LIMNOLOGY FIELD AND LABORATORY MANUAL:

METHODS FOR ASSESSING AQUATIC

PRODUCTION
by
J. P. Koenings Jim A. Edmundson
Gary B. Kyle John M. Edmundson

Number 71
LIMNOLOGY FIELD AND LABORATORY MANUAL:
METHODS FOR ASSESSING AQUATIC
PRODUCTION

J. P. Koenings Jim A. Edmundson

Gary B. Kyle John M. Edmundson
Number 71

Alaska Department of Fish and Game

~ i v i s i o nof Fisheries ~ehabilitation,
Enhancement, and Development

Robert D. Burkett, Ph.D.

chief, Technology and Development

P. 0. Box 3-2000
Juneau, Alaska 99802
February 1987
 LIMNOLOGY
FIELD & LABORATORY
MANUAL

ALASKA DEPARTMENT OF FISH & GAME

Fisheries Rehabilitation, Enhancement
& D e v e l o p m e n t D i v i s i o n (F.R.E.D.)
Limnology S e c t i o n
 PREFACE
This manual describes the limnological field techniques and
laboratory methods currently used by the ~imnologySection of
the Alaska Department of ~ i s hand Game (ADF&G), Fisheries
~ehabilitation,Enhancement, and Development (FRED) Division to
assess aquatic production. In order to develop a comprehensive
limnological data base for a specific lake system, its
physical, .biological, and chemical characteristics must be
determined. Since the limnology program, which involves many
geographically isolated personnel, is currently investigating
lakes statewide, it became apparent that a concise reference
describing the field and laboratory procedures should be made
available as a guide in limnological research. Thus, the
purpose of this manual is to provide statewide standardization
of both laboratory and field procedures in order to insure
consistency and quality in the collection and processing of
aquatic samples.
Although-the primary focus of the text is to describe the .
various procedures in use, brief introductions concerning the
importance or relevance of each limnological parameter and
principle have been included. Part I of the manual discusses
the sampling procedures used for sample collection and storage,
and for conducting field measurements. Part I1 constitutes the
laboratory section, which describes the techniques and
procedures for analyzing water quality samples, measuring
primary productivity, enumerating and identifying zooplankton,
and evaluating the food habits of juvenile salmon. Part I11
addresses instrument calibrations, and statistical evaluations
of analytical methodologies.
This manual is designed to support the ADF&G, FRED Division,
Lake ~nrichmentand Lake stocking Programs as well as those of
cooperative state, federal, and private agencies. The authors
intent is to provide a comprehensive procedural reference for
both field and laboratory personnel, as adoption of statewide
standard methodologies will further the consistent
interpretation of limnological results.
A preface permits the authors to express appreciation to those
colleagues who have influenced the contents. Most conspicuous
have been Robert Burkett and Paul Woods (U. S. Geological
Survey) whose support and advise on the format and scientific
content was much appreciated. The authors also thank ~enise
Cialek and Virginia Petanovitch for comments concerning
autoanalyzer procedures, Richard Yanusz for suggestions on the
chlorophyll g analysis and field procedures, and Dave Barto for
the staff gauge information. Finally, the authors are
extremely grateful to Carol Schneiderhan and Mary Whalen for
the preparation of figures and illustrations and to Sue Howell
for the seemingly endless task of typing this manual.
 TABLE OF CONTENTS
Section
INTRODUCTION ............................................. 1

PART I . SAMPLE COLLECTION TECHNIQUES AND

FIELD MEASUREMENTS ............................. 5

SAMPLE COLLECTION AND STORAGE ....................... 6

Cleaning Glassware and Plastics................ 6

General Sampling Protocol ...................... 7
Sample Preparation............................. 11
Preservatives for Sample Storage............... 15

FIELD MEASUREMENTS AND APPLICATIONS ................. 16

Temperature .................................... 16
Lake-Heat Budgets .............................. 16
Dissolved Oxygen ............................... 20
Photosynthetically Active Radiation (PAR)......
.
.

22
Underwater photometer..................... 22
Secchi disk ............................... 23
Stream Discharge............................... 26
Staff gauge calibration................... 29
Estimation of the hydraulic (water)
retention time .......................... 29

PART I1. LABORATORY TECHNIQUES AND METHODOLOGIES ........ 33

SAFETY CONSIDERATIONS...............................
General Procedures............................. 34
Handling Byproduct Materials ................... 35
Byproduct Waste Disposal....................... 37
Emergency Procedures........................... 38

GENE3RAL METHODOLOGIES ............................... 39

Solutes. Solvents. and Solutions ............... 39

Conductivity................................. 40
Salinity ...................................... 41
~ H . * . ~ . e e ~ e .................................... 42
Alkalinity ..................................... 43
Turbidity ..................................... 44
Color .......................................... 46
Total. Suspended. and Dissolved Solids......... 48
 TABLE OF CONTENTS
Section Paqe
METALS .............................................. 51

Calcium and Magnesium (titration).............. 51

Calcium (colorimeteric)........................ 54
'~agnesium(colorimetric)....................... 56
Total Iron (manual)............................
Total Iron (automated. tentative).............. 58
62
.......................................
Hardness 65

DISSOLVED GASES ..................................... 67

Dissolved Oxygen ............................... 67

Hydrdogen Sulfide .............................. 68

INORGANIC NUTRIENTS ................................. 71

~eactiveSilicon (manual)...................... 71
Reactive Silicon (automated)............*...... 74
Nitrogen ....................................... 78
Total Ammonia (manual)......................... 78
Total Ammonia (automated)...................... 81
Nitrate and nitrite (manual)................... 84
Nitrate and nitrite (automated)................ 90
Phosphorus..................................... 95
Total and Total Filterable
Phosphorus (manual).......................... 95
Filterable Reactive Phosphorus (manual)........ 100
Dissolved Phosphorus (manual).................. 102
Total Kjeldahl Nitrogen and Total
Phosphorus (automated)....................... 104

PARTICULATE NUTRIENTS ...............................

Filter Blanks .................................. 112
Organic Carbon ................................. 113
. Phosphorus (manual)............................ 115
Inorganic and Organic Phosphorus (manual)...... 117
Nitrogen and Phosphours (automated)............ 122
Phosphorus and Nitrogen in Fish Tissue......... 126

PRIMARY PRODUCTIVITY................................
Carbon-14 Assimilation ......................... 132
Field Methods ............................. 132
Laboratory Methods ........................ 135
Volumetric Productivity ~stimates .............. 137
Day-Rate Estimates ............................. 140
Areal ~stimates ................................ 143
 TABLE OF CONTENTS
Section Paqe
PRIMARY PRODUCTION .................................. 146

Chlorophyll a and Phaeophytin g

(spectrophotometric)......................... 146
'Chlorophyll a and Phaeophytin a
(fluorometric)............................... 148

SECONDARY PRODUCTION................................
Zooplankton Identification..................... 152
Volumetric and Areal Density Estimates ......... 153
Body-Size (length) Measurements ................ 155
Individual Body-Weight (biomass) Estimates..... 158
Total Wet/Dry Weight (biomass) Estimates ....... 161
Reference Slides............................... 163

JUVENILE SALMON EVALUATION..........................

Total Body-Burden of Oxytetracycline........... 166
Anesthetizing Fish ............................. 168
Stomach Content Analysis ....................... 170
Gastric Lavage............................ 171
Stomach Removal ........................... 171
Identification and Enumeration ............ 173
~lectivityIndex............................... 173
Diet Overlap ................................... 174
Freshwater Cohort Production................... 177

PART I11. QUALITY ASSURANCE. CALIBRATIONS. AND

STATISTICAL EVALUATIONS ........................ 184

QUALITY ASSURANCE ................................... 185

Laboratory Technique........................... 185

General Tests and Nutrients.................... 186
Primary Productivity/~roduction................ 190
Zooplankton.................................... 191

INSTRUMENT CALIBRATIONS............................. 192

Dissolved Oxygen/Temperature Meter ............. 192

Spectrophotometer.......................... 193
Autoanalyzer ................................... 193
Filter Fluorometer............................. 195
Planimeter..................................... 195
pH Meter ....................................... 197
Ocular Micrometer .............................. 197
 TABLE OF CONTENTS
Section Pase
METHODOLOGIES: INTERFERENCES AND STATISTICAL
EVALUATION ................................ 198

Interference ........................................ 198

Statistical Evaluation .............................. 199
APPENDIX ................................................. 204
 LIST OF TABLES
Table Paqe
Use of a planimeter to (A) estimate the
average temperature of five depth
increments; and (B) the calculation
of accumulated gram-calories (g-cal)
for each depth interval........................
Light levels found at specific depths
and use in calculating the
light-compensation point (CP) and the
light-extinction coefficient...................
Laboratory results comparing salinity
( % % ) , chlorinity (%%),and conductivity at
various temperatures...........................
~epresentativevalues used to establish
the relationship between abosrbances (abs)
at 400 nm and platinum-cobalt units (Pt) .......
The relationship between known
concentrations of calcium and the
volumes (ml) of EDTA titrant used to
calculate a standard curve in the
analysis of calcium ............................
The relationship between known
concentrations of magnesium and
volumes (ml) of EDTA titrant used
to calculate a standard curve in the
analysis of calcium............................
The relationship between known
---
concentrations of calcium and
absorbance~ (abs) at 520 nm used to
calculate a standard curve in the
colorimetric analysis of calcium ...............
The relationship between known
---
concentrations of magnesium and
absorbance~ (abs) at 540 nm used to
calculate a standard curve in the
colorimetric analysis of magnesium .............
The relationship between known
concentrations of iron and absorbances
(z;) at 562 nm (10-nun cuvette) used to
calculate a standard curve in the manual
analysis of total iron .........................
 LIST OF TABLES
Table Paqe
10 The relationship between known
---
concentrations of iron and absorbances
(abs) at 562 nm (40-mm cuvette) used
to calculate a standard curve in the
.analysis of total iron. ........................ 61
Comparisons of total iron (TFe) to
turbidity corrected iron (NFe) showing
an -1% turbidity error when using the
manual analysis, and turbidity (NTU) levels
in lakes influenced by glacial silt......... ... 62
The relationship between known
concentrations of iron and autoanalyzer
chart deflections (;a) used to calculate a
standard curve in the automated analysis
-

of total iron .................................. 65

Factors used to express various metal
cations as equivalent concentrations of
CaC03 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , . . . . . . . 65

The proportion of H2S and HS- as a

function of pH................................. 70

The relationship between known

---
concentrations of reactive silicon and
absorbance~(abs) at 810 nm used to
calculate a standard curve in the manual
analysis of reactive silicon................... 73

Comparison of reactive silicon (RSi)

levels to turbidity corrected silicon
(NRSi) showing <1% turbidity error when
using the manual procedure. Also shown
are reactive silicon levels determined by
the automated procedure, and a range of
turbidity levels (NTU) of Alaskan lakes........ 74

The relationship between known

concentrations of reactive silicon
and autoanalyzer chart deflections (c8)
used to calculate a standard curve in
the automated analysis of reactive silicon..... 77
 LIST OF TABLES
Table Paqe
18 The relationship between known
concentrations-sf nitrogen and
absorbances (abs) at 640 nm used to
calculate a standard curve in the manual
......................
.analysis of total ammonia 80
The percent unionized ammonia (l$H3) in
aqueous ammonia + ammonium (NH4 )
solutions (after Emerson et al. 1975) .......... 80
The relationship between known
concentrations of nitrogen and
autoanalyzer chart deflections (ca) used
to calculate a standard curve in the
automated analysis of total ammonia............ 84

Absorbances for nitrite and equivalent

nitrate standards after cadmium reduction
used to calculate the reduction efficiency
(R.E.) in the manual analysis of nitrate +
nitrite nitrogen............................... 89
The relationship between known
concentrations-sf nitrate nitrogen and
absorbances (abs) at 543 nm following
cadmium reduction used to calculate a
standard curve in the manual analysis of
nitrate + nitrite nitrogen ..................... 90
Chart deflections for nitrite and
equivalent nitrate standards after
cadmium reduction used to calculate
the reduction efficiency (R.E.) in the
automated analysis of nitrate + nitrite
nitrogen...................................... 93

24 The relationship between known

concentrations of nitrate nitrogen
and autoanalyzer chart deflections (ca)
used to calculate a standard curve in -
the automated analysis of nitrate +
nitrite nitrogen............................... 94

Comparison of phosphorus levels of

six fractions (see Definition of Terms)
summed to give total phosphorus (TP) and
directly determined TP for four lakes
with varying turbidity (NTU) ................... 97
 LIST OF TABLES
Table
26 The relationship between known
---of phosphorus and
concentrations
absorbance~ (abs) at 882 nm used to
calculate a standard curve in the manual
analyses of total and total filterable
-phosphorus ..................................... 99

comparison of total phosphorus (TP

manual) levels, turbidity corrected TP
(NTP), and of TP (automated) for lakes
with varying turbidity (NTU) ................... 100
The relationship between known
concentration5-gf phosphorus and
absorbances (abs) at 882 nm used to
calculate a standard curve in the analysis
of filterable reactive phosphorus.............. 102
The relationship between known
concentrations of nitrogen and
autoanalyzer chart deflections (;a) used
used to calculate a standard curve in
the automated analysis of total Kjeldahl
nitrogen....................................... 108
The relationship between known
concentrations of phosphorus and--
autoanalyzer chart deflections (cd)
used to calculate a standard curve in
the automated analysis of total phosphorus ..... 109
The relationship between known
concentrations of carbon and absorbances
(ass) at 440 nm used to calculate a
standard curve in the analysis of particulate
organic carbon ................................. 115
---amounts of
The relationship between known
phosphorus and absorbance~(abs) at 882 nm
used to calculate a standard curve in the
analysis of total particulate phosphorus ....... 117

---amounts of
The relationship between known
phosphorus and absorbance~(abs) at 882 nm
used to calculate a standard curve in the
analysis of inorganic particulate
phosphorus (IPP) ............................... 121
 LIST OF TABLES
Table Paqe
34 ---
The relationship between known amounts of
phosphorus and absorbance~(abs) at 882 nm
used to calculate a standard curve in the
analysis of organic particulate phosphorus
..........................................
..( OPP) 122

35 The relationship between known amounts

of nitrogen and autoanalyzer chart
deflections (;a) used to calculate a
standard curve in the automated analysis
of particulate nitrogen ........................ 125

The relationship between known amounts

of phosphorus and autoanalyzer chart
deflections (c8) used to calculate a
-
standard curve in the automated analysis
of total paticulate phosphorus ................. 126

Amounts (mg) of nitrogen and phosphorus,

and the nitrogen to phosphorus atom ratios
in (A) sockeye juveniles (fall fry) and
(B) smolts from Alaskan lakes during 1983...... 129

Carbon-14 incubation bottles used for

in-situ radiocarbon experiments and the
reactions occurring within each treatment...... 133

CPM from red (R) and green (G) channels

used to determine sample channels ratio
(SCR), and CPM (R) and DPM used to calculate
counting efficiencies (E) ...................... 137

Factors for the cpnversion of total

alkalinity [mg L- (CaCO ) 1 to milligrams
of carbon per liter (~auiderset al. 1962) ..... 139

Liquid scintillation spectrometer data

printout for a single carbon-14 uptake
experiment in Falls Lake.,...,........ ......... 140

Comparison of total pigment, chlorophyll

a (chl a ) , and phaeophytin a (phaeo g)
-
levels as determined by the trichromatic,
monochromatic, and fluorometeric methods....... 150

Student's t-statistic and sample sizes

(n) used to determine the number (N) of
zooplankters to be measured to achieve a
confidence level (CL) of 95%................... 158
 LIST OF TABLES
Table Pase
44 Regression equations showing the
relationship between individual body-size
(mm) and dry weight (mg) for seven
zooplankter species............................ 159
2
In-lake zooplankter biomass (mg/m )
calculated using density estimates,
and the relationship between zooplankter
dry weight and mean length at Packers
and Hidden Lakes ............................... 161
comparison of wet and dry weights, and
the moisture content of the zooplankton
communities in four Alaskan lakes during
the spring, summer, and fall periods ........... 163
The relationship between fluoresence
units (Zu) and amounts of oxytetracycline
(OTC) used to calculate a standard curve
for the fluorometric analysis of OTC in fish... 168
nesth he tics and generalized amounts used
to prepare immersion solutions to sedate
juvenile and adult salmonids................... 169
The proportions of food items (i) found
in sockeye juveniles (Y) and threespine
stickleback (X) used to calculate the
diet overlap coefficient (Cf) .................. 177
Summary of cohort production parameters
described by Allen (1951) and determined
either by planimetry or from equivalent
equations following ~illespieand Benke
(1979) ......................................... 180
Results from the 1982 U. S. Geological
Survey Standard Water Reference Sample
Program showing the constituents analyzed
in three reference samples, mean values,
and values determined by the limnology
laboratory with performance ratings ............ 187
Results from the 1983 U. S. Geological
Survey Standard Water Reference Sample
Program showing the constituents analyzed
in four reference samples, mean values,
and values determined by the limnology
laboratory with performance ratings ............ 188
 LIST OF TABLES
Paqe
53 Results from the 1984 U. S. Geological
Survey Standard Water Reference Sample
Program showing the constituents analyzed
in three samples, mean values, and values
'determined by the limnology laboratory
with performance ratings ....................... 189

Results from the 1985 U. S. ~eological

Survey Standard Water Reference Sample
Program showing the constituents analyzed
in two samples, mean values, and values
determined by the limnology laboratory
with performance ratings ....................... 190

55 Comparison of chl 2 and phaeo q levels

- in reference standards of the U. S.
~nvironmentalprotection Agency, as
determined using the spectrophotometric
and fluorometric procedures by the
limnology laboratory........................... 191

Fluorescence units before (Rb) and after

(Ra) acidification used to obtain acid
ratios (rs) and calibration factors (Sx)
at various sensitivity levels (S) for the
fluorometric analysis of chl q................. 196

statistical evaluations of analytical

methodologies used by the limnology
laboratory................,.................... 200
 LIST OF FIGURES
Pase
1 Generalized schematic of a lake ecosystem
emphasizing metabolic pathways or linkages
between trophic levels (see legend for
detailed description) .......................... 2

Water collection bottles used to sample

specific depths in the water column
without contamination from overlying
waters......................................... 9

Sample collection protocol for field

measurements and incubations; and
procedures for lake water processing
emphasizing three fractions
unfiltered, filtrate, and particulate.
Also shown are the analyses conducted
on each fraction and the recommended
-

storage procedures .............................

Apparatus used to process samples by
vacuum filtration to obtain particulates
and filtrate (A) using four separate
filtrate flasks with individual towers,
and (B) using a filtering manifold and
a single filtrate flask........................ 13

Isopleths of changing temperatures

within strata made from field measures.
of vertical temperature profiles on seven
dates over the growing season (isothermy
in the spring to fall) used to determine
the summer heat budget (4 C in the spring
to seasonal maximum temperature).. ............. 18

Nomograph for determining percent oxygen

saturation using field-feasurements of
dissolved oxygen (mg*L ) and
temperature (OC) ............................... 21
The relationship between the euphotic
zone depth (EZD) defined by the 1% light
level (determined by underwater photometer)
and the Secchi disk transparency (SD) for
glacial, organically stained, and
clear-water lakes.............................. 25
 LIST OF FIGURES
Fisure Paae
8 The relationship between depth and water
velocity in an open channel showing the
depths at which velocity can be measured
to obtain a mean flow rate..................... 27

Stream cross section partitioned into

segments and the segment boundaries at
which water depths and flow rates are
measured............................,.......... 28

The relationship of decreasing euphotic

zone depth (EZD) to increasing turbidity
(NTU), and the log-log transformation
and linear regression equation ................. 45

The relationship of decreasing euphotic

zone depth (EZD) to increasing lake-water
color, expressed as platinum cobalt units
(Pt), and the log-log transformation and
linear regression formula...................... 47

Autoanalyzer schematic showing reagent

lines with flow rates (ml/mm) , cam size
(number of samples per hour and sample to
wash ratio), mixing coils, and colorimeter
with specified flow cell (FC) and
wavelength (nm) used in the analysis of
total iron ..................................... 64

Autoanalyzer schematic showing reagent

lines with flow rates (ml/min), cam size
(number of samples per hour and sample to
wash ratio), mixing coils, and colorimeter
with specified flow cell (FC) and
wavelength (nm) used in the analysis of
reactive silicon ............................... 76

Autoanalyzer schematic showing reagent

lines with flow rates (ml/min), cam size
number of samples per hour and sample to
wash ratio), mixing coils, heating bath,
and colorimeter with specified flow cell
(FC) and wavelength (nm) used in the
analysis of total ammonia...................... 83
 LIST OF FIGURES
Fiqure Paqe
15 Cadmium column used $0 reduce nitrate
(NO-) to nitrite (NO2) in the manual
ana?ysis of nitrate + nitrite nitrogen.. ...... 86

16 'Autoanalyzer schematic showing reagent

lines with flow rates (ml/min), cam size
(number of samples per hour and sample to
wash ratio), mixing coils, cadmium
reduction (CD) column, debubbler, and
calorimeter with specified flow cell (FC)
and wavelength (nm) used in the automated
analysis of nitrate + nitrite nitrogen......... 92

17 Phosphorus fractions comprising the total

phosphorus (TP) in natural lake waters
- (see page 95 for definition of terms),
obtained either by direct measurement or
derived by difference.......................... 96

Apparatus used to process filtered water for

ultrafitrate ((10,000 MWCO), showing the
vacuum manifold and Millipore immersible
ultra-filters, used for analysis of
dissolved phosphorus fractions ................. 103

Autoanlyzer schematic showing reagent

lines and flow rates (ml/min), cam size
(number of samples per hour and sample to
wash ratios), mixing coils, heating baths,
and colorimeters with specified flow cells
(FC) and wavelengths (nm) used in the
simultaneous analysis of total phosphorus
and total Kjeldahl nitrogen.................... 107

20 Flow diagram of the analysis procedures

for determining (A) inorganic particulate
phosphorus (IPP) using fluoride
extraction, and (B) organic particulate
phosphorus (OPP) after acid digestion .......... 118

21 Autoanalyzer schematic showing reagent

lines and flow rates (ml/min), cam size
(number of samples per hour and sample to
wash ratio), mixing coils, heating baths,
and colorimeters with specified flow cells
(FC) and wavelength (nm) used in the
simultaneous analysis of particulate
nitrogen and phosphorus.. ...................... 124
 LIST OF FIGURES
Fiqure Pase
Plexiglass plate (A) with lock and key
cut-outs used to fasten incubation
bottles and (B) arrangement of a series
of plates in the water column used in
estimating carbon assimilation rates........... 134
The relationship of cumulative primary
productivity (expressed as a percent)
to day-length divided into five equal
periods (I-V). Also shown are ten
increments within each period (expressed
as a percent) used to locate the
initial and final carbon-14 incubation
times, and the corresponding proportion
(P) of cumulative productivity ................. 141
24 - Vertical distributions of day-rate
primary productivity estimates within
the euphotic zone used in three
procedu~esto obtain areal productivity
(mg C/m /day) in Falls Lake, 24 June

25 Vertical distributions of day-rate

primary3productivity estimates
(mg C/m /day) in three lakes, Falls,
Packers, and Snake, with varying photic
zones; and a comparison of areal
productivity estimates (A) as determined
by summation (a), planimetry (b), and
averaging (c). ................................. 145
26 Location of anterior and posterior
measuring points on the carapace of
cladocerans, and cyclopoid and calanoid
copepod-zooplankters used to determine
body-size (length) ............................. 157
27 The relationship between dry weight (mg)
and wet length (mm) for E~ishura
nevadensis derived using measured
zooplankters from both Hidden and
Packers Lakes.................................. 160
28 Manostat syringe apparatus, with teflon
tube used to remove stomach contents from
juvenile salmonids by gastric lavage ........... 172
 LIST OF FIGURES
Fisure Paqe
29 Range of zooplankter body-sizes from
0.2-1.64 mm (by 0.04 mm intervals)
showing the length interval of each
species selected by limnetic rearing
sockeye fry from Packers Lake. Also
shown are the total number of each
zooplankter consumed, and the overall
electivity index for each species .............. 175
Generalized Allen curve showing the
relationship between the number of fish
and mean individual weight used to
calculate production parameters (see
descriptors) for rearing fish. Biomass
(B) estimates shown represent the life
history stages of spring juveniles (B1),
late fall juveniles (B2), and smolts
(B3)........................................... 179
~escriptionof linear regression showing
(A) a positive correlation between
concentration and absorbance, and (B) a
negative correlation, and the general
form of the regression equation................ 202
 INTRODUCTION
The alteration of watershed habitat, the stocking of salmonid
fry, and/or the addition of nutrients to a lake system for the
purpose of increasing the standing stock of salmon, impact all
aspects of a lake ecosystem. Since few biological processes
are determined by independently operating factors, such
large-scale ecosystem manipulations become very complex and
produce mtmy changes within a lake. This is because a lake
system can be described as a complex mosaic of interacting
pathways joined by an array of complex yet concrete
compartments (Figure 1). It is the innate complexity of these
metabolic pathways that need to be understood in order for a
lake environment to either be maintained or manipulated. Each
choice demands knowledge, for without such understanding, lake
management would be impossible.
Lake productivity and production are both dependent on the
relative interaction of physical, biological, and chemical
features operating within a given system. The combination of
standing crop (compartments) and linkages (R) between them can
be thought of as a 'fingerprint' of a particular system (Figure
1) and, thus, are in themselves unique. However, the driving
mechanisms of lake production are ultimately linked to energy
supply (sunlight) and the seasonal cycling of nutrients (N +!.
.
Nutrient levels (e.g , phosphorus and nitrogen) fluctuatb m
availability over the course of a year and can become depleted
when demand (RZ) exceeds supply (R ) . During these periods,
generally over the summer, the prohction of phytoplankton is
reduced and/or the production is lost (R ) through nutrient
utilization by nuisance groups of phytoplankon. Either change
is felt first by the zooplankton, which directly feed (R ) on
specific phytoplankton groups, and then by successive tie$. in
the food chain, leading to a decrease in forage production (R )
for rearing salmon fry. The point where fish-forage producti%n
(R ) is exceeded by demand (R ) is the capacity point of a
lage, in terms of successfully Pearing salmon fry, and defines
rearing limitation of fish production.
Since t3e productive capacity of lakes, in terms of fish-forage
production, is independent of the capacity of the same lake
system to recruit rearing fry, specific lakes contain few, if
any, salmonid fry. In these systems either the actual spawning
area, the number of spawning adults (escapement), or both are
low. Here fish-forage production (R ) can exceed demand (R ) ,
and such lake systems define a recraitment limitation to ffsh
production.
In-lake manipulation techniques (lake enrichment or lake
stocking) are designed to either increase preexisting forage
production (R ) to satisfy the demands of rearing fry (R ) or
to increase tile demand (R4) on a high preexisting forage &upply
(Rj), respectively. That is, both techniques seek to balance
 Lake

PHYTOPLANKTON

OTHER PREDATORS,
AND LOSSES FROM

-
Figure 1. Generalized schematic of a lake ecosystem emphasizing metabolic pathways
or linkages between trophic levels (see legend for detailed description).
N1: Nutrient supply from the atmosphere, land runoff, R1: Rate of nutrient input to the lake system. Includes
and lake tributaries. atmospheric and watershed inputs as well as nutrients
supplied in carcasses of returning adult salmon.
N2: Nutrients imported in spawning anadromous fish.
R2 : Rate of nutrient uptake from dissolvq?3 available forms in
N3: Existing supply of available nutrients in the the water column to biomass as primary producers. This
euphotic zone. uptake rate is measured in minutes, and is reflected as
biomass of primary producer in 4 or 5 days.
Nq: Nutrients exported from the system in outmigrant
anadromous fish. R3: Rate of biomass change from primary to secondary producer.
This linkage of the food chain is the energy source for
N5: Nutrients lost to the system in the river outflow. production of rearing fish forage. It must be of proper
species composition in the proper location at the appro-
Phyto: Standing crop of primary producers, the priate time. Interacting factors necessary to achieve
phytoplankton. this may be critical e.g., temperature, and water clarity.
Zoopl: Standing crop of secondary producers, the Rate of biomass change for the rearing phase of lake
zooplankton. Note that this element in the populations of salmonids. Acceptable growth of rearing fish
food chain is instrumental in the linkage populations depends upon a continuous supply of food
between emergent fry and rearing juvenile. organisms. Again, for biomass to appear as the desired
If artificial nutrients are applied, they must salmonid linkage proper species and location must be accom-
be introduced at N1, so that zooplankton popula- plished through judicious application of fertilizer at the
tions are synchronized (species, timing, and N1 phase.
location) to entry of fry into limnetic rearing
areas. If this is not done properly, food R5 Rates of change to smolt outmigrant biomass as a result
chains might be stimulated that may have a of the survival and growth of the rearing salmonids. Out-
detrimental effect on targeted salmon or trout migrating smolts represent the desired end product of the
production. freshwater food chain.
Competitors: Includes non-target species that may be R7 : Rate of change of biomass to non-target elements within the
competing for food organisms. Examples are lake food web. For example, phytoplankton can be cropped
stickleback, white fish, etc.; this category may by zooplankton that are not appropriate as salmon food,
also include littoral communities (also in competitor or predator species of fish can crop zooplankton
competition for food supplies) utilizing lake or rearing salmonids; and nutrients can be side channeled-
nutrient supplies (N3). and bound into macrophytes.
For example, N3---=+ periphyton snail.
---I Such side channels from the desired food chain can produce
serious losses to lake fertilization programs, and may
Rearing Juvenile: Standing crop of rearing sockeye juve- reduce the benefit/cost ratio below feasible levels.
niles (modifications can be made for other species).
Su lary: Energy and nutrient flow in a lake is a product of
Smolt: Production from the lake represented in the out- nutrient supplies and the factors of sunlight, temperature,
migrant target species of fish. This could be and the nature of the lake basin. Successful production of
thought of as a final product of a lake fertili- targeted species will be enhanced by minimizing biomass side
zation project and, as ocean survival of smolts is channels (R7) such as large littoral communities and large
related to size (length), one system may respond populations of predator/competitor species. Other needs of
to a strategy of few large smolt while another the target species, such as spawning area, should be
system may respond to the strategy of many smaller considered.
smolts.
Successfully managed lake fertilization or fry stocking
Adult: The standing crop or biomass accrued outside of projects will have low standing crop levels and high rates
the system. Increase in this standing crop occurs of change with biomass moving rapidly through the food
by way of energy and nutrient sources in the ocean. chain to support growth of targeted fish populations. Little
Returning adults (spawners) may be an important or no change should be detectable in the other elements
source of nutrients for the lake system. within the lake's ecosystem.
the delivery of fry with the ability of a lake to produce
forage as a way to optimize (reduce R ) the use of a given
carrying capacity. However, the application of the proper
enhancement technique requires prior knowledge of where a lake
lies, relative to the balance point. The ranking of each lake,
relative to that point, can be assigned, given an understanding
of in-lake indicators, which for example, reflect the relative
magnitude of fry-feeding pressure on the zooplankton community.
Through a basic understanding of how production linkages are
made and what the key indicator variables are, empirical models
can be fabricated (Koenings and Burkett 1986a, 1986b; Koenings
et al. 1986). Through the use of such models, management of
lake systems for a given product can be undertaken with
increased likelihood of success. The field and laboratory
procedures described herein represent the latest methods and
techniques for comprehensive lake inventories or
research-project evaluations. It is our intent to provide a
basis for standardized limnological assessment in order to
study rearing-fry ecology through an understanding of lake
trophic-level dynamics.

REFERENCES
Koenings, J. P. and R. D. Burkett. 1986a. The production
patterns of sockeye salmon (Oncorh~nchus nerka) smolts
relative to temperature regimes, euphotic volume, fry
density and forage base within Alaskan lakes.
Proceedings, Sockeye 85, International Sockeye Salmon
Symposium. Naniamo, British Columbia, Canada. November
18-22, 1985. (In press) .
Koenings, J. P. and R. D. Burkett. 1986b. An Aquatic Rubic's
Cube: The return of the Karluk Lake sockeye.
Proceedings, Sockeye 85, International Sockeye Salmon
Symposium. Naniamo, British Columbia, Canada. November
18022,1985. (In press) .
Koenings. J. P., R. D. Burkett, G. B. Kyle, J. A. Edmundson,
and J. M. Edmundson. 1986. Trophic level responses to
glacial meltwater intrusion in Alaskan lakes. PP
179-194. In: Kane, D. L. (ed.). Proceedings: Cold
Regions Hydrology Symposium. American Water Resources
Assoc. Bethesda, MD. USA. 612 p.
 PART I.

SAMPLE COLLECTION TECHNIQUES

AND
FIELD MEASUREMENTS
 SAMPLE COLLECTION AND STORAGE

cleanins Glassware and Plastics

Three grades of water are used in the limnology laboratory:
Type I, Type 11, and distilled water. The primary water-
purificatian system is a Milli RO-20 system equipped with an
additional Milli Q reagent grade water filter.
Type I Water (DI) -
Type I or de-ionized (DI) water has been
filtered through a series of ion exchange cartridges of the
Milli Q system after reverse osmosis. The specific conductance
is less than 1.4 umhos/cm. All reagents and standards are
prepared using DI water.
Type I1 Water -Type I1 is tap water that has been purified by
the process of reverse osmosis (RO) using the Milli RO-20
system, and stored in an epoxy-lined, aluminum reservoir. RO
water has a specific conductance of less than 40 umhos/cm.
Distilled Water -
Distilled (DW) water is tap water purified by
distillation using a Corning Mega Pure still. Freshly
distilled water has a specific conductance of <2.0 umhos/cm,
but will increase to 2-4 umhos/cm within 48 hours.
Clean all glassware and plasticware, including nutrient sample
bottles, prior to use according to the following procedure:
1) Wash glassware and plastics with phosphate-free detergent
and rinse with tap water.
2) Rinse with 10% hydrochloric acid (HC1).
3) Rinse four time with Type I1 water.
4) ~ i n s etwice with DI water.
-
Carboys' Wash large containers with phosphate-free
and rinse three times with DI water.
detergent

Zooplankton bottles
detergent and rinse with
-
Wash bottles with
tap water.
phosphate-free
Bottles used for
zooplankton samples shoud not be re-used for nutrient samples.
Particulate nutrient filters -
Clean Whatman GF/F filters by
placing on the filtering apparatus and drawing 50 ml of DI
water through the filter. Clean circular or funnel filters by
rinsing with DI water using a wash bottle. Always use blunted
forceps when handling filters, and avoid using 10% HC1 to rinse
filters.
 General Samglins Protocol
In order to collect representative field data, maintain data
consistency, and avoid sample contamination, there are several
considerations to bear in mind. Routine field-sampling
techniques conducted at each lake station involve collection of
water samples, vertical zooplankton tows, measurements of
temperature, dissolved oxygen, light penetration, pH, and
Secchi-disk depth, and performing in-situ experiments with
carbon-14 to estimate primary productivity.
The establishment of permanent station markers is necessary in
order to conduct limnological sampling at the same location
over time. This is crucial, if changes in water quality are to
be accurately monitored and interpreted with confidence;
because at any one instance differences in chemical,
biological, and physical conditions may exist at different
areas within a lake. In addition, lake processes may vary
considerably over time. At least two sampling stations per
lake are-recommended;however, several stations may be required
to fully characterize a lake system consisting of several
basins or bays.
To closely monitor lake dynamics, field sampling should be
conducted approximately every three weeks during the ice-off to
ice-on period and once during mid-winter (ice cover) throughout
intensive sampling; e:g., prefertilization project phase.
During the less intensive feasibility phase, the same field
work is conducted less frequently, usually four times a year
(spring turnover, summer stratification, fall turnover, and
mid-winter).
The two most recurrent problems affecting water sample
collection are contamination and the delay in the initial
sample processing (filtering). By adhering to a few simple
procedures these problems can be eliminated. Prior to sample
collection make sure all carboys are cleaned (see cleaning
Glassware and Plastics, p. 6). Once at the sampling site,
rinse the carboys with sample water and discard before storing
the actual sample. The rinsing process permits !aging1 of the
vessel walls and prevents any subsequent loss of chemical
constituents from the water to the polyethylene (poly) surface.
Keep the carboys and sampling gear away from all petroleum
products. containers or sample bottles contaminated by
outboard fuel, gasoline, or oil are impossible to clean, and
must be discarded.
Keep all field equipment clean, dry, and fully charged. An
important note to remember is that battery power is dependent
on temperature; i.e., because an instrument runs well at 20 C
does not mean it will function properly at 0 C. Calibrations
should be conducted at each sampling station to insure the
accuracy of the measurements.
An additional point worth mentioning concerns communication
between field personnel and the laboratory. Sample analyses
are useless without complete and proper documentation.
Therefore, label all bottles and complete the inventory forms
prior to sending them to the laboratory. Moreover, if in doubt
concerning a meter reading, depth of sample, sample label,
etc., repeat the procedure. A little time lost at this point
is far better than trying to either decipher unclear notes or
interpret'-faultydata months later.
Finally, during sampling record general lake conditions. These
include weather observations, such as temperature, cloud cover,
precipitation, and wind speed as well as notes on lake surface
conditions, water color, ice depth, period of ice cover, etc.
These incidental notes may prove beneficial when examining the
field data at a later date.

Lake Water
Using either a Van Dorn sampler or Kemmerer bottle (Figure 2),
collect water samples from each sampling site at the 1.0-m
depth and the mid-hypolimnion. If the thermocline is absent,
take the deeper sample from 7 5 % of the lake depth. Collect 5
liters of water for each sample and pour it directly into a
clean carboy or other poly-container. Remember to rinse out
the carboy with sample water (-1 liter obtained from the first
cast) prior to sample collection. Discard any samples
containing sediment, and resample a few meters higher.
Keep the carboys (samples) cool (4 C) and in the dark, at least
out of direct sunlight, until they can be processed. In turn,
during winter sampling keep the samples from freezing. It is
important to filter the samples as soon as possible following
their collection. Delaying the filtering beyond 3-4 hours is
undesirable because of gross changes that may occur in the
chemical composition of the sample.

Dissolved Gases
Collect a water sample using a Van Dorn bottle with a length
(-10 cm) of tygon tubing attached to the outlet drain. Place
the tube outlet to the bottom of a 300-ml BOD bottle, and open
the valve, allowing the bottle to overflow. Carefully insert
the stopper without leaving an airspace, store in the dark at 4
C, and analyze as soon as possible.
 merrenger

meerenger

messenger

trip mechanlrm

trip mechanism

(PVC, b r a r r , or
nickle plated)
(pvc or acrylic)

drain valve

(horlzontsl conflguratlon) (vertical contlguratlon)

VAN DORN BOTTLE

KEMMERER SAMPLER

Figure 2. Water collection bottles used to sample specific depths in the water column
without contamination from overlying waters.
Suspend sets (2) of plexiglass plates (10 x 10 cm is suitable)
at the 1-m depth, and above a stable subtrate within the
littoral zone. After initial colonization and growth (-3-6
weeks), scrape the periphyton from both sides of each plate
into a funnel, and rinse the contents with tap water into a
250-ml polybottle. Store at 4 C in the dark, and return the
sample as'soon as possible to the laboratory. Reset the plates
for subsequent sampling at 3-6 week intervals throughout the
sampling season.
NOTE: Rough the surface of new plexiglass plates with steel
wool, and soak in tap water for several days to
'age1 prior to use.

Plankton
For phytoplankton identification and counting; use water
collected from the 1-m depth, as described above, during spring
turnover, summer stratification, fall turnover, and mid-winter.
Pour 100 ml of unfiltered lake water into a 125-ml polybottle.
For zooplankton identification and counting, use a 20-cm
diameter by 153-um mesh net for clear-water lakes or systems
with high zooplankton densities. Use a 0.5-m diameter net for
glacial lakes or systems with low zooplankton densities.
Collect two replicate tows from each sampling station. Lower
the net to 50 m or just off the bottom if the lake depth is
less than 50 m. Pull the net vertically to the lake surface at
a rate of 0.5 m/sec. Rinse the net by immersing several times
in the lake to just below the net collar. This washes
organisms caught in the mesh into the net bucket. Remove the
bucket from the net and using a wash bottle, rinse the contents
into a 125-ml polybottle. Label with the following
information: lake, station, date and time, depth of tow, tow
number, and type of net; i.e., diameter and mesh size.

