the States in Oct 6 Nov. 1970
0.5 ug/1-6.9 ug/1
Molybdenum
Mineral weathering
Mining and processing
molybdenum ore.
Effluents ot indus-
tries using molybdenum
in their operation
sucli as metallurgical,
glass, ceramics,
pigment producr.ion,
fertilizers and
other
Northeast Basin
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Pasin
Lower Mississippi R. Basin
Colorado R, Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Great Basin
Alaska Basin
Mean Positive:
0.025
0.033
0.015
0.025
0.070
0. 068
0. 088
0. 028
0. 083
0. 095
0. 130
0. 02«
0. 030
0. PU5
0. 1U5
0. 017
1962-67
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17
Sources
Agent
Natural
Man-Made
Concentrat ions
in U.S. Haters
basin Areas
Concentrations (mrj/l)
Selenium
Traces in ettltients
from plants usinq
silver to produce
sucn items as pig-
mentation tor paints,
dyes, glass, com-
ponents in rectifiers,
semi-conductors
Selected drinking water supplies in:
Connecticut 0.010
Maine 0.010
New Hampshire 0.010
Vermont 0.010
Delaware 0.010
New York 0.010
Pennsylvania 0.010
Kentucky 0.010
Maryland 0.010
North Carolina 0.010
Virginia 0.010
W. Virginia 0.010
Alabama 0.010
Florida 0.010
Georgia 0.010
Silver weathering of rocks
Trace amounts in
effluents trom in-
industrial plants
using silver such as
electroplating plants,
food and beverage
plants, photographic
chemicals producers,
ink manutacturing,
antiseptics for
medicinal purposes
Northeast Easin
North Atlantic Pasin
Southeast Basin
Ohio R. Basin
Lake Erie Basin
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R, Basin
Pacific Northwest Basin
Great Basin
Alaska Basin
Mean Positive:
0.0019 1962-67
0.0009
O.OOOU
0.0021
0.0053
0.003U
0.001U
0.0012
O.OOU3
0.0058
0.0009
0.0003
0.0011
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18
Sources
Agent:
Natural
Man-Made
Concent.r jt ions
in U.S. Waters
E'asin Areas
Concentrations
Uranluai Weathering of rack
Miniricj and proces-
sing of uranium in the
manufacture of atomic
weapons, the produc-
tion of radiojsotopes
in piles and reactors,
use of radioisotopes
in medical therapy,
scientific research
photography, and in
many chemical proces-
ses
Walkt-r K. Cilit.
Weber S., Utan'
Cottonwood Creek., Utah
Creek, Idaho
lllyt- Mountain, Calif.
Hell-Arches Naticnai Monunent, Utah
Slot Sprinq-Pyramid Lake, Nevada
Truchee R. , Nevada
Walker H., Nevada
Humi;olt R,, Nevada
Carson H., Nevada
Sevier R., Utah
Lake Tahoe, Calif.
Pyramid lake, Nevada (South end)
Pyramid Lake, Nevada (North end)
Walker Lake
Mono Lake
Principal rivers of U.S.
North Atlantic slope Basins (3dx at
Susquehanna R. at Harrisburq, Pa.
and Potomac R. at Hancock, Md.)
St. Lawrence R. Basin
Genessee R. at Rochester, N. Y.
South Atlantic Slope Basins (max at
Yadkin K. at Yadkin college, N.C.
and St. Johns R. at Christmas, Fla)
Gulf of Mexico Basin (max at
Missouri R. at Nebraska City, Neh.)
Colcrado R. at Yuma, Ariz.
Pacific Coast Basins (max at
San Joaquin H. at Vernalis, Calif.)
Yukon R. at Rampart, Alaska
Red R. (N) at Grand Forks, N.D.
n.0009
0.0013
0. 00?
0.0003
0. Ci, OOU
0.OC^C
o.oooos
0.0046
0.016
O.G072
O.GOU7
0.0081
0.0008
0 . 0 U.
O.C25
0.077
0. 139
none detected 1V5H-59
0.0004-0.0006 1960-61
G.OOOtt
19*0-61
0.000<»-0.0007 1960-61
0.0001-0.0006 1960-61
0.007';,
0.0004-0.008
o.oom
O.C01U
-------
19
Sources
Agent
Natural
Man-Made
Concentrat ions
in U.S. Waters
Basin Areas
Concentrations (mg/1)
Vanadium weathering of rock
Leachinq of soils.
Degradation of plants
and animals. Effluents
from plants making
steel, glass, mordant
used in dyeing and
printing fabrics,
and other products
Northeast Basin'
North Atlantic Basin
Southeast Basin
Tennessee R. Basin
Lake Erie Basin
Upper Mississippi R. Easin
Missouri R. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacific Northwest Basin
California Basin
Alaska Basin
Mean Positive:
0.009
O.U12
0.010
0.022
0.05'i
0.020
0. 171
0.025
0. 10?
0.009
0.013
0.030
0.012
1962-67
Zinc
Weathering of rock
Mining and processing
of ore. Effluents
from plants making
Pharmaceuticals, dyes,
insecticides, and many
other products
Northeast Basin
North Atlantic Dasin
Southeast Basin
Tennessee R. Basin
Ohio R. fiasin
La k e Er i e
Upper Mississippi R. Basin
Western Great Lakes Basin
Missouri P. Basin
Lower Mississippi R. Basin
Colorado R. Basin
Western Gulf Basin
Pacitic Northwest Easin
California Basin
Great Basin
Alaska Basin
Mean Positive:
0.09f>
0. OU9
0.052
0.028
0. 081
C.205
0.045
O.C2"
0.039
0.085
O.OM
0.092
C.CUO
0.016
19b2-67
C.02fi
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20
IV. Measurement and Classification of Waters 304 (a)(2)(C)
A. Measurement Techniques
Methods included within the following section cover a
variety of menstirenents and techniques. For some
pollutants, several methods are cited for measurement or
detection. Each method is summarized and presents a
synopsis and is not intended to give a detailed
presentation. Rather an outline of the technique is
followed immediately by the pertinent references for an
extensive review of the procedure.
The techniques are divided into three areas;
physical-chemical, biological, and bioassays. It is not
within the scope of this document to detail precisely or to
recommend a specific method. Often circumstances such as
sources and mixtures of pollutants, cost of test, and skill
of the personnel performing the test will dictate the
preferred procedure.
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21
ACIDITY
Principal Detection Techniquet Titration
Summary of Method; Sample is titrated to a final pH of 8.3.
Results reported as mg of CaC03 per liter.
Limitations;
Interferences; Hot applicable to acid samples from mine
drainage
Statistical Characteristics; Over Observed Range of;
(By 40 analysts in 17
laboratories)
Accuracy; Bias Acidity, as mg/1 C
+2.77% 20
+0.52% 21
Precision: Standard Dev.
1.79 mg/1 CaCOy 20
1.73 21
Time of Measurement (Maximum frequency, recovery period,
etc.); Not stated, but fast.
Data Outputs; Analog signal displayed on meter.
Special Sampling Requirements (Collection, Storage,
Handling); Refrigerate sample at 4*C.
Maximum holding time is 24 hours
References;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Annual Book of ASTM Standards, part 23 (Method
designation D1067). 1970. Society for Testing and
Materials. Philadelphia, Pennsylvania.
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22
ALiCALIMIYY
Name; of 'loasuronent Method; Eloctronctric Titratiou
Principal Detection Technique; Eloctrometric
Purpose of Measurement (Important Applications); Uriakiny
waters,ambient surface waters,' domestic and industrial
wastes, and saline waters. - '
Summary of Method; Unaltered sample is titrated to an
electromotrically determined end point of pil 4.5. Sample
must not be filtered, diluted, concentrated, or otherwise
altered.
Limitations;
Range of Applicability; All concentrations
Interferences; Salts of weak acids, oils and greases
Pitfalls; Special Precautions; Analyze sanple as soon
as possible after collection, preferably within a few
hours.
Statistical Characteristics; Over Observed Range of;
Accuracy; Bias Alkalinity in rag/1 CaCU,
+22.29% 9
- 3.19% 113
Precision; Std. Dev. , nig/1 CaCQ^
1.4 9
5.28 113
Time of Measurement (-Maximum frequency, recovery period,
etc.);:Hot stated, but rapid.
Data Outputs; Meter (analog voltage)
Special Sampling Requirements (Collection, Storagef Handling^;
Refrigerate" at 4°C. Maximum holding time 24 hours.
-------
23
References;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center ,
Cincinnati, Ohio.
2. Annual Book of ASTM Standards, part 23 (Method
designation D1067). 1970. Society for Testing and
Materials. Philadelphia, Pennsylvania.
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24
Total Alkalinity
Name of Measurement Method; Methyl Orange
Principal Detection Technique; Titration with colorimetric
detection of end point.
Purpose of Measurement (Important Applications); Surface .
waters,saline waters.
Summary of Method; A Technicon Autoanalyzer is used,
employing methyl orange as indicator. This is dissolved in
a weak buffer at pH of 3.1, just below the equivalence
point. Addition of alkalinity causes loss of color
proportional. Color measurement made by colorimeter at
approximately 550 nm.
Limitations;
Range of Applicability; 10 to 200 mg/1 expressed as
Statistical Characteristics;
Precision; Std. dev. was ±0.5 mg/1 CaCOj (in one lab)
using concentrations of 15, 57, 154, and 193 mg/1
Time of Measurement (Maximum frequency, recovery; period,
etc/H 30 min. warm-up, about 1 min. per deterrnintataon
Calibration Requirements; Requires preparation of standard
curve of peak heights vs. concentration
Data Outputs; Strip recorder (analog voltage)
Special Sampling Requirements (Collection, Storage, Handling)
Refrigerated at 4*C; sample analyzed as soon as possible.
References;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Technicon Autoanalyzer Methodology. 1961.
Bulletin 1261.
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25
ARSENIC
Name of Measurement Method^ Diethyldithiocarbamate method
Principal Detection Technique; Colorinetry
Purpose of Measurement (Important Applications); Most fresh
water
Summary of Method; Arsenic in sample is reduced to arsine,
AsH5 , in acid solution. The arsine is scrubbed to remove
sulfide, and is absorbed in a solution of diethyldithio-
carbamate dissolved in pyridine. The red complex thus
formed is measured in a spectrophotometer at 535 nm.
Limitations:
Range of Applicability; At or above 10 ug/1 As. (If
arsenic is organically bound, consult Standard Methods,
13th Edition).
Interferences; High concentrations of chromium, cobalt,
copper, mercury, molybdenum, nickel, or silver.
Pitfalls; Special Precautions; Difficulties may be
encountered with certain industrial v/astes containing
volatile substances. High sulfur content of wastes may
exceed removal capacity of lead acetate scrubber.
Statistical Characteristics; In 46 laboratories:
Over Observed Range Of;
Accuracy; Relative error = 0% 40 ug/1
Precision; Relative Std. Dev. = 13.8 40 ug/1 as As
Data Outputs; Chart or meter (analog voltage)
References;
1. Methods for Chemical Analysis of Water and Wastes.
1971. UFA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D. C.
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26
BIOCHEMICAL OXYGEN DEMAND (BOD)
Name of Measurement Method; Modified Winkler with Full
Bottle Technique.
Principal Detection Technique; Titration
Purpose of Measurement (Important'Applications); Ambient
surface water, domestic and industrial waste waters
(especially sewage treatment plant effluent).
Summary of Method; Sample of waste, diluted as appropriate,
is incubated for 5 days in darkness at 20eC. The reduction
in dissolved oxygen concentration during this period yields
a measure of the biochemical oxygen demand.
Limitations; Sec summary for Dissolved Oxygen (Modified
Winkler Method).
Statistical Characteristics:
Precision; Over Observed Range Of;
Seventy-seven analysts in fifty- An unspecified range with
three labs analyzed samples of mean value of 194 mg/1 BOD
natural water plus an exact
increment of biodegradable com-
pounds. At a mean of 194 mg/1
BOD, the standard deviation was
+ 40 mg/1
Time of Measurement (Maximum Frequency, Recovery Period, etc.);
Five to six days per determination.
Comments by Users; Because of local conditions, types of
samples to be tested, and variabilities of the bioassay
procedures, no specific standard test for BOD has been
selected by EPA.
References:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American
Public Health Association, Washington, D. C.
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27
BIOCHEMICAL OXYGEN DEMA1JD (BOD)
Hame of Measurement Method; DO Probe
Principal Detection Teclinigue; Electronetric method.
Purpose of Measurement (Important Applications): See
summary of BOD determination using Modified Winkler Method.
Summary of Method: See BOD determination using Modified
Uinkler Method.
Limitations; (See Summary for Dissolved Oxygen (Probe
Method)).
Statistical Characteristics; See DO determination using
Modified Winkler Method.
Comments by Usors^ Because of local conditions, typos of
samples to be tested, and variabilities of the bioassay
procedures, no specific standard test for BOD has been
selected by EPA.
Data Outputs^ Electrical signal displayed on meter.
Special Sampling Requirements (Collection, Storage, Handling)
Refrigerate at 4W C. Maximum storage tine - G hourj.
References;
1. Methods for Chemical Analysis of !7atcr and Wastes.
1971. EPA National Lnvironmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Uater and
Uastcwater. 1971. 13th Edition. 7unerican
Public Health Association, Washington, D. C.
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28
ORGANIC CARBON (TOTAL A1JD DISSOLVED)
Principal Detection Technique; Infrared spectres copy
Purpose of Measurement (Important Applications); Ambient
surface waters,domestic and industrial wastes,saline
waters; used for assessing potential oxygen-demanding load
of organic matter.
Suramary j3 f Method; A micro sample of the wastewater to be
analyzed is injected into a catalytic combustion tube wnich
is enclosed by an electric furnace thermostated at 950 C.
The water is vaporised and the carbonaceous material is
oxidized to Carbon dioxide (CO^.) and steam in a carrier
stream of pure oxygen or air. The oxygen flow carries the
steam and COjr. out of the furnace where the steam is
condensed and the condensate removed. The CO-j., oxygen, and
remaining water vapor enter an infrared analyzer sensitized
to provide a measure of CO2. The amount of COX present is
directly proportional to the concentration of carbonaceous
material in the injected sample.
Limitations:
Range of Applicability; 1 to 140 mg/1 total carbon.
Lnterforonces; Carbonates, bicarbonates
Pitfalls; Special Precautions; Since sample is injected
into apparatus by syringe, the needle opening liuits the
size of particles reaching combustor/detector.
Statistical Characteristics:
Twenty-eight analysts in twenty-one laboratories analyzed
distilled water solutions containing exact concentrations of
oxidizable organic compounds, with the following results:
Known Carbon Precision as Accuracy as
Cone, as TOC, Standard Deviation ijias, Bias,
mg/liter TOC, mg/liter % mg/liter
4.9 3.93 +15.27 + .75
107 8.32 + 1.01 +1.0J
Time of "easureraent; Hot stated; assumed to be rapiu (several
samples per hour).
-------
29
Special Sampling Requirements (Collection, Storage, Handling);
None indicated. Maximum holding time - 7 days.
References:;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American
Public Health Association, Washington, D. C.
3. ASTfl Standards. 1970. Part 23, Water, Atmospheric
Analysis.
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30
CHLORIDE
Principal Detection Tochnic^ue; Titration
Purpose of "loasurcnent (Important Applications) ; Drinking
v;aters, ambient surface v/aters. Domestic and industrial
wastes, saline waters.
Summary of Method; Dilute mercuric nitrate solution is
added to an acidified sample in the presence of mixed
diphenylcarbazone - bromophenol blue indicator. The end
point of the titration is the formation of the blue-violet
diphenylcarbazone complex.
Limitations:
Ran geof Applieability: All concentration ranges of
chloride
Interferences; Sulfites. If presence is suspected,
oxidize with hydrogen peroxide.
Statistical Characteristics; By 42 analysts in 13 laboratories
Accuracy; Percent bias ranged Over Observed Range Of:
from 13.50 (at 10 rag/1) to 17, 18, 91, 97, 392, 398
1.10 (at 398 mg/1). mg/1
Precision; Standard Deviations Same
ranged from 1.32 mg/1 (at 13 mg/1
C1-) to 11.3 mg/1 (at 393 mg/1 Cl~)
Data Outputs; Visual observation and manual recording (or
may use automated titration, yielding an analog voltage
recorded on chart).
Special Sampling Requirements (Collection, Storage, Handling);
None indicated. riaximum holding time - 7 days.
References:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and Waste-
v;ater. 1971. 13th Edition, American Public
Health Association, Washington, D. C.
3. ASTM Standards. 1970. Part 23, Water, Atmospheric
Analysis.
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31
CIILORID^
Mane of Measurement Mothodj 7uitorated rcrrioynnic'e Method
Principal Detection Techniqiie: Titratior., usincr colorireter
Purpose of Measurement (Inportant Applications) :
wa t drs"~ dones't'i c~ aridl"Tndus'trla!" wa"ste"&r~~sallne" w
Ambient surface
waters.
S urinary of Method: Thiocyanate ion (f?C?T) is liberated fron nprcuric
tHi^>cyana'te~ th'ro'ucfh sequestration of rorcury by chloritV ion to forn
un- ionized mercuric chloride ion. In the presence of. ferric ion the
thiocyanate SC11 forris highly colored ferric thiocyanate in
concentration proportional to the original chloride concentration.
The deternination nay he perfomer1 autonaticallv usina a ^echnicon
AutoAnnlyzer cnployinq a colorineter with 380 nn filter for enclpoint
detection.
Limitations :
Ranqe_of_ ApplJ.cahiJ.^i^tyj 1 to 250 rg/1 chloride
^»f-3-f*:if:fJ_Chara5^cri£>J:icR: Over Observed Ranee Ofj
In a single laboratory:
PrecisJ-onj Standard deviation 1; 100; and 250 pa/1
+T)~'3~ncf71~ chloride chloride
-j Hen_svirenent: About 2
ninutesrpe'r sar,pl'e~'"v7ith 30 nin.
warn-up.
. AnalofT voltaqe, recorded on strip chart
§PP52-f J- ^J'l.'PlJ-I1^. Re3i! j-jr1*1^!1^5 (Collection, Storaae, Hand lino) :
Hone . Tlax"inum~Tic)17l'ln~r time" -"V clays"
References :
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. O'Brien, J.E. 1962. Automatic Analysis of
Chlorides in Sewaqe. Waste Engineer ina, 33:
670-672.
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32
CULORITir
Principa_l_ Detection Techniaue: Colorimetry or Electronetrio
DeTiernination
Prrnose of_ Measurement (Inportanjt Applications) : To determine the
anount of chlorine to T>e add'ed" to sample to acTiieve certain desired
results, such as control of colifom densitites, destruction of
certain chcnical compounds, or establishment of specified chlorine
residuals .
The nethod given here is applicable to drinkina water, surface waters,
donestic and industrial wastes, and saline water.
Sunmary of Method: A solution of known chlorine content is added
incrementally ^;o a series of sanple aliauots. At the end of the
stipulated contact tine or when the desired result has been achieved
the residual chlorine is measured by the appropriate nethod.
Limitations :
Range of Applicability: Not single, specific procedure, but
varies wTEn purpose" or" result to be achieved.
Pj.t_fa_lls_;_Rpecia2_ Precautions: T7hen the purpose is to obtain a
speci"fTe7]'"c'nl.orine~'resl7lual~~'the sane nethod of chlorine
neasurencnt should be used for operational control and laboratory
testing.
All pertinent infornation should lie included in report of results,
such as: conditions of chlorination (p!I, temperature, contact
tine) ; nethod used for detemining the desired result; and the
chlorine renuired to produce result.
Statistical Characteristics:
Accuracy_and_Precisipnj Mature of this test precludes the use of
accuracy an7i~precTslon~ statenents .
Hot given, huh assumed to be r.nalocr voltage displayed
o"n ne"ter~o""chart .
References :
1. Methods for Cher.ical Analysis of Hater and Wastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
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33
2. Standard Methods for Exanination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D. C..
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34
CHEMICAL ox-rnr::i DEMAND (COD)
Principal Pet ect ion^ Techn igue ; Titratior - (Iliqh Concentration)
Purpose of Measurement (^Important Applications) : To determine
quantity o"f~oxygen required" to" oxT7!ize~ aTI~~org an ic natter ir a
wastewater sample under specified conditions.
Sunnary of Method: Organic substances ir the sample are oxidized by
po"tass'iui\r "eTchromate in 50% sulfuric acid solution at reflux
temperature. Silver sulfate is used as a catalyst and mercuric
sulfate is added to remove chloride interference. The excess
dichromate is titrated v/ith standard ferrous annoniun sulfate, using
orthophenanthroline ferrous corplex as an indicator.
Limitations :
Ranqe of Applicability: Organic carbon concentration of 15 rng/1
"•-"••• ^" *f •" T •• m •• •• ^ ^^ •• ^•^•^^ » ™ * — •
or higher.
Interferences; Chloride concentration over 2000 ng/1
Statistical Characteristics :
Preci^io^i: Eighty-nine analysts: Standard deviation of +27.5
ng71~COD at knov/n value 270 ng/1 COD.
Special Sar.pl ing Rcguirenents^ ^Collection , Storaae , n.
Adcl "5" nT~ Kj SO^ per liter oT^sanple" MaxlnTirrT s'to'rage~lIne~-"
7 days.
References :
Methods for Chenical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D. C.
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35
CHEMICAL OXYGEN DEMAND (COD)
Principal Detection Technique^ Titration - (Low
Concentration)
Purpose of Measurement (Important Applications); Ambient
surface water,domestic and industrial wastes with low
oxygen demand characteristics.
Summary of Method; Similar to that for COD-High Level,
except (1)that extreme care is exercised during sample
acquisition and handling and during analysis to insure that
no organic contaminants are introduced from glassware,
atmosphere, etc., (2) that highly pure reagents are used,
and (3) that chlorides are removed by complexing with
mercuric sulfato.
Limitations:
Range of Applicability; 5 to 30 mg/1 COD
Interferences; Organic contaminants; chlorides
Pitfalls; Special Precautions; Volatile materials may
be lost during sulfuric acid addition step.
Calibration Requirements; Standardize reagents daily.
Special Sampling Requirements (Collection, Storage, Handling)
Use glass sample bottles. Preserve with IUSO«j. Test
biologically active samples soon after acquisition.
References:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D« C.
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36
CHEMICAL OXYGEN DEMAND (COD)
Principal Detection Technique; Titration - (Saline Waters)
Purpose of Measurement (Important Applications): COD
Determination in saline waters.
Summary of Method; Organic and oxidizable inorganic
substances in an aqueous sample are oxidized by potassium
dichromate solution in 50 percent (by volume) sulfuric acid
solution. The excess dichromate is titrated with standard*
ferrous ammonium sulfate using orthophenanthroline ferrous
complex (ferroin) as an indicator. Mercuric sulfate is
added to complex the chlorides during digestion.
Limitations:
Range of Applicability: Minimum of 250 mg/1 COD when
chloride concentration exceeds 1000 mg/1. (The removal
of chlorides by MgSq, may not be complete in the case of
strong brines.)
Interferences; Extraneous organic matter
Special Sampling Requirements (Collection, Storage, Handling);
Tf
Use glass bottles if possible; preserve with
Reference:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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37
COLOR
Principal Detection^ Technigue_t Visual sensing
Purpose of Measur ement ( Import an t_ Appl icat ions ) : Measures color of
water derlveef f rom naturally occurring^ihaterials , e.g. vegetable
residues such as leaves, bark, humus, etc.
Summary of Method; Color is measured by visual comparison of the
sample with" platinum-cobalt standards. One unit of color is that
produced by 1 mg/1 platinum in the form of the chloroplatinate ion.
The Spectrophotometric and Tristimulus methods are useful for
detecting specific color problems. The usr of these methods, however,
is laborious and unless determination of the hue, purity, and
luminance is desired, they are of limited value.
Limi t.at_ions_:
Interferences! Turbidity; highly colored industrial wastes.
?itlfiisi. Special S-?~-u--°"-s Biological activity may change
color" characteristics a? ter" sample is acquired.
C a 1 Ibr a ti pn_ Regu j.r erne n ts j See Reference
Data Outputs^ Usually a visual observation, manually recorded.
Special Sampling Reguirements (Collection. Storage, Handling) :
Store sample atT*C. Maxlmum"Tiold"ing time -~^3" hours"! """"
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D. C.
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38
CYANIDE
Principal Detection Technique; Colorinetric
Purpose of Measurement (Important Applications); Ambient
surface water,domestic and industrial wastes, saline
waters.
Summary of Method; The cyanide as hydrocyanic acid (HCIJ) is
released from metallic cyanide complex ions by means of a
reflux-distillation operation and absorbed in a scrubber
containing sodium hydroxide solution. The cyanide ion in
the absorbing solution is then determined by volumetric
titration or coloriraetrically. The colorirnetric measurement
employs the pyridine-oyraxolone reaction in which the
cyanide is coupled with free chlorine to form cyanogen
chloride and then with the pyridine to a glutaconic
aldehyde. The aldehyde then reacts with l-paenyl-3-iaethyl-
5-pyraxolone to form a highly colored blue dye.
Limitations:
Range of Applicability; Cyanide concentrations below 1
mg/1(sensitiveto about 0.5 ug/1).
Interferences; Sulfides, oxidizing substances.
Pitfalls; Special Precautions; Material tested may be
highly toxic and should be treated accordingly. Sample
should be maintained at a highly basic pH.
Statistical Characteristics:
Determination by 47 analysts yielded:
Precision: Standard Deviation Known CM Concentration of;
0.020 mg/1 0.02 rag/1
0.306 mg/1 1.10 mg/1
Data Outputs; Analog signal (meter)
Special Sampling Requirements (Collection, Storage, Handling);
minimum sample size - 1 liter
adjust pH to 11 at time of sample collection
(using sodium hydroxide)
analyze as soon as possible after collection
(maximum holding time - 24 hours)
store at 4°c.
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39
References;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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40
CYAi:ii)E
Principal Detection Technique: Titration
Purpose of ileasuror.ient (Important Applications); Ambient
surface water,domestic and industrial wastes,Saline
waters.
Summary of Ilethod; The cyanide as hydrocyanic aciu (IICII) is
relt.asod from metallic cyanide complex ions by means of a
reflux-distillation operation and absorbed in a scrubber
containing sodium hydroxide solution. Tne cyanide ion in
tho absorbing solution is then determined by volumetric
titration or colorirnetrically. Tho titrinetric ineasurenent
uses a standard solution of silver nitrato to titrate
cyanide in the presence of a silver sensitive indicator.
Limitations:
Range of Anplicability; Concentrations of cyanide
exceeding 1 mg/1
Interferences; Sulfidas, oxidizing substances
Pitfalls; Special Precautions; See summary for Cyaniuo
Colorimetric
Statistical Characteristics:
Results by 47 analysts:
Precision: Standard Deviation Know;: CIJ Concentration of
0.035 mg/1 0.02 mg/1
0.333 mg/1 1.10 nig/1
Data Outputs; "lot stated. (Probably visual reading,
recorded manually).
Special Sampling Requirements (Collection Storage, Handling);
See summary for Cyanide, Colorimetric.
Reference:
1. Methods for Chemical /Analysis of Water and Wastes.
1971. EPA national Environmental Research Center,
Cincinnati/ Ohio.
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41
DISSOLVED OXYGKM (1)0)
Uarte of Measurement Method! Modified Winkler - Full Bottle
Technique
Principal Detection Technique; Titration
Purpose of Measurement (Important Applications); Most
ambient surface waters,most waste waters(but not donestic
sewage).
Summary of Method; The sample is treated with manqanous
sulfate, potassiun hydroxide and potassium iodide and
finally sulfuric acid. The initial precipitate of manganous
hydroxide combines with the dissolved oxygon in the sample
to form a brown precipitate, manganic hydroxide. Upon
acidification, the manganic hydroxide forms manganic sulfate
whicn acts as an oxidising agent to release free iodine from
the potassium iodide. The iodine, which is
stoiciiiometrically equivalent to tiie dissolved oxygen in the
sample is then titrated.
Limitations:
Interferences; Oxidizing or reducing materials
(especially sulfites, thiosulfates, polythionates,
chlorine, hypochlorite) nitrate ions, ferrous ions,
organic matter, high concentrations of suspended solids.
Pitfalls; Special Precautions; Sample may contain low
concentrations of ferrous ions (less than 1 mg/1) and
nitrates. High concentration of either interferes.
Statistical Characteristics; Exact data not available, the
following are approximate.
Precision; Reproducibility of 0.2 rag/1 of DO when known
concentration is 7.5 rag/1 DO.
Time of Measurement; Not stated, but a complex
procedure.
Commonts by Users; Most common interferences overcome by use
of DO probe.
Data Outputs; Visual observation or analog signal.
Special Sampling Requirements (Collection, Storage, Handling);
Grab sampling acquisition procedures are specified;also
preservative reagent to be added. Analysis should be completed
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42
within 4-8 hours after acquisition of sample.
Reference^
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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43
DISSOLVED OXYGEN (DO)
Name of Measurement Method; Probe Method
Principal Detection Technique; Electrometric
Purpose of .Measurement (Important Applications); Ambient
surface waters, domestic and industrial wastes.
Summary of Method; The most common instrumental probes for
determination of dissolved oxygen in water arc dependent
upon electrochemical reactions. Under steady-state
conditions, the current or potential can be correlated with
DO concentrations. Interfacial dynamics at the probe-sample
interface are a factor in probe response and a significant
degree of interfacial turbulence is necessary. For
precision performance, turbulence should be constant. The
probe method may be used under any circumstances as a
substitute for the modified Winkler procedure provided that
the probe itself is standardized against the Winkler method
on samples free of interfacing materials. The electronic
readout meter for the output from dissolved oxygen probes is
normally calibrated in convenient scale (0 to 10, 0 to 15, 0
to 20 rag/1, for example) with a sensitivity of approximately
0.05 mg/1.
Limitations:
Interferences; Sulfur compounds and certain reactive
gases(e.g., chlorine) may interfere. Dissolved
inorganic salts affect performance, but usually can be
compensated for. pH variation interferences with some
probes.
Pitfalls; Special Precautions; Probes may be sensitive
to temperature and may require temperature compensation.
Statistaical Characteristics; Manufacturers' Claims:
Accuracy; j^ 1% of true DO value
Precision; 0.1 mg/1 repeatability
Time of Measurement; Mot stated, but rapid.
Calibration Requirements; Not stated. (Should be
calibrated against standards with approximately the same
concentration of suspended solids as the sample).
Data Outputs; Electrical signal displayed on moter
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44
Special Sampling Requirements (Collection, Storage, Handling)
Hot stated. Can provide continuous, in situ measurements.
Reference:
1. Ilethods for Chemical Analysis of Water and Wastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
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45
FLUORIDE
Maine of Measurement Method; Automated Complexone Method
Principal Detection Technique; Photometric, automated
(Teclmicon AutoAnalyzer)
Purpose of Measurement (Important Applications); Ambient
surface water,domestic ana industrial wastes, saline waters.
Sunraary of Ilethod; Fluoride reacts with red cerous chelate
of alizarin complexone. A positive color is developed and
intensity is measured by a colorineter at about 650 nn.
Teclmicon AutoAnalyzer is used.
Limitations:
Range of Applicability; 0.05 to 1.5 rog/1 fluoride
Interferences; Aluminum
Statistical Characteristics;
Precision; At one laboratory, standard deviation of
measurement was 0.018 mg/1 F when actual concentrations
were 0.06, 0.15, 0.55, and 1.03 mg/1.
Tine of Measurement; 12 samples per hour after 30
minutes wanning.
Data Outputs; Analog electrical signal recorded on
stripchart.
Special Sampling Requirements (Collection, Storage, Handling)
Not stated. Can provide continuous, in situ measurements.
Reference:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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46
HARDNESS
Principal Detection Technique; Titrntion
Purpose of Measurement (Important Applications); Drinking
waters, ambient surface waters, domestic and industrial
wastes.
Summary of Method; Calcium and magnesium ions in the sample
are sequestered upon the addition of disodium dihydrogen
ethylenediamine tetraacetate (Lla-EDTA) . The end point of
the reaction is detected by means of Chrome Black T or
Calmagite, which has a red color in the presence of calcium
and magnesium and a blue color when the cations are
sequestered.
Limitations;
Range of Applicability; All concentrations of hardness.
Interferences; Excess concentrations of heavy metals
(may be removed by conplexing with cyanide)
Statistical Characteristics; 43 analysts in 17 laboratories
obtained the following results:
Precision Over Known Range of
Accuracy (Bias) (Std. Dev.) Hardness as CaCQj
-0.003 mg/1 (as 2.37 mg/1 31 mg/1
CaCOj )
-0.24 2.52 33
0.4 4.87 132
-2.0 2.98 194
-13.0 9.65 417
-14.3 3.73 444
Data Outputs; Not stated. Assume visual observation,
manual recording.
Special Sampling Iteguir orients (Collection, Storage, Handling)
Hone. riaximum holding "time, 7 days.
References;
1. Methods for Chemical Analysis of Water and VJastes.
1971. EPA National Environmental Research Center t
Cincinnati, Ohio.
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47
TOTAL HARDNESS
Name of Measurement Method; Automated Method
Principal Detection Technique; Colorinetry
Purpose of Measurenent (Important Applications); Surface
waters and saline waters
Summary of Method; The disodium magnesium EDTA exchanges
magnesium on an equivalent basis for any calcium and/or
other cations to form a more stable EDTA chelate than
magnesium. The free magnesium reacts with calmagite at a pli
of 10 to give a rod-violet complex. Thus, by measuring only
magnesium concentration in the final reaction stream, an
accurate measurement of total hardness is possible.
Method assumes the use of a Technicon AutoAnalyzer.
Colorimeter is equipped with 520 nru filter.
Limitations:
Ranges of Applicability; 10 to 400 mg/1 expressed as
CaC03
Statistical Characteristics:
Precision; Results from a single laboratory:
Gtd. Dev. Known Concentration
+ 1.5 mg/1 19 ing/1 CaCO..
+ 1.5 120 ^
+ 4.5 385
+ 5.0 366
Time of Measurement;
12 determinations per hour. 30 minutes warm up.
Calibration Requirements; Apparatus must be calibrated against
stock solutions of known concentration.
Data Outputs; Analog electrical signal, recorded on stripchart,
References;
1. Ilethods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Technicon AutoAnalyzer Methodology. 1960. Bulletin No. 2,
Channcey, New York,
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48
METALS
Name of Measurement Method; Atomic Absorption
Parameter(s) Measured; Metals
Aluminum Copper Potassium
Arsenic Iron Silver
Cadmium Lead Sodium
Calcium Magnesium Zinc
Chromium Manganese
Principal Detection Technique; Atomic Absorption
Spectrescopy.
Purpose of Measurement (Important Applications); Ra^id
determination of certain metals in ambient surface waters,
domestic and industrial water, and saline waters.
Summary of Method; Atonic absorption o^ectroocopy is
similar to flame emission photometry in that a sample is
atomized and aspirated into a flame. Flame photometry,
however, measures the amount of light emitted, whereas, in
atomic absorption spectrophotometry, a light beam is
directed through the flame into a monochromator, and onto a
detector that measures the amount of light absorbed. In
many instances absorption is more sensitive because it
depends upon the presence of free, unexcited atoms and
generally the ratio of unexcited to excited atoms at a given
moment is very high. Since the wavelength of the light beam
is characteristic of only the metal being determined, the
light energy absorbed by the flame is a measure of the
concentration of that metal in the sample. This principle
is the basis of atomic absorption spectroscopy.
Analytical procedures specific to each of the metals listed
above are given in the Reference cited.
Limitations:
Range of Applicability; Detection limits,
sensitivities, and optimum ranges of concentrations,
vary with make and model of atomic absorption
spectrometer. The following table provides some
indication of ranges of measurement. In many cases the
range can be extended higher or lower by instrument
adjustment, use of a different wavelength, or sample
pretreatment.
-------
.Metal
Aluninui.i
/vrsenic
Cadmiuu
Calciun
C'nroiniun
Copper
Iron
Lead
"lagnesiun
'langaneso
Potassium
Silver
Sodium
Zinc
Detection
Li nit
(IKJ/1)
0,1
0. 05
0.001
0.003
0. 01
0.005
0.004
0.01
0.0005
0.005
0.005
0.01
0.001
0. 005
r>enr;itivi ty
(no/D
0.4
1.0
0.00-1
0.07
0.02
0.04
0.006
0.06
0.005
0.04
0.01
0.05
0.003
0.02
G.-.>tii..ui.i Concentration
rinnge
(nuj/1)
Accuracy anc1 Precision Data:
Hctal
'lotal Concentration
(ug/1)
Direct Jetornination
Cadriiun
Chroniuu
Co-ver
Iron
May no si urn
.'lanijanese
Silver
Zinc
50
50
1000
300
200
50
50
500
Rolativo
Error
(Percant)
3.15
2.29
3.42
0.6-1
£.30
6.00
10.57
0.41
10
10
0.1
1
1
0.1
0.1
1
0.01 -
0.1
0.01 -
0.1 -
1
0.1
1000
100
2
200
200
10
20
10
2
20
2
20
200
2
Relative
Standard Deviation
(Percent)
21.62
26. 44
11.23
16.53
10.49
13.50
17.47
3.15
Extracted Gannlos
Cr.dniurn
Load
10
50
3.03
ID.00
72.77
23.46
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50
Time of Measurements Rapid
Calibration Requirononts; See Reference
Data Outputs; Electrical signals
Special Sampling Requirements (Collection! Storage, Handling)
See Reference
References:
1. iletliods for Chemical Analysis of Water and Wastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
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51
MERCURY
Marae o£ Measurement. Method; Flaraeless Atomic Absorption
Principal Detection Technique; Atonic Absorption
Spectroscopy
Purpose of Measurement (Important Applications); Ambient
surface waters, saline waters, wastewaters, and effluents.
Hay also be used for fish tissue, mud, sediments, and other
materials following proper digestion.
Summary of Method; The flameless AA procedure is a physical
method based on the absorption of radiation at 253.7 nm by
mercury vapor. The mercury is reduced to the elemental
state and aerated from solution in a closed system. The
mercury vapor passes through a cell positioned in the light
path of an atomic absorption spectrophotometer. Absorbance
(peak height) is measured as a function of mercury
concentration and recorded on a stripchart.