Stream Water
Generally 500 ml of unfiltered water are required for stream
samples. Collect the sample by submerging a 500-ml polybottle
upstream from where anyone has crossed or waded. ~ i n s e the
bottle with sample before storing in the dark at 4 C.
 Sample Preparation
Lake water contained in carboys, is processed (filtered) and
stored according to the type(s) of analysis to be conducted;
unfiltered refrigerated, unfiltered frozen, and filtrate frozen
water are saved for each sample (Figure 3). The filtering
procedure and a synopsis of samples to be returned to the
limnology laboratory are described below.
Clean all filter flasks, towers, and other necessary labware
(see Cleaning Glassware and Plastics, p. 6). A vacuum pump
capable of 15 psi negative pressure and sufficient to filter at
least 4 subsamples simultaneously is necessary. If the
apparatus is assembled as in Figure 4A, use one flask for the
filtrate, and a separate flask for filtering chlorophyll a
samples. If the apparatus is assembled as in Figure 4B use one
filter tower for all chlorophyll a samples, and the single
filter flask for filtrate. To avoid contamination, conduct all
the filtering and storage of one sample and rinse the filter
towers and filtrate flask with DI water before continuing to
the next.
Particulate nutrients - Place a clean 4.25-cm GF/F
(particle retention size of 0.7 um) Whatman filter on the
base of the filtering apparatus. Filter one liter of
lake water (if possible) at 515 psi using separate filters
for each particulate carbon (C), nitrogen (N), and
phosphorus (P) sample. Place each filter in a separate
petri-slide, label (including the volume filtered), and
store frozen.
2) -
Chlorophyll g (chl a) Follow the same procedure used
for particulate nutrients; however, as the last 50 ml of
sample is filtered, add - 5 ml of MgCO solution to the
filter. This prevents acidification 8f the sample and
subsequent conversion of chl q to phaeophytin (phaeo g).

3) -
Unfiltered refrigerated After rinsing a clean polybottle
with a small amount of sample, pour 500 ml from the carboy
directly into a 500-ml polybottle without leaving an
airspace. Store the sample in the dark at 4 C.
4) Unfiltered frozen - After rinsing a clean polybottle with
a small amount of sample, pour -200 ml from the carboy
directly into a 250-ml polybottle and store frozen.
Remember to leave an airspace within the bottle to allow
for expansion of the freezing water.
5) Filtrate frozen - Filter -100 ml of the sample to age the
filter flask, and discard. Filter the remaining -900 ml
of sample retaining both the particulate nutrient filter
 EPlLlMNETlC AND HYPOLIMNETIC ZONES

UNFILTERED ( f i e l d ) LAKE WATER UNFILTERED '(lab!

I
fllter a t 1Spsl
V
W conductlvity
dlsaolved oxygen PARTICULATES FILTRATE
temperature PH
alkalinity
v V turbldlty
p a r t i c u l a t e C. N. P. C h l g t o t a l ammonia
calclum
(fllter one l l t e r each nltrate - nltrlte
magnesium
and s t o r e frozen) fllterable reactlve-P
t o t a l Iron
t o t a l fllterable-P
reactlve sillcon
color
Incubate C-14 (500ml. r e f r i g e r a t e d
(450ml. frozen)
(In a i t u ) at 4 ' ~ )

v total phosphorus
FILTRATE FILTER ultraflltrate
total Kjeldahl

J
t o t a l dissolved-P
store waste at dissolved r e a c t l v e - P
place In labelled
p H >10 I n c l o s e d (200ml. f r o z e n )
sclntlllatlon vial
container
m ev
tals

(100m1, a c i d i f i e d
i -. _1
A -
Figure 3. Sample collection protocol for field measurements and incubations; and
procedures for lake water processing emphasizing three fractions, unfiltered, filtrate,
and particulate. Also shown are the analysis conducted on each fraction and the
recommended storage procedures.
 A
filter t o w e r

B
8 8-L--d:lq
-- --
vacuum

filter t o w e r s

vacuum container
manif old q~l
(Qr'
filtrate
flask pump

Figure 4. Apparatus used to process samples by vacuum filtration to obtain particulates

and filtrate (A) using four separate filtrate flasks with individual towers, and (B) using
a filtering manifold and a single filtrate flask.
 and filtrate. Pour -450 m l o f filtrate into a 500-ml
polybottle and store frozen.
NOTE: Remember to rinse all sample bottles with a portion
of the actual sample prior to storage, and fill out
all bottle labels completely.
Summarizing, to guarantee a complete water-quality analysis,
each carboy should yield the following samples, to be sent as
quickly as possible to the laboratory, for analysis (Figure 3).
1) 500 ml unfiltered refrigerated.
2) 200 ml unfiltered frozen.
3) 450 ml filtrate frozen.
4) 3 filters (1 liter each) for particulate C, N, and P.
5) 1 filter (1 liter) for chl g and phaeo q.
Alternately, our recent use of autoanalyzers reduces the sample
volume necessary for certain tests, and the use of the
fluorometric method for chl g reduces the volume of sample
needed on a filter. The following volumes can be used; but may
not allow retesting of inconsistent replicates.
1) 250 ml unfiltered refrigerated.
2) 200 rnl unfiltered frozen.
3) 225 ml filtrate frozen.
4) 3 filters (0.5 liter each) for particulate C, N, and P.
5) 1 filter (0.5 to 0.25 liters) for chlorophyll g and
phaeophytin a.

Collect samples for metal analysis twice a year: once before

lake stratification in the spring and once prior to fall
turnover. Pour -100 ml of unfiltered water into a 125-ml
polybottle, add 0.5 ml of 1:l nitric acid, and store at room
temperature.

Filter periphyton samples according to the procedure described

for chl a sample preparation (p. 11). Record the area of the
plexiglass plates on the petri-slide label.
 Phytoplankton
Add 2.0 ml'of Lugolls acetate solution to 100 ml of unfiltered
water in a 125-ml polybottle, and store at room temperature.

Zooplankton
Preserve .samples with 100% neutralized formalin (37%
formaldehyde) to make a final 10% volume/volume formalin/sample
solution (equivalent to a 3-4% neutralized formaldehyde
solution). Alternately, use a wash bottle containing 10%
neutralized formalin to rinse zooplankters from the collection
net into a 125-ml polybottle. Store at room temperature.

Preservatives for Sam~leStorase

1) Magnesium carbonate (MgCO ) - Mix 1 g of magnesium
carbonate-n-hydrate in 100 dl of DI water. Mix thoroughly
just before adding to the chl a samples because the MgC03
does not dissolve.
2) Lugolls acetate - Dissolve 10 g of potassium iodide, 5 g
of iodine, and 5 g of sodium acetate-trihydrate in 70 ml
of DI water.
- 3) -
10% Neutralized formalin Mix one part 100% formalin (37%
formaldehyde) with nine parts of DI water. This yields a
10% formalin or 3.7% formaldehyde solution. Neutralize
with 1 N sodium hydroxide (-1-2 ml per liter) until a pH
of 7-8 units is reached. As NaOH is usually supplied as
pellets, add -1 pellet per liter of 10% formalin.
4) Nitric acid ( 1 : - Carefully and slowly add
concentrated nitric acid to one part DI water.
one part
 FIELD MEASUREMENTS AND APPLICATIONS
Temperature
The pattern of heat intensity within a lake (as measured by
temperature) influences chemical reactions, nutrient cycles,
and, ultimately, productivity. Thus, temperature is one of the
most important physical aspects of an aquatic ecosystem.
In most lakes the increasing air temperatures and sunlight of
spring begin to warm the surface layers forming the epilimnion.
With increasing depth, the water column undergoes a rapid
decrease in temperature (>1 OC/m) forming a thermocline or
metalimnion which separates the colder layer below or
hypolimnion. When these discrete layers form, due to differing
densities, a lake is stratified and little or no mixing occurs
between layers. In some deep oligotrophic lakes, a well
defined thermocline may be absent, and instead, a gradual
warming of the upper water column occurs. During the fall,
which is-accompaniedby decreasing ambient air temperatures and
increasing winds, the epilimnion gradually cools to the
temperature of the hypolimnion. Eventually the entire water
column achieves a uniform temperature (isothermy), and the
water column slowly begins to mix from top to bottom
(turnover). This is one of the processes by which nutrients
are redistributed throughout the lake.
Procedure
Before taking temperature measurements, calibrate the
thermometer by placing the probe in a mixture of ice and water.
The ice-water mixture is 0 C. Record the temperature to the
nearest 0.5 C at 1 meter intervals when the lake is stratified
by lowering a 50-m cabled probe through the water column, and
at every other meter when the lake is isothermal.

Lake-Heat Budsets
In-lake temperatures can be used to calculate heat budgets
providing comparisons of thermal capacities and temperature
regimes among lakes. We are primarily concerned with the
summer heat budget because juvenile salmon growth is limited at
temperatures below 4 C. The summer heat budget is defined as
the accumulated calories (stored heat) within the lake from
isothermy at 4 C in the spring to the summer maximum
temperature. It is determined by both heat intensity and the
duration of the heating period. In general, the heat budget is
calculated after Birge (1915) by integrating the product of
temperature and the volume of water at selected depth
intervals.
Calculations
1) Construct a plot of lake temperatures (y-axis) using 4 C
as the origin versus the duration (days) of the growing
season (X-axis). The growing season is the number of days
above 4 C in the spring to isothermy at 4 C in the fall.
For each sampling date, plot the temperatures from a
minimum of 5 depth increments, beginning with the 1-m
strata, and continue to the lake bottom or to 4 C. Draw a
smooth curve through the points (Figure 5) forming five
depth contours.
2) Using a calibrated planimeter (see Instrument
Calibrations, p. 192), measure the area beneath each
depth-contour from 4 C in the spring to the maximum
temperature. Convert the planimeter reading to the number
of degree-days (Table la).
3) Divide the number of degree-days by the number of days
from 4 C in the spring to the summer maximum temperature
to determine the mean temperature of each depth increment
(Table la).
4) Use the mean temperature of an upper increment to
represent each depth interval. For example, the 5-m mean
temperature represents the 5 to 10-m depth interval
(Figure 5). Multiply the volume of water (ml) contained
within each depth interval by the mean temperature to
determine ml- C (Table lb).
5) Sum the results for each depth interval to obtain the
total accumulated ml-C (Table lb). As one calorie is
required to raise 1 g (1 ml) of water by 1 degree, ml- C
is equivalent to gram-calories (g-cal).
6) Divide the total number of g-cal by lake surface area
(cm ) to express theZsummer heat budget as g-cal per
surface area (g-cal/cm ) .
7) To calculate the seasonal mean lake temperature,
planimeter the area beneath each depth contour over the
entire growing season, and determine the average
temperature per depth interval (Steps 3 - 5 ) . Calculate the
weighted mean using interval volumes, and add 4 C.
Example
Our planimeter is calibrated as 90 degree-days per 146 units or
0.6164 degree-days per unit.
 TIME ( m o n t h s )
Figure 5. Isopleths of changing temperatures within strata made from field measures
of vertical temperature profiles on seven dates over the growing season (isothermy in
the spring to fall) used to determine the summer heat budget (4 C in the spring to the
seasonal maximum temperature).
Table 1. Use of a planimeter to (A) estimate the average
temperature of five depth increments; and (B) the
calculation of accumulated gram-calories (g-cal)
for each depth interval.

Depth planimeter Degree-days ~ i m eto maximum Average

increment units (P x 0.6164) temperature (d) temperature
(m) t P) (A) t B) (A/B)

1 834 513 74 6.9

5 681 420 64 6.6
10 177 109 64 1.7
15 60 37 44 0.8
Lake bottom 35 22 44 0.5
...............................................................

Average Interval
Depth interval temperature volume (ml) Gram-calories
f m) f C) t D) ( C x Dl

Total g-cal = 94.9~1012

Ths surface area of the lake equals 1.8 x lo6 m2 or 1.8 x 1010
cm . Therefore, the summer annual heat budget is:
94.9 x g-cal
= 5,772 g-cal/cm
2
.
1.8 x lolo cm2
 Dissolved Oxvqen
One of the most significant gases found in lakes is oxygen.
Dissolved oxygen (D.O.) is not only essential for aerobic
respiration, but it influences all biochemical activities
within an aquatic community. The distribution of D.O. is
affected by temperature, photosynthesis, decomposition, and
inorganic-chemical reactions. Oxygen solubility is inversely
affected by temperature; i.e., D.O. levels increase with a
decrease in temperature. iss solved oxygen is measured using a
combination D.O./temperature meter.
Procedure
Calibrate the meter (see Instrument calibrations, p. 192),
lower the probe while agitating if the probe is not equipped
wi h an automatic stirrer, and record D.O. concentrations (mg
L-1) and temperatures.
Calculatfons
Measurements of D.O. are often converted to percent oxygen
saturation using a solubility table, or more conveniently, by
using a nomograph (Figure 6). Placf a straight edge on the
nomograph, and match the D.O. (mg L- ) concentration with the
corresponding temperature. Read the percent saturation
directly from the graph.
 m
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 r~hotosyntheticall~
~ c t i v eRadiation (PAR)
Virtually all of the energy used in the metabolic processes
within lake ecosystems is derived from sunlight. Solar
radiation is converted to chemical energy by algal, bacterial
and macrophytic photosynthesis. In addition, water absorbs
light energy converting it to heat, thereby regulating the
thermal structure of a lake. Measurements of light penetration
are used Yo determine the 1% light level i.e., photosynthetic
compensation point (CP) or the euphotic zone depth (EZD). his
point refers to the maximal depth at which gross photosynthesis
exceeds respiration, and defines the depth of the trophogenic
zone. The CP depth varies seasonally and is affected by such
factors as precipitation, algal production, and silt input.

A. Underwater Photometer
Procedure
Using an underwater photometer (sensitive to wavelengths of
from 400-700 nm), record the incident light level by holding
the probe just above the lake surface. Next, place the probe
just below the surface (-5 cm), record the subsurface light
level, and continue taking measurements at 0.5-meter intervals
to a depth of 5 meters. Take additional readings at 1-meter
intervals (if possible) until reaching -1% of the subsurface
light reading.
Calculations
1) Light-compensation point -
Formulate a linear equation
(Table 2) by regressing depth (y-axis) against log (or In)
percent subsurface light (X-axas), and calculate the
coefficient of determination (r ) . The Y-intercept of
either regression formula gives the CP depth.
Alternately, convert measurements to percent subsurface
light, and plot on semi-log paper, the percent subsurface
light (Y-axis) versus depth (X-axis). The X-intercept is
the CP depth.
) Euphotic volume -
The penetration of 1% of the subsurface
PAR delineates the deepest extent of the trophogenic zone
and defines the euphotic zone depth (EZD). Multiply the
EZD by lake surface area to estimate euphotic volume
(trophogenic volume). Finally, the EZD is also used to
define the extent of the littoral zone in lakes.
3) -
Light-extinction coefficient The inverse slope of the In
regression line (Kd) is the light extinction coefficient
 (,-I) which can be obtained from conversion of the log
slope,to In values e.g., log slope/2.3 equals In slope.
Table 2. Light levels found at specific depths and use in
calculating the light-compensation point (CP) and
the light-extinction coefficient (Kd) .
Percent
Depth '- Light subsurface Log In
(Y (footcandles) lisht (X) (X) (X)
Incident 3,700 -- -- --
Subsurface 3,500 100.0 2.00 4.605
0.5 2,800 80.0 1.90 4.382
1.0 2,600 74.3 1.87 4.308
4.0 2,100 60.0 1.78 4.094
6.5 1,400 40.0 1.60 3.689
7.5 950 27.2 1.43 3.303
10.0 655 13.7 1.14 2.617
15.0 -- 295 8.4 0.92 2.128
22.0 145 4.1 0.61 1.411
30.0 30 0.9 -0.05 -0.105
...............................................................
Depth (m) = 29.2983 - 14.8851 (log % light)

Depth (m) = 29.3535 - 6.4746 (In % light)

B. Secchi disk
The Secchi disk is a black and white circular plate, 20 cm in
diameter, used to estimate the turbidity or degree of
visibility in natural waters. The Secchi disk provides a very
simple means of making transparency determinations in natural
waters. A measured line is attached to the center of the disk
by means of a special fitting that stabilizes the disk so that
it will be parallel to the surface. The disk is lowered into
the water until it just disappears from sight. The disk is
raised and lowered and readings are taken from the calibrated
line at the point where it disappears and reappears. The
average of the two readings is recorded as the Secchi disk
transparency (SD). Best results are obtained in the lee and/or
shaded side of a boat, and are usually obtained after early
morning and before late afternoon.
Transparency usually decreases in the summer when plankton,
silt, and organic matter are more likely to be prevalent. The
most transparent lakes are usually seepage lakes, because this
characteristic greatly reduces the amount of silt-bearing
influents. Drainage lakes carry more silt and usually are less
transparent. For example, a drainage lake has a SD of 1.0 to
1.4 m, but a seepage lake in the same area may give readings of
3.0 to 4.0 m. A high reading of 19 to 21 m would indicate
extreme clgrity; however, that same lake in the summer may read
only 10 m.
The SD is often used to estimate the euphotic zone depth (EZD)
or light-compensation point within lakes. However, SD
generally represents only a portion of the EZD which is
rigorously defined through the use of a submersible photometer.
The proportion of the EZD measured by SD varies by lake type
and should be validated in each lake. However, we have
developed regression equations that allow the EZD to be
estimated, by lake type, from SD readings (Figure 7 ) .
 30'

0
25. 9

20-

V
E
n 15-
N
W

stalned EZD = 1.0755 + 0.9864 SD r * = .71

glacial A E Z D = 1.1810 t 3.298 SD r 2 = -79

07 I
0 5 10 15 20
SD (m)

Figure 7. The relationship between the euphotic zone depth ( E Z D ) defined

by the 1% light level (determined by underwater photometer) and the Secchi
disk transparency ( S D ) for glacial, organically stained, and clear-waterlakes.
 Stream Discharse
Stream discharge is the volume of water passing a point during
a given period of time, it is related to flow rates and
stream-bed characteristics. Mean flow rates are used to
determine discharge and, when applied to an outlet stream,
annual discharge rates are used to estimate the flushing rate
or hydraulic retention time for lakes.
Procedure . *
1) Locate a fairly straight section of stream having a hard,
uniform bottom. This gives a symmetrical pattern of flow
within the stream.
2) Place a line or measuring tape across the stream to locate
the cross-section at which the stream flow or velocity is
to be determined.
3) with a current meter, measure the stream velocity at 60%
of the depth from the surface or at 40% of the depth from
the bottom (Figure 8) at selected intervals; i.e., divide
the stream into equal segments (Figure 9).
4) using the current meter instructions, determine the
velocity (m/sec).
5) From the depth and distance, calculate the surface area %f
each cross-sectional portion of the stream (m )
(Figure 9).
6) Average the stream velocity taken at the two siges of each
section, and calculate the average discharge (m /sec).
7) To determine total stream discharge, sum the discharges
for each section.

Flow of section C is to be measured at 0.6 x 1 m, or 60 cm from

the surface (40 cm from the bottom) and at 0.6 x 80 cm, or 48
cm from the surface (32 cm from the bottom) (Figure 9). If the
velocity measurements equalled 3 m/sec and 3.5 m/sec,
respectively, the discharge through Section C is calculated by:

The sum of discharges through sections A to J equals the total

discharge of the stream.
 6 0 meters

1Om 20m 30m -40 m 60m,.. . ........ ..,..,.,,

..e

stream bottom

Figure 9. Stream cross section partitioned into segments and the segment boundaries
at which water depths and flow rates are measured.
 A. Staff Gauge Calibration
Discharge (Q) measurements are time consuming and often require
several people to complete. Calibrated staff gauges offer a
viable alternative to frequent discharge measurements.
Briefly, a staff gauge is a strong rod, ruled in meters and
centimeters (feet-inches) which is permanently anchored into
the stream bottom. The anchorage is located in a quiet, but
flowing area allowing only water level to affect the metered
reading. calibration is achieved by determining stream
discharge and, at the same time, noting the water height on the
ruled gauge. Readings are taken at both high and low water
levels to provide for a broad range in discharges (Rantz et al.
1982).
Calculations
1) Formulate a linear equation by regressing discharges
(Y-axis) against staff gauge readings (X~axis), and
calculate the coefficient of determination (r ) .
2) To determine stream discharges, substitute staff gauge
readings into the regression equation.
Example
Discharges were measured and the water height recorded for a
staff gauge at the outlet of Falls Lake, Baranof Island, during
. . 1981 and 1985. The log of 20 measured discharges (Y-axis) were
regressed against staff gauge readings (X-axis) resulting in
the equation:
Log Q = -0.537 + 0.0339 (cm); r2 = 0.89
Using this equation, a 21.00 cm reading from tqe staff gauge
would result in an estimated discharge of 1.50 m /sec.

B. Estimation of the Hydraulic (Water) Retention Time

The hydraulic (water) retention or water residence time is
defined as the time required for the total volume of water
within a lake to be replaced.
The method utilizes multiple regression analysis of lake and
watershed characteristics versus known system discharges. The
analysis has been carried out by the U. S. Forest Service and
is delineated in the U. S. Forest Service Water Atlas
(Anonymous 1979).
Southeast
Mean Annual Flow: Q = 0.0312P 1.13A1.03 (r2 = 0.993)

Southcentral
Mean Annual Flow: Q = 0.0283P
1. 16A1. 02 (r2 = 0.997)

Q = mean annual flow (cubic feet per

second)
P = mean annual precipitation (inches)
A = watershed area (square miles)
[Q x 8.92 x lo5 = total lake outflow (TLO) (m3/yr)1.
The theoretical water residence time is calculated according to
the following:

= theoretical water resigen~etime (yr)

VTw = total lake volume ( 10 6m 4
TLO = total lake outflow ( 10 m /yr)

NOTE: This method can only be used for lakes in areas covered
by the U. S. Forest Service Water Atlas (1979).

REFERENCES
S a m ~ l eCollection and Storaae
Inland Waters Directorate, Water Quality Branch. 1982.
Sampling for water quality. Inland Waters Directorate
Water Quality Branch, Ottawa, Canada. 55 p.
MacDonald, R. W., F. A. McLaughlin, and J. S. Page. 1980.
Nutrient storage by freezing: data report and statistical
analysis. Institute of Ocean Sciences, Sidney, B. C.
69 p.
Wetzel, R. B. and D. F. Westlake. 1974. Periphyton. pp.
42-50. In: R. A. Vollenweider (ed.). A manual on
methods for measuring primary production in aquatic
environments. IBP Handbook 12. Blackwell Scientific
Publications, Oxford. 255 p.
Tem~erature, Dissolved Oxysen, Photosvntheticallv Active
Radiation
Birge, E. A. 1915. The heat budgets of American and European
lakes. Trans. Wis. Acad. Sci. Arts Letts. 18:166-213.
Brezonik, P. L. 1978. Effect of color and turbidity on Secchi
disk transparency. J. ~ i s h .Res. Board Can. 35:1410-1416.
Golteman, H. L. 1969. Methods for chemical analysis of fresh
water. IBP Handbook 8. Blackwell Scientific
~ublications,Oxford. 166 p.
Hutchinson, G. A. 1957. A treatise on limnology I.
Geography, physics and chemistry. John Wiley and Sons,
Inc., New York. 1115 p.
Reid, G. K. 1961. Ecology of inland waters and estuaries.
Reinhold Publishing corporation, New York. 373 p.
~chindle;, D. W. 1971. Light, temperature, and oxygen regimes
of selected lakes in the experimental lakes area,
northwestern Ontario. J. Fish. Res. Bd. Canada
28:157-169.
Wetzel, R. G. 1975. Limnology. W. B. Saunders Company,
Philadelphia, PA. 743 p.
Wetzel, R. G. and G. E. Likens. 1979. ~imnologicalanalyses.
W. B. Saunders Company, Philadelphia, PA. 357 p.
Stream Discharse and Hydraulic (Water) ~etentionTime
Anonymous. 1979. Water resources atlas. U. S. Department of
~griculture,Forest Service-Region 10. Juneau, Alaska.
7 P*
Hynes, H. B. N. 1972. The ecology of running waters.
University of Toronto Press. 555 p.
Rantz, S. E. and others. 1982. Measurement and computation of
stream flow. pp. 4-9. Volume 1: Measurements of stage
and discharge. Volume 2: computation of discharge.
Geological survey water supply paper 2175.
Zooplankton
Edmondson, W. T. and G. G. Winberg (eds.). 1971. A manual on
methods for the assessment of secondary productivity in
fresh waters. IBP Handbook 17. Blackwell Scientific
Publications, Oxford. 358 p.
Steedman, H. F. 1976. Zooplankton fixation and preservation.
Unesco Press. Paris. 350 p.
Tranter, D. J. and J. H. Fraser. 1968. Zooplankton sampling.
Unesco Press. Paris. 174 p.
 PART 11.

LABORATORY TECHNIQUES
AND
METHODOLOGIES
 SAFETY CONSIDERATIONS
The limnology staff recognizes the importance of laboratory
safety and has developed an overall safety program. This has
been accomplished and coordinated through an appointed safety
officer whose primary responsibilities are (1) to recognize and
eliminate both existing and potential health and safety
hazards; and (2) train personnel to become aware of health and
- safety hazards. Laboratory personnel are informed of the State
of Alaska's 'right to knowf law that states 'all hazards of
toxic and hazardous substances are evaluated and this
information is to be transmitted to personnel at any time'. In
compliance with this provision, the laboratory maintains
material safety data sheets (MSDS) for all hazardous and toxic
chemicals, and these are made available for inspection at any
time. More importantly, laboratory personnel are trained to
recognize safety hazards, and are instructed in the use of
safety equipment and procedures designed to minimize or
eliminate potential problems.
In addition, the limnology laboratory is equipped with basic
first-aid supplies, fire extinguishers, emergency eye-wash and
shower facilities. At least one person is-trainedin cardiac
pulmonary resuscitation (CPR), and emergency telephone numbers
(hospital, fire, poison-control center) are posted throughout
the lab. Although it is beyond the scope of this manual to
provide a complete laboratory health and safety reference, some
basic procedures are listed here regarding general laboratory
- practices and for handling radioisotopes.

General Procedures
1) Never remove labels from any chemical-substance container.
2) Store chemicals according to the instructions listed on
the manufacturerlslabel. Occupational Safety and Health
Administration (OSHA) approved storage cabinets are to be
used for flammables and corrosives. Bulk quantities of
other chemicals are stored in a ventilated and locked
storage bin.
3) Reagents, solutions, and chemicals routinely used for
analysis are kept on shelves below waist level.
4) Smoking is prohibited in the laboratory at all times.
5) Wear protective clothing (e.g., laboratory aprons or
coats, vinyl gloves, and protective eyewear) when handling
hazardous or toxic chemicals.
Be especially careful when preparing alcoholic-phenol or
dilute acids; protective clothing described above is
mandatory.
Prepare volatile solutions (e.g., acetone) and chemicals
only under the fume hood.
Always add concentrated acids or bases to water while
using the fume hood.
Wipe up chemical spills immediately using the appropriate
spill-control material.
Only autopipets are to be used for the transfer of
solutions.
Be aware of laboratory safety and health hazards. Let
your supervisors or safety officer know immediately if you
have any questions.
Handlins Bv~roductMaterials
While conducting experiments that utilize byproduct
materials (carbon-14 and/or phosphorus-32), you will be
required to use protective gear so marked; i.e., a
laboratory coat/apron plus vinyl gloves. In addition, any
transfer of the material in liquid form will require the
use of automatic pipets and/or syringes. In essence, use
prudent judgement in avoiding the possibility of direct
contact of the material with your person.
We are required to monitor the byproduct use area after
each experiment. The GM survey meter (model #493 and
serial #1713, with probe 489-35) will be calibrated yearly
by:
Victoreen Instrument, Inc.
NCR #34-0486-04
Repair and Calibration Dept.
10101 Woodland Avenue
Cleveland, Ohio 44104
(216) 795-8200
Records of the monitoring program are available from the
limnology laboratory manager and are physically located
within the limnology laboratory data files, Alaska
Department of Fish and Game, Kalifornsky Road, Soldotna.
Use of byproduct material will be confined to the
laboratory's sample preparation room using glassware so
designated. Byproduct materials will be stored in a
lockable refrigerator marked as containing radioactive
materials. Use and storage areas are both located within
 the sample-preparation room and are marked as containing
radioactive materials (9" x 12" 'caution radioactive
materials1 signs on exterior doors). Signs conform to CFR
20 regulations of the Nuclear Regulatory Commission
concerning color and appropriate warning.
4) You are required to follow good radiation safety practices
which include but are not limited to the following:
a) Use of byproduct material is restricted to the area
so designated under Item 3 above.
b) Smoking and the consumption of food and beverages is
prohibited in the designated-use area.
c) Contaminated areas will be cleaned using the
decontaminants provided (e.g., 'Count-off1 from New
England Nuclear) with solid wastes being disposed of
in the marked solid-waste containers. Potentially
- contaminated areas may be stained yellow to blue
because of the addition of iodine to all liquid
reaction vessels.
5) Each user will be issued personnel-monitoring devices
consisting of thermoluminesent wrist dosimeters. Records
of personnel-monitoring results (monthly) are kept in the
office of the laboratory manager with the monitoring
service provided by:
Eberline
P. 0. Box 2108
Santa Fe, NM 87501
(505) 471-3232
6) Records of byproduct material use, transfer, and/or
disposal will be kept and maintained within the
laboratory. Each record will be updated, following an
experiment, to include the location of the experiment,
byproduct material used, quantity, method of disposal, and
resultant total amount of byproduct remaining within the
laboratory. Current byproduct-use records are located
within the designated-use area, and all permanent records
are located in the office of the laboratory safety
officer.
7) Procedures for picking up, receiving, and opening packages
containing byproduct materials are:
a) Notify airline personnel that upon arrival of the
material, limnology laboratory personnel are to be
called at 262-5042 or 262-9368 from 8:00 a.m. to 4:30
p.m. or 262-5323 either before or after these hours.
 b) Upon receiving a call that materials have arrived,
pick up the container(s) within one working day.
c) As each package will contain no more than 10 mCi of
carbon-14 or 5 mCi of phosphorus-32, the package
exterior will not be monitored.
d) Transport the byproduct material to the laboratory by
.State of Alaska vehicle.
e) Shipping documents must accompany any and all
transfer of radioactive materials on public roads.
f) Maintain current records of any transfer of
radioactive materials either into or out of the
laboratory.
g) Open 'packages only within the designated-use area,
and inspect contents for leaks.
h) Open sealed containers containing byproduct material
within the designated-use area. Record any broken
ampules or glass vessels and clean or dispose
contaminated surfaces as outlined below.

By~roductWaste Disposal
1) Carbon-14 disposal will consist of:
a. Liquid waste (at 50.05 uCi per gm) will be returned
to the lake system (remote site lake); i.e., 69-383 u
Ci per lake per sampling period.

b. Liquid waste (at 10.05 u ~ iper gm) will be disposed

' o f down a specified sink not to exceed 1 ~i per yr.
and
c. Solid wastes (e.g., vials and filters) and disposable
items (e.g., gloves, absorbent materials, and pipet
tips) will be buried at a sanitary-disposal site
(50.0225 u Ci per filter).
and
d. Liquid scintillation fluor (toluene based) will be
allowed to evaporate from capped plastic vials placed
outside in a secured area.
2) Phosphorus-32 disposal will consist of holding all liquid
and solid wastes until decayed to background levels.

Emersencv Procedures
These instructions are required reading for all personnel
working within the limnology laboratory, regardless of whether
you are or are not actively using byproduct materials.
1) Notify the lab manager upon the contamination of a work
arena with carbon-14 in liquid form, and apply sodium
bicarbonate to the spill. Clean the area with
decontaminant scrubs located within the designated-use
area.
2) Upon contaminating your person, wash the affected area
with an alkaline detersent and co~iousamounts of water.
.
Use-decontaminant scrubs; e.g , bri-contrad, and notify
the lab manager or safety officer.
3) Use survey instruments and wipe tests as described below
to ensure that the area has been decontaminated:
a) Monitor the of general area with a survey instrument
sufficiently sensitive to detect at least 0.1 mR per
hr carbon-14.
b) Monitor bench tops, floors, etc., for contamination
using 2.4-cm absorbent pads wiped over an area of 100
cm , and count using the Packard Liquid scintillation
spectrometer.
c) As the designated-use area has its own water supply
and disposal area as well as a separate entry-exit
door, personnel pass-through will not be allowed
until the area is cleared by the laboratory manager.
d) Notify at least one of the following persons after
initial containment of the spill:
J. P. Koenings Jim Edmundson
219 Crest Street P. 0. Box 3155
Soldotna, Alaska 99669 Soldotna, Alaska 99669
(907)262-5323 (home) (907)262-4172 (home)
(907)262-9368 (907)262-5042
 GENERAL METHODOLOGIES

Solutes, Solvents, and Solutions

Laboratory methodologies require preparing reagents, solutions,
and standards from concentrated chemicals supplied either as
liquids or.*as solids (crystals, powders, and pellets). In
general, add the substance (solute) to an appropriate
volumetric flask and slowly add the solvent, while mixing,
until dissolved. Add additional solvent to reach the required
volume. In addition, it is common laboratory practice to
prepare a dilute (dil) solution from a concentrated (conc)
stock solution. For example, the methodology may require 250
ml of 2 N sulfuric acid; however, the only acid available is 36
N. To calculate the required volume of 36 N acid use the
expression:

The units of concentration (C) and volume (V) can vary, but
must be the same on both sides of the expression. Using the
above example, the calculation is:
36 N x V (ml) = 2 N x 250 ml

Therefore, dilute 13.9 ml of 36 N sulfuric acid to 250 ml with

DI water to yield the required volume of a 2 N sulfuric acid
solution.
Finally, samples may have concentrations exceeding the upper
limit of detection of the methodology i.e., analytical results
will be equivalent to the upper detection limit. Therefore,
sample dilution is necessary prior to analysis to reduce
concentrations to within operating limits. The correct
concentration is then calculated by multiplying the
concentration of the diluted sample by the dilution factor (DF)
using :
volume of sample + volume of diluent
volume of sample
For efample, Fred Lake has a Xjeldahl nitrogen level of -5000
ug L-l, but the upper limit of detection for the method is 3000
ug L . Diluting 5 ml of samplelwith 15 ml of water resulted
in a concentration of 1,376 ug L . The DF [ ( 5 + 15)/5] equals
4, and th~~correctconcentration of the original sample is
5,505 Ug L (1,376 X 4).
 Conductivitv
Specific conductance, conductivity, is defined as the measure
of a solutionfs ability to carry an electric current.
Resistance, the inverse of conductivity, is measured in ohms
with conductivity being expressed as mhos/cm. In addition,
conductivity can be expressed in Siemens ( S ) e l umhos/cm
divided by 10 is equivalent to 1 mS/m. Measurements of
conductivity are used to indicate levels of dissolved material
and, speci-fically, as an indication of potential nutrient
levels or fertility. As such, conductivity is often highly
correlated with the amount of dissolved solids, and has been
used in a procedure to determine salinity.
Accuracy
+ 0.2% full scale at 10-200 umhos/cm,
-
Apparatus
YSI model-32 conductance meter, 100-ml graduate cylinder,
100-ml beakers, and a magnetic stirrer.
Reasents
Sodium chloride standard -A standard supplied by Hach Chemical
Company has a conductivity of 1990 + 10 umhos/cm at 25 C.
Procedure
1) calibrate the meter according to the manufacturer's
instructions. Check the accuracy of the meter by
measuring the conductivity of the NaCl standard.
2) Pour 100 ml of sample into a beaker and place on a
magnetic stirrer. Immerse the probe and allow the meter
to reach equilibrium. Record the reading in umhos/cm.
3) Rinse the probe with DI water before continuing to the
next sample.
4) Not all conductivity meters are temperature compensated.
In addition, meters that are compensated for temperature
vary in regard to compensation temperature. Thus,
conversions are necessary to express conductivity at
various standard temperatures; e.g., 18 C and 25 C. If
the meter is not temperature compensated, use the
following procedure to convert to a standard temperature:
a) Determine the percent conductivity change per degree
centigrade ( % per C). Heat or cool a sample to
exactly 25.0 C, and heat or cool a duplicate sample
several degrees above or below 25.0 C.
 b) Record the temperature and conductivities of the
solutions, and calculate the slope from the
expression:

--

= slope = % per C
'25

Where, C25 = conductivity of solution at 25 C.

Cx == conductivity of solution x at T
,
.
25 C.
::5 = temperature of solution x.

4) If the slope lies within the range of 0-4%, conductivity

readings compensated to 25 C can be calculated from the
expression,
Conductivity (25 C) = sample conductivitv
slope/4% x (0.04-1) + 1

Salinitv
Salinity can be determined from conductivity measurements. The
ratio (R) of the sample conductivity at 15 C to that of a
- -. --standardhaving a salinity of exactly 35 parts per thousand
( % % ) is used in a polynomial expression which defines salinity
(Wooster et al. 1969). As conductivity increases with both
increasing temperature and salinity; Table 3 provides a
comparison of salinity, chlorinity, and conductivity at various
temperatures.
Procedure
1) Prepare a salinity standard by dissolving 31.77 g of
reagent grade NaCl in 1000 ml of DI water. This solution
has a chlorinity of 19.4%% and a salinity of 35%%.
2) Measure the conductivity of the standard and the sample.
Rinse the probe with DI water following the standard
measurement before continuing to the sample. Solutions
should be 15 C, and the meter's temperature compensator
turned off.
Table 3. Laboratory results comparing salinity ( % % ) ,
chlorinity ( % % ) , and conductivity at various
temperatures.

Salinity* ~hlorinity Conductivitv (umhos/cm)

(%%I (%%I 5 C 15 C 20 C 25 C

*Salinity is determined from the expression S%% = 1.80655 C1%%

(after Wooster et al. 1969).

R = Conductivitv of the sample

Conductivity of the standard
Substitute R into the following polynomial expression, to
define salinity in parts per thousand ( % % ) :

pH units are a measure of the acidity of a solution at a

specific temperature. Specifically, one unit of pH is equal to
the negative )og of the hydrogen ion concentration (pH = -log
[H ] where [H ] is the concentration of hydrogen ions in moles
per liter) .
pH is an important aspect of water quality, since almost every
chemical reaction is pH dependent. Measurements of pH are also
used in calculations of alkalinity and other acid-base
equilibria.
Precision
+ 0.02 p~ ( p range)
- ~ ; + 0.01 pH (pH expanded range) .
Accuracy
+ 0.05 p~ ( p range)
- ~ r + 0.005 pH (pH expanded range)
Orion model-399A/ion analyzer, 100-ml graduate cylinder, 100-ml
beakers, and magnetic stirrer.
Reasents
Buffer solutions of pH 4, 7, and 10.
Procedure '

1) calibrate the pH meter (see Instrument Calibrations, p.

192).
2) Allow the samplesto reach room temperature (-25 C).
3) Pour loo ml of sample into a beaker and place on a
magnetic stirrer. Immerse the probe, allow the meter to
reach equilibrium, and record the pH.
4) ~ i n s ethe probe with DI water before continuing to the
next sample

Alkalinitv
The alkalinity-of lake water is largely due to the presence of
carbonate (CO -) and bicarbonate HCO ) ions. The proportion
of each speci8s is a function of pa.
Since these ions are
converted to carbon dioxide (CO ) at pH 4.5, this procedure
determines the total amount of inorganic carbon available for
algal uptake (Table 40). Also, the amount of alkalinity
determines the ability of natural waters to resist changes in
pH. Such buffering capacity is important as many chemical
reactions e.g., photosynthesis, affect the pH.
Limit of Detection

pH meter, magnetic stirrier, 100-ml graduate cylinder, 100-ml

beakers, and a 10-m1 automatic buret (0.1 ml divisions).
Reasents
1) 1 N Sulfuric acid - Dilute 27.8 ml of concentrated H2S04
to 1 liter with DI water.
2) 0.02 N sulfuric acid standard - ~ilute10 ml of 1 N H2S04
to 500 ml with DI water.
3) Standard pH 4 and 7 buffers.
Procedure
1) Calibrate the pH meter (see Instrument calibrations, p.
192).
2) Pour 100 ml of sample into a beaker and place on a
magnetic stirrer. Immerse the pH probe in the sample.

3) Using the buret slowly add titrant (0.02 N H2S04) to a pH

of 4.5. Record the volume (ml) of titrant.
4) Rinse the probe with DI water before continuing to the
next sample.
Calculations
Total alkalinity (mg L-I as CaC03) = B x N x 50000
v
Where: B = ml titrant (0.02 N H SO )
N = normality of the titgane
V = sample volume (ml)

Turbiditv
Turbidity is primarily caused by suspension of inorganic
material (silt) and organic particles (algal cells) in the
water column. Measurements of turbidity are made by comparing
a sample's ability to scatter light to that of a known
reference. The unit of measurement is the nephelometric
turbidity unit (NTU). As suspended material acts to decrease
the depth of the euphotic zone (EZD), measurements of turbidity
are used to evaluate the photic qualities of lakes (Figure 10).
Apparatus
HF Instruments model-DRT 100 turbidimeter with reference
standard, and a 30-ml cuvette.
Procedure
1) Calibrate the turbidimeter with a reference standard
according to the manufacturer's instructions.
2) Invert an unfiltered sample several times and pour into a
cuvette.
3) After all air bubbles have dissipated, record the NTU
reading from the appropriate scale.
 3 0-
D

2 5- LOG E t D = 1.2270 - 0 . 6 6 3 6 ~ 0NTU

~

r = -94

b
20-

a8

15-
h
E t r
w

n
rtJ (I
W
10-

LOG NTU

6-

am. 1 a 1 -
m

0 I I I

0 10 20 30 40 50 60
NTU -
Figure 10. The relationship of decreasing euphotic zone depth ( E Z D ) to increasing
turbidity (NTU), and the log-log transformation and linear regression equation.
 Color
The presence of organic compounds and colloidal particles
impart true color to lake water, and both act to restrict light
penetration (Figure 11). In contrast, apparent color is
imparted to lakes by suspended inorganic material which also
causes turbidity.
The colorof filtered lake water is determined by comparing
absorbance at 400 nm to a standard containing a known amount of
platinum cobalt (Pt).
Spectrophotometer (400 nm) and 10-mm cuvettes.

Color standard - Dissolve 1.246 g of potassium chloroplatinate

(K PtC1 --) and 1 g of cobaltous chloride (CoC1 6H 0 ) in 500 ml
o f 2 ~ 1wgter containing 100 ml of concentratgd ~ $ 1and dilute
the mixture to 1 liter with DI water. Alternately, a 500
platinum cobalt unit standard is supplied by Hach chemical
Company.
Procedure
1) Prepare a serial dilution of the color standard (Table 4).
2) Measure the absorbance (abs) of standards at 400 nm
against a DI water blank.
3) Formulate a linear equation by regressing platinum cobalt
units (Y-axis) against absorbances Jx-axis) and calculate
the coefficient of determination (r ) .
4) Measure the absorbance of a filtered sample at 400 nm
against a DI water blank (see Instrument Calibrations,
p. 192).
Calculations
Calculate sample color (Pt units), by substituting sample
absorbances into the regression formula (Table 4).
 30-

-
2.0-
25-

1.5-
n
a rU
W
0
2 0- 2 1.0-

a
n 0.5-
E
w
n 15-
N 0 I
W 0 0.5 1.0 1.6 2.0
LOG Pt

1 0-
a

5-

01 1

0 10 20 30
COLOR (Pt)

Figure 11. The relationship of decreasing euphotic zone depth (EZD) t o increasing
lake-water color, expressed as platinum cobalt units (Pt), and the log-log transformation
and linear regression formula.
Table 4. Representative values used to establish the
relationship between absorbances (abs) at 400 nm
and platinum-cobalt units (Pt) .
Volume of Volume Platinum
color standard of DI cobalt units Absorbance
(ml) (ml) t Pt) (400 nm)

Pt units = 1.9104 + 1126.8922 (abs)

Total, Suspended, and Dissolved Solids

Total dissolved solids (TDS) is the residue formed upon
evaporation of filtered water, and total solids (TS) the
residue formed after evaporation of unfiltered water. As the
TDS determination includes dissolved (ions) as well as
suspended material, values can be correlated to both
conductivity and alkalinity. TS values can include sizeable
amounts of suspended solids (TSS) which impart turbidity.
Limit of Detection
2 mg L-I residue.
Apparatus
Electronic balance with minimal sensitivity of 0.0001 g, drying
oven, hot plate, and 30-ml petri dishes.
Procedure
1) Place a clean petri dish in the drying oven at 100 C for 4
hours.
 2) Desiccate for at least 4 hours, and weigh to the nearest
0.OOOl g (Wo).
3) Pour 30 ml (0.03 1) of filtered (TDS) or unfiltered (TS)
sample into the petri dish, and evaporate the water at 80
C on a hot plate. Evaporate an additional 30-ml aliquot
for a total sample volume of 0.06 1.
4) Place in the drying oven at 100 C for an additional 4
hours:
5) Desiccate the sample for 4 hours, and weigh (Wf).
Calculations
TDS and TS are calculated using the following expression:

NOTE: Suspended solids (TSS) can be determined by difference

using the expression TS = TDS + TSS.

REFERENCES
.. -+
Conductivity, DH, Alkalinity, Total Dissolved Solids. and Total
Solids
American Public Health Association, American Water Works
Association and Water ~ollutionControl Federation. 1985.
Standard methods for the examination of water and
wastewater. 16th ed. New York, N.Y. 1268 p.
Turbidity
American public Health ~ssociation,American Water Works
Association and Water Pollution Control Federation. 1985.
Standard methods for the examination of water and
wastewater. 16th ed. New York, N.Y. 1268 p.
Edmundson, J. A. and J. P. Koenings, 1985. The effects of
glacial silt on primary production, through altered light
regimes and phosphorus levels in Alaska lakes. pp: 3-20.
In: L. P. Dwight (ed.) , Resolving Alaska's Water
Resources Conflicts. University of Alaska-~airbanks,
Institute of Water Resources Report 108. 212 p.
Edmundson, J. M. and J. P. Koenings. 1985. The influences of
suspended glacial particles in the macro-zooplankton
community structure within glacial lakes. pp. 21-36.
In: L. P. Dwight (ed.), Resolving Alaska's Water
Resources Conflicts. University of Alaska-Fairbanks,
Institute of Water Resources Report 108. 212 p.
Koenings, J. P., R. D. Burkett, G. B. Kyle, J. A. Edmundson,
and J. M. Edmundson. 1986. Trophic level responses to
glaciql meltwater intrusion in Alaskan lakes. PP
179-194. In: Kane, D. L. (ed.), Proceedings: Cold
Regions Hydrology Symposium. American Water Resources
Assoc. Bethesda, MD. USA. 612 p.
Lloyd, D. S., J. P. Koenings, and J. D. LaPerriere. 1987.
Effects of turbidity in freshwater of Alaska. N. Amer. J.
of Fish Management 7: (In press).
Vanous, R. D., P. E. Larson, and C. C. Hach. 1982. The
theory and measurement of turbidity and residue. pp.
163-234. In: Minear, R. A. and L. H. Keith (eds.)
Water Analysis, Volume 1. Inorganic species, Part 1.
.
Academic Press, New York, N. Y. 287 p.
Color
American Public Health Association, American Water Works
Association and Water Pollution Control Federation. 1985.
Standard methods for the examination of water and
wastewater. 16th ed. New York, N.Y. 1268 p .
Brezonik, P. L. 1978. Effect of color and turbidity on Secchi
disk transparency. J, Fish. Res. Board Can. 35:1410-1416.
Salinity
Wooster, S. W., A. J. Lee, and G. Dietrich. 1969.
~edefinitionof salinity. Limnol. Oceanogr. 14:437.
 METALS

Calcium and Masnesium (titration)

Calcium (Ca) and magnesium (Mg) are two of the more important
elements contributing to water hardness. Both elements can be
- determines in the same sample by first precipitating out Mg:
and then determining Ca. Ca forms an orange compound with
glyoxal, but forms a stronger bond with EDTA. As EDTA is
added, the color changes from orange to yellow. ~ddition of
acid dissolves the Mg precipitate and destroys the Ca-EDTA
complex, allowing for a separate titrametric determination of
Mg
Standard Ranse
2.5 - 12.5 mg L-I Ca, 1.5 - 7.5 mg L-l Mg.
Uwger Limit of Detection
150 mg L-I Ca, 175 mg L-I Mg.
Lower Limit of Detection
~mpirical: 0.3 mg L-I Ca, 0.3 mg L-I Mg.
Predicted: 0.2 mg L-I Cat 0.2 mg L-' Mg.
Precision
11% at 5 mg L-l Ca, 21% at 3 mg L-l Mg.
Accuracy

pH meter, magnetic stirrer, graduate cylinder, 100-ml beakers,

and 10-ml automatic buret (0.1-ml divisions).
Reasents
1) 0.01 N EDTA - Dissolve 1.861 g EDTA-disodium salt into
-400 ml of DI water, and dilute to 500 ml.
2) Glyoxal indicator - Dissolve 0.3 g glyoxal bis-
(2-hydroxyanil) into 100 ml of reagent-grade methanol.
Warm gently to dissolve.
3) 0.1 N,NaOH - Dissolve 4 g of NaOH into -500 ml of DI
water, and dilute to 1 liter.
4) 1 N HC1 - Dilute 21.0 ml of concentrated HC1 to 250 ml
with DI water.
5) Borax buffer - Dissolve 10 g of sodium borate -
10 hydrate
in -200 ml of DI water. Add 2.5 g of NaOH, and 1.25 g of
sodiuin sulfide-9-hydrate previously dissolved in 25 ml of
DI water. Combine the two solutions, and dilute the
mixture to 250 ml with DI water.
6) Eriochrome Blue-Black B (C.I. 14640) indicator - Mix
together 0.4 g of Eriochrome Blue-Black B and 100 g of
sodium chloride.
Standards
1) Ca standard (1 mg ml-1 Ca) -
Dissolve 1.838 g of calcium
chloride-dihydrate into -450 ml of DI water, and dilute to .
500 ml.
2) Mg standard (0.6 mg ml-I Mg) - Dissolve 2.510 g magnesium
chloride-6-hydrate into -450 ml of DI water, and dilute to
500 ml.
Procedure
1) Prepare Ca and Mg standards in the same beakers according
to Tables 5 and 6.
2) Calibrate the pH meter using pH 4 and 10 buffers (see
Instrument Calibrations, p. 192).
3) Pour 40 ml of sample or combined standards into a beaker
and place on a magnetic stirrer.

A. Calcium
4) Add 5.0 ml of 0.1 N NaOH.
5) Add 3.0 ml of glyoxal indicator.
6) Allow 1-3 minutes for color development (orange-red).
7) Using the automatic buret, titrate the solution with 0.01
N EDTA until the color change of orange-red to yellow.
8) Record the volume (ml) of titrant and re-zero the buret.
 B. Magnesium
9) Add a' varying amount (-0.5 ml) of 1N HC1 using an
autopipet to pH 4.0.
10) Add 1 ml of borax buffer (pH should equal 1 0 1 ) and
-100 mg of Eriochrome Blue-Black indicator.
11) Titrate the solution with 0.01 N EDTA until the color
change of violet to blue, and record the volume (ml) of
titrant.
Calculations
1) Formulate separate linear equations for calcium and
magnesium by regressing concentrations (Y-axis) against
volumes (ml) of EDTA (x-ax4s), and calculate the
coefficient of determination (r ) .
2) Calculate sample concentrations by substituting volumes
(mf) of EDTA into the appropriate regression formula
(Tables 5 and 6).
Table 5. The relationship between known concentra-
tions of calcium and the volumes (ml) of
ETDA titrant used to calculate a standard
curve in the analysis of calcium.

Calcium Total Calcium EDTA

standard volume concentqtion titrant
(ml) (L) tms L 1 (ml)

Ca (mg L-I) = 50.1792 + 8.8737 (EDTA)

r = .9977
 Table 6. The relationship between known concentra-
tions of magnesium and volumes (ml) of EDTA
titrant used to calculate a standard curve in
the analysis of magnesium.

Magnesium Total Magnesium EDTA

standard volume concentrasion titrant
(ml) (L) (ms L- 1 (ml)

Calcium (colorimetric)
This method is adapted from Golterman (1970). The principle
involves the reaction of calcium with glyoxal to form an
orange-red compound. The color remains linear with calcium
concentration over a relatively small range; however, this
- procedugf is quite applicable for calcium levels not exceeding
- 5 m g ~ .
Standard Ranse
0.2-4.0 mg L-I.
Upper Limit of Detection
5 mg L-l Ca.
Lower Limit of Detection
~mpirical: 0.10 mg LI: Ca.
Predicted: 0.04 mg L Ca.
Precision
3% at 1.2 mg L-I ca.

Accuracy
+ 5% at 1.2 mg L-l Ca.
-
Spectrophotometer (520 nm), 15-1111disposable centrifuge tubes,
and 10-rnm cuvettes.
Reasents
1) Buffer - Dissolve 5 g of sodium hydroxide and 5 g of
sodiinn borate-10-hydrate into -450 ml of DI water, and
dilute to 500 ml.
2) Glyoxal indicator - Dissolve 0.5 g of glyoxal bis-
(2-hydroxyanil) into 100 ml of reagent-grade methanol.
3) Reagent alcohol, anhydrous.
Procedure
1) Pipet 5 ml of unfiltered sample or standard into a 15-1111
disposable centrifuge tube.
2) Add 0.5 ml of buffer.
3) Add 0.25 ml of glyoxal indicator.
4) Add 5 ml of reagent alcohol and invert to mix.
5) Allow 25 minutes for color development,
- and measure the
-- - -

absorbance at 520 nm against a DI water blank (see

Instrument calibrations, p. 192).
Standards
Primary standard (0.2 mg ml-I Ca) -
Dissolve 0.3676 g of
calcium chloride-dihydrate (CaC12-H20) into -450 ml of DI
water, and dilute to 500 ml.
Secondary standard (0.02 mg ml-l) - ~ilute10 ml of the primary
standard to 100 ml with DI water.
Calculations
1) Formulate a linear equation by regressing concentration
(Y-axis) against averaged absorbances minus the blank
axis) is), and calculate the coefficient of determination
Ir \

2) Calculate sample concentrations by subtracting the blank

value from the sample absorbances, and substitute into the
regression formula (Table 7).
Table 7. --- concentrations of
The relationship between known
Calcium and absorbance~ (abs) at 520 nm used to
calculate a standard curve in the colorimetric
analysis of calcium.

Secondary Total Calcium

standard volume -
concentrption Absorbance
(ml) (L) (mq L 1 (XI

Ca (mg L-')
---
=20.0141 + 5.3101 (abs)
r = .9999

Masnesium (colorimetric)
Magnesium reacts with Brilliant Yellow dye to form an
orange-colored complex (Taras 1948). The color remains linear
with magnesium concentration over a relatively small range;
however, this lprocedure is applicable for magnesium levels
below -5 mg L- .
Standard Ranse
0.4-4.0 mg L-l Mg.
U m e r Limit of Detection
5 mg L-l~ g .
Lower Limit of Detection
Empirical: 0.4 mg L-:~M~.
Predicted: 0.24 mg L Mg.
Precision
24% at 1.2 mg L-l Mg.
Accuracy
Spectrophotometer (540 nm), 15-ml disposable centrifuge tubes,
and 10-mm cuvettes.
Reasents
1) 1.0 N Sulfuric acid - Add 28 ml of concentrated H2S04 to
500 nil of DI water, cool, and dilute to 1 liter.
2) 0.1 N Sulfuric acid - ~ilute50.0 ml of 1 N sulfuric acid
to 500 ml with DI water.
3) 5 N Sodium hydroxide -
Dissolve 100 g of sodium hydroxide
into -400 ml of DI water, cool, and dilute to 500 ml.
4) calcium-aluminum solution - Dissolve 0.77 g of aluminum
sulfate (A12[S0 ] ) into -500 ml of DI water. Add 62.9 g
of calcium chfo3ide-dihydrate, 40 ml of concentrated
nitric (HN03)acid, and dilute to 1 liter with DI water.

5) 2% Starch solution - Add 10 g of soluble starch to 200-300

ml of DI water. Add 5 N sodium hydroxide while stirring
until the solution becomes clear, and add 1 ml of glacial
acetic acid. ~ilutethe mixture to 500 ml with DI water,
and filter through a ffasttpleated filter.
6) -
Brilliant Yellow (C.I. 24890) indicator Dissolve 0.017 g
- of Brilliant Yellow Dye into -450 ml DI water, and dilute
to 500 ml. Prepare fresh daily.
Mixed Reasents
1) Mixed reagent I (MR I) - combine equal volumes of 0.1 N
sulfuric acid and calcium-aluminum solution.
2) Mixed reagent I1 (MR 11) - combine equal volumes of 5 N
NaOH and 2% starch solution.
Procedure
1) Place 5 ml of sample or standard in a 15-ml centrifuge
tube.
2) Add 0.2 ml of MR I and invert to mix.
3) Add 1.0 ml of MR I1 and invert to mix.
4) Add 3.0 ml of the indicator solution and invert to mix.

5) Allow at least 20 minutes for full color development, and

measure the absorbance at 540 nm against a DI water blank
(see Instrument Calibrations, p. 192).
Standards
Primary standard (0.2 mg ml-I Mg) - Dissolve 0.508 g of
magnesium sulfate-heptahydrate (MgS04-7 H20) into 200 ml of DI
water, and dilute to 250 ml.
Secondary standard (0.02 mg ml-' Mg) - Dilute 10 ml of the
primary magnesium standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged absorbances minus the blank
(Xzaxis), and calculate the coefficient of determination
(r ) *
2) Calculate sample concentrations by subtracting the blank
from the sample absorbances, and substitute into the
regression formula (Table 8).
--- concentrations of
Table 8. The relationship between known
magnesium and absorbance~(abs) at 540 nm used to
calculate a standard curve in the colorimetric
analysis of magnesium.

Secondary Total Magnesium

standard volume Concentqtion Absorbance i z
(ml) (L) tms L 1 ( x) (;;-blank)

---
Mg (mg L-I) = 10.4248 + 27.3719 (abs)
r = .8912

Total Iron (manual)

Iron (Fe) is an essential nutrient for phytoplankton, and
chemically is closely coupled with the phosphorus cycle. In
natural waters iron exists in two oxidation states ferric (Fe
111) and ferrous (Fe 11); and in particulate as well as in
organic colloidal complexes. In natural water?, iron in
chemical equilibrium is present at levels (20 ug L .
 Total iron is determined by reducing ferric iron to ferrous
iron with +hydroxylamine and hydrochloric acid during heat
digestion. Ferrous iron reacts with Ferrozine to form a
violet-red compound which is measured colorimetrically.
Standard Ranse
10-200 ug L-I Fe.
Upper L i m G of Detection
7000 ug L-l Fe.
Lower Limit of Detection
Empirical: 4 ug L-l-~e.
Predicted: 3.3 ug L Fe.
Precision
17% at 40 ug L-I Fe.
Accuracy
+ 7% at 40 ug L-I Fe.
Apparatus
-. - - - Spectrophotameter (562 nm), autoclave (121 C, 15 psi), 50-ml
erlenmeyer flasks, and 10- or 40-mm cuvettes.
Reaqents
1) Acid-reductant - Dissolve 10 g of hydroxylamine-
hydrochloride into 100 ml of 1:l hydrochloric (HC1) acid.
2) 6 N sodium hydroxide -Dissolve 120 g of NaOH into -400 ml
of DI water, cool, and dilute to 500 ml.
3) Sodium acetate solution - Dissolve 75 g of sodium acetate-
trihydrate into -400 ml of DI water. Warm the solution to
dissolve, and dilute to 500 ml.
4) Ferrozine (iron reagent) - Commercially supplied by Hach
Chemical Company, the reagent is pre-mixed and ready to
use. Store in the dark at room temperature; however, a
precipitate may occur after prolonged storage, so
thoroughly mix prior to use.
Procedure
1) Pour 30 ml of unfiltered sample or standard into a 50-ml
erlenmeyer flask.
2) Add 1 ml of acid-reductant and swirl to mix.
3) ~over'the flask with a 15-ml polypropylene beaker and
autoclave (121 C, 15 psi) for 15 minutes.
4) Cool to room temperature and add in order: 1 ml of
~errozine,2 ml of sodium acetate, and 2 ml of 6 N sodium
hydroxide. Swirl to mix and allow 20 minutes for full
color development.
5) Measure the absorbance at 562 nm against a DI water blank
(see Instrument calibrations, p. 192).
Standards
Primary standard (0.03 mg ml-l Fe) - Dissolve 0.0726 g of
ferric chloride-6-hydrate into -400 ml of DI water containing
2.5 ml of 1:l sulfuric acid, and dilute to 500 ml with DI
water.
Secondary standard (0.3 ug ml-' Fe) - Dilute 1 ml of the
primary standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against the averaged absorbances minus the blank
(XZaxis), and calculate the coefficient of determination
(r I *
2) Calculate sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
regression formula (Tables 9 and 10).
Table 9. ---
The relationship between known concentrations of iron
and absorbance~ (abs) at 562 nm (10-mm cuvette) used
to calculate a standard curve in the manual analysis
of total iron.

Volume of
secondary Total Iron
standard - - volume concentr tion Absorbance ai3s
(ml) (L) (us L-PI (x) (:-blank)

Fe (ug L-I) =20.5903 + 2530.9843 (abs)

---
r = .9996

---known concentrations of
Table 10. The relationship between
iron and absorbance~ (abs) at 562 nm (40-mm cuvette)
used to calculate a standard curve in the analysis
of total iron.

Volume of Total Iron

standard volume concentrption Absorbance ~ 6 s
(ml) (L) (us L (x) (%blank)

Fe (ug L-I) = -A. 1344 + ---

583.7913 (abs)
r = .9994

correctins for Tubiditv

Glacial lakes, in particular, are characterized by large
concentrations of suspended silt particles causing turbidity
(>5-10 NTU). In the analysis of total iron (TFe), the
turbidity of unfiltered water could elevate sample absorbances
causing artificially high values. However, when we compared
estimates of iron made without turbidity blanks (TFe) to iron
levels found with turbidity blanks (NFe), we found an error of
only 1% (Table 11). For the range in turbidity and TFe tested,
no turbidity correction is warranted.
Table 11. Comparisons of total iron (TFe) to
turbidity corrected iron (NFe) showing an
-1% turbidity error when using the manual
.- analysis, and turbidity (NTU) levels in
lakes influenced by glacial silt.

Turbidity TFe-l NFe-l

Lake ( NTU 1 (us L 1 (us L 1
Silver
Kenai
Coghill
Upper Trail
Upper Trail
Tustumena
Tustumena
Tustumena
NFe = 0.4893 + 0.9871 TFe

Total Iron (automated, tentative)

This method is adapted from Skougstand et al. (1979). The
chemical principles are the same as those described for the
manual method.
Standard Ranse
5-500 ug L-I Fe.
Lower Limit of Detection
Empirical: 5 ug L-I Fe.
Predicted: Not available.

Autoanalyzer I1 equipped with 520-nm filters and 50-mm flow

cells, autoclave (121 C, 15 psi), and 30-ml culture tubes with
autoclavable caps.
 Reaqents ,

1) Ferrozine (iron reagent) - Premixed solution commercially

available from Hach Chemical Company.
2) Hydroxylamine-hydrochloric acid - Dissolve 100 g of
hydroxylamine hydrochloride into -500 ml of DI water
containing 40 ml of concentrated HC1, and dilute to 1
liter*with DI water.
3) Sodium acetate solution -Dissolve 250 g of sodium
acetate-trihydrate into -750 ml of DI water, and dilute to
1 liter.
4) Sample wash - Add 0.5 ml of Brij 35 wetting agent
(Technicon Industries) per liter of DI water.
Procedure
1) Pour 10 ml of unfiltered sample or standard into a 30-ml
culture tube, and add 0.3 ml of hydroxylamine-hydrochloric
acid.
2) Cover the tube and digest in the autoclave (121 C, 15 psi)
for 15 minutes.
3) Cool, and place a 4-ml aliquot into the sampler tray.
- 4) Arrange the Autoanalyzer (Figure 12), baseline, calibrate
(see Instrument Calibrations, p. 192), and pump the
standards and samples through the system.
Standards
Primary iron standard (50 ug ml-I Fe) - Dissolve 0.0605 g of
ferric chloride -6-hydrate (FeC1 -6H 0) into -200 ml of DI
water containing 2.5 ml of 1:l su?furic acid, and dilute to 250
ml with DI water.
Secondary iron standard (0.5 ug ml-' Fe) - Dilute 1 ml of the
primary standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged chart deflections minus the
blank (X-axis), and calculate the coefficient of
determination (r ) .
2) Calculate sample concentrations by subtracting the blank
value from the sample chart deflections, and substitute
into the regression formula (Table 12).
 ,
Colorimeter

0.32 ml/min AIR

520nm
F.C. 50mm c e l l 20x 20x 20x
oJI-Ll%l- 1.00 mllmin SAMPLE
1
0.23 ml/mln H Y D R O X Y LAMINE
-
0 . 4 2 ml/min SODIUM ACETATE

0 . 2 3 ml/min FERROZINE

2 . 0 0 ml/min WASH

WASTE 0.80 ml/min W A S T E

WASTE 0.42 ml/mitl DEBUBBLER

-
Figure 12. Autoanalyzer schematic showing reagent lines with flow rates (ml/min), cam
size (number of samples per hour and sample to wash ratio), mixing coils, and colorimeter
with specified flow cell (FC) and wavelength (nm) used in the analysis of total iron.
 Table 12. The relationship between known concentrations of
.iron and autoanalyzer chart deflections (ea) used
to calculate a standard curve in the automated
analysis of total iron.

Secondary Total Iron Chart

standard volume concentgation deflection 3
(ml) t L) (us L ) tx) (:-blank)

Hardness
Total hardness is computed as the sum of the cations
contributing to hardness and is expressed as equivalent
concentrations of calcium carbonate (CaC03). To calculate
- hardness, multiply the concentrations of the hardness producing
cations by the factofs listed in Table 13 and sum to obtain
total hardness (mg L- as CaC03). Report the results in terms
of the cations involved.
Table 13. Factors used to express
various metal cations
as equivalent concen-
trations of CaC03.

Cation Factor
 REFERENCES
Calcium and Masnesium
Golterman, H. L. 1969. Methods for chemical analysis of fresh
water. IBP Handbook 8. Blackwell Scientific
Publications, Oxford. 166 p.
Taras, M. --1948. Photometric determination of magnesium in
water with brilliant yellow. Anal. Chem. 20:1156-1158.
Iron
Golterman, H. L. 1969. Methods for chemical analysis of fresh
water. IBP Handbook 8. Blackwell Scientific
Publications, Oxford. 16 p.
Koenings, J. P. 1976. In situ experiments on the dissolved
and colloidal state of iron in an acid bog lake. Limnol.
Oceanogr. 21:674-683.
Skougstand, M. W., M. J. ~ishmen,L. C. rei id man, D. E. Erdman,
and S. S. Ducan, (eds). 1979. Methods for determination
of inorganic substances in water and fluvial sediments.
U. S. ~eological Survey. U. S. Department of the
Interior. 626 p.
strickland, J. D. H. and T. R. Parsons. 1972. A practical
- handbook of seawater analyses. Bull. Fish. Res. Board
Canada 167. 311 p.
Hardness
American Health Association, American Water Works Association
and Water Pollution Control Federation. 1985. Standard
methods for the examination of water and wastewater.
16th ed. New York, N.Y. 1268 p.
 DISSOLVED GASES

Dissolved Oxvsen
This method describes the Winkler procedure for dissolved
oxygen (D.O.) determination.
Ranqe

Limit of Detection
0.5 mg L - ~ .
Apparatus
10-ml automatic buret (0.1 ml divisions), 300-ml BOD bottles, .
and 250-ml erlenmeyer flasks.
Reasents
1) Manganese sulfate solution (I) - iss solve 365 g MnS04H20
into -500 ml DI of water, and dilute to 1 liter.
2) ~lkaline-iodidesolution (11) -
(1) iss solve 500 g of NaOH
into -400 ml of DI water and dilute to 500 ml, and (2) in
a mixing cylinder dissolve 300 g of potassium iodide in
-300 ml of DI water, and dilute to 450 ml. Carefully mix
parts 1 and 2 as the mixture will get hot.
3) Sulfuric acid - Concentrated reagent-grade H2S04.
4) 2% starch solution -
Suspend 2 g of soluble starch in
300-400 ml of DI water. Add an -20% NaOH solution while
stirring until the starch mixture becomes clear, and
dilute to 1 liter with DI water.
5) 0.025 N sodium thiosulfate -
Dissolve 6.20 g of sodium
thiosulfate into -500 ml of DI water, and dilute to 1
liter.
Procedure
1) Collect the sample in a 300-ml BOD bottle without trapping
air bubbles in the bottle (see Dissolved Gases, p. 8).
2) Add in order 2 ml each of solutions I and 11. Invert
several times to mix.
3) Allow the floc or precipitate to settle, mix again, and
allow .to resettle.
4) Add 2 ml of sulfuric acid and mix until the floc is
completely dissolved. The sample is now fixed and can be
analyzed later ( < 8 hr) if kept in the dark.
5) Pour 101 ml of the fixed sample into a 250-ml erlenmeyer
flask,
6) Using an automatic buret, titrate the sample with 0.025 N
sodium thiosulfate to a pale straw color.
7) Add 1-2 ml of the starch solution and complete the
titration until the blue-black color turns clear. Record
the volume (ml) of titrant used.
Calculations
1) F o r a 101ml fample, 1 m l of 0.025 N sodium thiosulfate
equals-2 mg L- of D.O.; i.e., volume of titrant (ml) x 2
= mg L of D.O.
2) For a 203 ml fample, 1 ml of 0.025 N sodium thiosulfate
equals-1 mg L- of D.O. ; i.e., volume of titrant (ml) x 1
= mg L of D.O.

Hvdrosen Sulfide
Sulfur is a nutrient required by phytoplankton. However, when
present as hydrogen sulfide (H2S), it is of even greater
interest in lake studies owing to its toxicity to aquatic life.
H S is produced by the decomposition of organic material under
&aerobic conditions. It exists in equilibrium with the
nontoxic $issocipted ion HS according to the reaction
H S c-.r H + HS
&centration
. This reaction is pH dependent as the
of H S increases with a decrease in pH.
Therefore, it is important to determine in-situ pH when
investipating sulfide levels. H S concentrations in excess of
3 ug L- are considered harmful ?o salmonids (Table 14).

s his procedure is modified from Strickland and Parsons (1972)

and Cline (1969). Sulfide is converted to a violet-colored
compound by its reaction with p-phenylenediamine and ferric
chloride.
Ranse
Limit of ~etection
Empirical: 1.5 ug L-l S.

Spectrophotometer (600 nm), 50-ml stoppered cylinders, and

10-mm cuvettes.
Reasents
1) P-phenylenediamine-HC1 -is solve 1 g of p-phenylene-
diamine into a mixture of 100 ml of concentrated HC1 and
300 ml of DI water, cool, and dilute to 500 ml with DI
water.
2) Ferric chloride solution -
Dissolve 16.6 g of ferric
chloride-6-hydrate (FeC1 -6H 0) into 100 ml of
concentrated HC1, carefulla aid to -450 ml of DI water,
and-dilute to 500 ml with DI water.
Procedure
1) Collect a 50 ml sample in a 300-ml BOD bottle without
trapping air bubbles inside (see Dissolved Gases, p. 8).
2) Pour 50 ml of sample into a 50-ml stoppered cylinder, and
immediately add 5 ml of the p-phenylenediamine-Hc~
solution and invert to mix.
3) Within 2 minutes, add 1 ml of the ferric chloride solution
and invert to mix.
4) After two minutes invert again. Color development is
complete within 30 minutes and is stable for days.
However, keep the cylinders cool and in the dark until
they can be analyzed.
NOTE: Prepare replicate reagent blanks from source
water known to be free of sulfide.
5) Measure the absorbance against reagent blanks at 600 nm
(see Instrument Calibrations, p. 192).
Calculations
Calculate the concentration of total sulfide (TS) using the
expression:
TS (ug L-l) = As - Ab x 1168
As = Sample absorbance
Ab = Blank absorbance
To determine if toxic levels of H S are present, find the
proportion 'of H2S based on the sample pH using Table 14, and
multiply by the concentration of total sulfide.
Table 14. The proportions of H S
and HS- as a functio4
of pH.

REFERENCES
American Public Health ~ssociation,American Water Works
Association and Water Pollution Control Federation. 1985.
Standard methods for the examination of water and
wastewater. 16th ed. New York, N.Y. 1268 p.
Cline, J. D. 1969. Spectrophotometric determination of
hydrogen sulfide in natural waters. Limnol. and Oceanogr.
14:454-456.
Smith L. L. Jr., S. J. Broderius, C. L. Ho, A . J. Mearns, and
J. B. Pearce. 1979. Sulfide-hydrogen sulfide. pp.
272-276. In: A review of the EPA Red Book: Quality
criteria for water. R. V. Thurston, R. C. Russo, C. M.
Fellerolf, Jr., T. A. Edsall, and Y. M. Barber, Jr. (Eds.)
Water Quality Section, American Fisheries Society,
Bethesda, MD. 313 p.
Strickland, J. D. H. and T. R. Parsons. 1972. A practical
handbook of seawater analysis. Bull. Fish. Res. Board
Canada 167. 311 p.
 INORGANIC NUTRIENTS

Reactive Silicon (manual)

silicon is a primary nutrient required by diatoms for the
formation of frustules. Should reactive silicon levels become
diminished, diatoms may be replaced by other algae.
This method measures reactive silicon, presumably the inorganic
form available for algal uptake. Silicon reacts with ammonium
molybdate to form a yellow-colored silicomolybdate complex
which is reduced by sodium sulfite to produce an intense blue
color.
Standard Ranse

U m e r Limit of Detection
5000 ug L-l Si.
Lower Limit of ~etection
Empirical: 20 ug LI: Si.
predicted: 27 ug L Si.
Precision

Accuracy /

Spectrophotometer (810 nm) wavelength, 15-ml disposable

centrifuge tubes, and 10-mm cuvettes.
Reasents
1) Ammonium molybdate - iss solve 2.0 g of ammonium molybdate-
4-hydrate in -150 ml of DI water. Add 6 ml of
concentrated hydrochloric acid, and dilute to 250 ml with
DI water. Store in a polyethylene wash bottle and discard
when a precipitate forms.
2) Metol-sulfite - is solve 2.4 g of sodium sulfite-
anhydrous into 200 ml of DI water. Add 4 g of
 p-methylaminophenol sulfate (metol) and dissolve. Prepare
fresh 'daily.
3) Oxalic acid - Dissolve 10 g of oxalic acid into -80 ml of
DI water, and dilute to 100 ml.
4) 1:l sulfuric acid - Slowly pour 250 ml of concentrated
H SO into -200 ml of DI water, cool, and dilute to
560 $2.
5) Reducing reagent - Combine 167 ml of metol-sulfite, 100 ml
of oxalic acid, 100 ml of 1:l sulfuric acid, and 133 ml of
DI water.
Procedure
1) Place 5 ml of unfiltered sample or standard into a 15-ml
centrifuge tube.
2) Add-2 rnl of rnolybdate reagent, invert to mix, and allow 15 .
minutes for color (yellow) development.
3) Add 3 ml of reducing reagent, invert 3 times, and allow 2
hours for full color (blue) development.
4) Measure the absorbance at 810 nm against a DI water blank
(see Instrument Calibrations, p. 192).
Standards
1) Primary standard (0.467 mg ml-I ~ i -) iss solve 0.782 g of
sodium fluosilicate (Na2SiF6) into -200 ml of DI water,
and dilute to 250 ml.
2) Secondary standard (4.67 ug ml-l) - ~ilute1 ml of the
primary standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(y-axis) against the averaged absorbances minus the blank
(Xzaxis) and calculate the coefficient of determination
(r I *
2) Calculate sample concentration by subtracting the blank
value from the sample absorbances, and substitute into the
regression formula (Table 15).
Table 15. ---
The relationship between known concentrations of
.reactive silicon and absorbance~ (abs) at 810 nm
used to calculate a standard curve in the
manual analysis of reactive silicon.

Reactive
Secondary Total silicon
standard . volume
tml) (L)
-
concentrption
(us L 1
Absorbance
(x)

Si (ug L-I) = -12.6923

---
+ 2366.6386 (abs)

correctins for ~urbiditv

Glacial lakes, in particular, are characterized by large
concentrations of suspended silt particles causing turbidity
(>5-10 NTU). In the manual analysis for reactive silicon
( R S ~ ) ,the turbidity of unfiltered water could elevate sample
absorbances causing artificially high values. However, when we
compared estimates of RSi made without turbidity blanks to
those made with turbidity blanks (NRSi), we found an error of
only 0.6% (Table 16) . For the range in turbidities and R S ~
tested, no turbidity correction is warranted.
Table 16. Comparison of reactive silicon ( R S ~ )levels to
.turbidity corrected silicon (NRSi) showing <1%
turbidity error when using the manual
procedure. Also shown are RSi levels determined
by the automated procedure, and a range of
turbidity levels (NTU) within Alaskan lakes.

RSi NRSi RSi

Turbidity (manual) (manmi) (automgled)
Lake t NTU ) tug L- 1 (ua L ) (us L )
Silver
Crosswind
Miners
Klutina
Kenai
Skilak
Tazlina
Miners
Tonsina --
Upper Trail
Tustumena
Petrof
NRSi (manual) = -36.9087 + 1.0064 RSi (manual)

Reactive Silicon (automated)

This method is adapted from Technicon Industrial Systems
(1977). Ascorbic acid is the reducing agent used to convert
silicomolybdates to molybdenum-blue compounds.
Standard Ranse
100-1750 ug L-I Si.
U m e r Limit of Detection
3000 ug L-l Si.
Lower Limit of Detection
Empirical: Si.
20 ug L I ~
Predicted: 33 ug L Si.
Precision
17% at 583 ug L-l Si.
Accuracy
+ 2% at 583 ug L-I Si.
-

Technicon Autoanalyzer I1 equipped with 660-nra filters, and

50-mm flow cells.
Reaqents
1) Ammonium molybdate -Dissolve 10 g of ammonium molybdate
4-hydrate in 1 liter of 0.1 N sulfuric acid.
2) Oxalic acid - Dissolve 50 g of oxalic acid into -500 ml of
DI water, and dilute to 1 liter.
3) Ascorbic acid -
Dissolve 17.6 g of ascorbic acid into -500
ml of DI water containing 50 ml of reagent grade acetone.
Dilute the mixture to 1 liter with DI water, and add 0.5
ml of Levor wetting agent (Technicon Industries).
4) Sample wash - Add 1 ml of Triton-X (Technicon Industries)
per liter of DI water.
Procedure
1) Arrange the autoanalyzer (~igure 13), baseline, and
calibrate (see Instrument Calibrations, p. 192).
2) Place 4 ml of unfiltered sample or standard into the
sampler tray and pump through the system.
Standards
Primary standard (0.7 mg ml-I Si) - Dissolve 2.335 g of sodium
fluosilicate (Na2SiF6) into -250 ml of DI water, and dilute to
500 ml.
Secondary standard (7 ug ml-l Si) - Dilute 1.0 ml of the
primary standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged chart deflections minus the
blank (X-axis),2 and calculate the coefficient of
determination (r ) .
 Figure 13. Autoanalyzer schematic showing reagent lines with flow rates (ml/min), cam
size (number of samples per hour and sample to wash ratio), mixing coils, and colorimeter
with specified flow cell (FC) and wavelength (nm) used in the analysis of reactive silicon.
2) Calculate sample concentrations by subtracting the blank
value from the sample chart deflections, and substitute
into the regression formula (Table 17).
NOTE: Because of possible contamination of silicon
especially from borosilicate glassware use
polyethylene containers for preparing and storing
reagents and standards.
Table 17. The relationship between known concentrations of
rgsctive silicon and autoanalyzer chart deflections
(cd) used to calculate a standard curve in the
automated analysis of reactive silicon.

Reactive
Secondary Total silicon Chart
standard
(mil
volume
(L)
concentrption
(ua L \
deflection
(xl
- a
(x-blanx)
In lqkes, nitrogen may exist as qrganic nitrogen, ammonium
(NH ) , ammonia (NH ) , nitrite (NO ) and nitrate (NO -)
varfous inorganic $ o m s can be u8ed as nitrogen saurces by
.
The
phytoplankton; however, ammonium is preferred. Ammonium, a
primary en@ product of organic decomposition reactions, can be
oxidized to nitrite and nitrate by bacteria (nitrification) in
well-oxygenated water. However, nitrate reduction
(denitrification) can also occur, but only during periods of
reduced oxygen. Thus, under aerobic conditions nitrate
dominates the inorganic nitrogen fractions,-yhile under low
oxygen or anaerobic conditions (<2 mg 1 02), ammonia
dominates.