Limitations:
Range of Applicability; Detection limit is 0.2 ug/1
mercury
Interferences; Sulfides, copper, high concentration of
chlorides, and certain volatile organics
Statistical Characteristics; Using an Ohio River composite
sample with a background mercury concentration of 0.35 ug/1,
spiked with concentrations of 1, 3, and 4 ug/1, the standard
deviations were ±0.14, +0.10 and +0.03 ug/1 respectively.
Standard deviation at tKe 0.35 level was 0.16. Percent
recoveries at the three levels were 89, 87, and 87%
respectively.
Calibration Requirements; Detailed calibration instructions
i
meluued in Reference below.
Data Outputs; Analog electrical signal, displayed on
stripchart.
Special Sampling Requirements (Collection, Storage, Handling)
Acidify sample to pll of 2 or lower.
Reference;
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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52
MERCURY
Name of Measurement Method; Cold Vapor Technique -
(Biological Materials)
Medium; Fish tissue (and other biological materials)
Principal Detection Technique; Atonic absorption
spectroscopy
Purpose of Measurement (Important Applications); To measure
total mercury(organic and inorganic)in fish and in other
biological materials.
Summary of Method; Weighed portion of sample is digested
with sulfuric and nitric acid and oxidized overnight with
potassium permanganate. Mercury content is measured by the
conventional cold vapor (Flaineless AA) technique summarized
in Method Summary for Mercury in Water (this compendium) and
described in References (1) and (2) of that Summary.
Limitations:
Range of Applicability; 0.2 to 5 ug/g. (May be
extended by varying sample size or through instrument
control).
Interferences; Most interferences are destroyed during
digestion and oxidation steps.
Pitfalls; Special Precautions; See "Sampling
Requi renents" below.
Statistical Characteristics:
Precision; The following standard deviations on
replicate fish samples were recorded at the indicated
levels: 0.19 ug/g+0.01; 0.74 ug/g+0.05; and 2.1
ug/g+0.07. The coefficients of variation at these
leveTs were 11.9 percent, 7 percent, and 3.6 percent
respectively.
Time of Measurement; Several hours.
Data Outputs; Electrical signals displayed or recorded,
Special Sampling Requirements (Collection, Storage, Handling):
Mercury is not uniformly distributed throughout the whole
fish and it is therefore necessary to decide in advance which
part of the fish is to bo analyzed. In the case of a large
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53
specimen, only a selected part of the fish may be examined.
(Ordinarily, only the edible flesh is analyzed). In any
event, the portion analyzed should be reported.
If it becomes necessary to freeze the fish before analysis,
the sample should not be allowed to thaw before weighing as
higher results may be observed.
References:
Mercury in Fish. 1972. Method Description received
from Analytical Quality Control Laboratory, EPA
national Environmental Research Center, Cincinnati,
Ohio.
lithe, J.F, , F.A.J. Armstrong and M.P. Stain ton*
Mercury Determination in Fish Samples by Wet
Diyestion and Flameless Atomic Absoprtion
Spectre-photometry. Jour. Fisheries Research
Board of Canada, 27:805.
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54
MERCURY
Name of Measurement Method; Gas Chronatography (In Fish)
Medium; Fish Tissue
Purpose of Measurement (Important Applications); To
determine concentration of mo thy 1 mercury in fish samples of
all typos.
Gur.unary of Method; ' A measured weight of fish is treated
with acid and bromide salt. Methyl mercury is extracted as
methyl mercury bromide with toluene. A cleanup operation is
performed by first extracting the toluene layer with aqueous
solution of sodium thiosulfate. The methyl mercury
thiosulfate complex formed in the aqueous layer is then
reacted with an excess of potassium iodide. Denzone is used
to extract any methyl mercury as the iodide salt. A portion
of the benzene extract is chromatographed directly and
response is compared to standard responses. The iodide is
detected using gas chromatography with an electron capture
detector.
Limitations:
Sensitivity; 0.01 ug/g
Interferences; Materials in sample will generally not
cause a problem. Toluene from extraction not removed by
cleanup step may interfere.
Pitfalls; Special Precautions; Potassium iodide
solution decomposes readily.Any free iodine may cause
interfering peaks on chromatogram.
Statistical Characteristics:
/vccuracy and Precision; When seven portions of a filet
of a large white perch were analyzed by this method, a
mean value of 0.37 ug/g was obtained, with a standard
deviation of 0.034 ug/g.
Recoveries of methyl mercury chloride injected into this
same fish at the 0.20 ug/g level averaged 95.5 percent.
Time of Measurement; Approximately one hour.
Calibration Requirements; No unusual requirements.
Calibration procedure given in Reference.
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55
Data Outputs; Analog electrical signal recorded as
chromatogram,
Special Sampling Requirements (Collection, Storage/ Handling);
Analysis should be performed oit the fish before decay has begun.
Fresh or frozen fish can be analyzed with confidence; results
on partially decomposed samples must be viewed with caution and
reported as such.
Fish samples can dehydrate rapidly unless protected during
handling. Defrosting and refreezing a frozen sample before
analysis, and permitting a sample to stand open at room
temperature before weighing must be avoided.
Selection of portions of the fish must be of a consistent
nature and properly identified as to location on the specimen.
References:
1. Methyl flercury in Pish. 1972. Provision Method
Description received from Analytical Quality Control
Laboratory, EPA National Environmental Research
Center, Cincinnati, Ohio.
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56
MERCURY
Medium; Sediment
Name of Measurement Method: Gas Chromatography (In
Sediment)
Principal Detection Technique; Electron capture gas
chromatography
Purpose of Measurement (Important Applications).; This
method is applicable to bottom samples such as mud, sludge,
silt and gravel.
Summary of Method; A measured weight of sediment is treated
with acid and bromide salt. Methyl mercury is extracted
with toluene as methyl mercury bromide. A cleanup operation
is performed by first extracting the toluene layer with an
aqueous solution of sodium thiosulfate. Thu methyl mercury
thiosulfate complex formed in the aqueous layer is then
reacted with an excess of potassium iodide. Benzene is used
to extract any methyl mercury as the iodide salt. A portion
of the benzene extract is chromatographed directly and
response is compared to standard responses. The iodide is
detected using gas chromatography with an electron capture
detector.
Limitations;
Sensitivity; 0.001 ug/g
Interferences; Materials in sample generally will not
cause a problem*
Pitfalls; Special Precautions; Potassium iodide
solution may decompose to free iodine, which may cause
interfering peaks on the chromatogram.
Statistical Characteristics;
Accuracy and Precision; Ten dfferent sediment samples
with a background level of less than 0.0005 ug/g were
spiked with 0.010 ug/g and measured with this teat. The
average was 0.00963 ug/g and the standard deviation was
0.00087 ug/g.
Calibration Requirements; Given in Reference below
Data Outputs; Analog electrical signal recorded as
chrornatogram.
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57
Special Sampling Requirement (Collection, Storage, Handling);
It should be understood that methyl mercury is generated
froia inorganic mercury by biological methylation. Since
this can occur at widely varying rates in samples stored in
a laboratory, analyses intented to determine the methyl
mercury concentration existing at the sampling site at the
time of sampling should be performed as quickly as possible.
Storing samples frozen or refrigerated before analysis is
acceptable but is a less desirable alternative.
References;
1. Ilethyl Mercury in Sediment. 1972. Provision Method.
Description received from Analytical Quality Control
Laboratory, EPA national Environmental Research Center,
Cincinnati, Ohio.
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58
ATOMIC ABSORPTION SPECTROSCOPY
IJame of Measurement Method; Mercury — Cold Vapor Technique
Purpose of .Measurement (Important Applications); This
metnod is applicable to surface waters,saline waters,
wastewaters, effluent and domestic sewage.
S ummary of Metho d; The atonic absorption procedure is a
physical method based on the absorption of radiation at
253.7 nrn by mercury vapor. The mercury is reduced to the
elemental state and aerated from solution in a closed
system. The mercury vapor passes through a cell positioned
in the light path of an atomic absorption spectrophotoraeter.
(Instruments designed specifically for the measurement of
mercury using the cold vapor technique are commercially
available and may be substituted for the atomic absorption
spectrophotometer). Absorbance (peak height) is measured as
a function of mercury concentration and recorded in the
usual manner.
In addition, inorganic forms of mercury, organic mercurials
may also be present in an effluent or surface water sample.
The organomercury compounds will not respond to the
flameless atomic absorption techniques unless they are first
broken down and converted to mercuric ions. Potassium
permaganate oxidizes many of these compounds but recent
studies have shown that a number of organic mercurials,
including phenyl mercuric acetate and methyl mercuric
chloride, are only partially oxidized by this reagent.
Potassuim persulfate has been found to give approximately
100 percent recovery when used as the oxidant with these
compounds. Therefore, a persulfate oxidation step following
the addition of the permanganate has been included to insure
that organomercury compounds, if present, will be oxidized
to the mercuric ion before measurement. A heat step is
required for methyl mercuric chloride when present in or
spiked to a natural system. For distilled water the heat
step is not necessary.
Limitations;
Range of Applicability; The range of the method may be
varied through instrument and/or recorder expansion.
Using a 100 ml sample, a detection limit of 0.2 ug Hg/1
can be achieved; concentrations below this level should
be reported as 0.2.
Interferences: Sulfides (interference can be eliminated
by addition of potassium permaganate), high
concentrations of copper and chlorides. (Certain
volatile organic materials may also interfere.)
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59
Statistical Characteristics :
Accuracy and Precision; Usiny an Ohio River composite
sample with a background mercury concentration of 0.35
ug/1, spiked with concentrations of 1, 3 and 4 ug/1, the
standard deviations were +0.14, +_0.10 and +0.03 u
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60
METIIYLEnr BLUE ACTIVE SUBSTA-JCLS (M1JAS)
Uarao of Measurement .'lotliod; "lethyleno Blue "lethod
Principal Detection Technique; Colorinetry
Purpose of Measurement (Important Applications):
Determination of concentration of detergents, phosphates,
surfactants in drinking water, ambient surface waters,
domestic and industrial wastes.
Summary of I lethod: The dye, muthylene blue, in aqueous
solution reacts with anionic-type surface active materials
to form a blue colored salt. The salb is extractable with
chloroform and the intensity of color produced is
proportional to the concentration of IIHAS.
Limitations;
Range of Applicability; 0.025 to 100 my/1 of M3AS
(expressed as linear alkyl sulfonate - LAS)
Interferences; Chloride at concentrations over 1,000
rug/1 sulfonates, carboxylates, phosphates, phenol,
cyanates, and thiocynaides (at concentrations higher
than normally encountered in water or wastev/ater).
Statistical Characteristics;
Accuracy; +1.2% to -11% bias (conditions as given for
Precision).
Precision; Determinations by 110 analysts exhibited
relative standard deviations of 10 to 15 percent for
known concentrations of :1BAS ranging from 0.27 to 2.94
mg/1 of LAS
Data Outputs; Analog electrical signal, displayed on ranter.
References; Methods for Chemical Analysis of Water and Wastes,
EPA National Environmental Research Center,
Cincinnati, Ohio (1971).
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61
NITROGEN
Name of Measurement Method; Ammonia Distillation Procedure
Principal Dectection Technique; Colorimetry
pose of Measurement (Important Applications); Ambient
face waters/ domestic and industrial wastewate.rs, and
Pur
sur
saline waters.
Summary of Method; The sample is buffered at pH of 9.5 x*ith
a borate buffer in order to decrease hydrolysis of cyanatea
and organic nitrogen compounds, and is then distilled into a
solution of boric acid. The ammonia in the distillate can
be determined either colorimetrically by nesslerization, or
titrimetrically with standard sulfuric acid with the use of
a mixed indicator, the choice between these two procedures
depending on the concentration of the ammonia.
Limitations;
Range of Applicability; 0.05 to 1.0 mg/1 ammoniacal
nitrogen
Interferences; Cyanates, certain alcohols, aldehydes,
and ketones may interfere. (Certain amines would
interfere if distillation step were not included.
Pitfalls; Special Precautions; Residual chlorine must
be removed. If sample is preserved by a mercury salt,
the mercury must be complexed.
Statistical Characteristics; 24 analysts in 16 laboratories
obtained;
Known Concentration Accuracy (Bias) Precision
in mg/1 Nitrogen mg/1 N (Std. Dev.) mg/1
0.21 -0.01 0.122
0.26 -0.05 0.070
1.71 -1-0.01 0.244
1.92 -0.04 0.279
Calibration Requirements; Set up a series of standards in
Nessler tubes. Care needed in calibrating for saline water
tests.
Data Outputs; Meter reading (analog electrical signal)
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62
SjGcial Sampling requirements (Collection., Storage/ Handling)
Preserve with mercuric chloride ami storo at4* C."
1. 'totiiods for Chemical Analysis of Water and Y7astQS.
1971. ]-]PA National Environmental Research Center/
Cincinnati/ Ohio.
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63
NITROGEN
Uaiae of Measurement Method; Ammonia
Principal Detection Technique; Titrimetric
Purpose of Measurement (Important Applications): Surface
waters, domestic and industrial wastewaters, and saline
waters.
Sunimajry of Sethodr .The" saznpl&risUauf fered at pH of 9 ..5. .wi±h«
Derate buffer in order-to decrease hydrolysis- of cynates and
organic nitrogen ammonia in the^distillate can be determined
either coloiTOtrically- by hesslerlzation or titrintrically
with*standard/sulf uric~acid and. the use of a mixed-ihdicator,
the*choice between-these two procedures'depending on the
concentration of the ammonia.
Limitations:
Range of Applicability; 1.0 to 25 mg/1 ammoniacal
nitrogen.
Interferences; Cyanatas, hydrozine and similar
compounds.
Pitfalls; Special Precautions: Residual chlorine and
mercury must be removed.
Sampling Sampling Requirements (Collection/ Storage/ Handling)
Preserve with mercuric chloride and store at 4"C.
References;
1. Methods for Cnemicai Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.'
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64
NITROOFN
Name of "oasurement -Method; Ammonia
Principal Detection Method; Automatic Colorimetric
Purpose of Measurement (Important Applications); Surface Waters,
salinewaters.
Summary of Method; The intensity of the indophenol blue color, formed
by the reaction of ammonia with alkaline phenol hypochlorite, is
measured. Sodium nitroprusside is used to intensify the blue color.
Uses Technicon AutoAnalyzer. Colorimeter is ecmipped with 360 or
650 filters.
Limitation;
Range of Applicability; .01 to 2.0 mq/1 nitrogen as NFL,
Pitfalls; Special Precautions; Harked variations in pH among
samples should be eliminated.
Statistical Characteristics;
Precision; In a single laboratory using surface water at water at
~~ concentrations of 1.41, 0.77, 0.59, 0.43 mg/1 of NIU-N,
the standard deviation was £.005 rag/1. ~
Calihcation Requirements; Requires calibration against samples, both
for fresh water and substitute ocean water of specified constituency.
Data Outputs; Analog electrical signal displayed on stripchart
Special Sampling Requirements (Collection, ftorage, Handling);
Preserve with mercuric chloride and refrigerate at 40*Max. holding
tine - 7 days
References;
1. Methods for Chemical Analysis of Water and Wastes. 1971. EPA
national Environmental Research Center, Cincinnati, Ohio.
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NITROGEN
Name of Measurement Method; Kjeldahl
Principal Detection Technique: Tritriraetric
Purpose of_JMeasurement (Important Applications): . Stir face waters,
domestic and industrial wastes,saline waters.
Summary of Method; Total Kjeldahl nitrogen is defined as the sum of
the free ammonia and organic nitrogen compounds which are converted to
ammonium sulfate under defined conditions of digestion. The procedure
converts nitrogen components of biological orgin such as amino acids,
peptides, and proteins to ammonia, but may not convert the nitrogenous
compounds of certain industrial wastes such as amines, hydrazones,
nitro compounds, and others.
The sample is heated in the presence of concentrated sulfuric
acid, Itj SO./) and HgSfy and evaporated until S0«j fumes are obtained
and the solution becomes colorless or pale yellow. The residue is
collected, diluted, and is treated and made alkaline with a hydroxide-
thiosulfate solution. The ammonia is distilled and determined after
distillation either by nesslerization or titrimetrically.
Limitations;
Range of Applicability! Concentration above 1 mg/1 of nitrogen.
Statistical Characteristics; Results obtained by 31 analysts in 20
laboratories
Known Cone, of Precision as Accuracy as
Nitrogen, Kjeldahl Standard Deviation Bias, Bias,
mg N/liter mg^N/lrter mg N/liter
+ .03
+ .02
+ .04
-.08
Special Sampling Requirements (Collection, Storage, Handling);
Unstable. Analyze soon after sample is acquired.
References;
1. Methods for Chemical Analysis of V7ater and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
0.20
0.31
4.10
4.61
0.197
0.247
1.056
1.191
+15.54
+ 5.45
+ 1.03
- 1.67
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66
NITROOFH
Name of Measurement Method; Kjeldahl
Principal Detection Technique: Automated Phenolate
Purpose of Measurement (Important Applications) ; Ambient surface
waters, saline waters, donest.i.c ana industrial wa s t e s .
Summary of Method ; The sample is autonatically digested with a
sulfuric acid solution containing potass.ium sulfate and mercuric as a
catalyst to convert organic nitrogen to ammonium sulfate. The
solution is then automatically neutralized with sodium hydroxide
solution and treated with alkaline phenol reagent and sodium hypo-
chlorite reagent. This treatment forms a blue color desiqnated as
indophenol. Sodium nitroprusside, v;hich increases the intensity of
the color, is added to obtain necessary sensitivity for measurement of
low level nitrogen.
Utilizes Technicon AutoAnalyzer. A colorimeter with 630 nm filter is
used.
Limitations:
Range of Applicability; Nitrogen concentrations from 0.05 to 2.0
Interferences ; Iron, chromium, or copper ions may interfere
Statistical Characteristics! Results from six laboratories analyzing
natural waters with known concentrations of Kjeldahl nitrogen:
Known' "Conc. of Precision as Accuracy as
Kjeldahl-Nitrogen Standard Deviation Bias Rir»s
mg N/liter _ Kjeldahl-N, mg N/liter % ma N/liter
1.89
2.18
5.09
5.81
0.54
0.61
1.25
1.85
-24.6
-20.3
-23.0
-21.9
- .46
- .62
-1.21
-1.27
Time of Measurement; About 20 samples per hour
Calibration Requirement; Requires preparation of standardizations
Data Outputs; Analog electrical signal recorded on stripchart
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67
Special Saraplinq_Requirenents (collection, storage/ handling);
Preserve with mercuric chloride; refrigerate at 4'C.Analyze
immediately after acquistion.
References;
1. Methods for Chemical Analysis of V7ator and TTantos. 1971. EPA
national Environnental Research Center, Cincinnati, Ohio.
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68
ITane of "easurcnent Method; Kjeld.ahl
Principal ^Detection Technique ; Colori'-'etrir
Purpose of Measurencnt (. Important Applications) ; Anhient surface
T 7.1. tors, saline waters, domestic anr! industrial v;a ste s .
^ of "othocl; Heo r-unnar" for ITitrorrnn, I'jnlc'ahl, r'
Color ir>e trie. Tor ?Te.nsleriz;ation, a spectronhonoter or color inetcr in
upod, filte>-orl at 400 to 525 nn.
Linitations;
Range of Applicability; ConcGntrationn of nitroqon helov 1 ma/1
Characteristics; Sep Tethocl Surnmary for nitrogen,
Kjolc^ahl, "otal (Ti trine trie)
Data utputs; "eto.r roarling (analon olectrical sianal), recorded
^eauirenonts (Collection, ftoracro,
Una tab In , analyse iTneylip.telv after sanplc is acquired.
References;
1. ;:ethorir> for Chemical Analysis of T^ter and barter;. 1971. FP71
national HnvironnRntal Research Center, Cincinnati, Ohio.
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Principal Detection Technique; Nitrite
Purpose of Measurement (Important Applications) ; Anb.ient surface
waters, saline waters, domestic ami industrial wa s te s .
Summary of Method ; The diazoniun cornound forned by dinzotation of
sul'f anITanide by nitrite in water under acid conditions is coupled
with IT- (l-naphtyl)-ethylenedianine to produce a reddish -purple color
which is read in a snectro photometer at 540nn against a blanl:.
Concentration of lio^-n is plotted aqainst optical density.
Limitations;
Range of Applicability; 0.05 to 1.0 mq/1 nitrite nitrogen
Interferences ; Strong oxidizing or reducing aqents or hirrh
alkalinity
Calibration Requirements ; Requires use of standard solutions
Data Outputs; lieter reading (analorr
Special Sampling Requirements (collection , Storage, Handling) ;
Preserve with mercuric chloride; refrigerate at 4feC. "axinun storage
tine - 7 days.
References;
1. Methods of Chemical Analysis of Uater and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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70
Mane of "casurernnt Method; nitrate-nitrite
Principal Detection Technique; Automated Cadmium Deduction T'ethod
Purpose of ileasureiuent (Important Applications); Determines nitrates
and nitriter;,n.i.n<7lv or combined, in surfaco. and saline waters.
nunnary of Method; The initj.nl ste^ ir? to reduce the nitrates to
nitritor; b^ using a cadniun-cop'inr catalyst. The nitrites (thosr
orrrinallv present plus roducerl nitratos) are tho.n reacted v:ith
snlfanilamdo to forn the c!iazo compound v-'l^ich in thon coupled in an
acid solution (p!1 2.0-2.5) with N-l nanhtylethylene^iamine
hydrochlorido to forri the azo dye. The azo values are readily
obtainable by carryina out the procedure—first with, and then
without, the initial Cd-Cu reduction step.
A Technicon AutoAnalyzer is used. The colorireter is equipped with
540 nn filters.
Limitations;
Range of Applicability; 0.05 to 10.0 mg/1 nitrogen present as
nitrate. *"
Interferences; Armenia and primary anines; some nctal ions
(mercury and copper ) nay produce interfering color complexes.
Statistical Charatoristics; Three laboratories analyzed four natural
water samples containing exact concentrations of inorganic nitrate,
with the following results:
Known Cone, of Precision as Accuracy as
nitrate nitrogen Standard Deviation Bias, Bias,
mg N/liter mg IJ/liter % mg N/liter
0.29 0.012 + 5.75 +.017
0.35 0.092 +18.10 +.063
2.31 0.313 + 4.47 +.103
2.48 0.176 - 2.69 -.067
Time of Hoasurenent; Not stated, but assumed to be rapid (i.e.,
several samples per hour.
Calibration^Reguireinonts: Calibration against standard solution is
necessary."Analysis of saline waters requires calibration against
substitute ocean water.
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71
Data Outputs: Analoa electrical signal disnlayml on stri^chart.
ne fanplincr ^eguirnnonts (collection , storage, handling); S
in not ntnble since anin^ in natural v;aterc. nay rnr.ct v.rth nitrites.
n shonlrl IP annl^^er1 an soon an possible after acnnqition.
References:
1. netliotiR for Chonical Annlvsis o^ T1ator nrv1 fantos. 1971. EPA
national Environmental Research Con tor, Cincinnati, Ohio.
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NITROGEN
Name of Measurement Methodt Nitrate and Nitrite
Principal Detection Technique; Automated Hydrazine Reduction Method
pvirpose of Measurement__(Important Applications) t Ambient surface
waters and domestic or industrial wastes.
Summary of Method; This method, using the Technicon AutoAnalyzer,
determines NO«-N by the conventional diazotization-coupling reaction.
The NQj-N is reduced with hydrazine sul^ate in another portion of the
sample and the. nitrite thus formed is determined in the usxial manner.
Subtraction of the NQ^-N orginally present in the sample from the
total NCU-N will give the orginal NOa-N concentration in terms of
NO^-N. - " *
Utilizes Technicon AutoAnalyzer.
Limitations;
Range of Applicability; Nitrite or nitrate nitrogen in
concentrations of 0.05 to 10.0 ing/liter.
Interferences; The following substances may interfere
(concentration below which interference will not occur are listed
in Reference); chlorides, phosphates, sulfides, ammonical
nitrogen, manganese, calcium, and ferric ions, and ABP.
Pitfalls; Special Precautions; Toxic reagents
Statistical Characteristics; Nine laboratories analyzed four natural
water sample's containing exact concentrations of inorganic nitrate,
with the following results:
Known Cone, of Precision as Accuracy as
Nitrate Nitrogen Standard Deviation Bias, Bias,
mg N/liter mg N/liter % mg N/liter
0.29
0.35
2.31
2.48
0.053
0.050
0.250
0.217
-0.8
+1.9
+3.0
-1.2
.002
.007
.07
.03
In a single laboratory, using surface water samples at concentrations
of 0.1, 0.2, 0.8, and 2.1 mg-N/1, the standard deviations were +0.04,
+0.05, and +0.05, respectively.
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73
Time of Measurement; About 20 samples per hour
Calibration Requirements; See Reference.
Data Outputs; Analog electrical sirrnal displayed on stripchart.
Special Sampling Requirements (collection, storage/ handling);
Preserve sample with mercuric chloride. Pefriqernte at 4*?C. Haxinum
holding tine - 7 days.
References;
1. .Methods for Chenical Analysis of T-Tater and TJastes. 1971.
National Environmental Research Center, Cincinnati, Ohio.
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74
NITROGEN
Name of Measurement Method; Organic Plus Ammonia
Principal Detection Technique; Automated Phenolate Method
Purpose of Measurement (Important Application): Ambient surface
waters and saline waters.
Summary of Method; Organic nitrogen is determined by manually
digesting the sample with potassium persulfate and sulfuric acid to
convert the organic nitrogen, and any ammonia present, to ammonium
sulfate. Subsequently, the automated phenol-hypochlorite procedure is
used to measure the ammonia nitrogen. Nitrate-nitrite nitrogen is not
measured by this procedure.
Utilized Technicon AutoAnal^'zer with colorimeter eauinped with 650 nr>
filter.
Limitations;
Range of Applicability; Nitrogen concentrations of 1.0 to 10.0
mg/1 ""
Time of Measurement; Not stable. Estimated to be between one and
two hours per determination.
Calibration Requirements; Calibrate against standard solutions
Data Outputs; Analog electrical signal recorded on stripchart.
Special Sampling Requirements (collection,storage,handling);
Preserve with mercuric chloride. Refrigerate at 4*jC. Maximum storago
time - 7 days.
Reference;
1. Methods for Chemical Analysis of Water and TTantes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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75
ACID
Name of T'oasureraent^ethod; Zinc-Zincon .Method
Principal Detection Technique; Colorinetry
Purpose of Measurement (Important Applications) Ambiont surface
waters (non-saline) .
Summary _of Method ; Zinc forms a blue-colored complex vith 2 carboxy-
2 • -hydroxy^S f -sul f o f ormazy Ibenzone (Zincon) in a solution buffered to
pll 9.2. When IITA is added, the Zn-Zincon complex is broken vhich
reduces the optical density in proportion of the anount of IITA
present.
A photoneter enuipped with 620 nn filter is used.
Limitations;
Range of Applicability; 0.5 to 10.0 mg/1 1TTA
Interferences ; Cations of conrcon netals (calciun, naqnesium,
zinc, copper iron, nanganesp, et al) .
Pitfalls; Special Precautions; Fost interfering cations can be
renoved by ion-exchanae resin.
Statistical Characteristics;
Precision; In a single laboratory, using spihed surface water
samples at concentrations of 0.5/2, 6, and 10 mg/1 NTA, standard
deviations were 4-0.17, 4-0.14, +0.1, and 4-0.16, respectively.
Calibration Requirements; Require stock solution
DataniOutj3uts; Analog electrical signal displayed on meter.
Special Sampling Requirements (collection storage , handling) ; Sample
should Ee analyzed as soon as possible after acquistion, since IITA is
biodegradable .
References;
1. Ilethods for Chemical Analysis of Uater and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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76
_jTjrr-,^TjJnTni.7V.crTic AC in (IITA)
Tlane of r.easurement Method; Automated Zinc-Zincon J'ethod
Principal Detection Technique; Color ipo try
Purpose of "easurenent (Important Applications) ; Ambient surface
v/atorn (non-nnline) .
Summary of "ethod; Zinc forms a blue-colored corplcr with 2-carboxv-
2 ' -hvdroxy-5 ' sulfof ornazylbFinzene (7incon) in a solution buffered to
nil 9.2. Uhen TITA is added, the Zn-Zincon conplex if? broken which
reduces the ontical density in proportion to the anount of ITT7\
present.
A Tehonicon AutoAnaly^er is used. The colorineter irs equipped with
600 to 625 nn filter.
Limitations!
of Applicability; 0.0^ to l.o mn/i Or 0.5 to 10.0 mq/1 TITA,
denendinq on tyr»c of nanifold sy
Interferences; Common cations (calcium, naqnesinm, copper iron,
nangaqnese) . Constituents of sewaqe cause sor.e interference also.
Pitfalls; Special rrecautions; Not applicable to saline water.
Statistical Characteristics;
Reproducihility; In a single laboratory, using surface water san.ples
at concentrations .of 0.1, 0.10, 0.27, and 0.44 ma/1, the standard
deviations v?ere +0.01, +0.004, +0.005, respectively. At
concentrations of 1.3, T. 0, 5.8, and 7.4 nq/1, the standard deviations
v;ere +0.05, +0.05, +0.07, and +0.1, respectively.
Time of Measurement; About 13 samples per hour.
Calibration Requirements ; Requires standard solutions.
Data Outputs ; Analog electric signal, recorded automatically.
Special Sampling Requirements Collection, Ptorage, Tandling) ; Analyze
soon after sample collection because of biodeqradability of HTA .
Reference;
1. Methods for Cheraical Analysis of Water and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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77
PHOSPHORUS
Name of Measurement .Method; Single Reagent Method
Principal Detection Technique; Colorimetry
Purpose of Measurement (Important Applications); Ambient surface
waters, drinking water, domestic and industrial wastes, saline waters
(may also be applicable to sediments, sludges, and algae blooms).
Summary of Method; Ammonium molybdate and potassium antimonyl-
tartraie react in an acid medium with dilute solutions of phosphorus
to form an antimony-phospho-molybdate complex. This complex is
reduced to an intensely blue-colored complex by ascorbic acid. This
color is proportional to the phosphorus concentration.
Only orthophosphate forms a blue color in this test. Polyphosphates
(and sone organic-phosphorus compounds) may be converted to the
orthophosphate form by sulfuric-acid-hyc'.rolysis. Organic phosphorus
compounds may be converted to the orthophosphate form by sulfuric acid
hydrolysis. Organic phosphorus compounds may be converted to the
orthophosphate form by persulfate digestion.
A spectrophotometer of filter photometer suitable for measurements at
880 nm is used.
Limitations;
Range of Applicability; 0.01 to 0.5 mg/1 phosphorus
Interferences; High concentrations of iron. Also, mercuric
chloride (when used as a preservative).
Pitfalls; Special Precuations; Avoid use of commercial detergents
for cleaning analyzed natural water samples containing exact
concentrations of organic phosphate, with the following results;
Known Cone, or Precision as Accuracy as
Total Phosphorus Standard Deviation Bias, Bias,
mg P/liter mg P/liter % mg P/liter
0.110
0.132
0.772
0.882
0.033
0.051
0.130
0.128
+ 3.091
+11.99
+ 2.96
- 0.92
+.003
+ .016
+ .023
-.008
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78
Twenty-six analysts in sixteen laboratories analyzed natural water
samples containinq exact concentrations of orthophosphatc, with the
following results:
Known Concen. ofPrecision an Accuracy as
Orthophocphate Standard Deviation Bias, Bias,
nq P/liter nq P/liter % ng P/liter
0.020
0.038
0.335
0.383
0.010
0.003
O.Olfl
0.023
-4.05
-P. 00
-2.75
-1.76
-.001
-.002
-.000
-.007
Calibration Requirements; Requires standard solutions
Data Outputs; Teeter (analoa voltaqe), nanually recorded
Special Sampling Requirements Collection,_ Storagey T^andling) ; If
stored nore than 0-10 hours, add mercuric "chloride or preservative anc!
refriqerate at 4^C.
References;
1. Methods for Chemical Analysis of TJater and Hastes. 1971.
national Environmental ?.e??earch Center, Cincinnati, Oliio.
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79
PHOSPHORUS
Name of neasurement_fTethocl: Automated flingle Reagent Method
Principal Detection Technirruet Colorinetry
Purpoi^e_of Measurement (Important^pplications) ; Ambient surface
waters^ "dome stic and industrial\vastes, saline waters. (May also
prove applicable to sediments, sludges.)
Summary of .Method; Ammonium molybd.ate and potassium antinonyl
tartrate react in an acid medium with dilute solutions of phosphorus
to form an antinony-phosphomolybdate cormlex. This complex is reduced
to an intensely blue-colored comple:: by ascorbic acid. The color in
proportional ot the phorsnhoruf? concentration.
A Technicon AutoAnalyzer is used with a colorimeter ecmipped vr.ith G50
nm filter.
Limitations;
Range ofApplicability; 0.01 to 1.0 mg/1 phosphorus
Interferences; Iron in hi
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80
Data Outputs; Analog electrical siqnal recorded on strinchart.
Special_f!anpling_Reguirement Collection,_rtorage, Handling) ; Avoid
benthic^leposits during collection.If not analyzed sane day as
collected, preserve with mercuric chloride and refrigerate at
References:
1. Methods for Chemical Analysis of Water and Uastos. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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81
PHOSPHORUS
Name of Measurement Method; Automated Stannous Chloride Method
Principal Detection Technique; Colorinetry
Purpose of f'-easurenont (Important Applications); Ambient surface
waters, don^stic and industrial wastes. (May prove applicable to
sedinent, sludge and alqae blooms).
nummary of Method; Phosphorus is determined by manually digesting the
samples with ammonium persulfate and sulfuric acid to convert the
various forns of phosphorus to the orthophosnhate form; and
measurement of this orthophosphate on a Technicon AutoAnalyzer, using
(NIL Jjjfto^with SnCl reduction to form color complex.
Limitations;
Range of App1i cab i1i ty; 0.01 to 1.0 mq/1 phosphorus
Pitfalls; Special Precautions; Avoid benthic deposits when
acquiring sample. Do not use commercial detergents for cleansing
apparatus.
Statistical Characteristics; On a single laboratory, using surface
v;ater sanples at concentrations of 0.06, 0.11, 0.48, and 0.62 ing
P/liter, the standard deviation was +0.004 (AOC Laboratory).
Tine of Measurement; About 15 samples per hour
Calibration Requirements; Requires standard solutions. See Ref. (1)
for details.
Data Outputs; Analog electrical signal recorded on stripchart.
Special Sampling Requirements Collection, Storage, Handling); Avoid
benthic deposits wJion sampling. If sample is not analyzed on day of
acquisition, preserve with mercuric chloride and refrigerate at
References;
1. Methods for Chemical Analysis of T7ator and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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82
SILIPA
Principal Detraction Tehrr.irmo!
ro Q-P 'Vi^rm. recent (Important Applications) ; Drinkinrr v.v.ter,
anhiont surface voters, domestic ant1 industrial wastes, saline waters.
of Kethod ; A vell-n.ixed sample in filtered throuah a 0.45u
nonbranc filter. Tho filtrate, unon the addition of nolyhflate ion in
acidic fiolutior, forns a qroftninh-i-ynllow color connlc:: proportional to
tho flirnolvGd silica in thn sf^nlo. Thf» color co"nlr>:: ir then
measured spectronhoton.etricnllv.
Linitations ;
Range of Applicability; 2 to 25 mg/1 silica
Interferences ; Excessive color or turbidity
Characteristics; Photonetric evaluations by the anino-
naphtho-sulfonic aci.d procedure have an estimated precision of +0.10
nq/1 in tho range from 0 to 2 rvf/1 (APT.M) . Photonetric evaluation of
the silica-riolyhdate color in the range from 2 to 50 na/1 have an
estimated precision of approximately 4 percent of the rruantity of
silica neasured
Data Outputs ; Analoa electrical sirmal
References;
1. Hethods for Chenical Analysis of T'Tater and Pastes .
1P71. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. APT1" Standards. 1970. Part 23, Atmospheric Analysis
Method DC 5 9-6 8.
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83
OIL AND GREASE
Name of Measurement Method; Extraction/Gravimetric
Purpose of Measurement (Important Applications); Measures hexane-
extractable matter (animal fats, non-volatile hydrocarbons, waxes,
grease$) in surface waters, industrial wastes, and domestic sewage.
Summary of Method; The sample is acidified to a low pH (4.3) and
extracted with hexane using a Soxhlet extraction. The solvent is
evaporated from the separated extract and the residue weighed.
Limitations;
Range of Applicability; 5 to 1000 mg/1 of extractahle matter
Statistical Characteristics; Precision and accuracy data not
available from reference below.
Time of Measurement; Not stated, but several hours.
Data Outputs; Read fron analytical balance.
References;
1. Methods for Chenical Analysis of Water and Wastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
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84
PH
Name of Measurement Method; Electronic trie
Principal Detection Technique^ Electronetric
Purpose of Measurement (Important Applications) ! DrinMnq water,
ambient surface water's, domestic and industrial waters, so linn waters,
Summary of Method; The pll of a sarmle is an eloctronotric
measurement, using either a glass electrode in combination x\'ith a
reference potential (saturated calomel electrode) or a combination
electrode (glass and reference).
Limitations:
Range of Applicability; Not stated, but assumed broad.
Interferencest Oil and grease.
Statistical Characteristics; Forty-four analysts in twenty
laboratories analyzed six synthetic water samplers containing exact
concentrations of hydrogen-hydroxyl ions, with the following results;
Known Cone. Precision as Accuracy as
as pll units Standard Deviation Bias, Bias,
pH units % pF units
3.5 0.10 -0.29 -0.01
3.5 0.11 -0.0
7.1 0.20 +1.01 +0.07
7.2 0.18 -0.03 -0.002
8.0 0.13 -0.12 -0.01
8.0 0.12 +0.16 +0.01
Time of Measurement; Not stated, but rapid (several per hour)
Calibration Requirements; Instrument must be initially standardized.
Comments by Users; Pield pR measurements made with comparable
instruments are reliable.