Total Ammonia (manuall

Ammonia+exists both as the unionized form NH and the ammoniula
ion NH . The proportion of NH increases with
an increase in
temperature and pH, andlis coilsidered toxic to salmonids at
levels above 0.016 mg L (Willingham et al. 1979). As the pH
of natural waters is typically between 5 and 8 units, NH is
present in far greater concentrations t h a ~NH .
In addftion,
concentrations of total ammonia (NH -f NH ) a h thought to be
harmful at levels exceeding 1.0 mg 2 wood 1983) .
Under basic conditions total ammonia (NH + NH + ) is converted
to NH and reacts with phenol and s o d i d hypoehlorite to form
b l u e Indophenol compounds.
Standard Ranae

U m e r Limit of Detection
200 ug L-' N.
Lower Limit of Detection
Empirical:
Predicted:
Precision
Accuracv
-
+ 8% at 15 ug L-' N.

Spectrophotometer (640 nm), 50-ml stoppered cylinders, and

100-mm cuvettes.
Reaaents
1) Phenol -Dissolve 25 g of crystalline phenol into
of reagent alcohol, and dilute to 250 ml with
-100 ml
reagent
alcohol.
2) Ferrocyanide solution -
Dissolve 1.55 g of potassium
ferrocyanide-trihydrate into -200 ml of DI water, and
dilute to 250 ml.
3) Hypochlorite solution -
h is solve 80 g of sodium
citrate-trihydrate and 4 g of sodium hydroxide into -300
ml of DI water. Add 100 ml of Clorox (5% sodium
hypochlorite), and dilute to 500 ml with DI water.
Procedure
1) Pour 50 ml of sample or standard into a 50-ml stoppered
cylinder .
2) Add 2 ml of phenol and invert to mix.
3) Add 2 ml of potassium ferrocyanide and invert to mix.
4) Add 5 ml of the hypochlorite solution, invert twice to
mix, and after 15 minutes invert again.
5) Allow 2 hours for full color development, and measure the
absorbance at 640 nm against a DI water blank (see
Instrument Calibrations, p. 192).
Standards
Primary nitrogen standard (0.2 mg ml-' N) -
iss solve 0.3824 g
of ammonium chloride (NH4C1) into -400 ml of DI water, and
dilute to 500 ml.
Secondary standard (0.5 ug ml-l
primary standard to 100 ml.
N) - Dilute 0.25 ml of the
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged absorbances minus the blank
(Xzaxis), and calculate the coefficient of determination
(r 1.
2) Calculate sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
regregsion formula (Table 18).
Table 18. ---
The relationship between known concentrations of
nitrogen and absorbance~ (abs) at 640 nm used to
calculate a standard curve in the manual analysis
of total ammonia.

Secondary Total Nitrogen

standard
tmll
volume
(L)
concentrption
(ua L 1
Absorbance
t x1
- G
(x-blank)

N (ug L-l) = -.0.2463

---
+ 85.9655 (abs)

3) Determine the concentration of toxic NH by finding the

percentage of MI , based on in situ t&aperaturo and pH
(Table 19), div$dinq by 100, and multiplying by the
concentration of total ammonia.
Table 19. The percent unionized ammonia (NH ) in aqueous
ammonia + ammonium (NH4+) solutioas (after Emerson
et ale 1975).

Temp. PH
_I C) 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
Fred Lake with a total ammonia concentration of 30 ug L-l, a
temperatuq of 10 C, and a pH of 7.5 has a toxic NH3 level of
0.18 ug L .

Total Ammonium (automated]

This procedure is a modification of Industrial Method No.
154-71W/B (Technicon Industrial Methods 1978) .
The
autoanalyzer arrangement is identical to the Technicon method,
except for reducing the sampling rate to 30/hr and using sodium
nitroferrocyanide as the catalyst. The chemistry involved is
the same as that described in the manual method, and the
procedure can be run simultaneously with the analysis of
nitrate and nitrite nitrogen.
Standard Ranse

U m e r Limit of Detection
500 ug L-' N.
Lower ~ i m i tof Detection
Empirical: 1 ug L-'-Y.
Predicted: 1.1 ug L N.
Precision

Accuracy

Technicon Autoanalyzer I1 equipped with 630-nm filters and

50-mm flow cells.
Reasents
1) Complexing reagent -
Dissolve 33 g of potassium sodium
tartrate, and 24 g of sodium citrate-dihydrate in -950 ml
of DI water. Adjust to a pH of 5.0 with -2 ml of
concentrated sulfuric acid, and dilute to 1 liter with DI
water. Add 0.5 ml of ~ r i j35 wetting agent (Technicon
Industries).
2) -
Phenol Dissolve 83 g of phenol in 50 ml of DI water.
Slowly add 180 ml of 20% sodium hydroxide (50 g of NaOH
diluted to 250 ml with DI water). Dilute the mixture to 1
liter with DI water.
3) -
Hypochlorite solution Dilute 200 ml of Clorox bleach (5%
hypochlorite) to 1 liter with DI water.
4) -
catalyst Dissolve 0.55 g of sodium nitroferrocyanide-
dihydrate in -750 ml of DI water, and dilute to 1 liter.
5) Sample wash - Add 0.5 ml of Brij 35 per liter of DI water.
Procedure
1) Arrange the autoanalyzer (Figure 14), baseline, and -
calibrate (see Instrument Calibrations, p. 192).
2) Place 4 ml of filtered sample or standard into the sampler
tray and pump through the system.
Standards
Primary nitrogen standard (0.5 mg ml-I N) -
Dissolve 0.9542 g
of ammonium chloride (NH4C1) in 250 ml of DI water, and dilute
to 500 ml.
Secondary standard (1.25 ug ml-l N)
primary standard to 200 ml.
- Dilute 0.5 ml of the

Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged chart deflections minus the
blank (X-axis), and calculate the coefficient of
determination (r ) .
2) Calculate sample concentrations by subtracting the blank
value from the sample chart deflections, and substitute
into the regression formula (Table 20).
 r

Cam

1.60ml/mlr SAMPLE WASH

0.32mllmir AIR

0.8Omj/mir COMPLEXINQ

0.42ml/min SAMPLE

0.42ml/mi PHENOL

0.32ml/mir HYPOCHLORITE

Colorim8t8r 0.42ml/mir SODIUM FERROCYANIDE

830nm
1.60ml/rnlr
60mmxl.6 . ) WASTE WASTE (
F.C.
I
k

Figure 14. Autoanalyzer schematic showing reagent lines with flow rates (ml/min),
cam size (number of samples per hour and sample to wash ratio), mixing coils, heating
bath, and calorimeter with specified flow cell ( F C ) and wavelength (nm) used in the
analysis of total ammonia.
Table 20. ,The relationship between known concentrations of
nitrogen and autoanalyzer chart deflections (Ea)
used to calculate a standard curve in the automated
analysis of total ammonia.

Secondary Total Nitrogen Chart

standard volume Concentrption deflections Ea
Imll ( L) (us L N) (XI I :-blank)

N (ug L-I) = 2.0053 + 0.9693 (z)

r2 = ,9971

it rate and Nitrite (manual)

Nitrate (NO -) is a primary nitrogen source for phytoplankton.
In oligotroahic systems, nitrite (NO ) is oxidized to nitrate
(nitrification) which is assfmilatea, after enzyme catalyzed
-
(NO -
redugtion,
NO -
during photosynthesis. Denitrification
NH ) is often characteristic of eutrophic
sysZams during peri8da of low-oxygen concentrations. As
nitrite is an intermediary in both reactions, its concentration
is usually insignificant.
his procedure measures nitrate plus nitrite. it rate is
reduced to nitrite by passing a sample through a column
containing cadmium granules coated with copper sulfate.
Nitrite passes through unchanged. Nitrite forms a pink azo dye
with sulfanilamide and NNED (Marshall's reagent) which is
measured colorirnetrically. Nitrite is determined on a separate
sample aliquot by omitting the reduction step.
Standard Ranse

UDser Limit of ~etection

1250 ug L-' N.
Lower Limit of ~etection
Empirical: 1 ug L-'-Y.
Predicted: 0.6 ug L N.
Precision
7% at 50 ug L-' N.
Accuracy
+ 2% at 50 ug L-I N.
-
Apparatus
Spectrophotometer (543 nm), 80-ml cadmium reduction columns,
50-ml stoppered cylinders, and 10-mm cuvettes.
Reaaents
1) Buffer solution -
Dissolve 100 g of ammonium chloride
(NH Cl), 20 g of sodium borate-10-hydrate, and 1 g of
N ~ - ~ D Tin
A -750 rnl of DI water, and dilute to 1 liter.
2) Sulfanilamide -
iss solve 6 g of sulfanilamide in 300 ml of
DI water and 100 ml of concentrated HC1, and dilute to 500
ml with DI water.
3) NNED -
Dissolve 0.6 g of N-1 naphthylethylenediamine
dihydrochloride in -250 ml of DI water, and dilute to 500
ml.
4) -
Cupric sulfate Dissolve 25 g of cupric-sulfate 5-hydrate
in -400 ml of DI water and dilute to 500 ml.
5) 10% Hydrochloric acid -
Slowly add 5 ml of concentrated
HC1 to 45 ml of DI water.
6) Cadmium - 40 to 60 mesh granules.
Procedure
1) Clean and repack the cadmium columns (~igure15) according
to the procedure 'described below.
2) Check the reduction efficiencies of the cadmium columns
according to the procedure described below.
3) Pour 50 ml of filtered sample or standard into a 50-ml
stoppered cylinder, add 5 ml of buffer solution and invert
to mix.
 t------------80ml reservoir

buffered sample

-3-way valve

cadmium granules

glass fiber wool

Figure 15. Cadmium column used to reduce nitrate (N0;)to

nitrite (NO:) in the manual analysi-s of nitrate + nitrite
nitrogen.
4) Pour 20 ml of buffered sample into the column reservoir.
allow the sample to drip through, and discard the
effluent .
5) Pour the remaining sample (-35 ml) into the column
reservoir and collect 30 ml of the effluent in the
original cylinder. Allow the remaining sample (-5 ml) to
drip through, and discard.
6) Add 0.5 ml of sulfanilamide to the 30-ml sample, and
invert twice to mix. Let sit for 5 minutes.
7) Add 0.5 ml of NNED solution, invert twice to mix, and
allow 15 minutes for full color development.
8) Measure the absorbance at 543 nm against a DI water blank
(see Instrument Calibrations, p. 192).

A. Cleaning and Repacking Cadmium Columns

1) Remove the cadmium granules by inverting the column into a
100-ml plastic beaker and tapping lightly.
2) Add 50 ml of 10% HC1 to the cadmium, swirl, and soak for
about 30 minutes.
3) Rinse the cadmium with DI water, add -30 ml of cupric
sulfate, and soak for 15 minutes.
4) Pour off the solution, rinse the cadmium with DI water,
and add 30 ml of cupric sulfate. Soak until the cadmium
is coated with copper; i.e., when the blue color
disappears. ~ i n s ethe cadmium thoroughly with DI water.
5) After cleaning the columns thoroughly, close the outlet
valve, and fill the column with DI water. Place a small
wad of glass fiber wool into the reservoir and allow it to
settle to the bottom of the column.
6) Using a DI wash bottle, rinse the cadmium into the column
so that the granules do not bypass the glass wool and the
column is not obstructed with air bubbles.
Standards
Primary nitrate standard (0.1 mg ml-' N) -
Dissolve 0.36101 g
of potassium nitrate (XN03) in -400 ml of DI water, and dilute
to 500 ml.
Secondary nitrate standard (1 ug ml-I N) - Dilute 2.5 ml of the
primary standard to 250 ml.
Primary nitrite standard (0.1 mg ml-' N) -
iss solve 0.24630 g
of sodium nitrite (NaN02) in -400 ml of DI water, and dilute to
500 ml.
Secondary nitrite standard (1 ug ml-l N) - Dilute 1 ml of the
primary standard to 100 ml.

B. ~eductionEfficiency
Prior to running the samples and/or standards through the
columns, determine the reduction efficiencies. If the
efficiencies are greater or equal to 95%, no correction of the
sample absorbances is necessary as essentially all of the
nitrate present in the sample has been reduced. If the
efficiencies are less than 95% or over 105% repeat the cleaning
and repacking procedure. However, the sample absorbances can
be divided by the reduction efficiency of the appropriate
column, and the corrected values used in the subsequent
calculations. The efficiency procedure should be conducted for
each column (Table 21) .
1) Prepare a reagent blank by adding 5 ml of buffer solution
to 50 ml of DI water in a stoppered cylinder.
2) Prepare two nitrate standargs by diluting 10 ml of the
secondary standard (1 ug ml N) to 5 0 ml with DI water,
and add 5 ml of buffer.
3) Prepare a nitrite standarfil by diluting 10 ml of the
secondary standard (1 ug ml N) to 50 ml with DI water,
and add 5 ml of buffer.
4) Pour the standards through the column in the following
order: blank, nitrate, nitrite, and nitrate.
Calculate the reduction efficiency (R.E.) of each column using
the following:
R.E. = (averaged absorbances of NO3
- standards) - blank
(absorbance of NOz standard) - blank
Table 21. Absorbances for nitrite and equivalent nitrate
.standards after cadmium reduction used to calculate
the reduction efficiency ( R . E . ) in the manual
analysis of nitrate + nitrite nitrogen.

Column I Column I1
. Concentfation Absorbance Absorbance
tua L N) (543 nm) ( 5 4 3 nm)

Blank 0 0.006 Blank 0.008

Nitrate 500 1.460 Nitrate 1.483
Nitrite 500 1.550 Nitrite 1.564
Nitrate 500 1.493 Nitrate 1.501
R . E . = 95%
...............................................................
R . E . = 96%

Column I11 Column IV Column v

Absorbance Absorbance Absorbance
(543 nm) (543 nm) (543 nm)
Blank 0.005 Blank 0.008 Blank 0.008
Nitrate 1.489 Nitrate 1.517 Nitrate 1.483
Nitrite 1.549 Nitrite 1.542 Nitrite 1.560
Nitrate 1.494 Nitrate 1.514 Nitrate 1.508
R.E. = 96% R . E . = 99% R . E . = 96%

Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged absorbances minus the blank
(X5axis), and calculate the coefficient of determination
(r I *
2) Calculate sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
regression formula (Table 22).
 Table 22. The relationship between known concqgtrations of
nitrate nitrogen and absorbances (abs) at 5 4 3 nm
'following cadmium reduction used to calculate a
standard curve in the manual analysis of nitrate +
nitrite nitrogen.

Secondary Total Nitrogen

standard volume concentfation Absorbance ZEi
(ml) + (L) (ua L N) t XI (:-blank)

0-0

N (ug L-l) = -2.2895 + 334.5656 (abs)

Nitrate and Nitrite tautomatedl

t his method is adapted from Stainton et al. (1977), and
involves the same principles described in the manual method.
--This procedure can be run simultaneously with the analysis of
ammonia nitrogen.
Standard Ranae

U m e r Limit of Detection

Lower Limit of Detection

Empirical: 1 ug ~-'-j[.
predicted: 3.4 ug L N.
Precision

Accuracy
Technicon ~utoanal~zer I1 equipped with 540-nm filters, 15-nun
flow cells, and cadmium reductor column.
Reasents
1) Prepare reagents 1-6 as described in the nitrate + nitrite
manual method (p. 85) .
2) Sample wash -
Add 1 ml of Brij 35 wetting agent (Technicon
Industries) per liter of DI water.
Procedure
1) Clean the cadmium granules (see p. 87) and repack the
reductor column (see below).
2) Arrange the autoanalyzer (Figure 16), baseline, and
calibrate the system (see Instrument Calibrations,
p. 192).
3) Determine the efficiency of the reductor column (see
below).
4) Place 4 ml of filtered sample or standard into the sampler
tray and pump through the system.

A. preparation of Reductor Column

Sift -25 g of clean 40-60 mesh cadmium granules through a fine
mesh screen. Using a 140-mm length of 4-rmn i.d. glass tubing,
place a small piece of glass fiber wool in one end, cap with a
plastic nipple, and fill with DI water. Add the cadmium to a
small weighing pan and rinse the granules into the tube with a
DI wash bottle. Place another piece of glass wool in the
opposite end, and cap with the same sized nipple.
Standards
Primary nitrate standard (0.1 mg ml-I N) -
Dissolve 0.3611 g of
.
potassium nitrate (KN03) in -400 ml DI water, and dilute to 500
ml
Secondary nitrate standard (1.0 ug ml-'
primary standard to 100 ml.
N) - Dilute 1 ml of the
primary nitrite standard (0.1 mg ml-I N) -
~issolvo0.2464 g of
sodium nitrite (NaN02) in -400 ml DI water, and dilute to 500
ml .
 Cam
>WASTE

1.60 ml/min 'WASH

DEBUBBLER
640nm CD
20x 6x REDUCTOR l5x
16mm 0.80 ml/min SAMPLE
X
2.0 I 0.10 ml/min BUFFER
F.C.
0.32 ml/min AIR
Colorimeter
0.10 mllmin SULFANILAMIDE

0.10 mllmin NNE~

0.42 ml/min AIR

0.60 mllmin
WASTE /,

0.42 ml/min
WASTE <

- A
i

Figure 16. Autoanalyzer schematic showing reagent lines with flow rates (ml/min),
cam size (number of samples per hour and sample to wash ratio), mixing coils, cadmium
reduction (CD) column, debubbler, and colorimetar with specified flow cell (FC) and
wavelength (nm) used in the automated analysis of nitrate + nitrite nitrogen.
Secondary nitrite standard (1.0 ug ml-I N) - Dilute 1 ml of the
primary standard to 100 ml.

B. Reduction Efficiency
Check the reduction efficiency (R.E.) of the pplumn a able 23)
by prepari.ng two nitrate standards (200 ug L ) and a nitrite
standard (200 ug L ) Pump the standards through the system
in the following order: blank, nitrate, blank, nitrite, blank,
and nitrate. Calculate R.E. using the expression:

(NO2 chaft deflection) -

blank
-
R.E. = (averaaed NO, chart deflection) blank

Table 23. Chart deflections for nitrite and equivalent nitrate

standards after cadmium reduction used to calculate
the reduction efficiency (R.E.) in the automated
_ analysis of nitrate + nitrite nitrogen.

Secondary Secondary
nitrate nitrite
standard standard Concansfation Chart
tmlI tmll tua L NI deflection

R.E. = 101%

Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged chart deflections minus the
blank (X-axis),2 and calculate the coefficient of
determination (r ) .
2) Calculate sample concentrations by subtracting the blank
values from the sample chart deflections, and substitute
into the regression formula (Table 24).
Table 24. The relationship between known concentrations of
nitrate nitrogen and autoanalyzer chart deflections
' (Ea) used to calculate a standard curve in the
automated analysis of nitrate + nitrite nitrogen.

Secondary
nitrate Total Nitrogen Chart
standard volume concentfation deflection Ea
(ml) . - (L) (us L N) (XI (:-blank)
Phosphorus is generally accepted as the primary nutrient
contrclling lake productivity. Soluble orthophosphate
(filterable reactive phosphorus) is the form most readily
available for algal uptake; however, it usually comprises only
a small portion of total phosphorus (TP). Other size fractions
(colloidal, particulate, etc.) may also be present, but
primarily filterable reactive and cellular (organic
particulate) phosphorus are considered biologically active.
All fractions should be determined individually in order to
define the phosphorus cycle operating within a given lake
(Figure 17). However, several of the colloidal and dissolved
fractions cannot be determined directly at this time and must
be derived by difference. However, our fractionation studies
have shown that the sum of the individual TP components
represent nearly 80% of directly determined TP (Table 25).
Definition of Phoswhorus IPI Terms
TP = Total P
NTP = Total P (corrected for turbidity)
TPP = Total Particulate P
IPP = Inorganic Particulate P
OPP = Organic Particulate P
TFP = Total Filterable P
TDP = Total Dissolved P
DRP = Dissolved Reactive P
DUP = Dissolved Unreactive P
FRP = Filterable Reactive P
FUP = Filterable Unreactive P
CoRP = Colloidal Reactive P
COUP = Colloidal Unreactive P

Total and Total Filterable Phos~horus(manual).

The method for total phosphorus (TP), and total filterable
phosphorus (TFP), are adapted from the procedure described by
~isenreich et al. (1975). Phosphorus in particulate,
colloidal, and organic forms is converted to inorganic
orthophosphate by a persulfate-sulfuric acid digestion.
Ammonium molybdate is then added to form a phosphomolybdate
complex which is reduced to molybdenum-blue by ascorbic acid.
Standard Ranse
2-20 ug L - ~P.
Upwer Limit of Detection
1100 ug t-I P.
 TP

I
I
I I
I
~ R P ] A
1
+OUP~A

by direct measurement
A by difference

TP=IPP + OPP + DRP + GORP + DUP + COUP

Figure 17. Phosphorus fractions comprising the total phosphorus ( T P ) in natural lake
waters (see p. 95 for definition of terms), obtained either by direct measurement or
derived by difference.
Table 25. Comparison of phosphorus levels of six fractions (see
Definition of Terms) summed to give total phosphorus (TP)
and directly determined TP for four lakes with varying
turbidity (NTU) .
TP TP
Lake NTU IPP OPP DRP CoRP DUP COUP (summation) (direct)

Hidden 1 0.4 2.5 2.0 -- 0.5 1.5 8.3 8.1

Hidden 1 0.7 2.0 2.1 -- 1.5 1.3 8.1 7.2

Packers 1 1.1 5.1 1.5 2.4 0.8 4.5 15.4 13.4

Packers 1 1.8 5.0 2.1 1.3 0.8 5.3 16.3 14.6

Ptarmigan 17 9.7 3.3 1.7 -- 2.8 -- 17.5 19.5*

Ptarmigan 18 10.9 4.0 1.6 0.2 3.6 -- 20.3 16.9*

Tustumena 50 21.8 11.8 3.9 1.0 0.8 5.2 44.5 32.8*

Tustumena 54 19.4 9.1 4.3 1.5 0.5 4.8 39.6 30.7*

*Corrected for turbidity.

Lower ~ i m i tof Detection
Empirical: 0.5 ug L - ~ ~ P .
Predicted: 0.25 ug L P.
Precision
5% at 6 ug L-l P.
Accuracv

Spectrophotometer (882 nm), autoclave (121 C, 15 psi), 50-ml

erlenmeyer flasks, and 100-mm cuvettes.
Reaaents
1) Antimony-tartrate solution -
Add 53.3 ml of concentrated
sulfuric acid (H SO) to -800 ml of DI water and cool.
Dissolve 0.748 g af antimony potassium tartrate trihydrate
in the H SO solution, and dilute to 1 liter with DI
water. ~ i l t i rthe solution when a precipitate forms.
2) Molybdate solution -
Dissolve 7.95 g of ammonium
molybdate-4-hydrate in -500 ml of DI water, and dilute to
1 liter.
3) 3.6 N H2S04 -
Add 50 ml of concentrated sulfuric acid
-400 ml of DI water, cool, and dilute to 500 ml.
to

4) Digestion reagent -
Dissolve 60 g of potassium persulfate
in -800 ml of DI water containing 100 ml of 3.6 N H2S04,
and dilute to 1 liter with DI water.
5) Mixed Reagents ( M R I) -
Combine 250 ml of both the
antimony-tartrate and molybdate solutions. Add 2 g of
ascorbic acid, dissolve, and dilute to 1 liter with DI
water.
Procedure
1) Pour 25 ml of unfiltered (TP) or filtered (TFP) sample or
standard in a 50-ml erlenmeyer flask.
2) Add 5 ml of digestion reagent, cover with a 15-ml
polypropylene beaker, mix, and digest in the autoclave for
30 minutes.
3) Cool to room temperature, and add 5 ml of MR I and mix.
4) Allow 20 minutes for full color development and measure
the absorbance at 882 nm against a DI water blank (see
Instrument calibrations, p. 192).
Standards
Primary phosphorus standard (0.05 mg ml-1 P) - D ~ S S O ~ 0.1389
V ~
g of potassium phosphate-dibasic (X2HP04) in -400 ml' of DI
water, and dilute to 500 ml.
Secondary Standard (0.5 ug ml-' P) - ~ i l u t e1 ml of the primary
standard to 100 ml.
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged absorbances minus the blank
(XZaxis), and calculate the coefficient of determination
(r I *
2) Calculate sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
regression formula (Table 26).
Table 26. The relationship between kno~-concentrations of
phosphorus and absorbances (abs) at 882 nm used to
calculate a standard curve in the analyses of total
and total filterable phosphorus.

Secondary Total Phosphorus

standard
tml)
volume
t Ll
concentrption
tug L 1
Absorbance
tx)
iz:
t z-blank)

P (ug L-l) = 0.0064 + 189.8708 (abs)

---

Correctina for Turbidity

In the analysis of TP, absorbance measurements of turbid
solutions are artificially high because of light backscattering
(Jullander and Brune 1950). As glacial lakes, in particular,
are characterized by large concentrations of suspended silt
particles causing turbidity (>5-10 NTU); TP levels need to be
corrected (Table 27). The additional absorbance due to
turbidity can be determined by analyzing a turbidity blank in
conjunction with the sample (see Interferences p. 198).
However, from a thorough analysis of glacial lakes; we found
that turbidity corrected TP (NTP) can be obtained using the
equaticn :
NTP (ug L - ~ )= 0.9229 + 0.5845 TP (ug L - ~ )
r = 0.902
Therefore,*only NTP of glacial lakes (4-12; Table 27) is
comparable to TP in clear-water or non-turbid lakes (1-3; Table
2 . Finally, the presence of iron concentrations above 10 mg
L inhibit color formation. This interference can be avoided
by an appropriate dilution of the sample prior to digestion.
Table 27. Comparison of total phosphorus (TP-manual)
levels, turbidity corrected TP (NTP), and of
TP (automated) for lakes with varying
turbidity (NTU) .
TP NTP TP
Turbidity (manupi) (manual) (automaffd)
Lake ( NTU 1 tua L 1 (ua L 1 (ua L 1
Silver
Crosswind
Miners
Klutina
Kenai
Skilak
Tazlina
Miners
Tonsina
Upper Trail
Tustumena
Petrof

Filterable Reactive Phos~horus(manual]

Filterable reactive phosphorus (FRP) includes both dissolved
(DRP) and colloidal reactive (CoRP) phosphorus (Figure 17).
Since the proportions vary according to lake type (i.e.,
glacial, organically stained, and clear), determination of FRP
may not accurately reflect the amount of soluble orthophosphate
in the sample.
Standard Ranse
1-10 ug L - ~P.

UDwer Limit of Detection

1100 ug L - ~P.

Lower ~ i m i tof Detection

~mpirical: 0.5 ug L-l P.
predicted: 0.12 ug L - ~P.
Precision
7% at 4 ug L-l P.
Accuracy
+
- 4% at 4 ug L-I P.

Spectrophotometer 50-ml erlenmeyer flasks, and 100-mm

cuvettes.
Reaaents
1) Prepare reagents 1-3 as described in the TP and TFP
methods (see p. 98).
2) Mixed reagent 11 (MR 11) -Combine in order, 250 ml of
ammonium-molybdate solution, 100 ml of 3.6 N H SO and
250 ml of the antimony-tartrate solution. ~ d d ~ 2 . 8 'of~
ascorbic acid, dissolve, and dilute to 1 liter.
Procedure
I) Pour 25 ml of filtered sample or standard into a 50-ml
erlenmeyer flask.
2) Add 5 ml of MR 11, and mix.
3) Allow 20 minutes tor full color development, and measure
the absorbance at 882 nm against a DI water blank (see
Instrument calibrations, p. 192).
Standards
Prepare primary and secondary standards as described in the TP
and TFP method (see p. 99).
Calculationg
The calculations are identical to that described in the TP
and TFP method (see p. 99); however, the standard curve is not
identical (Table 28) and needs to be determined separately.
Table 28. ---
The relationship between known concentrations of
phosphorus and absorbance~ (abs) at 882 nm used to
"calculate a standard curve in the analysis of
filterable reactive phosphorus.

Secondary Total Phosphorus

standard volume concentrption Absorbance aZ
(ml) (L) (ua L 1 (XI (z-blank)

P (ug j'-~
---
= 020851 + 171.6754 (abs)
r = .9997

Dissolved Phos~horus(manual)
Sample preparation involves ultra-filtration prior to analysis
using Millipore CX-10 immersible filters (MWCO 10,000).
Phosphorus in the ultra-filtrate can be analyzed for both DRP
and TDP (Figure 17).
Samrile Preriaration (ultrafiltration)
1) Assemble the apparatus (Figure 18), pass -25 ml of DI
water through the filter at 115 psi, and discard. This
cleans the filter of the glycerine preservative.
2) Filter -10 ml of sample, discard, and collect the
remaining (-100 ml) ultrafiltrate.
Procedure
1) Determine total dissolved phosphorus (TDP) using the TP
method (see p. 95) .
2) Determine total dissolved phosphorus (TDP) using the FRP
method (see p. 100).
 C

AI

CX-10
immersible filter

ultrafiltrate

used as vacuum manifold

?

Figure 18. Apparatus used to process filtered water for ultrafiltrate (110,000 MwCO),
showing the vacuum manifold and Millipore immersible ultra-filters, used for analysis
of dissolved phosphorus fractions.
 Total Kieldahl Nitrosen and Total Phos~horus (automated]
Total Kjeldahl nitrogen (TKN) refers to a procedure for
determining the organic nitrogen and total ammonia content of
water. Total phosphorus (TP) and total ~jeldahlnitrogen (TKN)
can be determined either simultaneously or individually using
this method.
Standard Ranqe

U m e r Limit of Detection
3000 ug L-l N; 100 ug L-l P.
Lower Limit of Detection
Empirical: 3 ug L-'-~; 1 ug L-' PL1
Predicted: 3.5 ug L N; 1.2 ug L P.
Precision

Accuracv

Technicon Autoanalyzer I1 equipped with 630 and 880-nm filters,

50-mm flow cells, BD 20 block digestor with 75-ml digestion
tubes, and a vortex mixer.

1) Digestion Reagent
(a) Dissolve 2 g of mercuric oxide (HgO) into 20 ml of
20% v/v sulfuric acid (20 ml concentrated H2S04
diluted to 100 ml with DI water).
(b) Dissolve 134 g of potassium sulfate (K SO ) into 500
ml of DI water, slowly add 200 ml 0 % doncentrated
sulfuric acid, and cool.
(c) Combine parts a and b, and dilute to 1 liter with DI
water.
2) Sample wash -Dissolve 13.4 g of potassium sulfate (K SO )
into -500 ml of DI water containing 15 ml of concentZatad
 sulfuric acid, and dilute the mixture to 1 liter with DI
water.
3) System wash - Add 1 ml of ~riton-x wetting agent
(Technicon Industries) per liter of DI water.

Total Kjeldahl reaaents

4) Complexing reagent - Dissolve 1 g of sodium chloride
(NaC1) and 3.2 g of sodium hydroxide (NaOH) into 500 ml of
DI water, dilute to 1 liter, and add 1 ml of Brij 35
wetting agent (Technicon Industries).
5) Buffer - Dissolve 83.5 g of potassium phosphate, dibasic
(K PHO*), 1 g of sodium citrate-trihydrate, 20.2 g of
soaium hydroxide (NaOH) and 9.3 g of Na-EDTA into 400 ml
of DI water. Dilute to 500 ml with DI water, and add 0.5
ml of Brij 35.
6) Phenate
(a) Add 25 ml of DI water to a new reagent bottle
containing 500 g of crystalline phenol, and warm to
dissolve by placing the bottle in hot water. Prepare
this phenol stock solution as necessary.
(b) Dissolve 2 g of potassium ferrocyanide-trihydrate,
12.5 g of sodium hydroxide (NaOH) into -400 ml of DI
water, and add 25 ml of phenol stock solution.
Dilute the mixture to 500 ml with DI water, and add
0.5 ml of Brij 35.
7) Hypochlorite solution - Dilute 20 ml of Clorox (5%
hypochlorite) to 100 ml with DI water, and add 0.1 ml of
Brij 35.
8) Catalyst -Dissolve 0.4 g of sodium nitroferrocyanide-
dihydrate into 500 ml of DI water, dilute to 1 liter, and
add 1 a1 of Brij 35.
Total ~hos~horus
reaaents
9) -
Molybdate solution Dissolve 12 g of ammonium molybdate
4-hydate, 0.25 g of antimony potassium tartrate into -500
ml of DI water. Add 250 rnl of 20% sulfuric acid, dilute
to 1 liter with DI water, and add 1 ml of Levor V wetting
agent (Technicon Industries). Allow the other phosphorus
reagents to pump through the autoanalyzer system for 5
minutes before introducing this solution.
10) ~scorbicacid -
Dissolve 20 g of ascorbic acid into
ml of DI water, dilute to 1 liter, and add 1 ml of
500
Levor
-
v.
11) -
Sodium chloride Dissolve 10 g of sodium chloride (NaC1)
into -500 ml of DI water, dilute to 1 liter, and add 0.5
ml of Levor V.
Procedure
1) Pour 20 ml of unfiltered sample or standard into a block
digestion tube. Reserve the same set of tubes for
standards.
2) Add 2 ml of digestion reagent and -10 teflon boiling
chips.
3) Place in the block digestor and digest for 1 hour at -
200 C.
4) F'urther digest the samples at 360 C for 20 + 1 minutes,
remove, and dilute to 20 ml with DI water while vortexing.
5) Withdraw a 4-ml aliquot using an autopipet, and add to the
sampler tray.
6) Arrange the autoanalyzer (Figure 19), baseline, and
calibrate the system (see Instrument Calibrations,
p. 192).
NOTE: The phosphorus line should be preconditioned by
pumping 1 N NaOH through the system for 5 minutes,
followed by a 5 minute wash with hydrogen peroxide
(20%) and then a ten minute system rinse with DI
water.
7) pump the standards and samples through the system.
Standards,
Primary nitrogen standard (0.2 mg ml-1 N) -
Dissolve 0.3824 g
of ammonium chloride (NH4C1) into -400 ml of DI water, and
dilute to 500 ml.
Secondary nitrogen standard (2 ug ml-I N)
primary standard to 100 ml.
- ~ilute1 ml of the

Primary phosphorus standard (0.5 mg ml-I P) -

Dissolve 0.1404 g
of potassium phosphate, dibasic (X2HP04) into -400 ml of DI
water, and dilute to 500.
Secondary phosphorus standard (0.50 ug ml-' P) - Dilute 1 ml of
the primary standard to 100 ml.
 2

Cam
-

Colorlmeter
3.4ml/mln WASH
(((

p
lox 2 0 ~ .32ml/min AIR
880nm
6Omm Jmn- 3a0c I .BOml/mln
.42ml/mln
NACL
P-LINE

L-ii
I .32ml/mln MOLYBOATE
.32ml/mln ASCORBIC
1OX .32ml/ml11 AIR
WASTE I .42rnl/min N-LINE
I .60ml/mln COMPLEXINQ
.32ml/mln AIR
)WASTE 2 0 ~ 2 0 ~
I .00ml/mln BUFFER
I .42ml/mln RESAMPLE N
630nm
6Omm
- .3l_ml/mln HYPOCHLORITE

F.C. .42ml/mln PHENATE

.42mllmln NITROPRU8810E
Colorimet@r
-
.8Oml/mln
WASTE (
1.60ml/mln
WASTE f 1
*

Figure 19. Autoanalyzer schematic showing reagent lines and flow rates (ml/min), cam
size (number of samples per hour and sample t o wash ratios), mixing coils, heating baths,
and colorimeters with specified flow cells ( F C ) and wavelengths (nm) used in the
simultaneous analysis of total phosphorus and total Kjeldahl nitrogen.
Calculations
1) Formulate separate linear equations for nitrogen and
phosphorus by regressing concentrations (y-axis) against
averaged chart deflections minus the blank (3-axis), and
calculate the coefficient of determination (r ) .
2) Calculate sample concentrations by subtracting gbe blank
value* from the sample chart deflections (cd), and
substitute into the appropriate regression formula (Tables
29 and 30).
Table 29. The relationship between known concentrations-gf
nitrogen and autoanalyzer chart deflections (cd)
used to calculate a standard curve in the
automated analysis of total Kjeldahl nitrogen.

Secondary
N Total Nitrogen Chart
standard volume concentrption deflection -a
(mll (L) tua L- Nl (x l (x-blank)
Table 30. The relationship between known concentrations of
' phosphorus and autoanalyzer chart deflections (Ea)
used to calculate a standard curve in the automated
analysis of total phosphorus.

Secondary
P Total Phosphorus Chart
standard . - volume concentrg$ion deflection zi
(ml) (L) (uq L 1 (xl [:-blank)

REFERENCES
Reactive Silicon
Stainton, M. P., M. J. Capel, and F. A. J. Armstrong. 1977.
The chemical analysis of fresh water. Can. Spec. Publ.
'No. 25, 2nd ed. 180 p.
Strickland, 3. D. H. and T. R. Parsons. 1972. A practical
handbook of seawater analysis. Bull. Fish. Res. Bd. of
Canada 167. 311 p.
Technicon Industrial Systems. 1977. Silicates in water and
seawater. Industrial method No. 186-72W/B8. Technicon
~ndustrialSystems, Tarrytown, New York.

Buckley, J. A. 1978. Acute toxicity of un-ionized ammonia to

fingerling coho salmon. Prog. Fish Cult, 40:30-32.
Crowther, J., B. Wright, and W. Wright. 1980. Semi-automated
determination of total phosphorus and total Kjeldahl
nitrogen in surface waters. Anal. Chem. 119:313-321.
Emerson, K.., R. C. Russo, R. E. Lund, and R. V. Thurston.
1975. Aqueous ammonia equilibria calculations: effect of
pH and temperature. J. Fish. Res. Bd. of Canada
32:2379-2383.
Haywood, G. P. 1983. Ammonia toxicity in teleost fishes: a
review. Can. Tech. Rep. Fish. Aquat. Sci. No. 1177.
35 Pa.*
Kuenzler, E. J., D. W. Stanley, and J. P. Koenings. 1979.
Nutrient kinetics of phytoplankton in the Pamlico River,
North ~arolina. University of North Carolina. Water
Resources Research Institute Report No. UNC-WRRI-79-139.
163 p.
Stainton, M. P., M. J. Capel, and F. A. J. Armstrong. 1977.
The chemical analysis of fresh water. Can. Spec. Publ.
No. 25, 2nd ed. 180 p.
~echniconIndustrial Systems. 1978. Ammonia in water and
seawater. Industrial Method No. 154-71W+/B. Technicon
Industrial Systems, Tarrytown,. New York.
Technicon Industrial Systems. 1978. Digestion and sample
preparation for the analysis of total Kjeldahl nitrogen
and/or total phosphorus in water samples using the
Technicon BD-40 block digestor. Industrial Method No.
276-75W/B. Technicon Industrial Systems, Tarrytown, New
York.
Technicon Industrial Systems. 1978. Individual/simultaneous
determination of nitrogen and/or phosphorus in BD acid
digests. Industrial Method No. 239-74W/B. Technicon
Industrial Systems, Tarrytown, New York.
Willingham, W. T., Colt, J. E., Fava, J. A., Hillaby, B. A.,
Ho, C. L., Katz, M., Russo, R. C., Swanson, D. L., and R.
V. Thurston. 1979. Ammonia. 6-18. In: A reveiw of the
EPA Red Book: Quality criteria for water. R. V.
Thurston, R. C. Russo, C. M. Felleroff, Jr., Edsall, T.
A , , and Y. M. Barber, Jr. (eds.) Water Quality Section,
American ~isheriesSociety, Bethesda, MD.