Data Outputs; Analog sianal displayed on meter.
Special Sampling Requirements (Collection, Storage, Handling)^
Analyze"~as soon as possible after collection. No holdinq.
References;
1. Hethods. for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washinqton, D. C.
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85
PHENOLICS
Name oj: f'eajsuremervt Method; 4-AAP Method with Distillation
Principal Detection Technique* t Colorimetry
Purpose of Measurement (Important Applications); Drinking water,
ambient surface waters, domestic ana industrial wastes, saline waters.
Summary of Method: Phenolic materials react with 4-aminoantipyrine in
the presence of potassium ferricyanide at a pH of 10 to form a stable
reddish-brown colored antipyrine'dye. The amount of color produced is
a function of the concentration of phenolic material.
Limitations:
Range of App1icabi1ity; 5 to 1000 g/1 phenol (with solvent
extraction)50 to 5000 g/1 phenol (without
solvent extraction)
Interferences; None stated. pH must be controlled
Pitfalls; Special Precautions; Method does not differentiate
different types of phenolic materials.
Statistical Characteristics; The following results were obtained by
analysts at six laboratories:
Known Standard Deviation
Concentration (ug/1) with
of Phnnol Solvent
(g/1) Extraction
9.6 0.99
48.3 3.1
93.5 4.2
Calibration Requirements; Color response varies with type of phenolic
material.Phenol is used as standard.
Data Qutnuts; Meter reading (analog electrical signal).
References;
1. Methods for Chenical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Uastowater. 1971. 13th Edition. American Public
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8G
Health Association, TTaRhin
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87
SOLIDS
(DISSOLVED)
Principal Detection Technique: Filtration/Hravinetric
Purpose of Iteasurenent (Important Applications)^ Ambient surface
waters, domestic and industrial wastes,saline waters.
Summary of Method; A well mixed sample is filtered through a standard
glass fiber filter. The filtrate is evaporated and dried to constant
weiqht at 180°C.
Limitations;
Range of Applicability; 10 to 20,000 mg/1 solids
Pitfalls; Special Precautions: Mineralized waters containing
calcium, magnesium, chloride and/or sulfate may be hygroscopic and
will require prolonged drying and quick weighinrr. Samples
containing bicarbonates require careful dryina at 180 C to insure
conversion to the carbonate.
Statistical Characteristics: Precision data not available at this
time. References belovr. Accuracy data on actual sample cannot be
obtained.
Data Outputs: Read from analytical balance.
SpecialSampling Requirements (Collection. Storage, Handling);
Sample should be analyzed as soon as possible after acquisition.
References:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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88
SOLIDS
(SUSPENDED)
Principal Detection Technique; Filtration/nravimotric
Purpose of Measurement (Important Applications) ; Ambient surface
waters, donestic and industrial wastes, saline waters.
Summary of Method; A well-mixed sample is filtered through a standard
glass fiber filter, and the residue retained on the filter is dried to
constant weight at 103-105°C. Non-filterable solids are defined as
those solids which are retained by a standard glass fiber filter and
dried to constant weiqht at 103-105°C.
Limitations:
Range of Applicability; 20 to '20,000 mg/1 solids
Pitfalls; Special Precautions; Too much residue on the filter
entrap water and require polonged drying .
Statistical Characteristics t Reproduction of the data not available
at this time. Accuracy data on actual samples cannot be obtained.
Data Outputs; Analytical balance readimr.
Special Sampling Requirements (Collection, Storage, Handling) ;
Preservation of sample is not practical; analysis should begin as soon
as practical. Non-homoqeneous particulates (sticks, fish, etc.)
should be excluded from sample.
References;
1. .Methods for Chemical Analysis of Water and T-Jastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
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89
SOLIDS
(TOTAL)
Principal Detection Technique: Filtration/Gravimetric
Purpose of Measurement (Important Applications); Ambient surface
waters, domestic and industrial wastes, saline waters.
Summary of Method; A well mixed aliquot of the test sample is
quantitatively transferred to a pre-v»eighed evaporating dish and
evaporated to dryness at 103-105°C. (Total Solids are defined as the
sum of the homogeneous suspended and dissolved materials in a sample),
Limitations;
Range of Applicability; 10 to 30,000 mg/1 solids
Pitfalls; Special Precautions; Floating oils and greases, if
present, should be dispersed with a blender. Large floating
particles should be excluded.
Statistical Characteristics; Precision and accuracy data not
available at this time.
Data Outputs; Read from analytical balance
References;
1. Methods for Chenical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
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90
SOLIDP
(VOLATILE)
Principal Detection Technique; Ignition/Filtration
Purpose of Measurement (Jimnortrint Applications) ; The test is useful in
obtaining a rbucrh annroximation of the amount of oraanic matter
present in the solid fraction of sewage, activated sludge, industrial
v/astes, or bottom sediments.
Summary of "ethod; The residue obtained fron the deternination o^
total, suspended, or dissolved solids is ignited at 550°C in a muffle
furnace. The loss of weiaht on ignition is renortect as mg/1 volatile
solids.
Linitations;
Pitfalls; Special Precautions: The test is subject to nany errors
due to loss of water of crystallization, loss of volatile ornanic
natter prior to combustion, incomplete oxidation of certain
conple:: orrranics, and decomposition of mineral snlts during
combustion.
Statistical Characteristics:
Renroducibility^ A collaborati^'e studv involving three
laboratories examining four samples by means of ten replicates
showed a standard deviation of +11 pa/1 at 170 mg/1 volatile
solids concentration.
Data Outputs; Read from analytical balance.
References:
1. Methods for Chemical Analysis o^ Tator and !7antes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
"antevrater. 1971. 13th Edition, American
Public Health Association, T'anhinaton, P. C,
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91
Principal Detection Technique; Colorinotry
Purpose of Measurement (Important Applications)! DrinMng water,
ambient surface waters, domestic and industrial was t e s.
Summary of Method: Sulfate ion is converted to a barium sulfate
suspension under controlled conditions. The resulting turbidity ic
determined by a photoelectric colorimeter or spectophotoneter and
compared to a curve prepared from standard sulfate solutions.
Limitations:
Ranae of Applic_a_biIj^ty; All concentrations of sulfate ion.
Higher concentrations" must be diluted such that sample aliquot
contain less than 40 nq/1 sulfate.
Interferences: Suspended matter and color interfere. Correct by
running blanks from which the barium chloride has been omitted.
Statistical Characteristics t Thirty-four analysts in 16 laboratories
analyzed six synthetic water samples containing exact concentrations
of inorganic sulfate with the following results:
Known Cone, of Precision as Accuracv as
Sulfate Standard Deviation Bias Bias
ma/liter mg/liter % ma/liter
8.6 2.30 -3.72 -.3
9.2 1.70 -0.26 -.8
110.n 7.86 -3.01 -3.3
122.0 7.50 -3.37 -4.1
188.0 9.58 •'•P.O/' +.1
199.0 11.8 -1.70 -3.4
Time of reasurenent; Rapid
Data Outputs; Meter output (analog electrical sianal), manually
References;
1. Methods for Chemical Analysis of "ater an<3 Wastes.
1971. EPA national Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Uastewater. 1971. 13th Edition. American
Public Health Association, Washington, D. C.
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92
RULFAT
Harm of veasurerient He they'll Autonnted CMoranilate J'ethod
Principal Detection ^echnirme; Color.lr.otrv
Purpose of '•eppurenent (Important Applications): AnMont surface
waters", donestic and industrial wastes, saline waters.
Runr.nry of I'.et3iorT; TTion solid bariun chloranilate in added to a
solution oontpinina sulfate, bariun sulfate is procinitatec!, releasing
tho l\irrhly colored acid chloranilate ion. The color intensity in the
resulting chloranilir acid is proportionate to the amount of sulfate
nresent.
I'ether* ernloy.n Technicon AutoAnal^'zor vrith colorin.eter eruipned \rith
nn filters.
Limitations;
Range of Applicability; 10 to 400 mg/1 sulfate
Interferences; Calciiim, aluninum, iron interfere, but nay he
removed by ion exchange.
Statistical Characteristics; In a single laboratory (A<">c) , using
surface water samples at concentrations of 39, 111, 188, and 194 mg
SO^/1, the standard deviations were +0.6, +1.0, +2.2, and +0.8,
respectively.
Tine of Measurement; About 15 samples per hour.
Calibration Requirements; Requires preparation of standard solutions
and calibration curve.
Data Output; Analog electrical signal, recorded on stripchart.
Special Sampling Requirements (Collection, Storage, Handling);
Refrigerate at*4 C.
References:
1. Methods for Cherdcal Analysis of !
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93
SULFIDE
Name of Measurement Method: Titrinetrie (Iodine) Method
Principal Detection Technique; Titration
Purpose ofMeasurement^(ImportantApplications) ; Drinkinrr waters,
ambient surf ace vjaters7 Domestic ~and industrial wastes, saline waters,
Summary of Method; Sulfides are stripped from the acidified sample
withTan inert gas and collected in a zinc acetate solution. Excess
iodine added to the zinc sulfide suspension reacts with the sulfide
under acidic conditions. Thiosulfate is used to measure unreacted
iodine to indicate- the quantity of iodine consumed by sulfide.
Limitations:
Range of Applicability; Concentrations of sulfide above one mg/1
Interferences; Sulfites, thiosulfates, hydrosulfates, and other
reel need sulfur compounds may interfere.
P_it falls; Special Precautions; Sample should have minimal contact
with air or oxyqon.
Statistical Characteristics; Precision and accuracy for this method
have not been determined, but it is claimed that the iodimetric
titration of the zinc sulfide is quite accurate.
Djita Outputst Not stated. Assumed to be visual observations manually
recorded.
Special Sampling Requirements (Collection, Storacre, Handling);
Minimize contact with air.Preserve with zinc acetate unless
analysis is performed, immediately.
References:
1. Methods for Chemical Analysis of Water and Wastes.
1971. EPA National Environmental Research Center,
Cincinnati, Ohio.
2. Standard Methods for Examination of Water and
Wastewater. 1971. 13th Edition. American Public
Health Association, Washington, D. C.
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94
TURBIDITY
Name of Measurement net-hod; Hephelometric
Principal Detection Techninue; Photometric (TTephelometer)
Purpose of Measurement (Important Applications): This method is
applicable to surface and saline waters in the ""range of turbidity fron
0 to 40 Jackson units.
Summary of Method; The nethod is based upon a comparison of the
intensity^ of liqht scattered hv the sample under definite conditions
with the intensity of light scattered by a standard reference
suspension. The hiaher the intensity of scattered liaht, the higher
the turbidity. Readinqs, in Jackson units, are made in a nepholometer
designed accordinq to specifications outlined in Reference cited
below. A standard suspension of Formazin, also prepared under closely
defined conditions, is used as the turbidity reference suspension for
water because it is more reproducible than other types of standards
previously used for turbidity standards. Desiqn criteria for
instruments are given in Reference.
Limitations: Range of Anplicability; 0 to 40 Jackson units.
Sensitivity should be 0.02 units or less in waters havinq
turbidity of less than one unit.
Interferences; Floating debris, coarse sediments, air bubbles, or
colored material on solution may interfere.
Pitfalls; Special Precautions; Care should be exercised in
selection of instrument that meets criteria given in Reference.
Statistical Characteristics:
Accuracy and Precision: Data not available at time of publication
of Reference.
Time of Measurement: Not stated. Assumed rapid but not
instantaneous, because sufficient time must be allowed after
shaking sample for air bubbles to disappear but not for all
suspended particles to settle. (Five minutes or less).
Calibration Requirements; Follow instrument manufacturer's
recommendations,but reliance on manufacturer's solid scatterinq
standard is not always acceptable. ' See Reference below.
Data Outputs: Photoelectric detector (analog sianal) with ammeter
readout.
Special Sampling Requirements (Collection, Storage, Handling);
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95
Samples taken for turbidity measurements should be analyzed as soon as
possible. Preservation of samples is not recommended.
References:
1. .Methods for Chemical Analysis of Water and Wastes. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
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TEMPERATURE
Principal Detection Technique; Thermonetry
Purpose of Measurement (Important Applications): Drinkinq waters,
ambient surface v/aters, cloncstic and industrial wastes, saline waters.
Summary of Methodt Temperature measurements nay be made x-rith any good
grade of mercury-filleri or dial-type centigrade thermometer, or a
thermistor.
Limitations;
Range of Applicability; Thermometers or thermistors can be
obtained to give valid results over almost any ranne of ambient
temperatures.
Pitfalls; rnecial Precautions; Measurement device should be
checked against a precision thermometer certified by the National
Bureau of Standards.
Statistical Characteristics; There is no accentable procedure for
determining the precision and accuracy of this test.
Time of Measurement; Rapid
Data Outputs; Usually a visual reading manually recorded.
Instruments producing analog voltages automatically recorded are
readily available.
References;
1. i"ethods for Chemical Analysis of Water and Vastcs. 1971. EPA
National Environmental Research Center, Cincinnati, Ohio.
2. Standard Methods for Examination of TTater anc? Nastewater. 1971.
13th Edition. American Public Health Association, Washington, D.C
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97
THRESHOLD ODOR
Name of Measurement Method; Consistent Scries Method
Principal Detection Technique*; Personal Sensing
Purpose of Measurement (Important Applications); This method is
applicable to the determination of threshold odor or finished waters.
surface waters, domestic and industrial wastes, and saline waters.
Summary of Method! The sample of water is diluted with odor-free
water until a dilution that is of the least definitely perceptible
odor to each tester is found. The resulting ratio by which the sample
has been diluted is called the "threshold odor number" (T.O.).
Peoole vary widely as to odor sensitivity, and even the same person
will not be consistent in the concentrations he can detect from day to
day. Therefore, panels of not less then five persons, and preferably
10 or more, are recommended to overcome the variability of usinq one
observer.
As an absolute minimum, two persons are necessary: one to make the
sample dilutions and one to determine the threshold odor.
Limitations t
Range of Applicability; Highly odorous samples are reduced in
concentration proportionately before being tested. Thus, the
method is applicable to samples ranging from nearly odorless
natural waters to industrial wastes with threshold odor numbers in
the thousands.
Interferences; Chlorine in tap water and some wastewaters may
affect results. It is sometimes desirable to determine the odor
of the chlorinated sample and the same sample after removal of the
chlorine. Removal can be accomplished by use of sodium
thiosulfate in stoichiometric proportions.
Pitfalls? Special Precautions; It is important to check a blank
to which a similar amount of a dechlorinating agent has been added
to determine if any odor has been imparted. Such odor usually
disappears upon standing if excess reagent has not been added.
Statistical Characteristics;
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98
Accuracy and Precision; Data not available at the tine of
publication of Reference cited below.
Tine of 1 Teasurenent_s: Several hours.
Calibration Recniirenents; See Reference
Data Outputs; Personal detection of odor
Special Sampling Requirements (Collection, Storage, Handling);
Water sampTos ~"must bo collected in glass bottles \*ith glass or Teflon
lined closures. Plastic containers are not reliable for odor samples
and nust not be used. Complete tent as soon as possible after sannle
collection. If storage is necessary, fill container to the top and
refrigerate.
References;
1. .Methods for Chenical Analysis of TJater and Wastes.
1971. EP7\. National Environmental Research Center,
Cincinnati, Ohio.
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99
SPECIFIC CONDUCTANCE
Principal Detection Technique; Electrometric
Purpose of Measurement (Important Applications) ; Drinking water,
ambient surface waters, domestic and industrial wastes, saline waters.
Summary of Method; The specific conductance of a sample is measured by
use of a self-contained conductivity meter, Wheatstone bridged-type, or
equivalent. Samples are preferably analyzed at 25°C. If not,
temperature corrections are made and results reported at 25°C.
Statistical Characteristics; Forty-one analysts in 17 laboratories
analyzed six synthetic water samples containing known concentrations
of inorganic salts, with hnown values of specific conductance. The
followino results were obtained;
Known Cone, of
Specific Conductance
mhos/en
ion
106
80S
848
1640
1710
Precision as
Standard Deviation
mhos/cm
7.55
8.14
66.1
79.6
106
119
7\ccuracv as
Bias Bias,
% mhos/cm
•2.02
•0.76
•3.63
•4.54
•r>.36
•5.08
-2.0
-0.8
-29.3
-3H.5
-37.9
In a single laboratory (7*OC) , using surface water sanples with an
average conductivity of 536 mhos/en at 25°C, the standard deviation
was +6.
Calibration Requirements; Peter reading (analog electrical signal,
manually recorded).
References;
1. Methods for Chemical Analysis of Water and \7astes. 1971. FPA
National Environmental Research Center, Cincinnati, Ohio.
2. Standard Methods for Examination of Water and T-Tantevater. 1971.
13th Edition. American Public Health Association, Washington, D.C.
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100
IDrNTIFICATTON OF WTATJTFRTD OR I^TT-TTATITFREn OILS
Medium; V7ater, sediments, and tissue
Name of fleasurenent Method; Woods Hole Oceanographic Institute .Method
Principal Detection Technirmes; Has chromatography (flane ionization)
Purpose of Measurement (Important.Applications); Passive tagging of
oils J"
Summary of Method: Oils are dissolved in carbon disulfide and
injected into the gas chromatocrraphy rn. The column is 50 feet x 0.02
inches (open tubular support coated—SCOT) column packed with nonpolar
liquid silicone OTr-ini/ rated at 25,000 effective plates. Oil
chronatoqrans are compared visually, and certain features are
abstracted, tabulated and connared to the chronatograns generated by
candidate unvreathernd oil sanples.
LinLtations;
Interferences! nannies which have undergone unusual bacterial
alteration contain hirrh levels of indiaenous hvdrocarbons;
nixtnres of oils and samples having under gone umisually prolonged
vreatherina reauire special treatment.
Pitfalls; Special Precautions; Tabulated indices of oils that
exhibit considerable v/eathering should not be compared directly
vrith the indices of umreathered oils.
Statistical Chracteristics;
Accuracy; 16 out of 17 unveathered oils used for this test
acconpanied 35 sinulated oil spill samples. Correct "definite
correlation" was achieved in 74 percent of the cases, and only one
"probable correlation" vras incorrect.
Precision; Tabulated indices of individual oils remain quite
stable firon column to column.
Calibration Requirements; I'.ethod requires frequent evaluation of
system performance by Tnjoctinq a standard oil that yields a
repeatable knovjn chromatogram.
Data Outputs; Analoa instrument readina, graphically recorded.
Special Sampling Requirements (Collection, Storage, Handling);
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101
Sample should be refrigerated if held for more than a tow days. Glass
or metal containers are required for sampling.
References;
1. Zaifirion, 0. M. Bluner and J. Myers. 1972. Correlation of Oils
and Oil Products by Gas Chromatography. !>7oods Hole Oceanographic
Institution Report 62-55.
2. Kawahara, Fred K.b 1969. Laboratory Guide for the Identification
of Petroleum Products, Federal Water Pollut5.on Control
Administration, Division of Water Quality Research, Cincinnati,
Ohio.
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102
PF.TP.OLW OILF
llane c>* Measurement Method; Quantitative Analysis of Oil in VTater
Diversion
Prj . ncir>a 1 De t e c t ion Techniones; Infrared Spectrophotoretry
Purpose of ^easnxenont (Important Applications); Identification of
the particular oTl^and its cruantitative determinations in the water
column are essential to proper I1" monitor and assess potential
biological damage renultinrr fron oil spill incidents.
of Ilethod; Halt and acid added to the sample which is then
extracted v/ith carbon tetrachloricle or Freon 113, using a separatory
funnel. The extract is measured by infrared spectrophotometry.
Limitations;
Sensitivity; Minimum detection limit 0.05 mg/1
Interferences; (Freon or CCl^) - Any solvent extractable
organics. ~~
Pitfalls; Special Precautions: Carbon tetrachloride has a TLV of
10 ng/1.:
Statistical Characteristics;
Time of .Measurement:
Ca 1 ibr at ion Renu i rements; Freon is not usable for preparing II?
standards of heavy oils (2).
Comments by Users; Carbon tetrachloride is more efficient than Freon
ll3 for extracting high concentrations of viscous oils (1000
centistokes at 100 F) from water dispersion.
Data Outputs; Analog instrumental reading.
Special ^amplinrr Perruirements (Collection, Storage. Handling);
Sample is collected in a glass-stoppered bottle ancl acldified at the
tirae of collection.
Reference s_;
1. Kawahara, Fred K. 1969. Laboratory Guide for the Identification
of Petroleum Products, Federal Water Pollution Control
Administration, Division of Water Quality Research, Cincinnati,
Ohio.
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103
Principal Detection Techniques: Electron Canture, Cas Linuicl
Chronatography
Purpose of Measurement (Important Applications); To determine
presence and identification of pesticides in waters.
Summary of Method; The v;ater sample is subject to multiple extraction
by ethyl ether/hexane. The combined extracts are dried vrith anhydrous
sodium sulfate, evaporated under a stream of nitrogen, and further
concentrated by heating. Aliquots of about 5 ul are used for the
initial electron capture gas liquid chronatorrraphy. Further
identification may be necessary through microcoulometry of thin layer
chromatography.
Limitations;
Interferences; If interferences are indicated in initial
chronatograns, it may be necessary to conduct a Floricil clean up
on the extract.
Calibration Requirements; See Reference
Data Outputs; Analog electrical signal displayed as chromatogron.
Special Sampling Requirements (Collection. Storage* Handling); Sampling
collection glassware should be scrupulously cleaned. If storage is
necessary samples should be kept in cool, dark place or preferably in
a refrigerator.
Reference;
1. Thompson, J. F. (Editor). 1971. Analysis of Pesticide Residues
in Human and Environmental Samples. EPA Primate and Pesticides
Effects Laboratory, Perrine, Florida.
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104
PESTICIDES
Parameter(s) Measured; Organochlorine Pesticides
Aldrin
BCH
Chlordane
ODD
DDE
DDT
Dieldrin
Endosulfan
Endrin
Heptachlor
Heptachlor Epoxide
Chlorodane (tech)
riethexychlor
Perthane
Sulphenone
under some
circumstances;
Keithane
Strobane
Toxaphene
Principal Detection Techniques:
Electron Capture (EC)
Microcoulometric Titration (MC)
Electrolytic Conductivity (ECD)
Purpose of :ieasurement_(Important Applications); For identification
and quantitation of various organocnlorine pesticides, certain
degradations products, and related compounds.
Summary of Method; The method offers several analytical alternatives,
depending on the complexity of pesticides in the sample and the nature
and amounts of interferences. In general, the pesticides in the
aqueous sample are extracted by organic solvents. (e.g., hexane or
hexane/ethyl ether mixtures), and the extract concentrated by careful
evaporation of the solvent. Removal of interferences (when necessary)
and pre-separation of pesticide mixtures are accomplished by column
chromatography, thin-layer chromatography, or liquid-liquid
partitioning. Identification of pesticides in the mixture is made by
selective gas chromatographic separations through the use of two or
more unlike columns. Detection and measurement of pesticides in the
sample are accomplished by electron capture, rnicrocoulometric, or
electrolytic conductivity techniques. Quantitative results are
obtained by measurement of areas under peaks in the resulting
chromatogram.
The reference cited describes the use of an effective solvent for
extraction of pesticides from aqueous samples. It provides
information on the selection of appropriate clean-up procedures and
detectors for various types of pesticide mixtures, and suggests
techniques for confirming qualitative identifications.
Limitations:
Range of Applicability; Specific concentration ranges for various
pesticides in environmental samples are not cited, but the
detection capabilities of four types of detectors are indicated
below.
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105
Sensitivity and Detection Limits;
Electron Capture Detector picoqram (110 grar1)
quantities of many orqanochlorine pesticides
f'icrocoulonetric Titration 5-20 ng o^ orqanochlorine
pesticides
Electrolytic Conductivity Sensitivity Detector
2 to 3 tines as qreat as microooulometric procedure
Flane Photonetric Detector Sub-nanogram quantities
of sulfur and phosnhorxis
Sample responses less then tvo tines the detector noise level
(II) should be reported as negative; responses greater than 2F
should bo quantified if possible.
Interferences; Polychlorinatod biphenyls; phthalate esters,
organonhosphorus pesticides. The presence of the latter two
classes of connounds are implicated in snnples that resnond to
certain detection technioues but not to others.
Pitfalls; Special Precautions; Contaminants in solvents,
reagents, glassware, and other equipnent nay yield results that
cause misinterpretation of chromatograms.
Statistical Characteristics;
Accuracy; Hot stated as percent relative error, but given as
percent recovery in Ref.
Precision; Results are given for four specific pesticides. Those
for aldrin are as follovrs;
Mean recovery Precision (ng/liter)
llo cleanup
Florisil cleanup
Calibration Requirements; Procedtiros are detailed in Reference.
(ng/liter)
10.42
79.00
17.00
64.54
Overall Sinqle Cperatio
4.86
32.06
9.13
27.16
2.59
20.19
3.48
R.02
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106
Connonts by Usf»rs; Reconnendod for une only b", or under close
supervision of, experienced residue analysts.
Data Outputs; Analog electrical signals recorded on strincharts.
Special Sampling Reguirenents (Collection, fltorago/ ITand.ling); T^ide-nouth
ipllnq
:h Tef
bottlon vrith Teflon-lined screv caps are used for sanplo collection.
References;
1. !*ethoc!s for Organic Pesticides in Uater and WaFtevater. 1P71.
EP.A National Environmental Research Center, Cincinnati, Ohio.
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2. BIOLOGICAL METHODS
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107
PHYTOPLANKTON AND PERIPHYTON
CELL COUNTS AND IDENTIFICATION
Principal Detection Technique; Visual Observation
Purpose of Measurement (Important Applications); Provide information
on the standing crops; indicator organisms and community diversity.
»
Summary of Method; A 24x60 nun, No. 1 coverglass is placed diagonally
across the S-R cell and a large bore pipet or eyedropper is used to
transfer a one/ml aliquot of a well-mixed sample into the open corner
of the Sedgwick-Rafter chamber. The S-R cell is allowed to stand for
at least 15 minutes to permit settling. The analysis may then proceed
in either of two ways: (1) depending on the density of organisms, two
to four "strips" the length of the cells are examined and all forms
that are totally or partially covered by the whipple-grid are
enumerated; or (2) a minimum of ten random whipple fields are examined
in at least two identically prepared S-R cells and forms that are
totally or partially covered by the whipple grid are enumerated.
Limitations;
Range of Applicability; Phytoplankton and periphyton
Interferences: In samples where algae concentrations are extreme
or where turbidity is high the sample must be diluted or
concentrated.
Pitfalls; Special Precautions; The depth of the counting chamber
precludes the use of the 45x or lOOx objectives. Collection of
phtoplankton by nets or pumps is not recommended.
Statistical Characteristics;
Precision; Provided reasonably reproducible information when used
with a calibrated microscope with an eyepiece measuring device.
Time of Measurement; 1 hour
Calibration Requirements; Each combination of oculars and objectives
is calibrated against a stage micrometer.
Data Outputs; Manual count
Special Sampling Requirements (Collection, Storage, Handling);
The pretreatment of the sample depends on the concentration of
organisms present. When phytoplankton densities are less then 500/ml,
approximately 6 liters of sample are required.
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108
References;
1. Biological Field and Laboratory Methods. 1972. iiPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. Preliminary Draft.
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109
VOLUME OF PERIPHYTON
Principal Detection Technique; Volumetric
Purpose of Measurement (Important Applications); Estimate of the
standing stock of periphyton.
Summary of .-lethod; A known volume of water is added to a thoroughly
drained sample. The difference between the volume of the periphyton
samples plus the added water and the volume of water alone is the
volume of the total amount of periphyton in the sample.
Limitations;
Range of Applicability; Periphyton/ only when large growths of
periphyton permit removal of excess water readily.
Pitfalls; Special Precautions; Excess fluid should be removed
from the sample.
Calibration Requirements; Graduated cylinder should be used for
volume measurements.~
Data Outputs; Volume measurement (visual)
Special Sampling Requirements (Collection/ Storage, Handling); The
preferred preservative is neutral or slightly basic formalin.
References;
1. Biological Field and Laboratory Method. 1972. EPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. Preliminary Draft.
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110
CELL COUNTS AND IDENTIFICATION OF PHYTOPLANKTON
Principal Detection Techniques; Visual observation
Purpose of Measurement (Important Applications); Provides information
on standing crop^indicator organisms and community diversity.
Summary of Method; The sample is introduced into cells which are
precisely matched glass slides with a finely-ruled grid on a counting
plate fitted with a specially ground cover slip. All forms which fall
within the griclded area of the cell are identified and counted. The
number of the various organisms found in the gridded area of the cell
is multiplied by the appropriate factor to obtain the count.
Limitations;
Pitfalls; Special Precautions; The analyst is advised to follow
carefully the specific directions accompanying the chamber or
cell. Collection of phytoplankton by nets or pumps is not
recommended. For statistical purposes a normal sample must be
either concentrated or a large number of mounts per sample should
be examined.
Statistical Characteristics;
Time or Measurement; 1 hour
Data C)utputs; Manual count
Special Sampling Requirements (Collection/ Storage, Handling;
Depends upon the location and type of sample. See Reference. Samples
must be concentrated when densities are below 500/ml. Approximately 6
liters of sample are required.
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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Ill
CELL COUNTS AND IDENTIFICATION OF PHYTOPLANKTON
Principal Detection Technique; Visual observation (Filter Method)
Purpose of Measurement (Important Applications); Provides information
on standing crop^indicator organisms and community diversity. Method
permits the use of high magnification for enumeration of small
plankton.
Summary of Method; A water sample of known volume is passed through
the membrane filter under a vacuum. The filter is allowed to clear
and the organisms enumerated. The occurrence of each species in 30
random fields is recorded and multiplied by a conversion factor to
obtain the total count of each species.
Interferences; Significant amounts of suspended matter may
obscure or crush the organisms.
Pitfalls; Special Precautions; In coastal and marine waters the
filter is rinsed with distilled water to remove salt. Collection
of phytoplankton by nets or pumps is not recommended.
Statistical Characteristics;
Precision; + 16% CL
Time of Measurement; Relatively rapid processing of samples, (1
hour) although total time required for the complete analysis is
48-50 hours.
Calibration Requirements; Use stage and ocular micrometers
Data Outputs; Manual count
Special Sampling Requirements (Collection, Storage, Handling;
Depends upon the location and type of sample taken.See Reference.
Samples must be concentrated when phytoplankton densities are less
than 500/ml. Approximately 6 liters of sample are required.
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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112
CULL COUNTS AND IDENTIFICATION OF PilYTOPWviJKTOIJ
Principal Detection Technique; Visual observation (Countiny Chamber Method^
Purpose of Measurement (Important Applications); Provides information
on algal standing crop^indicator organisms and community diversity.
Summary of Method; The method uses an inverted microscope in
conjunction with a cylindrical counting chamber with a clear glass
bottom. A sample is transferred to the counting chamber and allowed
to settle. The chamber is placed on the microscope stage and examined
using either the 20x, 45x or lOOx oil immersion lens. Either the
strip or random field counts are made.
Limitations;
Interferences; None identified
Pitfalls; Special Precautions; Collection of phytoplankton by
nets or pumps is not recommended.
Statistical Characteristics:
Time of Measurement; 4 hours per 10 mm of sample height in the
chamber is allowed for sedimentation. Count requires 1 hour.
Calibration Requirements; Use stage and ocular micrometers
Data Outputs; Manual count
Special Sampling Requirements (Collection, Storage, Handling);
Depends upon the location and type or sample taken. Se«* Rpfpr*»nrw».
References;
1. Biological Field and Laboratory Methods. 1972. UFA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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113
CELL VOLUME ESTIMATES OF PLANKTON AND PERIPHYTOH
Principal Detection Technique; Visual
Purpose of Measurement (Important Applications); Standing crop
estimate gives an indication as to water productivity.
Summary of Method; An aliquot of sample is concentrated and examined
wet at a 1000 x magnification with a microscope equipped with a
calibrated ocular micrometer. The optical measurements are made with
the micrometer. The average volume per organism is determined and
multiplied by the number of organisms per millilitar.
Limitations;
Range of Applicability; Phytoplankton, bacteria, periphyton
Calibration Requirements; The exact magnification with any set of
oculars must be calibrated.
Data Outputs; Visual examination
Special Sampling Requirement (Collection, Storage, Handling):
The preferred preservative is neutral or slightly basic formalin.
Reference:
1. Biological Field and Laboratory Methods, 1972. LJPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. {Preliminary Draft).
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114
PERIPIIYTON ATID Pr^
(Species Composition)
Principal Detection Technique; Visual examination
Purpose of Mea5urement (Important Application); Thr identification of
individual species of phytoplankton nay provide information on
indicator organisms, species diversity, and therefore the decree of
pollution.
£unnary of "nthocl; Folio'v.rv an initial examination of the sample to
obtain" rr> -rtirato of population etc., the phytoplankters are identified
to the desired taxonomic level and tallied under a standard system.
Limitations;
Range of Applicability; Plankton, periphyton
Pitfalls; Pnocial Precautions! Examination if? preferably done
before sample is preserved.The beginner is strongly vjarned
against the deceiving and nonvalid simplicity often found in the
identification of plankton.
Statistical Characteristics;
Time of .Measurement; 1 hour
Data Outputs; Numbers of species and the number of individuals per
species
Special .Sampling Requirement (Collection, Storage, Hand ling);
In cases; \7liere it has been shown that preservation has no effects on
the identification of organisms, a preservative should be used if
storage is necessary.
References:
1. Biological Field and Laboratory I'ethods. 1972. EPA national
Environmental Research Center /Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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115
DIATOM SPECIES IDENTIFICATION
Principal Detection Technique; Visual Observation
Purpose of Measurement (Important Applications); Provides information
on indicator organisms .and community diversity.
Summary of Method; The diatoms are concentrated by centrifugation
followed by sedimentation. The diatom concentrate is placed on an 18
mnrcpverglass and dried on a hotplate at 95°C followed by the
oxidization of organic matter. The hot coverglass is inverted and
placed on a drop of hyrax on a 25x75 mm microscope slide. A
protective coating of clear lacquer is sprayed on the frosted end of
the slide and the excess hyrax is scraped from around the coverglass.
Two hundred fifty diatoms are identified and counted at high
magnification under oil. If the slide has very feu diatoms the
analysis is limited to the number of cells encounted in 45 minutes of
scanning.
Limitations;
Range of Applicability; Diatoms
Interferences; Silt
Pitfalls; Special Precautions; If the dried sample is obscured by
soluble solids,the sample should be washed with distilled water.
Statistical Characteristics;
Time of Measurements; 1 hour
Calibration Requirements; Ocular and stage micrometer
Data Chitputs; Number of individuals per species
Special Sampling Requirements (Collections, Storage, Handling);
Depends upon the location and type of sample taken/ see Reference. If
plankton counts are less than 1000 per ml, the diatoms should be
concentrated from a larger volume of sample (one to five liters) by
allowing them to settle out.
References;
1. Biological Field and Laboratory Methods. 1972. LPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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116
CELT. COUIITF A*TD IPniTIFIC^TIOlT OF PITyTOPLATT7rTO!I
Principal Detection Techniques; Visual observation
Purpose of Measurement (Important Applications); Provides information
on standing crop indicator" organisms and community diversity.
Summary of Method; An aliquot of well nixed sample is introduced into
one of the '2x"5~~nm channels on either side of the circular Palner-
Ilaloney Hannop lank ton cell <-:'.th cover slip in place. After 10
minutes, the sanple is examined under the high-dry objective (45x)
and at least 20 v;hippie fields arc counted.
Limitations;
Range of Applicability! The circular chamber used v/as especially
designed for enumerating nannoplanJ'ton with a hiqh-dry obiective
(45:0.
Pitfalls; Special Precaiitions; The cell should not be used for
routine counting unless the samnles have counts exceeding
20,000/ml. Collection of phytoplankton by net or punps is not
recommended.
Statistical Characteristics;
Time of Iteasurenent; 1 hour
Calibratic-n Requirements; The microscope is calibrated using an
ocular and stage micrometer,
Comments by Users;
Data Outputs; .Manual counts
Special Sampling Requirement (Collection, Storage, Handling);
Depends upon the location and type of" sample. See Reference. .Camples
must be concentrated when densities are below 500/ml. Approximately G
liters of sample are reauired.
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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117
CHLOROPHYLL a OF
(In vitro)
Name of feanuromnnt Method; Monochromatic Method
Principal Detection Technique*; Fnectrophotometric
Purpose of Measurement (Important Applications); All algae contain
chlorophyll a. Measurement of this pigmpir.t can yield an estimate o*
standing crop.
Summary of .Method t Chlorophyll a can he cr.tinatnd independently of
the other chlorophylls by measuring the optical density of the pigment
extract of C>C<5 nr only, and inserting it into the followino equation:
C*,= 13.4D665. (P665 is the optical density, corrected for turbidity
bv substractinn the OP nm blank).
7eo
Linitations;
Pitfalls; Special Precautionn; Precautions should bo taJ^en to
min in i zo evapora tio n. Phebphyt in a natural dooradation product
of chlorophyll, has an absorption peaJ- in the sane region of the
visible spectrum as chlorophyll a and could be a source of error
in chlorophyll determinations.
Calibration Requirements; No unusual requirements
Data Outputs; Analog or digital electrical signal displayed on peter,
on tape or recorder.
Special Sampling Requirements (Collection, Ptoragn. Handlina);
Spec
The
type of sampling equipment used is highly dependent upon where and
how the sample is being taJ'en. If the analysis will be delaved, storo
the sample frozen in the darh to avoid, photochemical breakdown of tho
chlorophyll.
References;
1. Biological Field and Laboratory .Methods. 1972, EPA rational
Environmental Research Center, Analytical Puality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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118
CHLOROPHYLL a OF PHYTOPLANKTON
Principal Detection Technique; Spectrophotometric
Purpose of Measurement (Important Applications); All algae contain
chlorophyll a_. Measurement of this pigment can yield an estimate of
standing crop.
Summary of Method; Chlorophyll a_ can be estimated independently of
the other chlorophylls by measuring the optical density of the pigment
extract of 665 nm only, and inserting it into the following equation:
Ca,= 13.41)4,6,*- (D^^r is the optical density, corrected for turbidity by
subtracting the OD 750 nm blank).