Bray, R. H, and L. T. Kurtz. 1945. Determination of total,

organic, and available forms of phosphorus in soils.
American Journal of Soil Science 45:39-45.
Crowther, J:, B. Wright, and W. Wright. 1980. Semi-automated
determination of total phosphorus and total Kjeldahl
nitrogen in surface waters. Anal. Chem. 119:313-321.
Edmundson, J. A. and J. P. Koenings. 1985. The effects of
glacial silt on primary production through altered light
regimes and phosphorus levels in Alaska lakes. pp. 3-19.
In: L. P. might (ed.), ~esolving Alaska's Water
Resources conflicts. University of Alaska-Fairbanks,
Institute of Water Resources Report 108. 212 p.
~isenreich,S. J., R. T. Bannerman, and D. E. Armstrong. 1975.
A simplified phosphorus analysis technique. ~nvironmental
~etteks,9:43-53.
Jullander, I. and K. Brune. 1950. Light absorption
measurements on turbid solutions. Acta he mica 4:870-877.
Koenings, J. P. and F. F. Hooper. 1976. The influence of
colloidal organic matter on iron and iron-phosphorus
cycling in an acid bog lake. Limnol. Oceanogr.
21:684-696.
Koenings, J. P. 1977. The metabolism of nonparticulate
phosphorus in an acid bog lake. Ph.D. ~issertation,
university of Michigan, Ann Arbor. 212 p.
Koenings, J. P., R. D. Burkett, and 0 . B. Kyle. 1985.
Limnology and fisheries evidence for rearing limitation oi
sockeye production in Crescent Lake, Southcentral Alaska,
1979-1982. Alaska Department of ~ i s h and Game, FRED
Division Report Series No. 57. 82 p.
Kuenzler, E. J., D. W. Stanley, and J. P. ~oenings. 1979.
Nutrient kinetics of phytoplankton in the ~amlico River,
North Carolina. university of North ~arolina, Water
Resources Research Institute Report No. UNC-WRRI-79-139.
163 p.
strickland, J. D. H. and T. R. Parsons. 1972. A practical
handbook of seawater analysis. Bull. Fish. Res. Bd. of
Canada. 167:310 p.
~echniconIndustrial Systems. 1978. Digestion and sample
preparation for the analysis of total ~jeldahl nitrogen
and/or total phosphorus in water samples using the
Technicon BD-40 block digestor. Industrial Method No.
276-75W/B. Technicon Industrial Systems, Tarrytown, New
York.
Technicon ~ndustrialSystems. 1978. ~ndividual/simultaneous
determination of nitrogen and/or phosphorus in BD acid
digests. Industrial Method No. 239-74W/B. Technicon
Industrial Systems, Tarrytown, New York.
 PARTICULATE NUTRIENTS
The amounts of carbon (C), nitrogen (N), and phosPhorus(P)
contained on a 4.25 cm GF/F filter are reported as particulate
nutrients. The material retained by the filter consists of
detritus, bacteria, and phytoplankton: however, in oligotrophic
lakes phytoplankton are considered to be primary contributors.
Particulate nutrients, along with chl a, characterize the
standing crop of primary producers. In particular, the C:N:P
atom ratios are useful indicators of nutritional status with
ratios greater than 106:16:1 suggesting that phosphorus limits
biomass accrual (Kuenzler et al. 1979).

Filter Blanks
The amount of C, N, and P contained in a 4.25-cm GF/F filter
will be reported as a particulate nutrient, and is the filter
blank (FB). To avoid overestimating a particulate nutrient,
the FB is subtracted from the estimated amount of carbon,
nitrogen, and phosphorus. This correction is especially
important when estimating the extremely low nutrient levels in
oligotrophic systems.
Cleaned filters were analyzed for particulate C, N, and P to
determine the mean amount (ug) + 2 standard deviations (S.D.)
per filter, and are listed below. Each FB is used in the
calculation for the appropriate particulate nutrient. The
corrected amount of particulate nutrient and the volume
filtered (Vf) are used to calculate the in-lake concentration.
particulate Nutrient (PN)
Particulate Organic Carbon 46.2 (range 28.0-125.0)
Total Particulate Phosphorus (manual) 1.06 + 0.24
Total Particulate Phosphorus (automated) 0.785 + 0.264
Organic Particulate Phosphorus (manual) 1.229 + 0.742
Inorganic Particulate Phosphorus (manual) 0.352 + 0.348
Total Particulate Nitrogen (automated) 1.602 + 1.108

Particulant nutrient concentrations (ug L-I) are calculated

using the following general expression:
PN concentration (ug L-l) = (PN-FB)/Vf
 0ruanic Carbon (manual)
Measurements of particulate organic carbon provide a basis for
estimating the amount and the energy content of organic
material. This procedure measures reduced carbon by wet
oxidation using acid dichromate, but is referenced to
carbohydrate carbon. his technique has been criticized
because of an apparent inefficiency in oxidizing nitrogen-rich
compounds. However, Newel (1982) has shown that these
compounds.. are oxidized using dichromate. Thus, realistic
estimates of the total organic-carbon content are possible.
As carbon is oxidized by the dichromate mixture, the color of
the oxidant changes from yellow to green. The decrease in the
absorbance at 440 nm is linear with increasing carbon
concentration.
Standard Ranse

U m e r Limit of ~etection

Lower Limit of Detection

Empirical: 30 ug C.
Predicted: 24 ug C.

Precision

Accuracy

Spectrophotometer (400 nm), drying oven (100 C), centrifuge,

15-ml glass centrifuge tubes, 50-ml Pyrex erlenmeyer flasks,
and 10-mm cuvettes.
Reasents
I) Acid dichromate oxidant - Dissolve 4.84 g of potassium
dichromate (K Cr 0 ) in 20 ml of DI water. Slowly add the
dichromate so?ut3oi( to 500 ml of concentrated sulfuric
acid, cool, and dilute to 1 liter with concentrated
sulfuric acid.
2) Phosphoric acid - Concentrated reagent-grade phosphoric
acid (HjPOq).
NOTE: Use glass pipets and Pyrex containers for preparing and
storing all reagents.
Procedure
1) Place the particulate C filter, seston side down, into a
50-ml erlenmeyer flask.
2) Add 1.8 ml of DI water, 1 ml of phosphoric acid, and swirl
to mix.
3) Add 2 ml of the oxidant, swirl to mix, cover with aluminum
foil, and place in the drying oven at 100 C for 30
minutes. Swirl the flask and continue heating for an
additional 30 minutes.
4) Cool, add 45 ml of DI water, and vigorously mix using a
Teflon rod.
5) Allow the filter slurry to settle, decant 10 ml of the
solution into a 15-ml centrifuge tube, and centrifuge for
15-20 minutes at 2500 RPM.
6) Measure the absorbance at 440 nm against a DI water blank
(see Instrument Calibrations, p. 192).
Standards
Primary dextrose standard (30 mg ml-I C) -
Dissolve 18.75 g of
dextrose-anhydrous into 200 ml of DI water, and dilute to 250
ml .
Secondary standard (600 ug ml-'
primary standard to 100 ml.
as C) - Dilute 2 ml of the

Calculations
As absorbance at 440 nm decreases with an increasing carbon
content, absorbances of the standards and samples must be
subtracted from the blank.
1) Formulate a linear equation by regressing concentrations
(Y-axis) against averaged absorbances (X~axis), and
calculate the coefficient of determination (r ) .
2) Calculate concentrations by subtracting sample absorbances
from the blank value and substitute into the regression
formula.
3) Subtrgct the FB value, and divide by the volume filtered
(Table 31) .
Table 31. The relationship between known concentrations of
-0-

carbon and absorbances (abs) at 440 nm used to

calculate a standard curve in the analysis of
particulate organic carbon.

Secondary Total
standard volume Carbon Absorbance
tml) t L1 tual tx1

C
---
(ug) = 37.0358 + 3153.2679 (abs)
r = .9881

Phos~horus (manual1
Total particulate phosphorus (TPP) estimates the phosphorus
content of the limnetic phytoplankton, bacteria, and detritus.
In practice, TPP from oligotrophic lakes is considered to
represent phosphorus within the phytoplankton; and is
determined as reactive phosphorus after sulfuric
acid-persulfate digestion. TPP can be indirectly determined by
difference (TP-TFP) (Figure 17); however, the accuracy of
direct analysis is preferred given the extremely low phosphorus
levels within oligotrophic systems.
Standard Ranse

Lower Limit of Detection

Empirical: 0.05 ug P.
Predicted: 0.02 ug P.
Precision
Accuracy

Spectrophotometer (882 nm), autoclave (121 C, 15 psi)

centrifuge, 50-ml erlenmeyer flasks, 15-ml disposable
centrifuge tubes, and 10-mm cuvettes.
Reauents
1) Digestion acid (3.6 N H SO ) -
Slowly add 50 ml of
concentrated sulfuric acid2 t8 400 ml of DI water, cool,
and dilute to 500 ml.
2) Digestion solution - Dissolve 9.99 g of potassium.
persulfate (K S 08) into 250 ml of DI water. Add 16.7 ml
of digestion acidrand dilute to 1 liter with DI water.
3) Mixed reagent I (MR I) - See TP/TFP reagents (p. 98) .
Procedure
1) Place the particulate nutrient filter into a 50-ml
erlenmeyer flask seston side up.
2) Add 30 ml of digestion solution, swirl to mix, and cover
- with a 15-ml polypropylene beaker.
3) Autoclave (121 C, 15 psi) for 30 minutes, cool, and swirl
to mix.
4) Withdraw a 5-ml aliquot using an autopipet, place into a
15-ml centrifuge tube, add 1 rnl of MR I, and invert to
mix.
5) Centrifuge at 2500 r p m for 20 minutes during which time
color development is completed.

6) Measure the absorbance at 882 nm against a DI water blank

(see Instrument Calibrations, p. 192).
Standards
Primary standard (0.45 mg ml-I P) -
Dissolve 1.2640 g of
potassium phosphate-dibasic (K2HP04) in 500 ml of DI water, and
dilute to 500 ml.
Secondary standard (4.5 ug ml-I P)
standard to 100 ml.
- Dilute 1 ml of the primary
Calculations
1) Formulate a linear equation by regressing concentrations
(Y-axis) against the averaged absorbances minus the blank
axis) is), and calculate the coefficient of determination
(r I *
2) Determine sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
regression formula (Table 32).
3) Subtract the filter blank (FB) value, and divide by the
volume of lake water (liters) filtered.
Table 32. ---
The relationship between known amounts of phosphorus
and absorbance~ (abs) at 882 nm used to calculate a
standard curve in the analysis of total particulate
phosphorus.

Secondary Total
standard volume Phosphorus Absorbance GEG
(ml) (L) (uq) t XI (:-blank)

Inorsanic and Oruanic Phos~horus (manual1

Total particulate phosphorus (TPP) is comprised of both
inorganic (IPP) and organic (OPP) phosphorus. The proportion
of each varies according to lake type; i.e., glacial,
organically stained, or clear. For example, IPP (rock
phosphorus) has been found to dominate TPP in glacial lakes
(Edmundson and ~oenings1985). Both IPP and OPP are determined
on the same filter with acidified ammonium fluoride used to
first extract IPP (Figure 20), and then OPP being determined
after persulfate digestion.
Standard Ranae
0.675-6.75 ug P (IPP), 1.35-13.5 Ug P (OPP).
 PLACE FILTER I N
C-TUBE. ADD 6 m l
SHAKE TO DISSOLVE,
FILTER THROUQH --3IFfL]-3 DILUTE
- DILUTE' A l m l
ALIQUOT TO 10ml

3
TO 10ml WITH 1:1 EXTRACTINQ
EXTRACTING SOL. 26mm QF/F F I L T E R
SOLUTION AND WATER

FILTER
MEASURE ABS.
a 8 8 2 n m + ADD l m l
(2.6 HR)
MR I - ADD l m l
B O R1I C
0 %A C I D

OPP

ADD 30ml DlGESTlPN

SOLUTION TO FILTER
MASS
>- AUTOCLAVE 30mln
@ 121°C 161:,el - ADD l m l MR I
-
TO 8 m l A L I Q U O T
-CENTRIFUQE

MEASURE ABS.
@ 8 8 2 n m (16mIn)

Figure 20. Flow diagram of the analysis procedures for determining (A) inorganic
particulate phosphorus (IPP) using fluoride extraction, and (B) organic particulate
phosphorus (OPP) after acid digestion.
Lower Limit of ~etection
Empirical: 0.05 ug P (IPP), 0.05 ug P (OFF).
Predicted: 0.08 ug P (IPP), 0.03 ug P (OPP) .
Precision
12% at 2.25 ug P (IPP), 10% at 4.5 ug P (OPP).
Accuracv

Spectrophotometer (882 nm), autoclave (121 C, 15 psi), 25-mm

filter tower, centrifuge, 50-ml erlenmeyer flasks, 15-ml
centrifuge tubes, and 10-mm cuvettes.
Reaaents --
1) 1 N Ammonium fluoride -
Dissolve 37 g of NH4F into 500
of DI water, and dilute to 1 liter.
ml

2) 0.5 N Hydrochloric acid -

Add 20.8 ml of concentrated
to 400 ml of DI water, cool, and dilute to 500 ml.
HC1

3) Extracting solution -
Combine 15 ml of 1 N NH4F, 100 ml of
0.5 N HC1, and 385 ml of DI water.
4) Ammonium molybdate solution -
Dissolve 15 g of ammonium
molybdate-4-hydrate into 400 ml of DI water, and dilute to
500 ml.
5) Sulfuric acid solution - Slowly add 70 ml of
H2S04 to 450 ml of DI water.
concentrated

6) Ascorbic acid solution - Dissolve 5.4 g of ascorbic acid

in 100 ml of DI water. Prepare fresh daily.
7) 10% Boric acid solution -
Add 10 g of boric acid to 100 ml
of DI water, and filter the mixture through a 'fast'
pleated filter.
8) Potassium antimony-tartrate solution - is solve 0.34 g of
antimony potassium-tartrate-trihydrate into 200 ml of DI
water, and dilute to 250 ml.
9) Digestion solution -
Dissolve 9.99 g of potassium
persulfate (K S 0 ) into 250 ml DI water, add 16.7 ml of
'sad
digestion a c d , dilute the mixture to 500 ml.
 10) Special mixed reagent (SR) -
Combine in order, 20 ml of
ammonium molybdate solution, 50 ml of sulfuric acid
solution, 20 ml of ascorbic acid solution, and 10 ml of
the potassium antimony-tartrate solution.
11) Mixed reagent I (MR I) - See TP/TFP reagents 1, 2, and 5
(P* 98)
Procedure .*

1) Place a particulate nutrient filter into a disposable

15-ml centrifuge tube.
2) Add 5 ml of the extracting solution, cap, and shake
vigorously until the contents form a slurry (-1 minute).
3) Pour the slurry into a 25-mm filter tower, rinse the tube
with -2 ml of DI water, and filter the mixture through a
Whatman 2.5-cm GF/F glass-fiber filter.
4) Collect the extract (IPP) in a clean centrifuge tube,
dilute to 10 ml with DI water; and place the filter mass
into a 50-1111erlenmyer flask.
NOTE: Dilutions are required for extracts from glacial
waters; use a 1:l mixture of extracting solution
and DI water as the diluent prior to step 5.
-- . 5) - Add 1 ml of 10% boric acid solution to the IPP extract and
invert the centrifuge tube to mix.
6) Add 1 ml of SR, invert to mix, and allow 20 minutes for
color development.
7) Follow steps 2-5 of the TPP procedure (p. 116) for
determining the OPP held in the filter mass.
8) Measure the absorbances at 882 nm against a DI water blank
(see Instrument Calibrations, p. 192).
Standards
Primary standard (0.45 mg ml-I P) -
Dissolve 0.1264 g of
potassium phosphate dibasic (K2HP04) into 400 ml of DI water,
and dilute to 500 ml.
IPP secondary standard (2.25 ug ml-l P)
primary standard to 100 ml.
- Dilute 0.5 ml of the

OPP secondary standard (4.5 ug ml-I P) - Dilute 1 ml of the

primary standard to 100 ml.
Calculations
1) Formulate separate linear equations by regressing
concentrations (Y-axis) against averaged absorbances minus
the blank (X-axis), and calculate the coefficients of
determination (r ) (Tables 33 and 34) .
2) Calculate sample concentrations by subtracting the blank
value from the sample absorbances, and substitute into the
appropriate regression formula.
3) Subtract the appropriate FB values, divide by the volume
(liters) of the lake water filtered and, if necessary,
multiply by the dilution factor (p. 39).
Table 33. The relationship Qggween known amounts of phosphorus .
and absorbances (abs) at 882 rn used to calculate a
standard curve in the analysis of inorganic
- particulate phosphorus (IPP).

Secondary Total
standard volume Phosphorus Absorbance z g
(ml) (L) tua) (XI (&blank)

---
IPP (ug) = 020117 + 15.7434 (abs)
r = 1.0000
 Table 34. .The relationship b s w e e n known amounts of phosphorus
and absorbances (abs) at 882 nm used to calculate a
standard curve in the analysis of organic
particulate phosphorus (OPP).

Secondary Total
standard volume Phosphorus Absorbance G G
tml) (Ll (uq) (XI (z-blank)

-II-

OPP (ug) = 020709 + 51.2305 (abs)

r = .9997

Nitroaen and Phos~horus (automated]

particulate nitrogen, along with carbon and phosphorus, is a
primary nutrient of the plankton. After phosphorus, lack of
nitrogen is most likely to be limiting to primary production.
- - - Measurements of these two nutrients, along with carbon, allow
for the calculation of ratios which are useful indicators of
the nutritional status of the plankton.
This method is based upon the simultaneous determination of
total Kjeldahl nitrogen and total phosphorus (p. 104).
Standard Ranae
2-60 Ug N, 1-12 ug P.

Lower Limit of Detection

Empirical: 1 ug N, 0.6 ug P.
Predicted: 0.1 ug N, 0.4 ug P.
Precision

Accuracv
-+3% at 40.0 ug N, +7% at 4.5 ug P.
Technicon Autoanalyzer I1 equipped with 630- and 880-nm filters
and 50-mm flow cells, block digestor, vortex mixer, centrifuge,
15-ml disposable centrifuge tubes with caps, and 75-ml
digestion tubes.
Reasents
1) Prepare reagents 1-11 as described in the TICN/TP methods
(p. 104).
2) Acid diluent - Add 0.5 ml Levor V wetting agent (Technicon
Industries) per liter of DI water.
Procedure
1) Loosely fold the particulate nutrient filter with the
seston inside, place into a digestion tube, and add 20 ml
of DI water covering the filter.
2) Follow steps 2-4 of the TICN/TP procedure (p. 106).
3) Withdraw an 8 1 1 aliquot using an autopipet, place into a
15 ml disposable centrifuge tube, and cap.
4) Centrifuge at 2500 rpm for 15 minutes.
5) Pour -4 ml of sample or standard into the sampler tray.
7) Arrange the autoanalyzer (Figure 21), baseline, calibrate
(see Instrument calibrations, p. 192), and pump the
standards and samples through the system.
Standards
primary nitrogen standard (2 mg ml-' N) -
Dissolve 5.3619 g
glycine into -450 ml of DI water, and dilute to 500 ml.
of

Secondary nitrogen standard (20 ug ml-'

primary standard to 100 ml.
N) - ~ i l u t a1 ml of the

primary phosphorus standard (0.45 mg ml-I P) -

Dissolve 1.2640
g of potassium phosphate dibasic (K2PH04) into 400 ml of DI
water, and dilute to 500 ml.
Secondary phosphorus standard (22.5 ug ml-l P)
the primary standard to 100 ml.
- Dilute 5 ml of

Calculations
1) Formulate separate linear equations by regressing
concentrations (Y-axis) against the averaged chart
 Figure 21. Autoanalyzer schematic showing reagent lines and flow rates (ml/min),
cam size (number of samples per hour and sample to wash ratio), mixing coils,
heating baths, and colorimeters with specified flow cells (FC) and wavelength
(nm) used in the simultaneous analysis of particulate nitrogen and phosphorus.
 deflections minus the blank $X-axis), and calculate
coefficients of determination (r ) .
2) Calculate sample concentrations by subtracting the
appropriate blank value from the sample chart deflections,
and substitute into the appropriate regression formula
(Tables 35 and 36).
3) subtract the appropriate FB value, and divide by the
volume (liters) of lake water filtered.
Table 35. The relationship between known amounts of nitrogen
and autoanalyzer chart deflections (za) used to
calculate a standard curve in the automated analysis
of particulate nitrogen.

Secondary
N Total Chart
standard- volume Nitrogen deflection a:
(ml) (L) tua N) (XI (:-blank)
Table 36. The relationship between known amounts of phosphorus
% and autoanalyzer chart deflections (:a) used to
calculate a standard curve in the automated analysis
of total particulate phosphorus.

Secondary
P Total Chart
standard volume Phosphorus deflection a
(ml1 t L) tua) t XI (;-blank)

Phos~horusand Nitroaen in Fish Tissue

Nitrogen (N) and phosphorus (P) levels in sockeye (Oncorhvnchus
nerka) and coho a.
kitsutch) salmon are used to estimate
contributions of decomposing carcasses to in-lake nutrient
levels. In addition, rearing juveniles and smolts are also
examined to obtain both the N and P levels within the fish
community, and the quantity of nutrients leaving the lake,
respectively. Either whole-fish or fish-core samples are used
to determine the N and P content of fish tissue. The N and P
in fish tissue is determined after sulfuric acid digestion
using the TP/TKN method for water samples.

Technicon Autoanalyzer I1 equipped within 630- and 880-nm

filters BD-40 block digestor, 75-ml digestion tubes, autoclave
(121 C, 15 psi), autoclavable plastic bags, mechanical grinder,
and blender.
Reaaents
Prepare reagents as described in the particulate nitrogen and
phosphorus method (p. 122) .
Standards
Identical standards were used for whole and cored samples.
1) Primary nitrogen standard (2 mg ml-' P) -
Dissolve 2.6831
g of glycine into -200 ml of DI water, and dilute to 250
ml.
2) Secondary standard (20 ug ml-I N) - Dilute 1 ml of the
primary N standard to 100 ml.
3) Primary phosphorus standard (0.45 mg ml-I P) -
Dissolve
0.6322 g of potassium phosphate-dibasic into -200 ml of DI
water, and dilute to 250 ml.
4) Secondary standard (22.5 ug ml-I P)
primary P standard to 100 ml.
- Dilute 5 ml of

Prepare stangard curves with ranges of 10-3000 ug L - ~N, and

50-450 ug L P.

A. Whole Carcass Analysis

Procedure
1) Grind the carcass using a mechanical grinder, add to two
autoclavable bags, and add 500 ml of DI water to each bag.
2) Autoclave (121 C, 15 psi) for 30 minutes, cool, and
measure the combined volume of slurry.
3) Remove two 100-ml subsamples of slurry, place into 125-ml
polybottles, and freeze for later analysis.
4) Thaw, shake the sample well, withdraw a 5-ml aliquot using
an autopipet, place into a beaker, and add 45 ml of DI
water.
5) While stirring withdraw a 1-ml aliquot, place into a
digestion tube; add 20 ml DI water, 2 ml of digestion
reagent, and 10 teflon boiling chips.
6) Digest in the block digestor at 200 C for 1 hour. Include
several DI water blanks to be used as a later diluent.
7) Digest for an additional 20 +1 minutes at 360 C, and
dilute the residue with 20 ml oT DI water.
8) Withdraw a 0.05-ml aliquot, place in the sampler tray, and
add 3 ml of digested blank water.
9) Arrange the autoanalyzer (Figure 21), baseline, calibrate
(see Instrument Calibrations, p. 192), and pump the
standards and samples through the systems.
Calculations
1) Formulate separate linear equations by regressing
concentrations (Y-axis) against averaged chart deflections
minus the blank (X-qxis), and calculate the coefficients
of determination (r ) .
2) Calculate sample concentrations by subtracting the
appropriate blank value from the sample chart deflections
and substitute into the appropriate regression formula
(e.g., Tables 29 and 30) .
3) To estimate the N or P concentration in the slurry use the
following:
N or P ug per ml of fish slurry = N or P (ug L-I) x 16.2.
4) Multiply by the total volume (ml) of fish slurry (see
procedure, step 2) to obtain the amount of N and P in the
fish carcass.
5) Divide N by 14 and P by 31 and calculate an N : P atom ratio
(Table 37).

B. Cored Sample Analysis

Procedure
I) Remove several (-6) 1.25-cm diameter cores along the
mid-line of the weighed fish, combine the cores, and
weigh.
2) Place the cores into an autoclavable bag, add -100 ml of
DI water, and autoclave (121 C, 15 psi) for 3 0 minutes.
3) Pour the slurry into a blender, dilute to 1 liter with DI
water, and homogenize at high speed.
4) Remove two 100-ml subsamples of slurry, place into 125-1
polybottles, and freeze for later analysis.
5) Thaw, shake the sample, withdraw a 1-ml aliquot using an
autopipet, and place into a digestion tube.
6) Add 20 ml of DI water, 2 ml of digestion reagent, and 10
teflon boiling chips.
7) Follow the whole carcass procedure, steps 6 and 7.

8) withdraw a 0.2-m1 aliquot, place in the sampler tray, and

dilute to 4 ml with digested blank water.
 Table 37. Amounts of nitrogen and phosphorus, and the nitrogen to phosphorus atom
ratios in (A) sockeye juveniles (fall fry) and (B) smolts from Alaskan
lakes during 1983.

Phosphorus (mql Nitrosen [mq)

Percent Nitrite Percent
Life stage wet + wet TKN :TP
System (sample s i z e ) Total Reactive weiqht Ammonium Nitrate TKN* weisht [bv atoms)

I Redoubt A ( 6) 2.0 1.3 0.309 0.0 0.0 20.0 3.02 22:l

'-t Hidden A (15) 1.0 0.6 0.322 0.0 0.0 7.0 2.37 17 : 1
h)
w Larson A (18) 0.5 0.3 0.210 0.0 0.0 6.0 2.72 27:l
I
McDonald A (13) 6.0 1.7 0.425 0.0 0.0 28.0 2.00 22:l
........................................................................................
Tustumena B (24) 4.1 2.0 0.243 0.0 0.0 32.2 1.89 17:l
Falls B (19) 5.1 2.8 . 0.302 0.0 0.0 28.5 1.67 12:l
Larson B (19) 9.8 3.7 0.038 0.0 0.0 64.2 2.14 15: 1
Leisure B (10) 4.5 3.2 0.250 0.0 0.0 47.5 2.64 23:l

*Total Kjeldahl nitrogen

9) Arrange the autoanalyzer (Figure 21), baseline, calibrate
(see .Instrument Calibrations, p. 192), and pump the
standards and samples through the system.
Calculations
1) Formulate separate linear equations by regressing
concentrations (Y-axis) against averaged chart deflections
minus the blank (X-%xis), and calculate the coefficients
of determination (r ).
2) Calculate sample concentrations by subtracting the
appropriate blank value from the sample chart deflections,
and substitute into the appropriate regression formula
(e.g., Tables 29 and 30) .
3) To estimate the amount of N or P (ug) of core sample use
the following:
N or P (ug) per g of core - N or P tua L - ~ Ix 400.
core weight
4) Multiply by the weight (g) of the whole fish to obtain the
amount of N and P in the fish carcass.
5) Divide N by 14 and P by 31 and calculate a N:P atom ratio
(Table 37).

REFERENCES
Crowther, J., B. Wright, and W. Wright. 1980. Semi-automated
determination of total phosphorus and total ~jeldahl
nitrogen in surface waters. Anal. Chimica Acta
119:313-321.
Donaldson, J. R. 1967. The phosphorus budget of Iliamna Lake,
Alaska, as related to the cyclic abundance of sockeye
salmon, Ph.D. Thesis, Univ. Washington. 141 p.
Edmundson, J. A. and J. P. Koenings. 1985. The effects of
glacial silt on primary production, through altered light
regimes and phosphorus levels in Alaskan lakes. pp. 3-19.
In: L. P. m i g h t (ed.), Resolving Alaska's Water
Resources Conflicts. University of Alaska-Fairbanks,
Institute of Water Resources Report 108. 212 p.
Eisenreich, S. J., R. T. Bannerman, and D. E. Armstrong.
1975. A simplified phosphorus analysis technique.
Environ. Letters. 9:43-53.
Kuenzler, E. J., D. W. Stanley, and J. P. Koenings, 1979.
Nutrient kinetics of phytoplankton in the Pamlico River,
North Carolina. University of North Carolina, Water
Resources Resarch Institute Report No. UNC-WRRI-79-139.
163 p.
Nelson, P. P. and W. T. Edmondson. 1955. Limnological effects
of fertilizing Bare Lake, Alaska. Fish. Bull. 54:415-436.
Newel, R. I. E. 1982. An evaluation of the wet oxidation
technique for use in determining the energy content of
seston samples. Can. J. Fish. Aquat. Sci. 39:1383-1388.
Strickland, J. D. H. and T. R. Parsons. 1972. A practical
handbook of seawater analysis. Bull. Fish. Res. Bd. of
Canada 167. 310 p.
~echniconIndustrial Systems. 1978. ~ndividual/simultaneous
determination of nitrogen and/or phosphorus in BD acid
digests. Industrial Method No. 329-74W/B. Technicon
Industrial Systems, Tarrytown, N.Y.
 PRIMARY PRODUCTIVITY
Primary productivity is the rate of biomass accrual by
autotrophic organisms. These organisms include limnetic algae
and bacteria as well as littoral macrophytes and periphyton. A
majority of sockeye salmon nursery-lakes have littoral areas or
volumes that are extremely limited emphasizing the importance
of limnetic primary production. In the limnetic zone of
oligotrophic lakes, photosynthetic phytoplankton are
responsible for a majority of primary productivity; but rates
are low requiring a precise method of measurement. Thus,
estimating primary production through small increases in oxygen
(0 ) is replaced by determining the amount of carbon (C02) used
du$ing photosynthesis:

Measurements of primary productivity are made using carbon-14

(C-14) radioisotopes. Tracer amounts of C-14, added to lake
samples, are assimilated by algal cells over a known time that
the sample is incubated in situ. Following incubation, the
samples are filtered and the amount of C-14 on the filter is
determined using liquid scintillation spectrometry. Using
these rgsults, day-rate estimates of carbon assimilation
(mg C/m /day) are made at different depths, and when integrated
through the euph~tic~zone are used to estimate areal
productivity (mg C/m /day) .
Carbon-14 Assimilation

A. Field Methods

Van Dorn sampler, underwater photometer, temperature probe, pH

meter, vacuum pump, 0.25-ml autopipet, 125-ml BOD bottles,
plexiglass BOD bottle holders, 300-ml filter tower, 100-ml
graduate cylinder, and 20-ml polyethylene scintillation vials.
Reaaents
1) Carbon-14 isotope - 20.9 u Curies (uCi)/ml as sodium
bicarbonate, C-14.
2) Lugol's acetate solution
Storage, (p. 15).
- See Preservatives for Sample
3) 37% Formaldehyde.
 Procedure '

1) Determine the 1% PAR compensation point (see

Photosynthetically Active Radiation p. 22).
2) Collect water using an opaque sampler from three depths
per station: 1 m, mid-euphotic, and compensation depth.
Measure the pH and, if a temperature profile is not taken,
record the temperature of each sample.
3) Add 100 ml of lake water to four 125-ml BOD incubation
bottles for each depth sampled with two of the bottles
taped and/or painted to prevent light penetration (Table
38).
NOTE: Use of two dark and two light bottles provides for
replication of each treatment. However, one of the
dark bottleg can serve as a 'killed control' by the
- addition of formaldehyde and is used to separate
bacterial uptake from inorganic adsorption.
Table 38. Carbon-14 incubation bottles used for in-situ
radiocarbon experiments and the reactions occurring
within each treatment.

Sample
Bottle volume Formaldehyde
- - tme tmll (37%) (ml) Reactions
Light 100 0 Algal + inorganic + bacterial
C-14 uptake
Dark 100 0 ~norganic+ bacterial
C-14 uptake
Kill 95 5 ~norganicadsorption
C-14 uptake

4) Add 0.25 ml (5.2 u Ci) of C-14 isotope ( N ~ H C ~ to

~ O ~each
)
incubation bottle using an automatic pipet-
5) Attach the bottles into a notched plexiglass plate (Figure
22A) or similar device so that the light bottles are
unobstructed. Suspend each plate (Figure 22B) at the
depth from which the samples were collected, and record
the time (To) .
6) After -4 hr retrieve the samples, add 1 ml of Lugol's
acetate solution, invert to mix, and record the time (Tf).
If day rate estimates are necessary, see p a 140.
7) Invert each bottle several times, and filter the contents
through separate Whatman 2.4-cm GF/F filters at 15 psi.
 plexiglass plate

Figure 22. Plexiglass plate (A) with lock and key cut-outs used to fasten incubation
bottles and ( 8 ) arrangement of a series of plates in the water column used in estimating
carbon assimilation rates.
 place' each filter into a labeled polyethylene
scintillation vial and store frozen.
NOTE: Label onlv the cap of the scintillation vial and
avoid using tape.
8) Soak and scrub the incubation bottles, rinse several times
with-tapwater, DI water, and allow to drip dry.

B. Laboratory Methods
The amount of carbon-14 contained on a filter is quantified
using liquid scintillation spectrometry. As C-14 atoms
disintegrate they emit energetic electrons known as beta
particles. When these beta particles interact with a
scintillation cocktail photons are emitted (disintegrations), a
portion of which are counted by the sensitive photomultiplier
tubes of the spectrometer.

~iquidscintillation spectrometer and 10-ml dispensette.

Reasents
1) Aquasol I1 scintillation cocktail
Boston, MA.
- New England Nuclear,

2) 0.5 N Hydrochloric acid -

Slowly add 32 ml of concentrated
HC1 into -750 ml of DI water, and dilute to 1 liter.
Quench Curves and Countinu Efficiency
since the liquid scintillation spectrometer does not detect all
the disintegrations of a sample, the relationship between
sample counts (CPEI) and actual disintegrations (Dm) must be
established. The ratio of CPM versus DPM is the counting
efficiency (13).
E = -CpM X 100
DPM
However, counting efficiency is affected by quenching.
Quenching decreases the ability (efficiency) of the
spectrometer to detect beta particles, and is caused by a
number of chemical or optical effects such as sample
composition, color, vial type, or filter orientation. If
quenching is constant, counting efficiency remains constant,
but if quenching varies either the counting efficiency of each
sample must be determined or a quench curve derived to cover
the range of quenching. The varying yellow color of lake water
and Lugol'r acetate solution are primary quenching agents. We
derived a quench curve utilizing the sample channels ratio
(SCR) method described by Baille (1959), Herberg (1960), Bruno
and Christian (1962), and Bush (1976). A series of vials
containing varying amounts of Lugolls acetate solution and
equal amounts of carbon-14 (DPM) were counted at two different
energy levels [e.g., red (R) and green (G) channels]. A linear
relationship was derived from the ratio of the CPM in the two
channels (SCR) and counting efficiency (E) (Table 39) .
Procedure
1) Thaw the filters and place seston side up in
scintillation vials.
2) Add 0.3 ml of 0.5 N HC1 to saturate the filters and purge
inorganic 14C02 while in the fume hood (-2 hours).
3) Add 10 ml of scintillation cocktail (Aquasol 11) , number
the cap, and load the vials into the liquid scintillation
spectrometer.
4) Adjust the instrument to the settings listed below:
a) Depress the adjustable discriminator.
b) Red channel gain to 6.5%.
c) Red channel upper limit to 1000.
d) Red channel lower limit to 30.
e) Green channel gain to 6.5%.
f) Green channel upper limit to 1000.
g) Green channel lower limit to 300.
h) Cycle mode to 2.
i) Number of counts to XI.
j) Upper counting limit to 20,000.
k) Counting time at 10 minutes. (If the counts are
<1500 increase the counting time to 20 minutes.)
5) Depress 'load' and then Ioperate'. The spectrometer will
automatically load, count, and following completion of the
last sample, terminate the counting sequence.
NOTE: Record the discrimination settings used for each data
set on the actual printout.
Calculations
1) Formulate a linear equation by regressing E (Y-axis)
against SCR (X-qxis), and calculate the coefficient of
determination (r ).
2) Determine sample counting efficiencies by substituting
sample SCR values into the appropriate regression formula
(Table 39).
3) Divide sample counts from the red channel by E to derive
corrected disintegrations per minute (DPM).
Table 39..* CPM from red (R) and green (G) channels used to
determine sample channels ratio (SCR), and CPM (R)
and DPM used to calculate counting efficiencies (E).

R G
DPM (CPMI (CPM) SCR Et%)

If SCR 5.050:E = 72.919 + 262.7258 SCR r2 = .9691

If SCR >.050:E = 82.727 + 24.3955 SCR r2 = .9869

Volumetric Productivitv Estimates

Carbon uptake rates per liter or m3 of lake water measure the
fertility or algal productivity on a volumetric basis. Because
of changes in light, and often temperature, within the photic
zone; volumetric rates of photosynthesis change with depth
often reaching peak values near the mid-euphotic zone depth.
In addition, as the light compensation point often lies below
the metalimanion, considerable algal photosynthesis can occur
within the hypolimnion.
Calculations
Carbon (C-12) assimilation per cubic meter of lake water is
estimated using: temperature, pH, alkalinity (carbon), and the
C-14 DPM for each incubation bottle.
The C-12 assimilation rate is calculated using the expression:

B - Sample DPM divided by the total DPM added to the

incubation bottle.
C - Available carbon (C-12) from alkalinity (as
CaCO ) x 1000 x CF (CF is the temperature and pH
convarsion factor obtained from Table 40).
D - Incubation time (hr).
1.06 = Isotope discrimination factor (C-12/C-14).
Algal C-12 incorporation or uptake rate is calculated using:
Algal ug C-12/l/hr = - Ez
5 = Averaged C-12 uptake in light bottles
Eg = Averaged C-12 uptake in dark bottle.

The data printout (Table 41) for a single carbon-14 uptake

experiment in Falls Lake, June 24, 1981 is used in the
calculations. In addition, the following are necessary:
Temperature: 1 m = 12.4 , 5 m = 10.2, 10 m = 6.2
pH: 1 m = 5 m = 5.8, 10 m = 5.7
Alkalinity: 12 mg L as CaCO )
Carbon-14 added per bottle: 5 . 1 u Ci (11,569,925 DPH)
Incubation time: 1041 hr (To), 1441 hr (Ti)
For each incubation bottle, substitute the appropriate data

_
into the carbon uptake expression as shown for the 1 m, light
(L) bottle.
6.203 DPM X 12.840 ua C-12/1 x 1.06 1.82
C
-
12
'
1
'h
r a 11,569,925 DPM 4.0 hr
The carbon uptake rates for the remaining bottles comprising
the 1-m depth are 1.45 (L), 0.35 (D), and 0.24 (D) ug
C-12/l/hr. Finally, the algal carbon-12 uptake rate at the 1-m
depth equals 1.34 ug C/l/hr. Similarly the uptake rates for
the 5- and 10-m depths are 1.92 and 0.49 ug C/l/hr,
respectively.
Table 40. . F a c t o q for the conversion of total alkalinity
[mg L (CaCO ) ] to milligrams of carbon per
liter (saundess et al. 1962) .
 Table 41. ~iquidscintillation spectrometer data
printout for a single carbon-14 uptake
experiment in Falls Lake.

Depth Bottle
(m) type RCPM GCPM SCR

Dav-Rate Estimates
Day rate estimates of photosynthesis are facilitated if the
duration of experiments are timed proportional to day-length
(see Appendix)-. Day-length is divided into five equal periods
and by incubating in the second and third periods, 55%-60% of
the total day rate is produced. ~ccordingly, the error
introduced in estimating day-rate integrals of photosynthesis
from exposure during the second and third periods will be of
the order of + 10% (Vollenweider, 1965). In order to avoid
C-14 losses due to various processes (e.g., organic excretion)
incubation should not exceed 4-6 hours.
Calculations
1) Divide sunrise to sunset into five equal periods by
minutes, and incubate the samples for -4-6 hours during
the second and third periods (Figure 23).
2) Beginning with sunrise, add the number of minutes to T
and to Tf, and locate each within their respectiv8
periods.
3) Determine T , and T as a percent of their respective
periods, 18cate thgse percentages within each period
(X-axis), and read percent cumulative productivity
(Y-axis) for To and Tf.
 CUMULATIVE % FOR VOLLENWEIDER'S FIVE-PERIOD LIGHT DAY

sunset-sunrise = daylight
midday = 1/2 daylight'
. minutes/unit = daylight minutes
100
plot incubation period
working from midday
growth % = t f % - to%

Figure 23. The relationship of cumulative primary produc-

tivity (expressed as a percent) to day-length divided into
five equal periods (IV). Also shown are ten increments
within each period (expressed as a percent) used to locate
the initial and final carbon-14 incubation times, and the
corresponding proportion (P) of cumulative productivity.
4) .Subtract the percent cumulative productivity found for T
from that found for T which equals the percent (P) of
the total daily cdkbon-12 assimilated during the
incubation period.
5) Determine the amount of carbon-12 (mg C-12/m 3 ) assimilated
during the incubation period by multiplying the volumetic
carbon-12 assimilation rates (p. 137) by the duration of
.
incub.ation (hours)

6) Divide mg C-12/m3 by the proportion (P/100) of the daily

rate to obtain the day-rate estimate expressed as mg
C-l2/m/d.
NOTE: Vollenweider (1965) determined that the specific
shape of the photosynthesis depth curve is of much
less importance with regard to day-rate estimates
than had generally been thought. Nonetheless, C-14
in-situ exposure should take place at -40% of the
incident solar radiation when using single depth
determinations.
Example
During the Falls Lake experiment the total day-length on June
24 was 1097 minutes (sunrise 0352 hr, sunset 2209 hr), and the
five resultant 219 minute periods are:
Period 1 = 0- 219 minutes
Period 2 = 220- 438 minutes
Period 3 = 439- 658 minutes
Period 4 = 659- 878 minutes
Period 5 = 879-1,097 minutes
The incubation began (T ) at 1041 hours or at 408 minutes after
sunrise. The incubatioR ended at 1441 hrs or at 649 minutes
after sunrise. T (408) falls at 86% (408-220/219) of period
2, and Tf (649) f811s at 96% (649-439/219) of period 3.
T corresponds to a percent daily rate equal to 38% (Figure 23)
a!?d T corresponds to 68% of the daily rate. Thus, the percent
of thg day-rate productivity measured in this experiment was
30% (68%-38%). Finally to estimate the day-rate productivity
divide the total amount of algal carbon uptake by 0.3
(30%/100) .
The day-rate estimate equals:

similarly the day-rgte estimates for the 5- and lo-m depths are
25.1 and 6.5 mg C/m /day respectively.
 Areal Estimates
Areal primary productivity is defined as the rate of
autotrophic biomass ac~uralunder a unit area lake surface, and
is expressed as mg C/m /day.
Procedure
1) Plot day-rate estimates (mg C-12/m3/day) on the X-axis
versus depth on the Y-axis, and draw a smooth curve
through the points (Figure 24).
2) Calibrate the planimeter by measuring a known area (mg
C/m /day x m) to obtain a conversion factor of planimeter
readings to known area (see Instrument Calibrations,
p. 192).
3) Measure the area within the curve using a calibrated
planimeter, and convertZ planimeter readings to areal
productivity (mg C-12/m /day) by multiplying by the
conversion factor (Step 2).