Limitations;
Pitfalls; Special Precautions; Precautions should be taken to
minimize evaporation.Pheophytin a natural degradation product
of chlorophyll, has an absorption peak in the same region of the
visible spectrum as chlorophyll a, and could be a source of error
in chlorophyll determinations.
Calibration Requirements; No unusual requirements.
Data Outputs; Analog or digital electrical signal displayed on meter,
on tape or recorder.
Special Sampling Requirements (Collection, Storage, Handling);
Tho type of sampling equipment used is highly dependent upon where and
how the sample is being taken. If the analysis will be delayed, store
the sample frozen in the dark to avoid photochemical breakdown of the
chlorophyll.
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Uuality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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119
ZOOPLANKTOH VOLUME AND SPECIES IDENTIFICATION
Purpose of iMeasurement (Important Applications) ileasurement of
zooplankton. Volume provides an index to the standing crop (biomass)
of natural zooplankton population.
Summary of Method; The sample is screened, placed into a conical
container graduated in milliliters, allowed to settle for five
minutes, and the settled volume is recorded. The sample is then
stirred and a one ml subsample is withdrawn from the container with a
Stempel pipette. The subsample is examined under a dissecting
microscope for enumeration of zooplankton.
Limitations;
Range of Applicabilityt Rotifera, cladocera, copepods and other
large zooplankton forms.
Statistical Characteristics;
Tine of Measurement; Less than 1 hour
Data Outputs; Visual observation, manual reading
Special Sampling Requirements (Collection, Storage, Handling);
Depends upon location and type of sample taken. See Reference.
Reference;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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120
III SITU PRODUCTIVITY OF PHYTOPLAMKTOH
Principal Detection Technique; Radioactive assay
Purpose of Measurement (Important Applications)! Phytoplankton
productivity measurements indicate the rate of uptake of inorganic
carbon by phytoplankton during photosynthesis and are useful in
determining the effects of pollutants and nutrients on the aquatic
community.
Summary of Method; A solution of radioactive carbonate is added to
light and dark bottles which have been filled with aarnples taken from
preselected depths in the euphotic zone. Following in situ incubation
(up to four hours) the plankton is collected on a membrane filter,
dried in a desiccator, and assayed for radioactivity. The quantity of
carbon fixed is proportional to the fraction of radioactive carbon
assimilated. If measurements are required for the entire photoperiod,
samples may be taken at overlapping four-hour periods or the data may
be adjusted based on the solar radiation profile.
Limitations;
Range of Applicability; Phytoplankton
Pitfalls; Special Precautions; Carbon in the filtered sample
should yield the number of counts required for statistical
significance.
Statistical Characteristics;
Sensitivity; Method is more sensitive than the oxygen method, but
fails to account for the organic materials that leach from cells
during the incubation period.
Time of Measurement; Sample should be incubated for at least two
hours.
Comments by Users; Method affords a direct measurement of carbon
uptake and measures only photosynthesis.
Data Outputs; Instrument count
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121
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center/ Analytical quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
2. Standard Methods for the Examination of Water and Wastewater.
1971. 13th Edition. American Public Health Association,
Washington, D. C.
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122
IN SITU PRODUCTIVITY OP PHYTOPLANKTON
Name and Measurement Method; Oxygen method
Purpose of Measurement (Important Applications); Phytoplankton
productivity measurements indicate the rate of uptake of inorganic
carbon by phytoplankton during photosynthesis and are useful in
determining the effects of pollutants and nutrients on the aquatic
community.
Summary of Method! Samples are taken from preselected depths in the
euphotic zone and placed in duplicate clear, darkened and initial
analysis bottles. The duplicate clear and darkened bottles are
suspended at the depth from which the samples are taken and allowed to
incubate. At the end of the exposure period the dissolved oxygen of
the samples is measured. The increase in oxygen concentration in the
light bottle during incubation represents net production and the loss
of oxygen in the dark bottles is an estimate of respiration.
Limitations;
Range of Applicability; Phytoplankton
Pitfalls, Special Precautions; The incubation period should not
be long enough to allow oxygen gas bubbles to form in the clear
bottles or dissolved oxygen to be depleted in the dark bottles.
The solar radiation profile in addition to the photosynthetic rate
during the incubation period should be used to adjust data to
represent productivity for the entire photoperiod.
Statistical Characteristics;
Time of Measurement; Samples should be incubated for at least two
hours.
Comments by Users; Chief advantages of the oxygen method are that it
provides estimates of gross and net productivity and respiration and
that the analysis can be performed both with inexpensive laboratory
equipment and common reagents.
Data Outputs; Visual observation (titrimetric) or analog or digital
electrical signal (electrometric DO method).
Special Sampling Requirements (Collection, Storage/ Handling);
If the dissolved oxygen is not measured immediately, it should be
fixed and protected from direct sunlight.
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123
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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124
PHYTOPLAUKTOIJ AND PLRIPHYTON SPECIES COMPOSITION
Phytoplankton and Periphyton Species Composition
Principal Detection Technique; Visual examination
Purpose of Measurement (Important Applications); The identification
of individual species of phytoplankton may provide information on
indicator organisms, species diversity, and therefore the degree of
pollution.
Summary of Method: Following an initial examination of the sample to
obtain an estimate of population density, etc., the phytoplankton are
identified to the desired taxonomic level and tallied under a standard
system.
Limitations;
Range of Applicability; Plankton, periphyton
Pitfalls; Special Precautions; Examination is preferably done
before sample is preserved. The beginner is strongly warned
against the deceiving and nonvalid simplicity often found in the
identification of plankton.
Statistical Characteristics;
Time of Measurement; 1 hour
Data Outputs; Number of species and the number of individuals per
species
Special Sampling Requirements (Collection, Storage,^ Handling]I ;
In cases where it has been shown that preservation has no effects on
the identification of organisms, a preservative should be used if
storage is necessary.
References;
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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125
CHLOROPHYLL a, b, AND c OF PHYTOPLANKTON
AND PERIPHYTON
Name of Measurement Method; In Vitro - Trichromatic Method
Principal Detection Technique; Spectrophotometrie
Purpose of Measurement (Important Applications); All algae contain
chlorophyll.Measurement of these pigments can yield an estimate of
standing crop and taxonomic congestion.
Summary of Method; The sample is concentrated/ macerated, steeped in
90% acetone, at 4*C for 24 hours, and clarified by centrifugation.
The optical density of the decanted extract is determined at 750, 663,
645, and 630 nm using 90% acetone blank. The 750 nm reading is used
to correct for turbidity and the other readings are inserted into
SCOR/UNESCO equations for calculating chlorophyll a, b, and c_.
Limitations;
Pitfalls; Special Precautions; Precautions should be taken to
minimize evaporation.Phebphytin, a natural degradation product
of chlorophyll, has an absorption peak in the same region of the
visible spectrum as chlorophyll a and could be a source of error
in chlorophyll determination.
Statistical Characteristics;
Calibration Requirements; No unusual requirements.
Data Outputs;
Data Outputs; Analog or digital electrical signal
Special Sampling Requirements (Collection, Storage, Handling);
If the analysis will be delayed, store fro2en.The stored samples
must be kept in the dark to avoid photochemical breakdown of the
chlorophyll. Maximum storage time is 30 days.
References;
1. Biological Field and Laboratory Methods. 1972. LPA National
L'nvironmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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126
CELL SURFACE AREA OF PHYTOPLANKTON
Principal Detection Technique; Visual observation
Purpose of Measurement (Important Applications); Determination of
cell surface area of phytoplankton is another indicator of plankton
abundance.
Summary of Method; Measure the dimension of several representative
individuals of each major species microscopically. From the linear
dimensions compute the average surface area per species. Multiply the
area per species by the number of organisms per mi Hi liter.
Limitations;
Range of Applicability; Phytoplankton.
Calibration Requirements; Ocular and stage micrometric
Data Outputs; Visual observation
Special Sampling Requirements (Collection, Storage, Handling);
Depends upon the location and type of sample taken, see Reference.
References:
1. Biological Field and Laboratory Methods. 1972. EPA National
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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127
BIOMAES OF MICROPHYTES
Name of Measurement Method; Dryweight biomass
Principal Detection Techniques; Gravimetric
Purpose of Measurement (Important Applications)! Estimates of growth
rates related to pollution such as nutrient stimulation, can be
accomplished by standing crop estimates at predetermined intervals.
Summary of Method; A sample is taV-en from a small defined area with
conspicuous borders. The wet weight of material is obtained after the
plants have drained for a period of time. The sample is then dried
for 24 hours at 105°C and reweiohed. The dry weight of vegetation per
unit area is then calculated.
Data Outputs; Mechanical scale reading
Special Sampling Requirements (Collection. Storage, Handling);
Before beginning a miantitative investioation it is desirable to have
a statistical study design which will assist in determining the best
sampling procedure, sampling area size, and number of samples.
References:
1. Biological Field and Laboratory Methods. 1972. FPA national
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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128
ALOAT, GROWTH POTENTIAL
Name of Measurement; Bottle test method
Principal Detection Techniques; Volumetric, Gravimetric, etc.
(depends on method used to indicate algal rrrowth).
Purposeof Measurement (Important Applications); Alqal assays are
conducted to determine the effects of various discharges on the growth
of algao or to determine whether or not varionr, compounds or water
samples are toxic or inhibitory to algae,
Summary of M.ethnd; 7^fter inoculation of test organisms the sample is
incubated at 24 4- 2°C under cool white fluorescent lighting. During
incubation, algaT grov/th is determiner! by cell counting, "or algal
hionass or the chlorophyll analynis methods at predetermined
intervals. The collected data are used to determine the maximum
standing crop and the maximum specific growth rate.
Limitations;
Pitfalls, Special.. Pro cautions; T'Then trace nutrients are being
studied special galssware such as Vycor or polycarbonate
containers should be used.
Statistical Characteristics;
Precision; Excellent aareer.ent in data using this method was
obtained by eight participating laboratories.
Time of .Measurement! The analysis continued until there is less
than 5 percent per day increase in biomass.
Calibration Requirements; Fee individual method in Reference cited
below.
Data Outputs; Visual counts
Special Sampling Requirements (Collection, Storage, Handling);
Sample pretreatment is~'"required when determinations are being made
6f (1) growth limiting soluble nutrients or (2) amount of algal
biomass then can be grown from all nutrients in waters.
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129
References;
1. Biological Field and Laboratory Methods. 1972. FPA national
Environmental Research Center, Analytical Quality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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130
CHLOROPHYLL a OF PlIYTOPLAiJiC'TON
(Fluorescence)
IJane of Measurement Ilethod; In vivo method
Purpose of Measurement (ImportantApplications); All algae contain
chlorophyll a_. Measurement of this pigment can yield an estirate of
standing crop.
Limitations;
Rangu of Applicability; Scale deflection should be between 15 and
90 units.
Interferences; Phaeohytin
Pitfalls; Special Precautions; This method ia less efficient than
the extraction method, yielding about one-tenth as much
fluorescence per unit weight as the same amount in solution.
Statistical Characteristics;
Sensitivity; Method is more sensitive than the spectrophotometer
method.
Precision; The precision of the method using natural population
shows for ten samples, a maximum variation of 15 percent.
Time of Measurement; Less than 2 hours.
Calibration Requirements; The fluorometer should be calibrated with a
chlorophyll extract which has been analyzed with a spectrofluorometer.
Comments by Users;
Data Outputs; Analog or digital electrical signal
Special Sampling Requirements (Collection, Storage. Handling);
Prolonged storage or extracts(greater than 5 days;under
refrigeration and darkness should be avoided. Fluorescence of
extracts kept at room temperatures and in light are stable for at
least ten hours.
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131
Hcferences;
1. Yentsch, C. G. and D. W. Menzel. 1963. A Method for the
Determination of Phytoplankton Chlorophyll and Phaeophytin by
Fluorescense. Deep-Sea Research 10; 221-281.
2. Biological Field and Laboratory Methods. 1972. iiPA National
Environmental Research Center, Analytical Duality Control
Laboratory, Cincinnati, Ohio. (Preliminary Draft).
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3. BIOASSAY PROCEDURES
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132
3. BIOASSAYS
The purpose of this section is to discuss some available bioassay
procedures for determining "safe" levels of pollutants. Bioassays are
essential to evaluate a qiven pollutant in terms ot existing water
quality, including environmental variables as well as pollution
already present. Pertinent to this stance is the fact, that the
majority of specific pollution problems are those involving discharge
of unknown and variable composition. Almost without exception, more
than one toxicant or stress is present. It is believed that "safe"
levels of toxic pollutants, predicated upon bioassays with application
factors, adequately protect, aquatic life, if the basic physical
parameters, e.g., dissolved oxygen, temperature-, and pH are within the
limits recommended. Tf the latter parameters are outside recommended
limits, appropriate alterations in the criteria for toxicants must be
made.
Acute ly^Lethal^Versus^^Sate^Cgncentrat ions
Harmful effects of pollutants may be divided into a number of
categories which overlap somewhat; most could be described accurately
by using one or two of the following terms, all of which have good
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133
dictionary meanings. In the present context of toxicity to aquatic
life, the terms might be briefly defined as follows:
t
acute - involvinq a stimulus, severe enough to bring about a
response speedily, usually within four days for fish;
subacute - involving a stimulus which is less severe than an acute
stimulus, and which produces a response in a longer time; may become
chronic
chronic - involvinq a stimulus which is lingering or continues for
a long time; often signifying periods of about one-tenth of tne life
span or more;
lethal - causing death, or sufficient to cause it, by direct
action;
sublethal - below the level which directly causes death;
cumulative - brought about, or increased in strength, by
successive additions.
threshold - the point at which a physiological effect begins to be
produced.
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These and other terms have sometimes been used in careless or
conflicting ways, but they should be used with precision. In general,
two broad categories of effects may be distinguished; acute toxicity,
which is usually lethal, and chronic toxicity, which may be lethal or
sublethal (1).
Most of the available acute toxicity data are reported as the
median tolerance limit (TLm) or median lethal concentration (LC50) .
Either symbol signifies the concentration at which 50 percent of the
test organisms survive within a specified time span, usually in 96
hours. The customary 96-hour (four day) time period is recommended as
adequate for most routine tests of acute toxicity with fish and the
lethal threshold concentration attained within this time is usually
reported (2). Mortality tests of a week or longer are necessary at
times to determine the threshold concentrations. The lethal threshold
concentration, as well as absence of any threshold concentration,
should be reported. The lethal threshold concentration is more useful
for comparative purooses than the arbitrary LC50.
This system of reporting is merely a convenient reference point
for expressing the acute lethal toxicity of a given toxicant tor the
test animal. Usually, the "safe" levels (those which permit
reproduction, growth, and all other normal life-processes in the
fish*s natural habitat) are much lower than the LCSO.
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135
Data on both lonq-term effects and safe levels are available for
only a few toxicants. Some information is now becoming available on
the effect of toxicants on reproduction, a very important aspect of
all long-term toxicity tests. Also important is the sensitivity of
the various life staqes of organisms. Many organisms are most
sensitive in the larval, nymphal, molting, or fry stage; some may be
most sensitive in the egg and sperm stage.
It would be desirable if a single, universal, rapid, biological
test could be used to directly measure sublethal effects of a
pollutant. A large number of sublethal responses of fish have been
used, i.e. specific physiological and biochemical changes in various
body systems, and histological studies. A review of these shows that
unfortunately, no single test is meaningful for all kinds of
pollutants (3).
variabl,e_ConcentraMon
Criteria for continuously acceptable concentrations must be lower
than the intermittent higher concentrations that may be reached
occasionally but briefly without causing damage. One way in which to
resolve this difficulty is to use both maximum short-time
concentrations and also a more restrictive range of safe
concentrations for continuous exposure. It is recognized that
extremes do limit organisms, but, within these extremes there are
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ranges of concentration that can be tolerated, and are sate for
prolonged periods of exposure. Most of r.h^> criteria recommended in
Volume I of this report are those thouqht to be safe for continuous
exposure.
In field situations and industrial operations, average 21-hour
concentrations can be determined by using a small water pump to
collect small aliquots of a few ml every few minutes, or to collect a
slight trickle continuously. After 21 hours, the sample is mixed and
analyzed and the concentration found represents the average
concentration. Samples obtained this way are more reproducible and
easier to secure than t.he maximum instantaneous concentration. But
average concentrations are of little significance if fish are killed
by a sharp peak of concentration; hence the need to determine maximum
concentrations.
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METHODS OF BIOASSAY
The use of bioassays to det^rmiac.- the to::icitv of. «« material or
waste is the most effective and accurate method of predicting or
assessing potential danger. In use of a bioasray, no assumptions need
be made concern inq the chemical structure or form of the pollutant,
nor does the investigator have to hnov; the constituent substances.
The effects of water quality on toxicii-y also rray be measured. of a
toxicant in water, the more precise the assay can be.
li233say^_to_Determine_AcuteJ.Y_Lethal_Concent rations
While there are many types of assays, two are in general use: the
static bioassay in which the organisms are held in a -t-.ank of standing
test-solution, which may or may not be changed during the period of
the test; and the continuous flow or flow-through bioassay in which
the test solution is renewed continually, occasionally the difference
between the two types is not very great, but sometimes one has clear
advantages over the other.
A recommended outline of methods for routine bioassays is given by
"Standard Methods" (U) . Methods for more searching tests and for
research purposes have been critically reviewed (3,5,6). The items
which should be described in bioassay reports have been published
(7,8).
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The procedure for acute bioassay with fish is becoming
standardized. Almost, without exception, present-day tests
incorporate:
1) a series of test containers, each with a different but constant
concentration of the toxicant;
2) a qroup of similar fish, usually ten, in each container;
3) observations of fish mortality or other detrimental responses
during exposures which last between 1 day and about 1 week, often four
days; and
1) final results expressed as concentration tolerated by the
median or "average" fish.
Beyond this, there are many other factors which make for good
practice, and these are summarized in the references cited above. In
essence, the items cited below serve as a checklist of good bioassay
procedure.
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Species
A fish or other aquatic organism of local importance snould he
used in bioassays. Preferably the test organism should be a game or
panfish, since they are generally among the more sensitive species.
Ability to duplicate an experiment would be enhanced if one used a
selected strain of fish (9). A selected strain would also enable one
to determine the difference between toxicants more reliably and to
better determine the cause for variation in results due to different
test apparatus. However, a selected strain does account for different.
sensitivities due to the response to other stresses such as disease,
population stress, etc., which may be effecting a resident population.
Differences between fish species in susceptibility to a toxicant
are generally less *-han miqht be expected - sometimes no greater than
the variability within a single species tested in different types of
water. For example, trout and carp are about equally resistant to
some toxicants if tests continue for several days, giving the "coarse"
species time to react (10,11). On the other hand, larvae and
juveniles are most often more sensitive than adults and may be
preferred for the test procedure. For projecting entire ecosystems,
criteria suitable for the selected test fish will often also be
suitable for other aquatic animals and plants. Thera are exceptions
to this generalization. For example, ccpper is guite damaging to
algae and molluscs, and insecticides are especially dangerous to
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aquatic arthropods. There are data to allow one to predict these
situations. When they are expected, bioassays should also be run with
invertebrates and algae (12).