The day-rate estimates for Falls Lake (June 24, 1981) equalled
17.2 (1 m), 25.1 (5 m), and 6.5 (10 m) mg C/m /day (Figure 24).
The area within the curve is represented by 317 planimeter
units, and when multiplied by the conversion fqctor of 0.6152
resulted in an areal productivity of 195 mg C/m /day.
If a planimeter is unavailable, two alternative methods are:
1) Sum the mg c/m3/day for each 1-m lfyer (where the area
witgin each 1-m layer equals mg C/n /day x 1 meter or mg
C/m /day) using the mid-strata value for each layer
(Figure 24).
2) Average the day rate estimates at the various depths, and
multiply by the depth of the euphotic zone.

Finally, areal productivity estimates using the three methods

were compared for Falls Lake (June 24, 1981) (Figure 24), and
for three lakes with varying photic zones (Figure 25).
t J

L A K E SURFACE

I
- - - - - -1 00
I----
7
1- -- 66

2-

3-

4-
W
5 --
z --
0 21
N I-
6- -
0
I-
I
2
E
Y 0 J

I 7;
I W
I- P 0
n 3 a
W l
L
8- a
3
cn
a
9- a
cn
ae
10- -- 3
7
11- /
L

'
12- /
/
7-
13- /

14 1 , 1
0 15 30
MG C / M ~ / D A Y

b) PLANIMETRY: 195 MG C / M ~ / I J
c) AVERAGING: 227 MG C / M ~ / D

Figure 24. Vertical distributions of day-rate primary pro-

ductivity estimates within the euphotic zone used in three
procedures to obtain areal productivity (mg c/m2/day) in Falls
Lake, 24 June 1981.
. i

MG C / M ~ / D A Y
0 25 50 75 100

--

15-
n
Ah
E
Y

x
I-
n
W
'L
0 20-

'A

25 -
FALLS LK PACKERS LK SNAKE LK
Ah - - - t - - - - t
a) lQl 234 113

30 - A = { b) 196 238 125

c) 227 206 127

A = MG C / M ~ / D A Y x 1M=MG C / M ~ / D A Y

35 I I 1

0 25 50 75 100

Figure 25. Vertical distributions of day-rate primary pro-

ductivity estimates (mg c/m2/day) in three lakes, Falls, Packers,
and Snake, with varying photic zones; and a comparison of areal
productivity estimates (A) as determined by summation (a),
planimetry (b), and averaging (c) .
 PRIMARY PRODUCTION
Phytoplankton standing crop can be represented by the
concentration of algal pigments. The composition of the algal
pigments varies depending upon physiological status and the
relative classes making up the phytoplankton community.
However, only chlorophyll g (chl g) is considered here since
concentrations of chl $ and chl p are negligible by comparison.
chlorophyii samples rapidly degrade to phaeophytin g (phaeo a)
upon changes in light, temperature, and pH. As the inactive
phaeo g pigment absorbs and fluoresces light at the same
wavelengths as chl g, large amounts of phaeo g bias estimates
of chl g. Thus, both the spectrophotometric and fluorometric
analyses require an acidification step to correct for
phaeophytin. By taking spectrophotometric and/or fluorometric
measurements before and after acidification, estimates of both
chl a and phaeo a concentrations can be made.
The procedures for determining chl q and phaeo 3 are modified
from Strickland and Parsons (1972). Chl g is extracted from
algal cells after grinding the filter in 90% acetone. The
absorbance of the extraction solution is then measured before
and after acidification with dilute HC1 (Reimann 1978).

Chloro~hvlla and Phaeo~hvtina ts~ectro~hotometric)

Limit of Detection
0.04 ug L-I (lake concentration) .
Spectrophotometer (750 mm, 6 6 5 nm, 663 nm, 6 4 5 nm, 630 n m ) ,
tissue grinder, teflon serrated pestle, grinding vessel,
centrifuge, 15-ml glass centrifuge tubes, and 10-mm cuvettes.
Reaaents
1) 90% neutralized acetone - Dilute 900 ml of reagent-grade
acetone to 1 liter with DI water, add 1 ml of 1 N NaHC03,
and filter through a 'fastt pleated filter.
2) 2 N HC1 - Dilute 42 ml of concentrated hydrochloric acid
to 250 ml with DI water.
3) 1 N Sodium bicarbonate
into 100 ml DI water.
- Dissolve and dilute 8.4 g NaHC03
Procedure .
1) Thaw filters, place into a grinding vessel, and add -2 ml
of 90% acetone.
2) Grind while moving the vessel up and down against the
serrated pestle until the contents form a slurry.
3) ~inse'the pestle with -4 ml of 90% acetone, transfer the
slurry into a 15-ml centrifuge tube, rinse the vessel with
-4 ml 90% acetone, and add the contents to the centrifuge
tube.
4) Cover with aluminum foil, and refrigerate the samples for
2-3 hours to complete chl g extraction.
5) Centrifuge for 40 minutes at 2500 rpm, and decant the
supernatant into another 15-ml centrifuge-tube, and dilute
to 12 ml with 90% acetone.
6) Invert, split into two equal parts, and to one tube add
0.05 ml of 2 N HC1. Invert the acidified tube to mix.
7) Measure the absorbances of the unacidified fraction
against a 90% acetone blank at 750 nm, 665 nm, 663 nm, 645
nm, and 630 nm.
8) Measure the absorbance of the acidified fraction against a
90% acetone blank at 750 nm and 663 nm.
NOTE: While analyzing keep the samples in the dark or
under subdued light as much as possible. Wash all
glassware with 902 acetone and avoid using 10% HC1.
Calculations
The trichromatic and monochromatic methods can both be used to
determine chlorophyll concentration. Careful attention must be
given to the interpretation of the results. The trichromatic
method estimates total pigment (chl g plus phaeo a ) ; whereas
the monochromatic method estimates chl a and phaeo a
separately. The trichromatic equation (S%R/UNESCO) uses
absorbances before acidification, and is modified here to yield
in-lake pigment concentration.
[ll.64 (663,) -2.16 (64S0)+. 10 (6300)]VS
chl g + phaeo g (ug L-1) =
L x Vf
 (6630) = absorbance 663 nm - absorbance
750 mu
(645,) = absorbance 645 nm - absorbance
750 nm
(6300) = absorbance 630 nm - absorbance
750 nm
Vs = total volume (ml) of sample extract (Step 5).
L = pathlength (cm) of cuvette.
Vf = volume (1) of lake water filtered.

The monochromatic method (Strickland and Parsons 1972) uses

separate equations for chl g and phaeo a. These equations are
both modified to yield in-lake pigment concentrations.

phaeo (ug L
-
'
) =
26.7 [1.7 (665,) - 6650] x VS
L x Vf
(6650) = absorbance 665 nm
acidification)
(665,) = absorbance 665 nm
. --
absorbance 750 nm (before
absorbance 750 nm (after
acidification).
Vs = total volume (ml) of sample extract (Step 5).
L = pathlength (cm) of cuvette.
Vf = volume (1) of lake water filtered.
AS periphyton samples (see p. 10) often contain very high chi a
concentrations, we recommend using the spectrophotometric
(monochromatic) method in order to avoid large dilutions of the
sample extract. Periphyton chl q and phaeo a concentrations
are reported as. mg/m by removing the volume filtered ( V f l from
the monochromatic equations and substituting the area (m ) of
the plexi-glass plates.

Chloroahvll a and Phaeoahvtin a ffluorometric)

Algal pigments extracted in acetone fluoresce when subjected to
a specific wavelength of light. Instrument readings both
before and after acidification are required to determine chl a
and phaeo g levels. The fluorometric method is preferred
because of its increased sensitivity. Pigment concentrations
are based on sample fluorescense, relative to known chl
standards.
~ i m i tof Detection
0.04 ug LO' chl 1 (lake concentration), 0.05 ug L-' phaeo q.
Turner model 111 or 112 filter fluorometer equipped with a
F4T5-B source lamp, corning CS 5-50 excitation filter (420 nm)
and a Corning CS2-64 emission filter, tissue grinder, serrated
pestle, centrifuge, 0.05-ml autopipet, 15-ml centrifuge tubes,
12- x 75-mm borosilicate-glass culture tubes (cuvettes).
Reasents
Prepare the reagents as described in the spectrophotometric
method (p. 146).
Procedure
1) Follow steps 1-5 of the spectrophotometric procedure
(p. 147).
2) Calibrate the fluorometer (see Instrument calibrations,
p. 192).
3) Pour -4 ml of the extract into a glass culture tube, and
record fluorescense units (Rb) and the appropriate
sensitivity setting (S). If the reading is greater than
100 on the S window, dilute the sample with 90% acetone,
and calculat& a dilution factor (DF). If the reading is
less than 10, increase the sensitivity setting.
4) Acidify the extract in the culture tube with two drops of
2 N HC1 using a 0.05 ml autopipet, and mix. wait 30
seconds, record the fluorescence (Ra) and the appropriate
sensitivity setting.
Calculations
In-lake concentrations of chl and phaeo g are calculated
using:

Chl 3 (ug LW1) = Sx(Rb-Ra) ( VF

.012) tDF)

Phaeo a

Sx = calibration factor.
rs = acid ratio.
Rb = fluorescence units before acidification.
Ra = fluorescence units after acidification.
DF = dilution factor.
VF = volume (1) of lake water filtered.
In summary, there are three methods that can be used to analyze
for total pigment, chlorophyll a and phaeophytin a. These are
the (1) trichromatic, (2) monochromatic, and (3) fluorometric
methods (Table 42). Low chlorophyll a levels should be
dpfemined using the fluorometric method, while higher (>lo ug
L ) in-lake concentrations, and chl a in macrophytes and
periphyton should be analyzed using spectrophotometric
methods.
Table 42. Comparison of total pigment, chlorophyll g (chl g),
and phaeophytin g (phaeo g) levels as determined by
"the trichromatic, monochromatic and fluorometric
methods.

Trichromatic Monochromatic Fluorometric

Sam~le Total pianent Chl a Phaeo a Chl a Phaeo a
Grant Lake 0.21 0.16 0.06 0.11 0.15
Packers Lake 1.35 1.28 0.06 1.24 0.67
08Malley~-
Lake 4.35 3.52 1.60 2.75 2.23
Bear Lake 18.68 20.8 <O. 04 16.52 1.13
EPA spectro-
photometric
reference 7200 5794 2561 6024 4453
Sigma
reference
Macrophyte
extract

NOTE: The results are expressed as ug L-l with tho exception

of the macrophyte extract which is expressed as mg per g
dry weight of plant material.

REFERENCES
Baker, K. S., R. C. Smith, and J. R. Nelson. 1983.
Chlorophyll determinations with filter fluorometer:
larnp/filter combination can minimize error.
Oceanogr. 28:1037-1040.
Limn01 .
Baille, L. A. 1960. Determination of liquid scintillation
counting efficiency by pulse height shift. Inter. J.
Applied Rad. and Isotopes 8:l.
Bruno, G. A. and J. E. Christian. 1962. Corrections for
quenching associated with liquid scintillation counting.
Anal. Chem. 33:650-651.
Bush, E. T. 1963. A double ratio technique as an aid to
selection of sample preparation procedures in liquid
scintillation counting. Anal. Chem. 35:447-450.
Herberg, &. J. 1965. Channels ratio method of quench
correction in liquid scintillation counting. Packard
Technical Bulletin 15. 1-8.
Reimann, B. 1978. Carotenoid interference in the
spectrophotometric determination of chlorophyll
degradation products from natural populations of
phytoplankton. Limnol. Oceanogr. 23:1059-1066.
Saunders, G. W., F. 8 . Trama, and R. W. Bachmann. 1962.
valuation of a modified 14C technique for shipboard
estimation of photosynthesis in large lakes. Univ.
Michigan, Great Lakes Res. Div., Spec. Rep. No. 8. 61 p.
Strickland, J. D. H. and T. R. Parsons. 1972. A practical
handbook of seawater analysis. Bull. Fish. Res. Bd. of
Canada 176:310 p.
Vollenweider, R. A. 1965. Calculation models of
photosynthesis-depth curves and some implications
regarding day rate estimates in primary production
measurements. Mem. 1st. Ital. Idrobiol. Suppl:18.
425-457.
Wetzel, R. B. and G. E. Likens. 1979. Limnological analyses.
W. B. Saunders Company, Philadelphia, PA. 357 p.
 SECONDARY PRODUCTION
The limited littoral area typical of sockeye salmon nursery
lakes emphasizes the importance of limnetic zooplankton
production for juvenile survival and growth. As the
zooplankton community serves as the primary forage base for
sockeye juveniles, the characteristics of zooplankton
populations are important indicators to gauge either present or
potential rearing (forage) conditions for limnetic feeding
salmon fry'; and to evaluate results of try stocking and
nutrient enrichment programs. Important zooplankter
characteristics include species or community composition,
body-size, biomass, and seasonal timing and fluctuations in
density. The amount of zooplankton that is actually available
as food for rearing salmon fry is often a sub-fraction of the
total because fry are very selective feeders. By electing to
feed on certain species and body-sizes, fry exert a predation
pressure that can structure zooplankton communities. In
general, zooplankter communities with few or small cladocerans
are consistent with a heavy predator pressure. In contrast,
communities with high densities of large cladocerans may have
little or no predatory pressure from fish.
Such generalizations need to be applied with caution, as recent
studies (Gliwicz 1986, ~oeningset al. 1986) have shown that
environmental conditions can also reduce or eliminate
cladocerans from the zooplankton community. ~ppropriate fish
enhancement strategies can be formulated from a knowledge of
how fish predation and other components of the environment
structure zooplankton communities.

The zooplankton community consists of three major groups,

cladocerans, copepods, and rotifers. Each has evolved
different life history strategies in response to predator
pressures, food availability, and environmental stimuli.
Briefly, rotifers are small, non-prey items for salmonids, and
have a rapid reproductive rate. Cladocerans also have a rapid
reproductive rate; but are larger, non-evasive and are
preferred prey items for salmonids. The copepods are of
similar size to the cladocerans; but have a slower reproductive
rate, are extremely evasive, and are only occasional forage for
salmonids. That is, cladocerans survive by simply
out-producing predators while copepods simply out-maneuver
them.
Zooplankters are identified to species using either a stereo
dissecting or compound microscope, and various taxanomic keys.
Cladocerans can be easily identified using only a dissecting
microscope. However, copepods must be speciated using adult
specimens with identifying parts dissected out, mounted on
glass slides, and examined under a compound microscope.

Bausch and Lomb stereo dissecting microscope, 1-ml

Hensen-Stemple pipet, 1-ml Sedgewick-Rafter counting cell, and
taxanomic.keys for freshwater invertebrates.
Procedure
1) Place a section of 130-u mesh plankton net into a funnel,
and empty the contents of the sample bottle onto the net.
Rinse the sample bottle several times with tap water and
pour onto the net.
2) Invert the net over a 250-ml beaker, and using a wash
bottle rinse into the beaker. The zooplankters are now
formalin free and can be diluted to a known volume with
tap water for density estimates (see below).
3) Withdraw a 1-ml aliquot of sample using a Hensen-Stemple
pipet, add to a Sedgewick-Rafter counting cell, dissect
for identification if necessary, and place under the
appropriate microscope.
4) Identify Da~hniausing Brooks (1957), Bosmina and other
cladocerans using Brooks (1959), and copepods using Wilson
(1959), Yeatman (1959), and Harding and Smith (1974).
NOTE: The common cladocerans found in Alaskan lakes
are Bosmina, Da~hnia,Holo~edium,Chvdorus, and
Polv~hemus. The copepods include Cvclo~s,
Dia~tomus,and E~ishura. Rotifers include
Kellicottia, As~lanchia,Karetellq,
Conochiloides, and Filinia.
5) Replace the tap water in the beaker with 10% neutralized
formalin if the sample is to be reused or stored.

Volumetric and Areal Density Estimates

Density estimates measure standing crop at any moment in time,
and differ from rates of biomass accrual or production measured
overtime. As such, standing crop estimates represent a balance
between removal by predators and accrual by reproduction.
Zooplankters peak at different times (in response to
environmental stimuli), occupy different depths in the water
column, and vary in susceptibility to vertebrate and
invertebrate predation pressures. Thus, synchrony of peak
densities of zooplankters peferred by limetic feeding fry is
critical for optimizing freshwater growth and survival.
In-lake density estimates are derived from counting
zooplankters within 1-ml subsamples withdrawn from a jknown
volume of preserved sample; and fro9 either the volume (m ) of
lake water filtered qr diameter (m ) of the zooplankton net.
Areal density (per m ) estimates the number of zooplankters
beneath one square meter of lake surface regardless of verticgl
distribution in the water column. volumetric density (per m )
estimates imply an equal dispersion of the zooplankters
throughout the water column.

Bausch and Lomb stereo dissecting microscope, 1-ml

Hensen-Stemple pipet, 1-ml Sedgwick-Rafter counting cell.
Procedure
1) See Zooplankter ~dentificationprocedure Steps 1 and 2
(p. 152).
2) Dilute the contents of the beaker with tap water so that a
1-ml aliquot will contain -100-150 organisms.
NOTE: If the 250-1111dilution is still too concentrated,
the sample must be split or divided into equal
parts.
3) Connect a number (-7) of triangular containers together
and place on a record turntable. Pour the sample into a
'closed funnel, and rinse the beaker with 5 0 ml of tap
water.
4) Rotate the turntable, open the funnel, and allow the
sample to distribute into the containers.
5) Remove any three containers, pour into three beakers, and
dilute each to a known volume.
6) Mix, withdraw a 1-ml subsample with a Hensen-Stemple
pipet, and expel onto the sedgewick-Rafter cell. Apply a
cover glass, count all the plankters in the cell, and
return the subsample to the beaker.
7) Count a total of three 1-ml aliquots, and if the sample is
to be saved, reconcentrate and preserve.
calculations
1) Average the number of each species per ml and multiply by
the total volume (ml) of subsample. If the original
 250-ml sample was split, first multiply by the number of
containers (-7) and then by 250 ml.
2) Divide by the number of tows to obtain the number of
plankters per tow.
3) Divide the plankton per tow eitger by the area (m2 ) of the
net opening or by the volume (m ) towed:
number in entire sample
Zooplankter per m2 =
net area (m2)

number in entire sample

Zooplankter per m3 =
2
depth (m) of tow x net area (m )
General Notes
a) The counts from the three 1.0-ml subsamples (aliquots)
should be similar. If one count differs, recount an
additional subsample, and substitute for the biased
subsample.
b) If the 1-ml subsample contains less than 100-150
zooplankters, additional counts from a concentrated sample
may be necessary to achieve consistent counts.
c) Count copepod egg sacs, and report as a separate total.
d) Etched lines on the Sedgewick-Rafter cell facilitate
systematic counting.

~odv-Size(lensth) Measurements
Macro-zooplankters ranging in body-size (length) from 0.2 to
>2.0 mm are represented by cladocerans and copepods. Juvenile
fish feeding on large zooplankters grow faster and more
efficiently; however, continual pressure from size selective
predation restructures the zooplankton community. That is, the
remaining zooplankters are small-sized and are species that are
more evasive. Both responses confer a resilience to further
predation. Moreover, as zooplanker body-sizes are lowered,
fecundity drops, and densities decline. Thus, zooplankton
communities in lakes with planktivorous fish differ
substantially from communities in similar lakes without
planktivores; and the standing crop remaining after predation
reflects both the degree of predator pressure and the feeding
nature of the predator.
Body-size measurements are made with an ocular micrometer using
a stage micrometer and the power-magnification adjustment for
calibrating the micrometer.

Bausch and Lomb stereo dissecting microscope, ocular

micrometer, stage micrometer, 1-ml Hensen-Stemple pipet, and a
1-ml Sedgwick-Rafter counting cell.
Procedure
1) Calibrate the ocular micrometer (see Instrument
Calibrations, p. 192) for the appropriate (0-2 mm or 1-10
mm) scale.
2) In each of three 1-ml subsamples, measure the distance -
from the top of the head to the end of the carapace
(Figure 26) of the first five individuals of each species
encountered.
3) Record lengths to the nearest 0.01 mm when using the 0-2
mm scale and to the nearest 0.1 mm when using the 1-10 nun
scale.
4) Determine the required number of organisms to measure with
a precision of + 10% at the 95% confidence level using the
following:

a) Calculate the mean length (L) and standard deviation

(SD) of the first 15 zooplankters of a species, and
determine n by substituting into the formula:

b) Determine the number (N) of zooplankters to be

measured by using n-1 and the t-statistic at a
confidence level of 95% (Table 43), and substituting
t, SD, and L into the formula:

N = number to be measured
Calculation
Determine a weighted mean length, from the numbers of
zooplankters within each size class (0.5 mm intervals), per
species for each set (2) of samples.
 Figure 26. .Location of anterior and posterior measuring
points on the carapace of cladocerans, and cyclopoid and cal-
anoid copepods used to determine body-size (length).
Table 43. Student's t-statistic and sample sizes (n) used to
-determine the number (N) of zooplankters to be
measured to achieve a confidence level (CL) of 95%.

t-statistic t-statistic t-statistic

n-1 (95% CL) n-1 (95% CL) n-1 (95% CL)

Individual Bodv-Weiqht (biomass1 Estimates

As zooplankton biomass is dependent on both the body-size
(weight) and density (numbers), seasonal changes in density may
not be consistent with changes in biomass. Since the
zooplankton community serves as the primary forage base for
rearing sockeye juveniles, the carrying capacity of lakes is
dependent on the biomass of the zooplankton. More precisely,
rearing capacity depends on the biomass of the specific
zooplankters that are used by rearing juveniles. As this may
be only a fraction of the total, the biomass of only the
preferred prey species can serve as an index to carrying
capacity. Individual zooplankters are routinely identifed,
enumerated, and sized; but not weighed because of time
constraints. However, from an empirical correlation between
individual dry weights and body-sizes (wet); routine size
measurements can be converted to weights and then to biomass.
Individual zooplankters of each species are dried, and then
weighed on a Mettler UM3 micro-balance to the nearest 0.0001
mg. The resulting regression equations, between each
zooplanktar length and dry weight are used to calculate
zooplankter biomass from individual lakes.

Mettler UM3 micro-balance, Basch and Lomb dissecting

microscope, 1" x 3" glass microscope slides, and needle probes.
Procedure .
1) Place a representative size range of individual
zooplankters of known lengths onto a labeled 1" x 3"
microscope slide, and carefully agitate while drying (-3-4
minutes) with a needle probe (-10 zooplankters can be
placed on a slide).
2) place an individual zooplankter on the balance pan using
the needle probe, and weigh to the nearest 0.0001 mg.
3) Record dry weights along with corresponding wet lengths
for each zooplankter.
Calculations
1) Regress individual dry weights (mg) (Y-axis) against
length (mm) (X-axis), formulate either a linear or a powsr
curve, and calculate the coefficient of determination (r )
for each species (Figure 27).
2) To calculate biomass, substitute the weighted mean length
(mm) of each species into the appropriate regression
formula (Table 44), calculate the mean weight (mg) of each
species, and multiply by areal densities (Table 45).
Table 44. Regression equations showing the relationship
between individual body-size (mm) and dry weight
(mg) for seven zooplankter species.

Coefficient of
determination
Species Rearession eauation 2

Bosmina lonairostris mg=- 0.0033+0.0118(mm) 0.80

D a ~ h n i qlonairemis mg=o. 0043 (mm)2.26 0.84
Holo~ediulggibberurn mg=0.0114 (mm)2.44 0.81
Dia~tomusgribilofensis mg=0.0042(m) 2.77 0.91
E~ischuranevadensis mg=0.0039 (mm)3.05 0.93
C v c l o ~ scolumbianus mg=O. 0036 (mm)1.99 0.84
C v c l o ~ svernalis mg=0.0048 (mm)3.01 0.82

3) Sum the biomass of 4ach species to yield the total dry

weight biomass (mg/m ) of the zooplankton.
Table 45. . In-lake zooplankter biomass (mg/m2) calculated
using density estimates, and the relationship
between zooplankter dry weight and mean length
at Packers and Hidden Lakes.

Mean Mean DrY

wet dry Density weight
length weight (n-er/ biomaqs
Lake Date (nun) (ma) m 1 tma/m I
Da~hnialonairemis
Packers 06/06/84 0.68 -0018 18,047 32.5
Packers 06/27/84 0.96 ,0040 77,495 310.0
Packers 06/3 0/ 84 0.89 ,0030 81,476 244.4
E~ishuranevadensis
Hidden - 06/25/8 1.42 .0114 50,375 574.3
Hidden 07/16/8 0.84 .a018 5,921 10.7
Hidden 09/01/8 1.13 .0060 2,400 14.4

Packers 06/06/84 0.71 .0016 131,370 210.2

Packers 06/27/84 1.00 .0042 180,467 758.0
Packers 08/30/84 1.15 .0063 87,315 550.0
Bosmina loncfirostris
Packers 06/06/84 0.36 .0010 16,189 16.2
Packers 09/19/84 0.44 .0018 8,623 15.5
Packers 10/11/84 0.50 .0021 2,389 5.0

Total Wet/Drv Weiaht tbiomassl Estimate*

Material retained by a 130 u mesh zooplankton net includes
macro- and micro-zooplankters as well as detritus and
filamentous algae which bias weights determined directly from
the netted material. However, in oligotrophic systems
zooplankton are considered to constitute a majority of the
total weight so actual bias may be minimal.
The biomass of the total zooplankton community is estimated
from the dry weight of organisms retained on a preweighed
filter, and is expressed on the basis of net area or towed
volume.
Vacuum pump (115 psi), analytical balance with a resolution of
0.0001 g, Millipore RAWP 04700 filters, aluminum weighing pans
or small glass petri-dishes, drying oven (80 C), filter
forceps, and a desiccator.
Procedure
1) place-a filter on the balance pan using blunt filter
forceps, record the weight (Wd), and place the filters in
numbered weighing pans.
2) Using the filtering apparatus (~igure3), draw -100 ml of
tap water through the filter at a vacuum of 115 psi, weigh
(W,), and return to the numbered weighing pan.
NOTE: Using RAWP filters, we have found a <lo%
difference in weight between filters that are
hydrated, dried, and desiccated; and untreated
filters.
3) Remove the formalin by straining a sample through a
section of 153-u mesh netting, and rinsing with tap water.
4) Remove the zooplankton from the net by inverting over a
beaker, and rinsing with -50 ml of tap water.
5) Filter the contents of the beaker through a pre-weighed
(W and W ) filter, and rinse the tower with a small
voPume or Vater.
6) Remove the filter and weigh, record the weight (Wwf), and
return to the numbered weighing pan.
7) Place the weighing pan into the drying oven at 80 C for -3
hours, and cool in a desiccator for -2 hours.
8) weigh the desiccated filter and record the weight (Wdf).
NOTE: Weigh immediately after desiccation as the filter
rapidly adsorbs moisture from the air.
Calculations
1) Determine wet and dry weights using the following:
Wet weight (g) = Wwf - Ww
Dry weight (g) = Wdf - Wd
2) Divide the2wet and dry weights gy the area of the net
opening (m ) and by the volume (m ) of lake water filtered
to obtain biomass estimates.
3) Determine the percent moisture content (Table 46) using
the following:

" Moisture =
Wet weight - Dry weight x 100
content Wet weight
(%I
Table 46. Comparison of wet and dry weights, and the moisture
content of the zooplankton communities in four
Alaskan lakes during the spring, summer, and fall
periods.

wet Dry
weight weight Moisture content
Lake Date (ms) (ms) (%I
Karluk 05/10/86 82 8
06/28/86 621 54
11/21/86 305 29

Redoubt 05/10/83 46 2
07/16/83 164 23
11/20/83 46 3
Packers 03/06/81 54 1
07/09/81 430. 48
10/13/81 203 21

Frazer 05/14/86 62 3
08/28/85 258 19
10/02/85 213 11

Reference Slides
Reference slides are used to confirm the identification of
zooplankters, particularly the copepods. Reference sllides are
sent to the Smithsonian Institution, Washington D.C. for
zooplankter identification, and have been added to the
Smithsonianfs permanent reference collection.

4-dram glass vials, microscope slides, cover slips, needle

probe, disposable pasteur pipets, and a hot plate.
Reaaents
1) Rose Bengel - Dissolve -0.2 g of Rose Bengel stain
45440) into 100 ml of DI water.
(C.I.

2) Mounting medium - Permount, Fisher Scientific Company.

3) 70% Reagent alcohol -
~ i l u t e 70-ml of reagent
alcohol-anhydrous to 100-ml with DI water.
4) Glycerin
5) Glycerin jelly
Procedure
1) Add -2 drops of Rose Bengel to cover zooplankton in a
glass vial, and stain overnight.
2) Pour off the excess stain, add tap water, decant, and
repeat until clear.
3) Place the specimens in 70% reagent alcohol, add liquid
glycerin to make a 20% solution, and leave the vial
uncapped until the alcohol and water evaporate ( - 2 days).
4) Place a specimen on a microscope slide, add one drop of
liquid glycerin, add one or two drops of warmed glycerin
jelly, and quickly realign the specimen using a warm
needle probe.
5) Cover with a warmed cover slip, allow to congeal for 30
minutes, and cement the outer edge of the cover slip with
Permount.

REFERENCES
Brooks, J. L. 1957. The systematics of North American
Da~hnfa. Mem. Conn. Acad. Arts. Sci. 13:l-180.
Brooks, J. L. 1959. Cladocera. pp. 5 8 7 - 6 5 6 . In: W. T.
Edmondson [ed.], Fresh-water biology, 2nd. edition. John
Wiley and Sons, New York.
Edmondson, W. T. and G. G. Winberg (ed.). 1971. A manual on
methods for the assessment of secondary productivity in
fresh waters. Mem. Inst. Ital. ~drobiol., 18 Suppl:
358 p.
Edmundson, 3 . M. and J. P. Koenings. 1986. The influences of
suspended glacial particles on the macro-zooplankton
community structure within glacial lakes. Alaska
Department of Fish and Game. FRED Division Report Series
No. 67. 22 p.
~liwicz,M. Z. 1986. Suspended clay concentration controlled
by filter-feeding zooplankton in a tropical reservoir.
~ a t u r e323:330-332.
Harding, J. P. and W. A. Smith. 1974. A key to the British
freshwater Cyclopoid and Calanoid Copepods. Sci. Publ.
Freshwater Biol. Assoc. 18:l-54.
Koenings, J. P., R. D. Burkett, G. B. Kyle, J. A. Edmundson,
and J. M. Edmundson. 1986. Trophic level responses to
glacial melt-water intrusion in Alasakan lakes. pp.
179-194. In: Kane D. L. (ed.). Proceedings: Cold
Regions Hydrology Symposium. American Water Resources
Assoc. Bethesda, MD. USA. 612 p.
Pennak, R. W. 1978. Fresh-water invertebrates of the United
States, 2nd edition. John Wiley and Sons, New York.
803 p.
United Nations ~ducational,scientific and Cultural
organization (UNESCO)
sampling. 174 p.
(pub.) . 1968. Zooplankton

United Nations ~ducational, scientific and Cultural

Organization (UNESCO) (pub.). 1968. Zooplankton fixation
.and preservation. 350 p.
Wilson, M. S. 1959. Calanoida. pp. 738-794. In: W. T.
Edmondson [ed.], Fresh-water biology, 2nd edition. John
Wiley and Sons, New York.
Yeatman, H. C. 1959. Cyclopoida. pp. 795-815. In: W. T.
Edmondson [ed.], Fresh-water biology, 2nd edition. John
Wiley and Sons, New York.
 JUVENILE SALMON EVALUATION

Total Bodv Burden of Oxvtetracvcline

The antibiotic oxytetracyline (OTC) is added to hatchery fish
food and used as a prophylactic medicine prior to release.
Once digested, OTC is absorbed and deposited in the skin,
cartilage, and skeleton of juvenile salmon. OTC held in bone
as a calcium-complex is retained for the life of the fish.
Koenings et al. (1986) have shown the feasibility of using OTC
as an internal mark for hatchery-reared sockeye salmon.
OTC extracted from biological material using trichloroacetic
acid (TCA) forms a fluorescent complex with calcium that is .
measured by fluorometry.
Standard Ranse
0.47 - 9.4 ug OTC.
Lower Limit of Detection
-1
Empirical: 0.031 ug ml-l.
Predicted: 0.012 ug ml .
Shaker table, centrifuge, spectrofluorometer (excitation 390
nm; emission 520 nm) 50-ml centrifuge tubes with teflon lined
caps, blender with micro-cell, and disposable 15-75 nun
borosilicate culture tubes (cuvettes).
Reaaents
1) 5% TCA -
Dissolve 50 g of trichloroacetic acid (TCA) into
900 ml of DI water, and dilute to 1 liter.
2) Ethylacetoacetate - Ultrapure.
3) Ethylacetate solution -
Dilute 13 ml of ethylacetoacetate
to 1 liter with ethyl-acetate.
4) 0.5 M CaC12 -
Dissolve 36.7 g of CaC12 into 450 ml of DI
water, and dilute to 500 ml.
5) 7 N NH OH - Dilute 437.5 ml of concentrated
N H ~ O Hto i liter with DI water.
reagent-grade
Procedure .
1) Homogenize individual fish in 15 ml of 5% TCA using a
blender, pour into a 50-ml centrifuge tube, and rinse the
slurry using 5 ml of TCA into the tube.
NOTE: Soak centrifuge tubes overnight in 10% HCl,
. thoroughly wash with phosphate-free detergent, and
rinse with 10% HcI.
2) Place the tubes on a shaker table, and agitate for 1 hour
in the dark.
3) Centrifuge at 2000 rpm for -30 minutes, pipet 15 ml of the
TCA extract into a clean 50-ml centrifuge tube, and add 15
ml of ethylacetate solution.
4) Agitate on a shaker table -10 minutes, and centrifuge at
2000 rpm for 5 minutes.
5) Withdraw the top layer using an autopipet, place into
another 50-ml centrifuge tube and add 0.5 ml of 0.5 M
CaC12 followed by 15 ml of 7 N NH40H.
6) Agitate for -10 minutes and centrifuge at 2000 rpm for 5
minutes.
7) After -45 minutes withdraw a 5-pl aliquot from the top
layer, add to a cuvette, and measure the fluorescence
against a reagent blank (see manufacturer's instructions).
NOTE: Rinse autopipet tips with ethylacetate prior to
use.
Standards
Primary standard (940 ug ml-l) -
Dissolve 1 g of
oxytetracycline-hydrochloride (Sigma Chemical) into -900 ml DI
water, and dilute to 1 liter.
Secondary standard (9.4
standard to 100 ml.
ug ml-l) - Dilute 1.0 ml of primary

Calculations
1) Formulate a linear equation by regressing concentations
(Y-axis) against averaged fluoresence units minus the
reagent blank (XIaxis), and calculate the coefficient of
determination (r ) .
2) Calculate sample concentrations by subtracting the blank
value from the sample fluorescence, and substitute into
the regression formula (Table 47).
Table 47. The relationship between fluoresence units (zc) and
*amounts of oxytetracycline (OTC) used to calculate
a standard curve for the fluorometric analysis of
OTC in fish.

Secondary Total Fluoresence --

standard volume OTC units fu
Iml) t L) t us) I x) (:-blank)

OTC (ug per fish) = 0.0625 + 1.1092 (?c)

Anesthetizins Fish
Rearing salmonids (especially sockeye smolts) are extremely
prone to handling mortality resulting from scale loss,
desliming, and excitation. As both juveniles and smolts have
to be captured, processed for length, weight, and implanted
hand1 ing .
with coded-wire micro-tags; the fish must be sedated prior to

The degree of sedation and the rates of anesthetization are

controlled by concentration of either 2-phenoxyethanol or
ethyl-m-aminobenzoate-methane sulfonate (MS-222). Moreover,
the action of the anesthetic is slower at cooler gpmperatures,
in weakly buffered water [alkalinity of (10 mg 1 (taco,)1 t
and for larger fish. Consequently, preliminary tests of
varying concentrations should be made against small numbers of
fish to determine optimum exposure and recovery times.
Apparatus
Measuring (g) spoons, autopipets, thermometer, and immersion
tubs.
Reasents
1) MS-222 - Technical grade.
2) 2-Phenoxyethanol - Reagent grade.
3) MS-22.2 stock solution -Dissolve -10 g of MS-222 into 100
ml of DI water, and neutralize to pH 7 with 0.1 N NaOH.
Procedure
1) Dissolve or dilute anesthetic (Table 48) in lake water as
required, immerse the fish(s), and anesthetize (-2-4
minutes).
2) Process the fish(s) immediately after sedation, revive by
immersing (5-15 minutes) in clean aerated water, and
release to the system.
Cautions
As MS-222 is a skin irritant and can cause disturbances of
the central nervous system, wear rubber gloves when
handling the chemical. -lAlso, adding to poorly buffered
[alkalinity of 510 mg L (CaC03)], low pH (4.5-5.5) lake
water further lowers the pH. This can be avoided by
preparing a fresh stock solution, neutralizing, and
dispensing as a liquid. This also reduces in-field
problems associated with dissolving MS-222 at lower water
temperatures. Finally, the U. S. Food and Drug
Administration (FDA) has approved MS-222 for use with food
fishes.
2) 2-phenoxyethanol does not lower the pH of the
anesthetizing water, but does not readily dissolve in
cooler (-4 C) water; however, the anesthetic will dissolve
with vigorous stirring. Finally, the FDA has not approved
2-phenoxyethanol for use with food fishes, i.e., fish that
are potentially consumed by humans.
Table 48. Anesthetics and generalized amounts used to prepare
immersion solutions to sedate juvenile and adult
salmonids,

Tem~erature Amount
esthetic (C) (F) ( ~ e rliter) ( ~ e r
sall
1) Ethyl-m-aminabenzoate-
methane sulfonate
(MS-222) 7-12 46-54 0.10 g 0,40 g
2) MS-222 stock solution 4-12 39-54 1 ml 4 ml
 Stomach Content Analysis
Stomach contents are used to compare forage selected by fish to
that available in the natural habitat. This comparison is used
to assess the cause-and-effect relationship between predator
and prey. Rearing salmonid juveniles are obligate (sockeye)
and facultative (coho, trout) planktivores having differing
abilities to forage on zooplankters. In general, facultative
fishes choose larger-sized zooplankters (11.00 mm), and, if not
present, move to other food sources. In contrast, obligate
planktivores have refined the ability to exploit limnetic
forage and can feed on forms as small as 0.4 mm. Thus, actual
prey (zooplankton) body-sizes as well as those of non-prey are
used to evaluate size-selective feeding. As fishery
enhancement projects seek to increase either the production of
potential prey items (nutrient enrichment) or the number of -
predators (lake stocking); the success of enhancement depends
on defining the linkage between predator and prey.
Many methods of removing stomach contents have been designed
and tested; however, the preferred method is gastric lavage,
Extraction efficiencies and survivals are >go%, and the
technique is easy to conduct in the field by one person. The
technique is similar to that described by ~ i g h tet al. (1983)
which uses a pressurized flow of water from a Manostat syringe
to remove stomach contents.

10-ml Manostat syringe with 2.5-3.5-cm long piece of 1.0-mm

blunted teflon tubing, 50-ml plastic centrifuge tubes with
caps, funnel, wash bottle, fine tipped wash bottle, and 20-ml
polyethylene scintillation vials.
Bausch and Lomb stereo dissecting microscope, 1-ml
Hensen-Stemple pipet, 1-ml Sedgewick-Rafter counting cell, 0.0
to 2.0-mm ocular micrometer, and zooplankter and aquatic insect
taxanomic keys (see References).
Reaaents
1) - See Anethetizing Fish (p. 168).
nesth he tic
2) 15% Neutralized formalin - Dilute 250
formaldehyde to 1 liter with tap water.
ml of 37%
 A. Gastric Lavage
Procedure
1) Anesthetize fish with either MS-222 or 2-phenoxyethanol
before processing.
2) Hold.the fish over a funnel attached to a sample vial, and
carefully insert the teflon tubing into the stomach until
resistance is felt (Figure 28).
3) Flush the stomach contents using two 5-ml injections of
water from the Manostat syringe into the sample vial.
4) Measure, weigh, and then revive the fish.
5) Add neutralized formalin to the sample vial to make a 15%
solution (Sturgess and Nicola 1975).

B. Stomach Removal
Procedure
1) Narcotize or kill the fish in a solution of MS-222 (-150
mg/liter) or 2-phenoxyethanol (-3 ml/liter) to prevent
regurgitation of the stomach contents, and then store
whole in 15% neutralized formalin.
2) Dissect the stomachs from the fish, and preserve in
individual vials.
3) Label each vial with appropriate age, length, and catch
data.
4) Open the stomach, and carefully wash the contents into a
small petri-dish using a fine tipped wash bottle.
5) Rinse the contents into the labeled vial, and add 15%
neutralized formalin.
NOTE: We have determined lavage efficiencies (E) of -go%,
for coho or sockeye juveniles, using the
formula:

La = Number of items removed from lavaged stomachs.

LaD = Number of items removed from lavaged and
dissected stomachs.
 7

- stomach

15 % buffered formalin

v .
Figure 28. Manostat syringe apparatus, with teflon tube, used to remove stomach
contents from juvenile salmonids by gastric lavage.
 C. Identification and Enumeration
Procedure
1) Identify and enumerate zooplankters (see ~olumetric and
Areal Density Estimates, steps 1-7, p. 153).
2) Identify insects to genus, and count as whole organisms
only'distinguishable parts, e.g., head capsule.
3) Measure zooplankter body-size [See Body-size (length)
measurements, p. 1551.
Calculations
1 Average the number of each zooplankter in the three 1-ml
subsamples, and multiply by the total diluted volume (ml)
to obtain the total number of each zooplankter consumed.
2) Sum to obtain the total number of zooplankton found in
each stomach.
3) Sum the counts for each insect taxa found to obtain the
total number of insects in each stomach.
4) Determine the number of fish containing forage and divide
by the total number of fish sampled.
5) ~eterminepercent incidence of each item by dividing the
number of fish containing each item by the total number of
fish sampled (x 100) .
6) Determine percent numerical composition by dividing the
number of each item by the total number of items (x 100).

Electivitv Indey
The electivity index (Ivlev 1961) has a range of -1 to +I;
negative values indicate either avoidance or inaccessibility of
a prey item, zero indicates random selection, and positive
values indicate active selection. There are variations of the
electivity index that compensate for bias introduced when
either the abundance of prey in the environment differs
substantially from prey found in the fish or when predator-prey
habitats differ (Paloheimo 1979; Strauss 1979). Regardless of
the version, selectivity or prey preference based on the
electivity index is a relative measure until other phenomena
such as probability of prey capture, and distribution of prey
are better understood.
Procedure
1) Capture rearing juveniles (230) using tow nets (Gjernes
1979), and minnow traps or beach seines for sockeye and
coho respectively.
2) Remove stomach contents, identify, and enumerate (see
Stomach Content Analysis, p. 170).
3) collect zooplankters using vertical paired tows (sea
Plankton, p. lo), identify, and enumerate (see Zooplankter
Identification, p. 152).
NOTE: Rearing juveniles and zooplankton must be from
concurrent samples and habitats.
Calculations
The electivity index (E) or the degree of selection of a
particul-ar prey item by a predator is defined as:

where r , represents the relative abundance of prey item i in

the stohach of the predator expressed as a proportion or
percentage of the total stomach contents, and p represents the
relative abundance of the same prey item in &he environment
expressed as a proportion or percentage of the total density
(Figure 29).

Diet over la^

Considerable interest exists among biologists in the
measurement of resource overlap among co-existing species e . g . ,
sockeye salmon juveniles and stickleback. The measurements
obtained can be, and have been, of more than academic interest.
Unfortunately, they have also been used in the mistaken belief
that the degree of overlap between the diets of two species can
be directly used to assess competition. However, simply
demonstrating an overlap in resource use by two species in
nature can be evidence either for or against inter-specific
competition. Two definitions are important:
Overlap is the use, typically at the same time, by
more than one organism of the same resource
regardless of resource abundance.
 31 August

Figure 29. Range o f zooplankter body-sizes from 0.2-1.64 mn (by 0.04 mm

intervals) showing the length interval of each species selected by limnetic
rearing sockeye fry from Packers Lake. Also shown are the total number of
each zooplankter consumed, and the overall electivity index for each species.
 Competition is the demand, typically at the same
time,'by more than one organism for the same
resource in excess of immediate supply.
Thus, to show inter- or intra-specific competition for a
resource, decreased growth or survivorship, of one or both
species is necessary in addition to diet overlap. In other
words, if overlap in diet is high fish can be using the same
resource,.but are not 'limited1 by such overlap as food is not
of llimitingl supply.
Procedure
1) Capture rearing juveniles (230) using tow nets (~jernes
1979), and minnow traps or beach seines for sockeye and
coho respectively.
2) Remove stomach contents, identify, and enumerate (see
Stomach Content Analysis, p. 170).
3) Collect zooplankters using vertical paired tows (see
Plankton, p. lo), identify, and enumerate (see Zooplankter
Identification, p. 152).
NOTE: Rearing juveniles and zooplankton must be from
concurrent samples and habitats.
Calculations
Using the terminology from Morisita (1959) and Horn (1966), we
have characterized subsamples xo and yo from populations X and
Y respectively. Out of a total number of food categories from
both samples, species i is represented xi times in X and y.
times in Y .If sample sizes of X and Y are relative&
then the dyet overlap coefficient (Cf) equals:
equai

Cf = n n 2
sum (xi2) + sum (Yi )
i-1 111
This formula is also appropriate where the data are expressed
as the proportions xi and y of the respective samples composed
of species i. This empiri&al measure has an upper limit of
exactly 1, and ranges between 0 (no overlap) to 1 (complete
overlap). Zaret and Rand (1971) considered a Ci 2 0.60 to
indicate Isignificantl overlap.
Example
Samples were taken, by tow net, of the limnetic rearing
juveniles in Fred Lake, and stomach contents removed by gastric
lavage. Our subsamples of the rearing fishes came from the
same habitat and were taken at the same time. We compared the
diet of sockeye juveniles and threespine stickleback by
calculating the diet overlap coefficient (Cf) using food type
proportions (Table 49).
Table 49. The proportions of food items (i) found in sockeye
juveniles (Y) and threespine stickleback (X) used
to calculate the diet overlap coefficient (Cf).

Fish group X Fish group Y

Food Item

Cvclows

Sum

Freshwater Cohort Production

Freshwater salmon production is commonly expressed as gross
yield of smolt biomass (kg). However, because of differences
in either stocking time, initial fish densities and mean sizes,
or all three; it is useful to present the yield of smolts as a
net change in biomass over time (t). That is, introduced (fry
plants) or initial in-lake biomass (estimated by hydroacoustic
techniques or by mark-recapture) subtracted from the gross
yield of smolts equals net biomass produced. The net change in
biomass (kg or kg/ha) allows comparisons of freshwater salmon
production between lakes, and within the same lake but between
years.
Methods used to estimate cohort prc
numbers (N ) and individual weights (
stages. $lan (1951) graphically descr
from a curve relating the number of
individualweights. The area beneath
using planimetry and can be used to
production. Alternately, Gillespie anc
production equations based on the Allen
be used to calculate cohort production.
Procedure
1) Estimate the number of juveniles t
estimate in-lake populations (N) k
mark-recapture, and the mean indivi
and Koenings 1985).
2) Estimate the number of in-lake juv
of the growing season (October to
geographical location) hydroa
mark-recapture, and the mean indivi
and Koenings 1985).
3) Determine the number of smolts
individual weight (W ) (Blackett et
~ebida1984, Flagg 1385, and Koenin
Calculations
1) Construct a plot of numbers of
against mean individual weight (X-a
2) Determine, with a calibrated plan
Calibrations, p. 192), the individu
X, Y, and Z (Figure 30), used to
parameters (Table 50).
3) Alternately, calculate the areas of s
Z (Figure 30), and determine the F
(Table 5 0 ) .
 DESCRIPTORS
IN P U.T (8.1 Beginning biomass (81) = NIW1=X+V
- I- Gross yield (B3) = N3W3= Z + V
-- = 83 --&=(z+v)-(x+v) 82-x
Net change in biomass (AB)
Net yield z B3- V = (Z+V) - V=Z
Production = Z+Y
Directly recycled production = Y
Production efficiency (gross) = (Z+V) / (z+ Y)
Production efficiency (net) = Z / (2+Y)

I I I
w1 W2 W3
MEAN INDIVIDUAL WEIGHT (increasing-
Figure 30. Generalized Allen curve showing the relationship between the number
of fish and mean individual weight used to calculate production parameters (see
descriptors) for rearing fish. Biomass ( B ) estimates shown represent the life
history stages of spring juveniles (Bl), late fall juveniles ( B 2 ) , and smolts ( B 3 ) .
Table 50. Summary of cohort production parameters described
by Allen (1951) and detemined either by planimetry
or from equivalent equations following Gillespie and
Benke (1979).

Planimetered Equivalent Production

area calculation ~arameters
X + V N1 W1 Initial biomass (B1)
z + v N3 W3 Gross yield (B3)
Z W3 - W1 x N3 Net yield
Y (W3 - W1) (N1 - Nj) Recycled production

Z + Y .- Net yield + recycled Total production

production
(z+v)/ (z+y) Gross yield/total Gross production
production efficiency
z/ (z+y) Net yield/ total Net production
production efficiency

REFERENCES
Total Body Burden of the Antibiotic Oxvtetracvcline
Koenings, 3 . P., J. Lipton, and P. McKay. 1986. Quantitative
determination of oxytetracycline uptake and release by
juvenile sockeye salmon. Trans. Amer. Fish. Soc.
115: 621-629.
Stomach Content Analvsis
Borror, D. J., D. M. De Long, and C. A. Triplehorn. 1981. An
introduction to the study of insects. Fifth edition.
Saunders College Publishing, New York, 827 p.
Brooks, J. L. 1957. The systematics of North American
Da~hnia. Mem. Conn. Acad. Arts Sci. 13:l-180.
Harding, J. P., and W. A. Smith. 1974. A key to the British
freshwater cyclopoid and calanoid copepods. Sci. Publ.
Freshwater Biol. Assoc. 18:l-54.
Light, R. W., P. H. Adler, and D. E. Arnold. 1983.
Evaluation of gastric lavage for stomach analysis. N.
Amer. Jour. Fish Manag. 3:81-85.
.
Merritt, R. W., and K. W. C u m i n s (eds.) 1978. An
introduction to the aquatic insects of North America.
Kendall/Hunt Publishing Company. Dubuque, Iowa. 440 p.
Pennak, R..,W. 1978. Fresh-water invertebrates of the United
States, 2nd edition. John Wiley and Sons, New York. 803
P a
Ross, H. H., C. A. Ross, and J. R. P. Ross. 1982. A textbook
of Entomology. Fourth edition. John Wiley and Sons, New
York. 666 p.
Sturgess, J. A. and S. J. Nicola. 1975. Preparation of fish
for identification and preservation as museum and
specimens. Res. Agenc. Calif. Dept. Fish and Game.
Inland Fish. Inform. Leaf. No. 29. 7 p.
Wilson, M. S. 1959. Calanoida pp. 795-815. In: W. T.
Edmondson [ed.], Fresh-water biology, 2nd edition. John
Wiley and Sons, New York.
Yeatman, H. C. 1959. Cyclopoida. pp. 795-815. In: W. T.
Edmondson [ed.], Fresh-water biology, 2nd edition. John
Wiley and Sons, New York.
Electivitv Index
Gjernes, T. 1979. A portable midwater trawling system for use
in remote lakes. Dept. Fish. and Oceans Tech. Report
Series. 10 p.
Ivlev, V. S. 1961. Experimental ecology of feeding of fishes.
Yale Univ. Press, New Haven USA. 302 p.
~aloheimo,J. E. 1979. Indices of food preferences by a
predator. J. Fish. Res. Board Can. 36:470-473.
Strauss, R. E. 1979. Reliability estimates for Ivlev's
electivity index, for forage ratio, and a proposed linear
index of food selection. Trans. Amer. Fish. Soc.
108:344-352.
Diet Overlap
Morisita, M. 1959. Measuring of interspecific association and
similarity between communities. Memoirs of the Faculty of
Science, Kyusha Univ., Series E (Biology) 3:65-80.
Horn, H. S. 1966. Measurement of 'overlapt in comparative
ecological studies. The American Naturalist.
100:419-424.
Zaret, T. M., and A. S. Rand. 1971. Competition in tropical
stream fishes: Support for the competitive exclusion
principle. Ecology. 52:336-342.
Freshwater Cohort Production
Allen, K. R. 1951. The Horokiwi stream - a study of a trout
population. New Zealand Marine Dept., Fish Bull. 10, 238
P-
Blackett, R. F., A. Daun, and P. A. Russell. 1969. Kodiak
Island sockeye salmon investigation, 1969. Alaska
Department of Fish and Game Annual Technical Report PL
89-304. AFC Project 8-3:194 p.
Crone, R. A. 1981. Potential for production of coho salmon
(Oncorhvnchus kisutch) in lakes with outlet barrier falls,
Southeastern Alaska. Ph.D. Dissertation. University of
Michigan. 388 p.
Crone, R. A:, and J. P. Koenings. 1985. Limnological and
fisheries evidence for rearing limitation of coho salmon,
Oncorhvnchus kisutch, production from Sea Lion Cove Lake,
Northern Southeast Alaska (1981-1983). Alaska Department
of Fish and Game, FRED Division Report Series No. 54.
74 p.
Flagg, L. B., P. shields, and D. C. Waite. 1985. Sockeye
. salmon smolt studies Kasilof River, Alaska, 1984. Alaska
Department of Fish and Game, FRED Division Report Series
No. 47. 43 p.
Gjernes, T. 1979. A portable midwater trawling system for use
in remote lakes. Dept. Fish. and Ocean Tech. Report
Series. 10 p.
Gillespie, D. M., and A. C. Benke. 1979. Methods of
calculating cohort production from field data - some
relationships. Limnol. Oceanogr., 24(1):171-176.
Koenings, J. P., T. McDaniel, and D. Barto. 1985.
Limnological and fisheries evidence for rearing limitation
of sockeye salmon, Oncorhvnchus nerka, production from
Lake Tokun, Lower Copper River (1981-1984). Alaska
Department of Fish and Game, FRED Division Report Series
NO. 55. 82 p.
Kyle, G. B. 1983. Crescent Lake sockeye salmon smolt
enmaration and sampling, 1982. Alaska Department of Fish
and Game, FRED ~ i v i s i o nReport series No. 17. 24 p.
Lebida, R. C. 1984. Larson Lake sockeye and coho salmon smolt
enumeration and sampling, 1982. Alaska Department of Fish
and Game, FRED Division Report series No. 35. 3 1 p .
 PART 111.

QUALITY ASSURANCE, CALIBRATIONS,

AND
STATISTICAL EVALUATION
 QUALITY ASSURANCE

Laboratory Technicme
Quality assurance in the limnology laboratory is achieved by 1)
use of highly purified (Type I) water, 2) properly cleaned
glassware, 3) use of reagent-grade chemicals, 4) frequent
rnaintenanee and calibration of instruments, 5 ) daily
preparation of standards, and 6) replicated standard and sample
analysis. While no single factor determines precise and
accurate results, each adds a fundamental part to internal
quality control.
To assure precision and accuracy of analytical results, it is
crucial that the laboratory have a source of water free of
contaminants for preparing standards and reagents, and for .
washins slassware. Use of Type I water is the most
signifk a n t factor affecting methodological capabilities and is
especially critical when analyzing for dilute concentrations.
Thus, laboratory water is monitored daily for purity, and
appropriate maintenance measures taken when water quality falls
below Tyle I standards.
Laboratory analysis procedures require glassware and
plasticware for preparing and transferring standards, reagents,
and samples. To avoid contamination it is essential to adhere
to the cleaning procedures outlined in Part I (p. 6) to assure
consistent and accurate analytical results.
Chemicals and reagents are manufactured with different levels
of purity. That is, product containers with the same chemical
name or formula may not be of similar quality. Use only
substances certified by the American Chemical Society and of
analytical-reagent grade. Use of more inferior grades e.g., a
technical quality, may result in a reduction of analytical
accuracy.
Laboratory instruments are generally regarded as being reliable
and stable; however, minor adjustments of components, cleaning,
and carefully conducted calibrations with known reference
materials are routinely required. Specific calibration
procedure8 are detailed in the section: Instrument Calibrations
(p. 192).
Analysis of standards is used to assess levels of precision and
accuracy of methodologies. calibration standards are prepared
by adding known quantities of pure chemical to D1 water, and
then analyzed under the same conditions as the samples, The
results are used to formulate a calibration curve to determine
sample concentrations. By comparing calibration curves it is
possible to detect error and biases in the methodology.
Systematic and random error are minimized by using a
calibration curve when analyzing each test lot.
For each analysis, two aliquot8 of sample and standard are
analyzed and the mean result reported as the best estimate of
the constituent; however, a third aliquot is analyzed and
averaged in place of a biased or erred value.
While the., laboratory techniques described above are key to
quality assurance, it is essential that analytical results be
confirmed by an independent testing agency. confirmation
provides confidence and reliability in analytical results and
more importantly, allows data to be comparable with that of
other laboratories. The limnology laboratory utilizes several
outside agencies for confirmation of internal quality control.

General Tests and Nutrients

Each year the limnology laboratory participates in the U. S.
Geological Survey (USGS) Standard Reference Water Sample
Program which distributes reference samples to laboratories
nationwide. In 1985, over 100 laboratories participated thus,
providing a broad database to evaluate laboratory capabilities
and performance. Analytical results are submitted to the USGS
which list the results from each laboratory, provides a
statistical evaluation, and scores the performance for each
reference value as follows:
Standard Deviations
Performance Ratinq from the mean value
4 (excellent)
3 (good)
2 (satisfactory)
1 (questionable)
0 (poor)
USGS reference values as determined by participating
laboratories, the values reported by the limnology laboratory,
and the parformance rating for each parameter are summarized-fn
(Tables 51-54). Nutrients and metals are reported in mg L ,
pH in units, and conductivity in umhos/cm. Finally, turbidity
determinations are referenced to the U. S. Environmental
Protection Agency (EPA) Environmental Monitoring and Support
Laboratory (Cincinnati, Ohio) standards.
Table 51. .Results from the 1982 U. S. Geological Survey
Standard Water Reference Sample Program showing the
constituents analyzed in three reference samples,
mean values, and values determined by the limnology
laboratory with performance ratings.

Value determined Mean value

by the limnology of all
Sam~le laboratory laboratories Ratinq

Conductivity 8.9 9.25 4

PH 6.05 6.20 4
Calcium 1.5 0.8 0
Magnesium
Nitrate
<0.3
0.123
0.121
0.122
-4
Total ammonia
............................................................... 0.002 0.054 3

Reactive phosphorus 1.2 1.08 4

Total phosphorus 1.8 1.68 4
Organic nitrogen 0.4 0.93 1
Nitrate 4.3 4 28 4
Nitrite 0.21 0.20 4
Total ammonia 0.79 0.365 1
--"------"---------------------------------------'------------

Conductivity
PH
Alkalinity
Calcium
Magnesium
Silica
Total phosphorus
Nitrate
Nitrite
Table 52. Results from the 1983 U. S. Geological Survey
Standard Water Reference Sample program showing the
z constituents analyzed in four samples, mean values,
and value determined by the limnology laboratory
with performance ratings.

Value determined Mean Value

by the limnology of all
Sam~le laboratory laboratories Ratinq

Conductivity
PH
Calcium
Magnesium
Total ammonia
Nitrate

Conducitivity 839 859 4

PH 8.1 8.13 4
Alkalinity 150 150.7 4
Calcium 65 70.6 3
Magnesium 22 28 0
Silica 13.2 12.62 4
Total phosphorus 0.54 0.497 3
Nitrite 0.01 0.011 4
Nitrate
.....................................................
4.61 3.975 1
N-10

Total phosphours 1.58 1.432 3

Reactive phosphours 0.98 1.00 4
Organic nitrogen 0.51 1.342 3
Total ammonia 1.53 1.191 2
Nitrate 2.55 2.478 3
Nitrite 0.004 0.008 3
---------------------------
N-ll
Total phosphorus 0.54 0.513 4
Reactive phosphorus 0.49 0.487 4
Organic nitrogen 0.18 0.732 2
Total ammonia 0.29 0.16 1
Nitrate 4.14 3.962 3
Nitrite 0.013 0.014 4
Table 53. Results from the 1984 U. S. Geological Survey
. Standard Water Reference Sample Program showing the
constituents analyzed in three samples, mean values,
and values determined by the limnology laboratory
with performance ratings.

Value determined Mean value

by the limnology of all
sample laboratory laboratories Ratinq

Ammonia
Nitrite
Nitrate
Organic nitrogen
Total phosphorus
Reactive phosphorus
-------------------

Conductivity 8.8 9.96 3

PH 6.00 6.42 2
Calcium 1.05 0.91 3
Magnesium c0.34 0.17 0
Ammonia 0.01 0.03 3
Nitrate 0.05 0.06 3
-------------------------------------------.-*----.------------

Alkalinity
Calcium
Magnesium
itr rite
Nitrate
Total phosphorus
Silica
Table 54. Results from the 1985 U. S. Geological Sunrey
Standard Water Reference Sample Program showing the
constituents analyzed in two samples, mean values,
and values determined by the limnology laboratory
with performance ratings.

Value determined Mean value

by the limnology of all
Sam~le, laboratory laboratories Ratinq

Conductivity 110 111.2 4

PH 7.31 7.488 4
Alkalinity 28.0 27.2 4
Calcium 11.2 11.4 4
Magnesium 2.9 2.95 4
Nitrite c0. 00 0.005 0
Nitrate 0.188 0.18 4
Total phosphorus 0.019 0.01 3
Silica 7.18 3
...............................................................

Ammonia 0.55
Nitrite 0.00
Nitrate 0.96
Organic nitrogen 0.738
Total phosphorus 0.77
Reactive phosphorus . 0.191

Carbon-14 (C-14) isotopes are supplied by ICN Pharmaceuticals,

Irving, California and stipulated to have a true activity
between the limits of 25% at the 95% confidence level. Liquid
scintillation spectrometer calibration is based on the
efficiency of detecting Beta-particle emission of a C-14
standard prepared by New England Nuclear, Boston,
Massachusettes. We have consistently detected activity or
disintegrations per minute (DPM) within +5% of the known
reference.
Algal standing crop measurements are referenced to chlorophyll
a (chl a) and phaeophytin a (phaeo g) standards of algal
-
extracts provided by the EPA (Table 55). In addition, the
standards are used for both the spectrophotometric and
fluorometric procedures and for calibrating the filter
fluorometer. As EPA reference standards may not be available,
chl q and* phaeo g extracts obtained from sigma Chemical
Company, St. Louis, ~issourican be used to prepare standards.
Finally, algal enumeration and identification is conducted
using phase microscopy at magnifications of 450x and lOOOx by
Eco-Logic Ltd., West Vancouver, ~ritishColumbia.
Table 55. Comparisons of chl q and phaeo g levels in
reference standards of the U. S. Environmental
'-ProtectionAgency, as determined using the
spectrophotometric and fluorometric procedures by
the limnology laboratory.

EPA Limnology
reference Confidence laboratorv4

valq limitp valuel

Constituent Method (uc3 L (ua L 1 (uq L I
Chl a Fluorometric 3.2 2.4 - 4.0 3.7
Phaeo a_
Chl
Fluorometric 1.4 0.8 - 2.0 1.9
Fluorometric 16.7 14.2 -19.2 18.0
Phaeo a
Chl a
Fluorometric
Trichromatic
-0.1
7.76
-2.4 - 2.1
7.57- 7.96
2.1
7.45
Chl a Trichromatic 0.6 0.47- 0.74 0.74
Chl a Trichromatic 1.29 1.14- 1.44 1.18
Chl a Monochromatic 6.29 5.85- 6.73 5.95
Phaeo g Monochromatic 2.11 1.34- 2.88 2.19

Species identification is determined through the use of

accepted taxanomic keys as listed in the methodology. In
addition, reference slides are prepared of each species and
submitted for verification by Thomas E. Bowman, Director of
Crustacea, at the Smithsonian Institute, Washington, D. C.
 INSTRUMENT CALIBRATIONS
Proper use, maintenance, and calibration of laboratory
instruments is critical to the precision and accuracy of
analytical results. Equipment must be cleaned, adjusted and
properly calibrated against quality reference standards at
intervals specified in the methods or as recommended by the
manufacturer.

Inspect the probe membrane for any air bubbles, holes or

creases, and replace if necessary. It is important to
calibrate the meter at each sampling station to insure accurate
D.O. measurements. The preferred calibration technique is to
compare the meter reading (D.O. probe at subsurface depth) with
the D.O. value of the subsurface depth determined by the
~inkler-method. Air calibration should be used only if
recommended by the manufacturer. calibration uses a Hach
chemical D.O. kit for the method outlined below.
1) Fill a 60-ml BOD bottle and cap making sure no air bubbles
are trapped inside.
2) Add 1 pillow of D.O. I reagent and shake well.
3) Add 1 pillow of D.O. I1 reagent and shake. Allow the floc
to settle until the upper -half of the solution is clear.
Mix again and let settle.
4) A d d 1 pillow of D.O. I11 reagent and dissolve. The
solution will turn a clear yellow or gold in the presence
of oxygen.
5) Add a few drops of standard (2%) starch solution. The
sample will turn blue-black.
6) Divide the sample and titrate each 30-ml subsample with
0.019 N phenylarsine oxide (PAO) using a 5.0-ml syringe-
The solution will become clear when the endpoint is
reached.
7) Calculate the D.O. from the appropriate expression:
D.O. = ml titrant x 5.81, for a 15-ml sample.
mg L - ~
mg L - ~D.O. = ml titrant x 2.9 for a 30-ml sample.
Average the results of the 2 subsamples.
8) Immerse the D.O. probe and set the meter reading to the
value*obtained from the Winkler titration.

Throughout the manual the term absorbance (abs), synonymous

with optical density or light extinction, is used as the
measure ' of color intensity in spectrophotometric
determinations. Absorbance measurements can also be converted
to percent transmission (T) using:
log T = 2 - abs or abs = -log (T x .01)
Spectrophotometers are calibrated using the procedure described
below:
1) Allow the instrument to warm up for 15 minutes, select the
wavelength specified in the method, and switch to
absorbance mode.
2) Clean two quartz-glass optical cuvettes (capable of
transmission at wavelengths of 200-900 nm), fill with DI
water, and place in the reference and sample compartments.
3) Adjust the blanking control until the reference cuvette
reads .000 absorbance and, without changing the control,
measure the absorbance of the sample cuvette. If the
absorbance*differs by a reading of + .003, the cuvettes
are considered optically matched and can be used to read
sample and standard absorbances.
5) Read standards and sample absorbances, and rezero the
cuvette as necessary between samples using the blanking
control.
NOTE: Matched cuvettes for one wavelength does not assure
they are optically matched at a different
wavelength. Therefore, calibrate and rezero the
instrument each time the wavelength is changed.

Autoanalvzer
1) Pump DI water through the system for 10 minutes.
2) Set the toggle switch inside the calorimeter to IDg
(direct chemistry). Turn the rotary switch to gzerog and
to 'full scaleg making sure the recorder pen responds, and
adjust the set screws if necessary.
3) Set the standard calibration control to 1.0 and the
baseline control midway. Establish a water baseline by
opening (clockwise rotation) both apertures and if the pen
is below zero, slowly rotate aperature A counterclockwise
until the pen is at zero. If the pen is above zero,
slowly rotate B counterclockwise to adjust.
4) Place the reagent feed lines in their containers and pump
through the system for 10 minutes, and note the reagent
deflection line. An abnormal deflection line indicates
either one or more reagents or pump tubes need replacing.
Zero using the baseline control if the deflection line is
comparable to previous reagent calibrations.
5) Pump the highest standard through the system. When the
probe returns to the sample wash, pump for 5 minutes, and
repeat twice. As the highest standard appears on the
chart recorder, rotate the standard calibration control to
maximum chart deflection. Lock the control and record the
standard calibration value.
6) Pump the standards through the system from lowest to
highest concentration, and return the probe to the sample
wash for 2 minutes to avoid contamination of the first
sample.
7) Run the samples, and following completion, place the
reagent lines in the system wash or DI water, and continue
operation for 15 minutes. Remove the pump platen to
prolong pump-tube life.

calibration procedures using known chl q standards should be

conducted quarterly, following a lamp change, and after
adjusting the attenuator.
Standards
-
Primary standard Dissolve -1 mg of pure chl a (sigma chemical
Company, St. Louis, MO) into -80 ml of 90% neutralized acetone,
and dilute to 100 ml with 90% acetone.
-
Secondary standard Dilute 1 ml of the primary standard to 100
ml with 90% acetone.
Procedure
1) Using the spectrophotometer, measure the absorbances of
the secondary standard against a 90% acetone blank at 663
 nm, and calculate the chl a concentration using the
monochromatic method (p. 148).
2) Prepare serial dilutions of the secondary standard with
90% acetone in 20-ml glass vials covered with aluminum
foil so that fluorescence readings are between 15 and 85
units for at each sensitivity level (Table 56).
3) Pour -4 ml of each diluted standard into separate
cuvettes, and measure the fluorescence (Rb) three times
during a one minute period at the appropriate sensitivity
level (s) .
4) Add 2 drops of 2 N HC1 to each cuvette using a 0.05-ml
autopipet, invert, and after 30 seconds measure the
fluorescence (Ra) three times during a one minute period.
Calculations
1) Divide the averaged Rb value by the averaged Ra value for
each standard read at that sensitivity level to obtain the
acid ratio (rs) for each sensitivity level.
2) Divide the concentation (ug L - ~ )of the diluted standard
(Step 1) by the averaged Rb value for each concentration
to obtain a conversion factor (FS) for each sensitivity
level.
3) Calculate a calibration factor (Sx) for each sensitivity
level using: --
--

sx = FE rs
-
(rs 1)

Planimeter
The planimeter is used to integrate the area beneath a curve;
however, the instrument reading is not directly equivalent to
actual units of area. Therefore, the planimeter must be
calibrated to convert instrument readings to measured area.
1) Place the paper with the area to be measured on a smooth
flat surface, and securely fasten with tape.
2) Rule off a known area ( A ) , carefully trace 3 times, and
record each planimeter reading (P).
3) Average the readings, and determine a calibration factor
(F) as:
 i

Table 56. Fluorscence units before (Rb) and after (Ra) acidification used to obtain
acid ratios (rs) and calibration factors (Sx) at various sensitivity
levels (S) for the fluorometric analysis of chl 8 .

Volume of
Sensitivity secondary Total Standard rs Fs Sx
level standard volume chl al
(S) (ml) (L) (us L 1 35 Ei (%/m (chl a / s ) rFs(rs/rs-1) 1

chi a (ug L-I) = Sx (Rb-Ra)

Phaeo g (ug L-l) = Sx (rs x Ra - Rb)
NOTE: Chl g and phaeo indicated here are not in-lake concentrations.
4) Trace the unknown area three times, average P, and
multiply by F to obtain the measured area.

pH Meter
1) Allow the meter to warm-up for 5 minutes while preparing
the $robe(s) according to the manufacturers instructions.
2) Select 2 buffers; pH 7, and a second buffer of either pH 4
or 10 to cover the range of the unknown solutions. Pour
100 ml of each into separate beakers, and measure the
temperature of the solutions.
3) Place the pH 7 buffer on the magnetic stirrer, immerse the
probe, and rotate the calibration control until the meter
reading equals 7.0.
4) ~ i n s ethe probe with DI water, immerse into the second
buffer, and rotate the temperature compensator until the
meter reading equals the pH of the second buffer.
5) Rotate the slope indicator until the temperature
compensator points to the temperature of the buffers and
read the slope value. If the slope value is >90% the
meter is calibrated. If the slope is <90% either the
probe is defective or one of the buffers is contaminated.

Ocular Micrometex
The Bausch and Lomb (B&L) model 31-16-43 micrometer is
calibrated with a dual-scale stage micrometer prior to
measuring zooplankter body-lengths. Calibrate the ocular
micrometer using the 0-2.00-mm (0.02 divisions) stage scale
when measuring body-lengths (2.00 mm. Calibrate the ocular
micrometer using the 0-10 mm (0.1 divisions) stage micrometer
scale when measuring body-lengths >2.00 mm.
1) Insert the ocular micrometer into one of the oculars and
position the stage micrometer.
2) Set the zoom control to 5X to calibrate the 0-2.00 mm
scale, and 1X to calibrate the 0-10.0 mm scale. Focus on
the appropriate scale until the divisions of both
micrometers are superimposed.
 METHODOLOGIES: INTERFERENCE AND STATISTICAL EVALUATION

Interference
~nterfering substances artificially enhance or reduce the
analytical level of another substance. In general, there are
two types of interference, 1) turbidity which effects
colorimetric methods by enhancing absorbances, and 2) chemical
which affects colorimetric and titrametric methods by reducing
absorbances or titrant volumes respectively. Interference can
be eliminated by use of either turbidity blanks, spiking with
equivalent amounts of interfering substance or sample
dilutions.

Turbidity
Turbidity, due to the presence of suspended particles e.g.,
silt, causes increased spectrophotometric absorbances and
autoanalyzer chart deflections. If left uncorrected,
analytical results will be overestimated: however, corrections
can be made using turbidity blanks. Turbidity blanks are
prepared by substituting DI water for the indicator or
color-producing reagent and then analyzed. For example, an
equal volume of DI water is added in place of ammonium
- molybdate in the analysis of reactive silicon and total
phosphorus.
To correct for turbidity, subtract the absorbances or chart
deflections of both the turbidity blank and the reagent blank
from the sample absorbance. Substitute the corrected value
into the regression formula as described in the specific
methodologies.

Chemical
Typically, interfering chemicals lower analytical results by
either reacting with the constituent being analyzed forming an
unreactive complex or reacting with the reagents to prevent the
substance from being anplyzed. For example, iron
concentrations above 10 mg L reduce color formation in the
analysis of total phosphorus. One method of compensating is to
add the interfering substance to the calibration standards.
That is, if the concentration of the interfering substance is
known, add the same amount to the standards.
Alternately, a series of sample dilutions can be made prior to
the analyscis to reduce the concentration of the interfering
substance. If interference is occurring, diluted samples will
have increased absorbances or chart deflections. To obtain the
correct concentration, multiply the concentration of the
diluted sample by the appropriate dilution factor (see p. 39).

Statistical Evaluation
Standard Ranse
The standard range is defined by the highest and lowest
standard concentration routinely used to formulate a
calibration curve. preparing standards using these limits is
generally sufficient to accomodate those concentrations found
for Alaskan lakes. The standard range is not equivalent to the
operating range which is defined by the lower and upper limits
of detec-tion (Table 57).
U m e r Limit of Detection
The upper limit is the maximum concentration that the method is
capable of detecting. Sample concentrations exceeding the
upper limit of detection must be diluted prior to analysis.
Lower Limit of Detection
The lower limit of detection is the lowest concentration
detected as significantly greater than the reference blank.
Detection limits are derived empirically or statistically
(Table 57). Empirical detection limits were derived by
decreasing standard concentrations until absorbances, titrant
volumes, and chart deflections were significantly different
from the reagent blank value. The statistical or predicted
limit was obtained by formulating an aggregate linear
regression using the results of 30 representative calibration
curves. The Y-intercept of the upper limit of the 95%
confidence interval is the predicted lower limit of detection.

Precision (P) refers to the reproducibility of test results

under operating conditions, and is expressed as the coefficient
of variation (CV) :

P=CV=
standard deviation
mean
Although the level of precision may vary at different
concentrations, the CV reported is for a mid-range standard
concentration (Table 57) .
 Table 57. S t a t i s t i c a l e v a l u a t i o n s of a n a l y t i c a l m e t h o d o l o g i e s u s e d by t h e l i m n o l o g y
laboratory.

Lower
Standard l i m i t s of detection
Methodology range Empirical Predicted Precision Accuracy

Calcium ( t i t r a t i o n ) 2.5-12.5 mg/l 0.3mg/l 0.20mg/l 11%@ 5mg/l + 5 % @ 5mg/l

Calcium ( c o l o r i m e t r i c ) 0.2- 4 . 0 mg/l 0 . 1 mg/l 0.04 mg/l 3% @ 1 mg/l T 6% @ 1 mg/l
Magnesium ( t i t r a t i o n ) 1.5- 7.5 mg/l 0.3 mg/l 0 . 2 0 mg/l 21% @ 3 mg/l T 11%@ 3 mg/l
Magnesium ( c o l o r i m e t r i c ) 0.4- 4 . 0 mg/l 0.4rng/l 0.24mg/l 24%@ 1mg/l i 2 0 % @ 1mg/l
T o t a l i r o n (manual) 10- 200 ug/l 4.0 u g / l 3.30 u g / l 17% @ 40 u g / l + 7% @ 40 u g / l
T o t a l i r o n (automated) 5- 500 ug/l 2.0 ug/l N. A. N.A. N.A.
R e a c t i v e s i l i c o n (manual) 100-4000 ug/l 20.0 u g / l 27.00 u g / l 6 % @ 1868 u g / l +- 2% @ 1868 u g / l
I
Reactive s i l i c o n (automated) 100-1750 ug/l 20.0 u g / l 33.00 u g / l 1 7 % @ 583 u g / l +
- 2 % @ 583 u g / l
N K j e l d a h l n i t r o g e n (automated) 50- 300 ug/l 3.0 ug/l 3.50 u g / l 8% @ 100 u g / l 5 3% @ 1 0 0 u g / l
o T o t a l ammonia ( m a n u a l ) 1- 50 ug/l l.Oug/l 0.25ug/l 1 5 % @ 15ug/l 8 % @ 15ug/l
0
I
T o t a l ammonia ( a u t o m a t e d ) 12.5- 100 ug/l 1.0 ug/l 1-10 ug/l 13% @ 20 u g / l +
- 7% @ 20 u g / l
N i t r a t e + n i t r i t e (manual) 2- 200 ug/l 1.0 ug/l 0.60 u g / l 7% @ 50 u g / l + 2% @ 50 u g / l
N i t r a t e + n i t r i t e (automated) 20.0- 1 0 0 ug/l 1.0 ug/l 3.40 u g / l 9% @ 50 u g / l +
- 3% @ 50 u g / l
T o t a l phosphorus (manual) 2- 20 ug/l 0.5ug/l 0.25uq/l 5 % @ 6ug/l +
- 3 % @ 6ug/l
T o t a l phosphorus (automated) 2.5- 50 ug/l 1.0 ug/l 1.20 ug/l 12% @ 13 ug/l +- 9% @ 13 ug/l
R e a c t i v e phosphorus (manual) 1- 1 0 ug/l 0.5ug/l 0.12ug/l 7 % @ 4ug/l 5 4 % @ 4ug/l
Organic c a r b o n (manual) 60- 600 ug 30.0 ug 2 4 . 0 0 ug 25% @ 300 ug +
- 9% @ 300 ug
Total particulate
phosphorus (manual) 1- 1 4 ug 0.05 ug 0.02 ug 3 % @ 5ug +
- 3 % @ 5ug
Total p a r t i c u l a t e
phosphorus (automated) 1- 11 u g 0 . 6 0 ug 0.40 u g 6 % @ 5ug +- 7 % @ 5ug
Inorganic p a r t i c u l a t e
phosphorus 1- 7ug 0.05 ug 0.08 ug 1 2 % @ 2.25 ug + 3% @ 2.25 ug
Organic p a r t i c u l a t e phosphorus 1- 1 4 ug 0.05 ug 0.03 ug 10% @ 5 ug T
- 4 % @ 5ug

N.A. = Not a v a i l a b l e
Accuracy
Accuracy (A) is defined as the difference between the
analytical result and the true value. That is, the difference
between a prepared standard (true value), and the result of the
analysis (observed) (Table 5 7 ) . Thirty determinations were
made, the mean absolute deviate calculated, and expressed as a
percent oS the true value:
A = 1 observed - true value(/true value x 100
Procedures
Levels of precision and accuracy permit acceptance or rejection
of analytical results. In general, analytical data are
accepted if error, measured as accuracy, is of the order of
+lo%. We have statistically determined the accuracy of our
iieth~d~lo~iesto be <+lo% with the exception of the magnesium
analysis (Table 57). The methodological capabilties and their
analytical limits were derived from thirty individual
calibration curves for each procedure, and were carried out by
several technicians over a period of two years. As such, the
results of the statistical evaluations (Table 5 7 ) represent
realistic limits for routine testing, and do not result from
tspecializedt procedures. Consequently, the analytical
determinations using our methodologies can be expected to yield
results within the given statistical limits.
Calculations
Colorimetric analyses are based on the principle of Beer's law
which states that optical density (absorbance) at a specific
wavelength can be correlated to the concentration of a specific
substance. In addition, titrant volumes can also be correlated
to concentrations. Standards are analyzed, and results used to
formulate a linear equation by regressing concentrations
against the color intensity (absorbances or chart deflections),
and titrant volumes. These types of correlation are
mathematically derived using linear regression (Figure 31).
Linear regression is detg~inedby the method of least squares,
which minimizes (Y Y)- .
The slope of the regression (m)
refers to the steepness of the line, and may be either
positive, describing an increase in absorbance with
concentration (Figure 31A) or negative, describing a decrease
in absorbance with concentration (Figure 31B). The Y-intercept
is the intersection of the regression line on the Y-axis, and
can be positive, negative, or zero. Thus, the regression line
is mathematically described by the slope and the Y-intercept.
The coefficient of determination (r2) is the square of the
correlation coefficient and measures hqw well the data 'fitst
the regression line. For example, an r value of 1 means that
 ABSORBANCE

= dependent variable (concentration)

1% = y ax18 intercept

Y A m = slope
yh = b-mX x = independent variable (absorbance)
r2 = .9990 r2 = coefficient of determination
tb

0 .
0 >
X
ABSORBANCE

Figure 31. Description of linear regression showing (A) a

positve correlation between concentration and absorbance, and
( B ) a negative correlation, and the general form of the regres-
sion equation.
all the data points lie on the regression line; i.e., 100% of
tpe variation in Y is accounted for by its regression on X. An
r value of 0 means there is no correlatioq between the X and Y
variables. For our analytical methods, r values >.9990 are
desired.
Finally, the relationship between the X and Y variables deviate
from linearity above or below certain concentrations due to
limitations in the analytical method; e.g., reagent strength,
sample volume, and instrumentation. Therefore, it is crucial
to use the linear portion of the calibration curve when
calculating sample concentrations.