When possible, the bioassay should te conducted in waters obtained
from the water body of concern since characteristics of me water,
e.g., hardness, are included in the test results. Work ot less
immediate application, for example, when attemptinq to predict the
toxicity of a new waste under various conditions, a selection of water
types should be tried. *here is considerable merit in using both hard
and soft, synthetically prepared dilution waters (13) or varying the
salinity of marine systems. There is great danger in using "tap
water" for dilution, if it has been chlorinated. Even d ech lor i nation
procedures do not always remove the last traces of chlorine, and much
of the past toxicity research is accordingly suspect.
Tap water can contain dangerous concentrations of copper, zinc or
lead from plumbing.
Variations in other physical-chemical characteristics of water
such as temperature, organic content and pH commonly atfect toxicity
of pollutants. Effects of five such environmental entities on the
lethal threshold of ammonia were illustrated a decade ago (13, 11).
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Water hardness is particularly important in toxicity of metals.
Hydrogen ion concentration is a most important modifying factor for
ammonia and cyanide. Higher temperatures sometimes increase toxicity
of a pollutant, but recent work shows that phenol, hydrogen cyanide,
ammonia, and zinc may be more toxic at low temperatures (15).
In static tests there should be two or three liters of water per
gram of fish, changed daily, or increased proportionally in volume for
the number of days of the test. In continuous flow tests, the flow
must provide at least two or three liters of water per gram of fish
per day, and it must egual test-volume in five hours, giving 90
percent replacement in half a day or less.
Acclimation
Acclimation of the test organism to the new environmental
situation encountered before the bioassay begins may have a marked
effect upon the outcome. Abrupt changes of quality of holding water
should be avoided. Time for acclimation of the organisms to the new
test conditions including temperature, should be as generous as
possible and dependent on life-span, at least two weeks is recommended
for fish.
Test Methods
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These must be adequately described when the results are given and
the easiest way is to follow one of the recommended bioassay
procedures in Table 1.
Controls
Adequate and appropriate control tests must always be performed
(2). Survival of control organisms is a minimum indication of the
quality of test organisms. In addition, levels of survival and health
in holding tanks should be indicated and the conclusions recorded.
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TABLE 1
LITERATURE SOURCES FOP BIOASSAY AND BIOMONITORING
PROCEDURES WITH VARIOUS AQUATIC ORGANISMS
Kind of Type of
§E Response
Appropriate Situations
for_use
Reference
Fish and
Macroin-
vertebrates
96 hour lethal
concentration
To measure lethal toxicity
of a waste of known compostion,
to serve as a foundation
for extrapolating to pre-
sumably safe concentrations.
Fish and
Macroin-
vertebrates
Lethal threshold
concentration
For research applications
for document lethal thres-
holds.
2, 5
Fish and
inverte-
brates
Incipient lethal
temperatures and
ultimate incipi-
ent lethal
temperatures
Research to determine
lethal temperature ranges
of a given species.
17, 18
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TABLE 1 (CONT'D)
Kind of
Type of
Appropriate Situations
for_Use
Reference
Fish
Respiratory
movements as
acute sublethal
Quick (1 day) indication of
possible sublethal effects.
Research and Monitoring.
19
Pish (iAe_.
fathead
minnows,
brook trout,
bluegill)
Reprodution,
growth, and
survival
Chronic tests for research
on "safe" concentrations.
20-25
Daphnia
Survival 6
Reprodution
Rapid completion of chronic
tests for testing special
susceptibility of crustaceans
25
Diatoms
Survival, growth
and reproduction
A sensitive, rapid, chronic
test for research, pre-
diction, or monitoring*
26
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TABLE 1 (CONT'D)
Kind of
Organism
Type of
Appropriate Situations
for Use
Reference
Marine
Crustacean
Larvae
Mollusks
Survival, growth
and development
through immature
stages.
A sensitive, rapid, chronic
test, for research, pre-
diction, or monitoring*
27
* Requires an operator with some specialized biological training.
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Environment
The supply of dilution wat>?r must be adequate to maintain constant
test conditions. In both static and continuous-flow tests, a
sufficiently large volume of test water must be used, and it must be
replaced or replenished frequently. This is to provide oxyqen tor the
organism, dilution of metabolic wastes, limit changes in temperature,
pH, etc., and to compensate for degradation, volatilization, intake
and sorption of the toxicant. In static tests, there should be 2 or 3
liters oxygen per gram of fish, changed daily or else the volume
proportionally increased for the number of days of the test. In
continuous-flow tests, the flow must: (a) provide at least 2 or 3
liters oxygen per gram of fish per day, and (h) flow must equal test-
volume in five hours or less, giving 903? replacement in half a day or
less.
The problem of maintaining dissolved oxygen concentrations
suitable for aquatic life in the test chamber can be difficult. The
suggestions above on test-volume and replacement times should provide
for adequate oxyqen in most cases. However, with some wastes,
insufficient oxygen may be present in the test water because BOD or
COD may consume much of the available dissolved oxygen. Aeration or
oxygenation may degrade or remove the test material. Devices for
maintaining satisfactory dissolved oxygen in static tests have been
proposed and used with some degree of effectiveness, and have been
described (28).
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Periodic measurements of concentration of the toxicant should b
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14P
time versus LCSO could then be constructed on loqarithroetic axes. The
lethal threshold concentration could then be estimated in many cases
(3, 5, 6) and this usually provides a more valid single number for
description'of acute toxicity than the arbitrary-time 96-hour LC50.
Test_Organis m s
For important bodies of water, there is a strong case for testing
several kinds of aquatic organisms, not only fish. A comparative
study of 20 pollutants on fish, snails, and diatoms found that no
single kind of organism was most sensitive in all situations (10).
The short-term bioassay method for fish can also be used for many
of the larger invertebrate animals. Few changes in method are
necessary except those of size, and a much greater volume of test
water and/or rate of flow in relation to weight of th» animals, since
their metabolic rate is hiaher on a weight basis.
Metho^s^or_ysin2_Diatoms_as_fiioa^say__gr.ganisms_i.n_the Laboratory
Algae and other microorganisms are uspful for bioassays because a
chronic test can actually be done in a week, since cycles of
reproduction and growth occur in that time (34). It is important in
selecting one to remember that a kinetic rather than a two-point
measure of growth rate is necessary to avoid errors introduced by lag
and recovery ohenomona commonly encountered.
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Short-term or acute toxicity tests do not indicate concentrations
of a potential toxicant that are harmless under conditions of long-
term exposure. It is desirable, therefore, to multiply the 96-hour
values by factors, to estimate concentrations of the constituent in
question that is safe in the receiving water. Such factors have been
called application factors. An application factor is not a safety
factor since it makes no extra allowance of safety for unknown
factors. It is merely a fractional or decimal factor applied to a
lethal or effective concentration in a short term test, in order
to estimate the safe concentration as accurately as possible.
Ideally, an application factor should be experimentally determined
for each pollutant. To do this, it wculd first be necessary to
determine the lethal concentration of the constituent (s) in question
according to the bioassay procedures outlined above. To obtain the
application factor it would be necessary to determine the truly "safe"
concentration of the same constituent(s) on the same species by
thorough research on physiological, biochemical, and behavioral
effects, and by studying growth, reproduction, and production in the
laboratory and field. The safe-to-lethal ratio obtained can then be
used in the future as an application factor, to work from measured
LCSO's of similar kinds of water, to predict the safe concentration.
Such bioassays should be repeated at least monthly.
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For example, if the 96 hour LC5C is 0.5 tng/1 and the concentration
of the constituent found to be safe is 0.01 mg/1, the ratio would be:
Safe Concentration 0.01 1
= = = 0.02
96-hour LC50 0.50 50
In this instance, the safe-to-lethal ratio is 0.02, which can be
used as an application factor in other situations. Then, in a given
situation involving this constituent, the safe concentration in the
receiving stream would be found by multiplying the four-day LC50 by
0.02.
This predictive procedure based on lethal concentrations is useful
since the safe level of many pollutants is not known, because of the
uncertainty about the toxicity of mixed pollutants because ot the
difference in sensitivity among fish and fish food organisms. The
various considerations involved in developing application factors are
discussed in the literature (25). Results of studies in which
continuous exposure was used reveal that the safe-to-lethal ratio
which permits spawning ranges over nearly two orders of magnitude (20-
21) . It is recognized that exposure in most natural situations will
not be constant and that higher concentrations usually can be
tolerated for short periods.
Lethal threshold concentrations, which may reguire more than 96-
hour exposure, may be beneficially used to replace 96-hour LCSO's
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in the above procedures, and there is a current trend to use such
threshold concentrations (2U).
Mixtures of Two or More Pollutants
For the lethal action of a mixture, the t.oi-al toxicity may be
estimated by expressing the actual concentration of each toxicant as a
proportion of its lethal threshold concentration (= 96 hour LC^
usually)r then addinq together the resulting number for all the
toxicants. If the total is 1.0 or greater, the mixture is predicted
to be lethal.
The system of "adding up" different toxicants is based on the
premise that their lethal actions are simply additive, one with the
other. This simple rule has been found to govern the combined lethal
action of many pairs and mixtures of quite dissimilar toxicants and is
true for such diverse toxicants as copper and ammonia: and zinc and
phenol in both laboratory (36-38) and field studies (39). The method
of addition is useful and reasonably accurate for predicting
thresholds of lethal effects in mixtures. Also, there are cases where
the combined actions of two or more toxicants exceed the simple
additive effects of the individual toxicants. This phenomenon is
commonly referred to as synergism. No technique is known for
predicting increased lethality of mixtures of toxicants acting
synergistically. Conversely there is evidence of mixtures of two or
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153
more compounds havinq a lethal effect that is less than the single
additive effects of -t-.he individual toxicants. This phenomenon is
commonly referred to as antagonism. Techniques for predicting the
lethality of mixtures of antagonistic consitituents also are not
known.
Sublethal or chronic effects of mixtures are of great importance
for water quality criteria. Sublethal concentrations of different
toxicants should also be assumed to be additive in effect. Here
again, it would be expected that for any giver toxicant there would be
gome low concentration which would have no deleterious etfect on an
organism, and would not contribute any sublethal toxicity to a
mixture. Research is extremely scanty on this topic.
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154
1. Alderice, D. F. 1967. "The detection and measurement
of water pollution - biological assays". Canadian
Dept. of Fish. Kept. No 9. 33-39
2. Sprague, J. B. 1969. Measurements of pollutant
toxicity to fish. I, Bioassay methods for acute
toxicity. Water Res. 3_: 793-821
3. Sprague, J. B. 1971. Measurement of pollutant toxicity
to fish. III. Sublethal effects and safe con-
centrations. Water Pes. 5: 245-266
U. American Public Health Association, American Water
Works Association, and Water Pollution Control
Federation (1971). Standard Methods for Examination
of Water and Wastewater. 13th ed. American
Public Health Association, Washington, D. C.
87U p.
5. Sprague, J. P., p. p. Eicon and R. L. Saunclers, 1965.
Sublethal copper - zinc pollution in a solnon river:
A field and laboratory study, Air Water Pollution
9_: 531-543
6. Sprague, J. B. 1971. Measurement of pollutant toxicity
to fish. II. Utilizing and applying bioassay
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155
results. Water Res. U: 3-32
7. Cairns, J. 1969. Fish bioassays - reproducibility
and rating. Rev. Biol. 7: 7-12
8. Cope, 0. B. 1861. Standards for reporting fish
toxicity tests. Progr. Fish Cult. 23: 187-189
9. Lennon, R. E. 1967. Selected strains of fish as
bioassay animals. Progr. Fish Cult. 29: 129-132
10. Ball, I. R. 1967a. The relative susceptibilities of
some species of freshwater fish to poisons.
I. Ammonia. Wat*»r Research 1, 767-775
11. Ball, I. R. 1967b. The relative susceptibilities of
some species of freshwater fish to poisons.
II. Zinc. Water Research 1, 777-783
12. Patrick, R., J. Cairns and A. Scheier. 1968.
The relative sensitivity of diatoms, snails and fish
to 20 common constituents of industrial wastes.
Cult. 30, 137-140
13. Lloyd, R. 1961. The toxicity of ammonia to rainbow
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156
trout (Saimo aairdnerii^ Richardson) . Water Waste
Treat. fl: 278-279
1U. Lloyd, R. 1961. Effect of dissolved oxygen con-
centrations on the toxicity of several poisons to
rainbow trout (Saljrio gairdnerii Richardson) .
J. Exp. Biol. 38 447-455
15. U.K. Ministry of Technology. 1969. Some effects of
pollution on fish. II. Notes on Water Pollution
NO. U5: 5.
16. Brown, V. M. 1968. The calculations of the acute
toxicity of mixtures of poisons to rainbow trout.
Water Res. 2: 723-733
17. Fry, F. E. J. 19U7. Effects of the environment on
animal activity, u. of Toronto studies, Biol.
Serv. No. 55. Pub. Ont. Fish Pes. Board No. 68.'
62.
18. Brett, J. P. 1952. Temperature tolerance of young
Pacific salmon, genus Oncorhynchus J. Fish.
Res. Board Can. 9: 265-323
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157
19. Schamhurg F. D., T. E. Howard, C. C. Walden 1967.
A method to evaluate the effects of water pollutants
on fish resperat.ion. Water Res. 1: 731-737
20. Mount, D. I. 1968. Chronic toxicity of copper to
fathead minows (Pi.me2ha.ies promelas, Rafinesquo) .
Water Res. 2: 215-223
21. Mount, D. I. and C. E. Stephen 1967. A method of
establishing acceptable toxicant limits for fish:
malathion and butoxyethanol ester of 2, 4-D.
Trans. Amer. Fish Soc. 9ji: 185-193
22. Brungs, W. A. 1969. Chronic toxicity of zinc to the
fathead minnow, PimeEhalas B!2me,l.a.§ Rafinesque.
Trans. Amer. Fish Soc. 98: 272-279
23. McKim, J. M. and D. A. Benoit 1971. Effects of
long-term exposures to copper-on survival, growth,
and reproduction of brook trout. (Salvelinus fontinalis)
J. Fish. Res. Board Can. 28: b"55-CG2
2«». Eaton, J. G, 1970. Chronic malathion toxicity to
the bluegill (Leponms SiaciSClJiUSS' Rafinesque)
Water Res. U: 673-G84
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158
25. Anderson, B. G. 1950. The apparent thresholds of
toxioity to Daphnia magria for chloride of various
metals when added to Lake Erie waters.
Trans, Amer. Fish Soc. 78: 96-113
26. Patrick, R. 196R. Standard methods for tests for
evaluating inhibitory toxicity of industrial waste
waters in American Society for Testir.q and Materials
Book of Standards, part 23: Industrial Water; Atmospheric
analysis (American Soc.-for Testing and Materials,
Philadelphia, Pa.) pp 657-665
27. Woelke, c. E. 1967. Measurements of water quality
criteria with Pacific oyster embryo hioassay.
Amer. Soc. Test. Mater. Spec. Tech. Publ no. U17: 112-120
28. Doudoroff, P. , B. G. Anderson, G. E. Burdick, P.S. Galtsoft,
W. B. Hart, R. Patrick, E. R. Strong, E. W. Surber; and
M. W. VanHorn. 1951.. Bioassay methods for the evaluation
of accute toxicity of industrial wastes to fish. Sewaye
Indust. Wastes. 23; 1380-1397
29. Brunqs, W. and D. I. Mount, 1967. A device for continuous treatment
of fish in holding chambers. Trans. Am. Fish. Soc.
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159
96: 55-57
30. Stark, G. T. C. 1967. An automatic dosir.q aoparatus made
with standard laboratory ware. Lab. Pract. 16: 594-595
31. Mount, D. I. and Warner. R. E. 1965. A serial-dilution
apparatus for continuous delivery of various conentrations
of materials in water. U.S. Publ.Health. Service,
Pub no. 999-WP-23, 16pp.
32. Mount, D. I. and Brunqs, W. A. 1967. A simplified
dosing apparatus for fish toxicology studies.
Water Research 1: 21-29.
33. Litchfield, J. T. and F. Wilcoxon, 1949. A simplified method of
evaluating does-effect experiments.
J. Pharmac. Exper. Therapeutics, ^6: 99-113
3«». Patrick, Ruth, 196U. Tentative method of test for
evaluating ingihitory toxicity of industrial waste
waters. American Soc. of Testino and Materials.
pp 517-525
35. Henderson, C. 1957. Applicaiton factors to be applied
to bioassays for the safe disposal of toxic wastes.
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Biological Problems in water Pollution. Trans, of 195h
Seminar U.S. Public Health Servic_, P.A. Taft Sanit. Enynq.
Center, Tech. Dept. W60-3 : 31-37
36. Herbert, D. W. M. and Van Dyke, Jennifer M. 196U. The
toxicity to fish of mixtures of poisons-IT. copper-
ammonia and zinc-phenol mistures. Ann. Anpl. Biol.
53 : 415-5121
37. Jordan, D. H. M. and Lloyd R. 196U. The resistance
of rainbow trout (Sa^mo Gairdnerii, Richardson) and roach,
(Rutilus rutilus L.) to alkaline solutions.
Tnt. j. Air Wat. Pollut. 8 : 405-U09
38. Brown, V. M. Jordan, D. H. M. and Tiller, B. A. 1969.
The acute toxicity to rainbow trout, of fluctuating
concentrations and mistures of ammonia, phenol and
zinc. J.Fish Biol. 1:1-9
39. Herbert. D. W. M. 1965. Pollution and fisheries.
In Ecology and the Industrial society, 5th Symposium
British Ecological Society, pp 173-195. Blackwell
Scientific, Oxford.
40. Lloyd, R. and Orr, Lydia, D. 1969. The diuretic
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response by rainbow trout, to sub-lethal concentrations
of ammonia. Water Research 3; 335-3UU.
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B. Classification of Waters 30U(a) (2) C
Classification of waters, depending upon the suitability for
•
the protection of human health and for the propagation ot fish and
aquatic life is categorized as follows:
1. Protection^gf^Human Health
a. Acceptable: Waters which are suitable
for contact recreation.
b. Treatable (Acceptable with treatment):
Waters which, although not suitable for contact recreation,
are acceptable for drinking water supply intake and can be
treated to acceptable potable water meeting human health
standards.
c. Unacceptable: Waters which contain
substances such as hydrocarbons (grease, oils) and hazardous
substances which cannot be removed and thereby render the
water unsafe for recreation or drinking water intake.
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2. Protection of Fish and Aquatic Life
a. Acceptable: Waters which are suitable
for the propagation of the most important sensitive species
of aquatic biot.a.
b. Unacceptable: Waters that contain
concentrations of pollutants that, will not sustain "the most
important sensitive species" of aquatic biota.
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V. Identification of Pollutants Suitable for Maximum Daily Loads
30U (a) (2)D
All pollutants described by Volume I, Water Quality Criteria, are
EStentially suitable for maximum daily load restriction; however, the
existence of Water Quality Standards is a prerequisite tor making this
determination. Only those pollutants which have a speciiic limitincr
value in the standards or those pollutants whose effects are
specifically limited in the standards are suitable for maximum daily
load.
«U.S. COVBRNMENT PRINTING OFFICEM971 54b-3i.VI4* 1-1
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