REFERENCES
American Public Health ~ssociation,American Water Works
Association and Water Pollution Control Federation. 1985.
Standard methods for the examination of water and
wastewater. 16th ed. New York, N.Y. 1268 p.
Friedman, L. C. and D. E. Erdman. 1982. ~ualityAssurance
practices for the chemical and biological analyses of
water and fluvial sediments. U. S. ~eological Survey,
Washington, D. C. 181 p.
Johnson, R. R. 1976. Elementary statistics. 2nd ed.
Wadsworth Publishing Co., Belmont, CA. 550 p.
Winefordner, J. D. and G. L. Long. 1983. ~ i m i tof detection.
A closer look at the IUPAC definition. Anal. Chem.
55:712A-724A.
 APPENDIX
DAILY TIMES FOR SUNRISE AND SUNSET AT VARIOUS
GEOGRAPHIC LOCATIONS WITHIN ALASKA:
USED TO CALCULATE THE DAY LENGTH FACTOR
IN DETERMINING DAY RATE ESTIMATES
OF PHOTOSYNTHESIS
 TABLES OF SUNRISE AND SUNSET
SECOND PRINTING
The table on the other side of this sheet may be used in any year of
the twentieth century and within the geographical boundary of the stated
place with a n error not exceeding 2 minutes and generally less than 1
minute. It may also be used anywhere in the vicinity of the stated place
with a n additional error of less than 1 minute for each nine miles, reck-
oned from the station of the U.S.Weather Bureau for the stated place,
or reckoned from the nearest boundary in cases when no station of the
Weather Bureau existed for the stated place.
Tables are available for almost all stations of the U.S. Weather
Bureau, and ail cities of over 50,000 population (1950 census) which
a r e sufficiently remote from other cities of like size to require a separate
computation.
The standard time shown is in conformity with time zone boundaries
specified by the Interstate Commerce Commission a s of 1 June 1969.
Eastern Standard Time is the local time of the 75th meridian.
Central Standard Time is the local time of the 90th meridian. Mountain
Standard Time is the local time of the 105th meridian. Pacific Standard
Time is the local time of the 120th meridian.
Sunrise and sunset a r e considered to occur when the upper edge of
the disk of the Sun appears to be exactly on the horizon. The times of
sunrise and sunset given in this table a r e for a n unobstructed horizon,
with normal atmospheric conditions, a t zero elevation above the Earth's
surface in a level region.
The computations a r e based on a constant semidiameter of the Sun
of 16 minutes of arc, a n adopted refraction a t the horizon of 34 minutes
of arc, and the path of the Sun for the year 1966.
Should greater precision be required, corrections for elevation of
the observer, angular elevation of the visible horizon, deviations from
standard atmospheric conditions, and for a specific year may be derived
from ''Tables of Sunrise, Sunset and Twilight," Supplement to the Ameri-
can Ephemeris, 1946, obtainable from the Superintendent of Documents,
U.S. Government Printing Office, Washington, D.C., 20402.
 SUNRISE AND SUNSET AT ANCHORAGE. ALASKA NO.1005
STANDARD TIME OF THE 150th MERIDIAN WEST.

JAN. FEB. MAR. APR. MAY JUNE JULY AUG; SEPT. Om. NOV. DEC.
DAY ' Rise Set Rise Set R h Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rlsk Set Rise Set
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.

1 9 14 2 54 8 21 4 07 7 01 5 25 5 24 6 45 3 52 8 04 2 37 9 20 2 28 9 39 3 32 8 39 4 51 7 07 6 06 5 32 7 27 3 59 8 45 2 53
2 9 13 2 55 8 18 4 10 6 58 5 27 5 21 6 48 3 49 8 07 2 35 9 22 2 29 9 38 3 34 8 36 4 54 7 04 6 08 5 29 7 30 3 56 8 47 2 51
3 9 12 2 57 8 15 4 13 6 55 5 30 5 17 6 51 3 46 8 09 2 34 9 23 2 30 9 37 3 37 8 33 4 56 7 01 6 11 5 26 7 33 3 53 8 49 2 50
4 9 12 2 59 8 13 4 16 6 52 5 33 5 14 6 53 3 43 8 12 2 32 9 25 2 32 9 36 3 40 8 31 4 59 6 57 6 13 5 22 7 35 3 51 8 51 2 49
5 9 11 3 01 8 10 4 19 6 49 5 35 5 11 6 56 3 40 8 15 2 31 9 27 2 33 9 35 3 42 8 28 5 01 6 54 6 16 5 19 7 38 3 48 8 53 2 48
6 9 10 3 02 8 08 4 21 6 46 5 38 5 08 6 58 3 38 8 17 2 30 9 29 2 35 9 33 3 45 8 25 5 04 6 51 6 19 5 16 7 41 3 45 8 55 2 47
7 9 09 3 04 8 05 4 24 6 43 5 41 5 05 7 01 3 35 8 20 2 28 9 30 2 36 9 32 3 47 8 22 5 06 6 48 6 21 5 13 7 44 3 43 8 57 2 46
8 9 08 3 06 8 02 4 27 6 39 $ 43 5 02 7 04 3 32 8 23 2 27 9 32 2 38 9 31 3 50 8 19 5 09 6 45 6 24 5 10 7 46 3 40 8 59 2 45
9 9 06 3 09 8 00 4 30 6 36 5 46 4 59 7 06 3 29 8 25 2 26 9 33 2 40 9 29 3 52 8 16 5 11 6 42 6 26 5 07 7 49 3 38 9 01 2 44
10 9 05 3 11 7 57 4 33 6 33 5 49 4 55 7 09 3 27 8 28 2 25 9 34 2 42 9 28 3 55 8 14 5 14 6 38 6 29 5 04 7 52 3 35 9 02 2 43
11 9 04 3 13 7 54 4 36 6 30 5 51 4 52 7 11 3 24 8 30 2 24 9 35 2 43 9 26 3 58 8 11 5 16 6 35 6 31 5 01 7 55 3 33 9 04 2 43
12 9 02 3 15 7 51 4 38 6 27 5 54 4 49 7 14 3 21 8 33 2 24 9 37 2 45 9 24 4 00 8 08 5 19 6 32 6 34 4 58 7 57 3 30 9 05 2 42
13 9 01 3 17 7 48 4 41 6 24 5 56 4 46 7 17 3 19 8 36 2 23 9 38 2 47 9 22 4 03 8 05 5 21 6 29 6 37 4 55 8 00 3 28 9 07 2 42
14 8 59 3 20 7 46 4 44 6 21 5 59 4 43 7 19 3 16 8 38 2 22 9 39 2 49 9 21 4 05 8 02 5 24 6 26 6 39 4 52 8 03 3 25 9 08 2 41
15 8 57 3 22 7 43 4 47 6 18 6 02 4 40 7 22 3 14 8 41 2 22 9 39 2 51 9 19 4 08 7 59 5 26 6 23 6 42 4 49 8 05 3 23 9 09 2 41
16 8 56 3 25 7 40 4 49 6 14 6 04 4 37 7 24 3 11 8 43 2 21 9 40 2 54 9 17 4 10 7 56 5 29 6 19 6 44 4 45 8 08 3 21 9 10 2 41
17 8 54 3 27 7 37 4 52 6 11 6 07 4 34 7 27 3 09 8 46 2 21 9 41 2 56 9 15 4 13 7 53 5 31 6 16 6 47 4 42 8 11 3 19 9 11 2 41
18 8 52 3 30 7 34 4 55 6 08 6 09 4 31 7 30 3 06 8 48 2 21 9 41 2 58 9 13 4 16 7 50 5 34 6 13 6 50 4 39 8 13 3 16 9 12 2 41
19 8 50 3 32 7 31 4 58 6 05 6 12 4 28 7 32 3 04 8 51 2 21 9 42 3 00 9 10 4 18 7 47 5 36 6 10 6 52 4 36 8 16 3 14 9 13 2 41
20 8 48 3 35 7 28 5 00 6 02 6 15 4 25 7 35 3 01 8 53 2 21 9 42 3 03 9 08 4 21 7 44 5 38 6 07 6 55 4 33 8 19 3 12 9 13 2 42
21 8 46 3 37 7 25 5 03 5 59 6 17 4 21 7 38 2 59 8 56 2 21 9 42 3 05 9 06 4 23 7 41 5 41 6 04 6 58 4 30 8 21 3 10 9 14 2 42
22 8 44 3 40 7 22 5 06 5 55 6 20 4 18 7 40 2 57 8 58 2 21 9 42 3 07 9 04 4 26 7 38 5 43 6 00 7 00 4 28 8 24 3 08 9 15 2 43
23 8 42 3 43 7 19 5 09 5 52 6 22 4 15 7 43 2 55 9 00 2 22 9 42 3 10 9 01 4 28 7 35 5 46 5 57 7 03 4 25 8 26 3 06 9 15 2 43
24 8 40 3 45 7 16 5 11 5 49 6 25 4 12 7 45 2 52 9 03 2 22 9 42 3 12 8 59 4 31 7 32 5 48 5 54 7 06 4 22 8 29 3 04 9 15 2 44
25 837 348 7,13 514 546 627 409 748 250 905 223 942 314 857 434 729 551 551 708 419 831 302 915 245
26 8 35 3 51 7 10 5 17 5 43 6 30 4 06 7 51 2 48 9 07 2 23 9 42 3 17 8 54 4 36 7 26 5 53 5 48 7 11 4 16 8 34 3 00 9 16 2 46
27 8 33 3 54 7 07 5 19 5 40 6 33 4 04 7 53 2 46 9 09 2 24 9 41 3 19 8 52 4 39 7 22 5 56 5 45 7 14 4 13 8 36 2 59 9 16 2 47
28 8 30 3 56 7 04 5 22 5 36 6 35 4 01 7 56 2 44 9 12 2 25 9 41 3 22 8 49 4 41 7 19 5 58 5 41 7 16 4 10 8 38 2 57 9 15 2 48
29 8 28 3 59 7 03 5 24 5 33 6 38 3 58 7 59 2 42 9 14 2 26 9 40 3 24 8 47 4 44 7 16 6 01 5 38 7 19 4 07 8 41 2 55 9 15 2 49
30 826 4 02 5 30 6 40 3 55 8 01 2 40 9 16 2 27 9 40 3 27 8 44 4 46 7 13 6 03 5 35 7 22 4 04 8 43 2 54 9 15 2 50
31 8 23 4 05 5 27 6 43 2 39 9 18 3 29 8 41 4 49 7 10 7 24 4 02 9 14 2 52
Add one hour for Daylight Saving Time if and when in use.

Prepared by
NAUTICAL ALMANAC OFFICE
UNITED STATES NAVAL OBSERVATORY
WASHINGTON, D.C. 20390
 SUNRISE AND SUNSET AT ANNETTE ISLAND,ALASKA NO. 1006
PACIFIC STANDARD TIME

JAN. FEB. MAR. APR. MAY JUNE JULY 4UG. SEPT. OCT. NOV. DEC.
DAY'Rise Set Rise Set h Set Rise Set Rise Set Rise Set R
im Set Rise Set Rise Set Rise Set: Rise Set Rise -Set
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.
1 9 11 4 29 8 38 5 23 7 37 6 22 6 18 7 23 5 06 8 22 4 15 9 14 4 11 9 28 4 54 8 49 5 52 7 39 6 48 6 22 7 50 5 09 8 47 4 23
2 9 11 4 30 8 36 5 25 7 34 6 24 6 16 7 25 5 04 8 23 4 14 9 15 4 12 9 28 4 56 8 47 5 54 7 37 6 50 6 20 7 52 5 07 8 49 4 22
3 9 11 4 31 8 34 5 27 7 32 6 26 6 13 7 27 5 02 8 25 4 13 9 17 4 13 9 27 4 58 8 45 5 56 7 34 6 52 6 17 7 54 5 05 8 50 4 21
4 9 11 4 32 8 32 5 29 7 29 6 28 6 11 7 29 5 00 8 27 4 12 9 18 4 14 9 27 5 00 8 43 5 58 7 32 6 54 6 15 7 56 5 03 8 52 4 21
5 9 10 4 34 8 30 5 31 7 27 6 30 6 08 7 31 4 58 8 29 4 11 9 19 4 15 9 26 5 02 8 41 5 59 7 29 6 56 6 12 7 58 5 01 8 53 4 20
6 9 10 4 35 8 28 5 34 7 24 6 32 6 06 7 33 4 56 8 31 4 10 9 20 4 16 9 25 5 03 8 39 6 01 7 27 6 58 6 10 8 00 4 59 8 55 4 20
7 9 09 4 37 8 26 5 36 7 22 6 34 6 03 7 35 4 54 8 33 4 10 9 21 4 17 9 25 5 05 8 37 6 03 7 24 7 00 6 07 8 02 4 57 8 56 4 19
8 9 08 4 38 8 24 5 38 7 19 6 36 6 01 7 37 4 52 8 35 4 09 9 22 4 18 9 24 5 07 8 35 6 05 7 22 7 02 6 05 8 04 4 55 8 58 4 19
9 9 08 4 40 8 22 5 40 7 17 6 38 5 58 7 39 4 50 8 37 4 08 9 23 4 19 9 23 5 09 8 33 6 07 7 19 7 04 6 02 8 06 4 53 8 59 4 18
10 9 07 4 41 8 20 5 42 7 14 6 40 5 56 7 41 4 48 8 39 4 08 9 24 4 20 9 22 5 11 8 31 6 09 7 16 7 06 6 00 8 08 4 52 9 00 4 18
11 9 06 4 43 8 18 5 44 7 12 6 42 5 53 7 43 4 46 8 40 4 08 9 25 4 22 9 21 5 13 8 29 6 11 7 14 7 08 5 57 8 10 4 50 9 01 4 18
12 9 05 4 44 8 16 5 46 7 09 6 44 5 51 7 45 4 44 8 42 4 07 9 25 4 23 9 20 5 15 8 27 6 13 7 11 7 10 5 55 8 12 4 48 9 02 4 18
13 9 04 4 46 8 14 5 48 7 07 6 46 5 48 7 47 4 42 8 44 4 07 9 26 4 24 9 19 5 16 8 24 6 14 7 09 7 12 5 53 8 14 4 46 9 03 4 18
14 9 03 4 48 8 11 5 50 7 04 6 48 5 46 7 48 4 40 8 46 4 07 9 27 4 25 9 18 5 18 8 22 6 16 7 06 7 14 5 50 8 16 4 45 9 04 4 17
15 9 02 4 50 8 09 5 53 7 02 6 50 5 43 7 50 4 39 8 48 4 06 9 27 4 27 9 16 5 20 8 20 6 18 7 04 7 15 5 48 8 18 4 43 9 05 4 18
16 9 01 4 51 8 07 5 55 6 59 6 52 5 41 7 52 4 37 8 49 4 06 9 28 4 28 9 15 5 22 8 18 6 20 7 01 7 17 5 45 8 20 4 41 9 06 4 18
17 9 00 4 53 8 05 5 57 6 57 6 54 5 39 7 54 4 35 8 51 4 06 9 28 4 30 9 14 5 24 8 15 6 22 6 58 7 19 5 43 8 22 4 40 9 07 4 18
18 8 59 4 55 8 03 5 59 6 54 6 56 5 36 7 56 4 34 8 53 4 06 9 29 4 31 9 13 5 26 8 13 6 24 6 56 7 21 5 41 8 24 4 38 9 08 4 18
19 8 58 4 57 8 00 6 01 6 52 6 58 5 34 7 58 4 32 8 55 4 06 9 29 4 33 9 11 5 28 8 11 6 26 6 53 7 23 5 38 8 26 4 37 9 08 4 18
20 8 56 4 59 7 58 6 03 6 49 7 00 5 31 8 00 4 30 8 56 4 06 9 29 4 34 9 10 5 29 8 08 6 28 6 51 7 25 5 36 8 28 4 35 9 09 4 19
21 8 55 5 01 7 56 6 05 6 47 7 02 5 29 8 02 4 29 8 58 4 06 9 29 4 36 9 08 5 31 8 06 6 29 6 48 7 27 5 33 8 30 4 34 9 10 4 19
22 8 54 5 03 7 53 6 07 6 44 7 04 5 27 8 04 4 27 8 59 4 07 9 30 4 37 9 07 5 33 8 04 6 31 6 46 7 29 5 31 8 32 4 33 9 10 4 20
23 8 52 5 05 7 51 6 09 6 41 7 06 5 24 8 06 4 26 9 01 4 07 9 30 4 39 9 05 5 35 8 01 6 33 6 43 7 31 5 29 8 34 4 31 9 11 4 20
24 8 51 5 07 7 49 6 11 6 39 7 07 5 22 8 08 4 24 9 03 4 07 9 30 4 41 9 04 5 37 7 59 6 35 6 40 7 34 5 27 8 35 4 30 9 11 4 21
25 8 49 5 09 7 46 6 13 6 36 7 09 5 20 8 10 4 23 9 04 4 08 9 30 4 42 9 02 5 39 7 56 6 37 6 38 7 36 5 24 8 37 4 29 9 11 4 22
26 8 48 5 11 7 44 6 16 6 34 7 11 5 18 8 12 4 22 9 06 4 08 9 30 4 44 9 00 5 41 7 54 6 39 6 35 7 38 5 22 8 39 4 28 9 12 4 22
27 846 513 742 618 631 713 515 814 420 907 409 930 4 4 6 859 543 752 6 4 1 6 3 3 7 4 0 520 841 427 9 1 2 423
28 8 44 5 15 7 39 6 20 6 29 7 15 5 13 8 16 4 19 9 09 - 409 9 29 4 47 8 57 5 45 7 49 6 43 6 30 7 42 5 18 8 42 4 26 9 12 4 24
29 8 43 5 17 7 38 6 21 6 26 7 17 5 11 8 18 4 18 9 10 4 10 9 29 4 49 8 55 5 46 7 47 6 44 6 28 7 44 5 16 8 44 4 25 9 12 4 25
30 841 5 19 6 24 7 19 5 09 8 20 4 17 9 11 4 11 9 29 4 51 8 53 5 48 7 44 6 46 6 25 7 46 5 13 8 46 4 24 9 12 4 26
31 8 39 5 21 6 21 7 21 4 16 9 13 4 53 8 51 5 50 7 42 7 48 5 11 9 12 4 27
Add one hour for Daylight Saving Time if a n d when in use. I certify that the above d a t a a r e the result of a n a c c u r a t e a n d true com-
putation by the Nautical A l m a n a c Office, United States Naval Observatory,
a n agency charged by Federal Statute (9 Stat. L 374, 375) with the duty
of making such computations a n d publishing the results.

E. W. WOOLARD
Director Nautical Almanac
U. S. Naval Observatory
C. G. CHRISTIE
P . .A - T l P l r
 SUNRISE AND SUNSET AT CORDOVA, ALASKA NO. 1011
STANDARD TIME OF THE 150th MERIDIAN WEST

JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. NOV. DEC.
DAY Rise Set Rise Set Set Rise Set Rise Set Set Rise Set Set Rise Set Set kise Set
Rise Rise Klse Rise Rise st.
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.
1 849 2 42 7 59 3 53 6 42 5 08 5 07 6 26 3 38 7 42 2 26 8 54 2 18 9 13 3 19 8 16 4 35 6 47 5 47 5 15 7 06 3 44 8 21 2 41
2 849 2 44 7 56 3 56 6 39 5 11 5 04 6 29 3 35 7 45 2 25 8 56 2 19 9 12 3 2 1 8 13 4 38 6 44 5 50 5 11 7 09 3 4 1 8,2- 2 39
3 848 2 46 7 54 3 59 6 36 5 13 5 01 6 31 3 32 7 47 2 23 8 58 2 20 9 11 3 24 8 11 4 40 6 4 1 5 52 5 08 7 11 3 39 8 25 2 38
4 847 2 47 7 51 4 0 1 6 33 5 16 4 58 6 34 3 29 7 50 2 22 9 00 2 22 9 10 3 26 8 08 4 42 6 38 5 55 5 05 7 14 3 36 8 27 2 37
5 846 2 49 7 49 4 04 6 30 5 19 4 55 6 36 3 27 7 52 2 2 1 9 01 2 23 9 09 3 29 8 06 4 45 6 35 5 57 5 02 7 17 3 34 8 29 2 36
6 845 2 51 7 46 4 07 6 27 5 2 1 4 52 6 39 3 24 7 55 2 15 9 03 2 25 9 08 3 3 1 8 03 4 47 6 32 6 00 4 59 7 19 3 3 1 8 31 2 35
7 844 2 53 7 44 4 10 6 24 5 24 4 48 6 41 3 2 1 7 58 2 18 9 04 2 26 9 06 3 33 8 00 4 50 6 29 6 02 4 56 7 22 3 29 8 33 2 34
8 843 2 55 7 41 4 12 6 2 1 5 26 4 45 6 44 3 19 8 00 2 17 9 06 2 28 9 05 3 36 7 57 4 52 6 26 6 05 4 53 7 25 3 26 8 34 2 33
9 8 42 2 57 7 39 4 15 6 18 5 29 4 42 6 46 3 16 8 03 2 16 9 07 2 29 9 04 3 38 7 55 4 54 6 23 6 07 4 50 7 27 3 24 8 36 2 33
10 8 41 2 59 7 36 4 18 6 15 5 3 1 4 39 6 49 3 13 8 05 2 15 9 08 2 31 9 02 3 4 1 7 52 4 57 6 19 6 10 4 47 7 30 3 21 8 38 2 32
11 8 40 3 01 7 33 4 20 6 12 5 34 4 36 6 51 3 11 8 08 2 15 9 09 2 33 9 01 3 43 7 49 4 59 6 16 6 12 4 44 7 33 3 19 8 39 2 31
12 8 38 3 03 7 30 4 23 6 09 5 36 4 33 6 54 3 08 8 10 2 14 9 10 2 35 8 59 3 46 7 46 5 02 6 13 6 15 4 41 7 35 3 16 8 40 2 31
13 8 37 3 05 7 28 4 26 6 06 5 39 4 30 6 56 3 06 8 13 2 13 9 11 2 36 8 57 3 48 7 43 5 04 6 10 6 17 4 38 7 38 3 14 8 42 2 31
14 8 35 3 08 7 25 4 29 6 02 5 4 1 4 27 6 59 3 03 8 15 2 13 9 12 2 38 8 56 3 51 7 41 5 06 6 07 6 20 4 35 7 40 3 12 8 43 2 30
15 8 34 3 10 7 22 4 3 1 5 59 5 44 4 24 7 02 3 0 1 8 17 2 12 9 13 2 40 8 54 3 53 7 38 5 09 6 04 6 22 4 32 7 43 3 10 8 44 2 30
16 8 32 3 12 719 4 34 5 56 5 46 4 21 7 04 2 58 8 20 2 12 9 14 2 42 8 52 3 56 7 35 511 6 01 6 25 4 29 7 46 3 07 8 45 2 30
17 8 30 3 15 717 4 37 5 53 5 49 4 18 7 07 2 56 8 22 2 12 9 14 2 44 8 50 3 58 7 32 514 5 58 6 27 4 26 7 48 3 05 8 46 2 30
18 8 29 3 17 714 4 39 5 50 5 5 1 4 15 7 09 2 54 8 25 2 11 9 15 2 46 3 48 4 0 1 7 29 516 5 55 6 30 4 23 7 51 3 03 8 47 2 30
19 8 27 3 19 711 4 42 5 47 5 54 4 12 7 12 2 5 1 8 27 2 11 9 15 2 49 8 46 4 03 7 26 518 5 52 6 32 4 20 7 53 3 01 8 48 2 31
20 8 25 3 22 708 4 45 5 44 5 56 4 09 7 14 2 49 8 29 2 11 9 16 2 51 8 44 4 06 7 23 521 5 48 6 35 4 18 7 56 2 59 8 48 2 31
2 1 8 23 3 24 7 05 4 47 5 4 1 5 59 4 06 7 17 2 47 8 32 2 12 9 16 2 53 8 42 4 08 7 20 5 23 5 45 6 37 4 15 7 58 2 57 8 49 2 31
22 8 21 3 27 7 02 4 50 5 38 6 01 4 03 7 19 2 45 8 34 2 12 9 16 2 55 8 40 4 11 7 17 5 26 5 42 6 40 4 12 8 01 2 55 8 50 2 32
23 8 19 3 30 6 59 4 53 5 35 6 04 4 00 7 22 2 43 8 36 2 12 9 16 2 57 8 38 4 13 7 14 5 28 5 39 6 43 4 09 8 03 2 53 8 50 2 32
24 8 17 3 32 6 57 4 55 5 32 6 06 3 58 7 24 2 4 1 8 38 2 13 9 16 3 00 8 35 4 16 7 11 5 31 5 36 6 45 4 06 8 05 2 51 8 50 2 33
25 8 15 3 35 6 54 4 58 5 29 6 09 3 55 7 27 2 39 8 4 1 2 13 9 16 3 02 8 33 4 18 7 08 5 33 5 33 6 48 4 03 8 08 2 50 8 51 2 34
26 8 13 3 37 6 51 5 00 5 25 6 11 3 52 7 30 2 37 8 43 2 14 9 15 3 04 8 31 4 20 7 05 5 35 5 30 6 50 4 01 8 10 2 48 8 51 2 35
27 8 10 3 40 6 48 5 03 5 22 6 14 3 49 7 32 2 35 8 45 2 14 9 15 3 07 8 28 4 23 7 02 5 38 5 27 6 53 3 58 8 12 2 46 8 51 2 36
28 8 08 3 43 6 45 5 06 5 19 6 16 3 46 7 35 2 33 8 47 2 15 . 9 15 3 09 8 26 4 25 6 59 5 40 5 24 6 56 3 55 8 15 2 45 8 51 2 37
29 8 06 3 45 6 44 5 07 5 16 6 19 3 43 7 37 2 3 1 8 49 2 16 9 14 3 11 8 24 4 28 6 56 5 43 5 2 1 6 58 3 52 8 17 2 43 8 50 2 38
30 8 04 3 48 5 13 6 2 1 3 40 7 40 2 30 8 5 1 2 17 9 13 3 14 8 21 4 30 6 53 5 45 5 18 7 0 1 3 49 8 19 2 42 8 50 2 39
3 1 8 01 3 51 5 10 6 24 2 28 8 53 3 16 8 19 4 33 6 50 7 04 3 47 8 50 2 41
Add one hour for Daylight Saving Time if a n d when in use. I certify that the above d a t a a r e the result of a n a c c u r a t e a n d true com-
putation by the Nautical A l m a n a c Office, United States Naval Observatory,
a n agency charged by Federal Statute (9 Stat. L 374, 375) with the duty
of making such computations a n d publishing the results.

E. W. WOOLARD
Director Nautical A l m a n a c
U. S. Naval Observatory
C. G. CHRISTIE
 SUNRISE AND SUNSET AT FAIRBANKS, ALASKA NO. 1012
STANDARD TIME OF THE 150th MERIDIAN WEST.

JAN. FEB. MAR. APR. MAY J UNE JULY ~UG. SEPT. OCT. NOV. DEC.
DAY . Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rlse t Rise Set Rise " Set
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.
1 9 55 1 56 8 38 3 34 7 01 5 08 5 09 6 44 3 19 8 20 1 34 10 07 1 11 10 37 2 50 9 02 4 31 7 10 6 00 5 20 7 39 3 3 1 9 19 2 02
2 9 53 1 58 8 34 3 37 6 58 5 11 5 05 6 47 3 15 8 24 1 3 1 10 10 1 13 10 35 2 54 8 59 4 34 7 06 6 03 5 17 7 42 3 27 9 22 2 00
3 9 52 2 00 8 31 3 41 6 54 5 14 5 01 6 50 3 12 8 27 1 2 8 10 13 1 16 10 33 2 57 8 55 4 37 7 03 6 07 5 13 7 45 3 24 9 25 1 57
4 9 51 2 03 8 28 3 44 6 51 5 17 4 58 6 53 3 08 8 31 1 2 5 10 17 1 19 10 3 1 3 01 8 52 4 40 6 59 6 10 5 09 7 49 3 20 9 28 1 55
5 9 49 2 05 8 25 3 48 6 47 5 20 4 54 6 56 3 05 8 34 1 2 3 10 20 1 2 1 10 28 3 04 8 48 4 43 6 55 6 13 5 06 7 52 3 17 9 30 1 54
6 9 47 2 08 8 21 3 51 6 43 5 24 4 50 6 59 3 01 8 37 1 2 0 10 22 1 2 4 10 26 3 07 8 45 4 46 6 52 6 16 5 02 7 55 3 14 9 33 1 52
7 9 45 2 11 8 18 3 55 6 40 5 27 4 47 7 03 2 58 8 4 1 1 18 10 25 1 2 7 10 23 3 11 8 41 4 49 6 48 6 19 4 58 7 59 3 11 9 36 150
8 9 43 2 14 8 15 3 58 6 36 5 30 4 43 7 06 2 54 8 44 1 15 10 28 1 3 0 10 21 3 14 8 37 4 52 6 44 6 22 4 55 8 02 3 07 9 38 148
9 9 41 2 17 8 11 4 01 6 33 5 33 4 39 7 09 2 50 8 48 1 13 10 30 1 3 3 10 18 3 17 8 34 4 55 6 41 6 25 4 51 8 06 3 04 9 40 147
10 9 39 2 19 8 08 4 05 6 29 5 36 4 36 7 12 2 47 8 5 1 111 10 33 1 3 6 10 15 3 21 8 30 4 58 6 37 6 28 4 48 8 09 3 01 9 43 146
11 9 37 2 23 8 04 4 08 6 25 5 39 4 32 7 15 2 43 8 55 1 09 10 35 1 3 9 10 12 3 24 8 27 5 01 6 33 6 31 4 44 8 12 2 58 9 45 144
12 9 35 2 26 8 01 4 12 6 22 5 42 4 28 7 18 2 40 8 58 1 0 7 10 37 1 4 2 10 09 3 27 8 23 5 04 6 30 6 34 4 40 8 16 2 54 9 47 143
13 9 32 2 29 7 58 4 15 6 18 5 45 4 25 7 21 2 36 9 02 1 05 10 39 1 4 6 10 06 3 31 8 19 5 07 6 26 6 37 4 37 8 19 2 5 1 9 49 142
14 9 30 2 32 7 54 4 19 6 14 5 49 4 21 7 25 2 33 9 05 1 0 3 10 41 1 4 9 10 03 3 34 8 16 5 10 6 22 6 40 4 33 8 23 2 48 9 5 1 142
15 9 27 2 35 7 51 4 22 6 11 5 52 4 17 7 28 2 29 9 09 1 02 10 43 1 5 2 10 00 3 37 8 12 5 13 6 19 6 43 4 30 8 26 2 45 9 52 141
16 9 25 2 38 7 47 4 25 6 07 5 55 4 14 7 31 2 26 9 12 1 0 1 10 44 156 9 57 3 40 8 09 5 16 6 15 6 47 4 26 8 30 2 42 9 54 141
17 9 22 2 42 7 44 4 29 6 04 5 58 4 10 7 34 2 22 9 16 1 00 10 45 1 59 9 53 3 44 8 05 5 19 6 11 6 50 4 23 8 33 2 39 9 55 1 40
18 9 2 0 245 740 432 600 601 406 7'37 219 91912591046 202 950 347 801 522 608 653 419 837 236 956 140
19 9 1 7 249 737 435 556 604 403 741 216 92312591047 206 947 350 758 525 604 656 416 840 233 957 140
20 9 14 2 52 7 33 4 39 5 53 6 07 3 59 7 44 2 12 9 26 12 59 10 48 2 09 9 44 3 53 7 54 5 28 6 00 6 59 4 12 8 43 2 30 9 58 140
21 9 11 2 55 7 30 442 5 49 6 10 3 55 7 47 2 09 9 30 12 59 1 0 4 8 2 13 9 40 3 56 7 50 5 31 5 57 7 02 4 09 847 2'27 9 59 141
22 9 08 2 59 7 26 4 45 5 45 6 13 3 52 7 50 2 06 9 33 12 59 10 48 2 16 9 37 4 00 7 47 5 34 5 53 7 06 4 05 8 50 2 25 9 59 141
23 9 05 3 02 7 23 4 48 5 42 6 16 3 48 7 54 2 02 9 37 12 59 10 47 2 19 9 34 4 03 7 43 5 37 5 49 7 09 4 02 8 53 2 22 9 59 142
24 9 02 3 06 7 19 4 52 5 38 6 19 3 44 7 57 1 5 9 9 40 1 0 0 10 47 2 23 9 30 4 06 7 39 5 40 5 46 7 12 3 58 8 57 2 19 10 00 143
25 8 59 3 09 7 16 4 55 5 34 6 22 3 41 8 00 1 56 9 44 1 0 1 10 46 2 26 9 27 4 09 7 36 5 43 5 42 7 15 3 55 9 00 2 16 10 00 144
26 8 56 3 13 7 12 4 58 5 31 6 25 3 37 8 04 1 5 2 9 47 1 02 10 45 2 30 9 23 4 12 7 32 5 46 5 38 7 19 3 51 9 03 2 14 9 59 1 4 5
27 8 53 3 16 7 08 5 01 5 27 6 29 3 34 8 07 1 4 9 9 51 1 04 10 44 2 33 9 20 4 15 7 28 5 49 5 35 7 22 3 48 9 06 2 11 9 59 1 4 6
28 850 320 705 505 523 632 330 810 146 954 1051043 237 916 418 725 551 531 725 344 910 209 959 148
29 8 47 3 23 7 03 5 07 5 20 6 35 3 26 8 14 1 4 3 9 57 1 07 10 4 1 2 40 9 13 4 22 7 21 5 54 5 27 7 29 3 41 9 13 2 06 9 58 1 50
30 8 44 3 27 5 16 6 38 3 23 8 17 1 4 0 10 01 1 0 9 10 39 2 44 9 09 4 25 7 17 5 57 5 24 7 32 3 37 9 16 2 04 9 57 1 51
31 841 3 30 5 12 6 41 1 37 10 04 2 47 9 06 4 28 7 14 7 35 3 34 9 56 1 53

Add one hour for Daylight Saving Time if a n d when in use.

Prepared by
NAUTICAL ALMANAC OFFICE
UNITED STATES NAVAL OBSERVATORY
 SUNRISE AND SUNSET AT JUNEAU, ALASKA NO. 1013
PACIFIC STANDARD TIME

IAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. OCT. NOV. DEC.
DAy-R~~oht R~Y El RISE Sn Rise Sct ~ i s e Sat Rise Set Rise %I Rise hl Rise St Rise Set .'Rise St Rlsc iSt
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. PM. A.td. P.hi. A.M p.:.$.
1 9 47 4 17 9 04 5 2 1 7 54 6 20 6 26 7 39 5 05 8 4 8 4 02 9 51 3 57 10 07 4 49 9 19 5 57 7 5 8 7 02 6 3 2 8 14 5 09 9 2 1 4 1 4
2 9 46 4 19 9 02 5 23 7 51 6 31 6 24 7 42 5 02 8 50 4 01 9 52 3 58 10 06 4 51 9 17 5 59 7 55 7 05 6 30 8 16 5 07 9 22 4 13
3 9 46 4 20 9 00 5 26 7 49 6 33 6 21 7 44 5 0 0 8 52 4 00 9 54 3 59 10 06 4 53 9 14 6 01 7 53 7 07 6 27 8 18 5 05 9 24 4 12
4 9 45 4 22 8 58 5 28 7 46 6 35 6 18 7 46 4 57 9 55 3 59 9 55 4 00 10 05 4 55 9 12 6 03 7 50 7 09 6 24 8 21 5 02 9 26 4 11
5 9 45 4 23 8 55 5 3 0 7 43 6 38 6 15 7 49 4 55 8 57 3 58 9 56 4 01 10 04 4 57 9 10 6 06 7 47 7 11 6 21 8 23 5 00 9 28 4 10
5 9 44 4 25 8 53 5 33 7 4 0 b 4 0 6 12 7 51 4 52 8 59 3 57 9 50 4 02 10 03 5 00 9 07 6 0 8 7 4 4 7 13 6 1 8 8 25 4 58 '1 29 3 0';.
7 9 43 4 27 8 51 5 35 7 38 6 112 6 09 7 53 4 50 9 01 3 56 9 53 4 03 10 02 5 02 9 05 6 1 0 '7 4 1 7 1 6 h 1 6 8 28 4 55 9 3 1 4 09
8 9 42 4 28 8 48 5 38 7 35 6 45 6 07 7 55 4 40 9 03 3 55 1 0 00 4 05 10 01 5 04 9 02 6 1 2 7 38 7 1 8 ti 13 8 3 0 1 53 9 32 4 17E
9 9 41 4 30 8 46 5 40 7 32 6 47 6 04 7 58 4 45 9 06 3 54 10 0 1 4 06 10 00 5 06 9 00 6 1 4 7 3 6 7 20 t , 1 0 8 3 3 4 5 1 :) 34 4 0;.
10 9 43 4 32 8 44 5 43 7 29 6 49 6 0 1 8 00 4 43 9 08 3 54 1 0 02 4 08 9 59 5 08 8 57 6 1 6 7 33 7 22 b 07 8 3 5 4 49 35 1 0,
11 9 39 4 34 8 41 5 45 7 26 6 52 5 58 8 02 4 41 9 10 3 53 1 0 03 4 09 9 57 5 11 8 55 6 19 7 30 7 25 04 8 37 4 47 9 37 .I 0 7
12 9 36 4 36 0 39 5 48 7 23 6 54 5 55 8 0.4 4 38 9 12 3 52 1 0 04 4 11 9 56 5 1 3 8 52 6 21 7 27 7 27 b 02 0 4 0 4 45 9 38 4 06
13 937 438 036 550 721 656 553 807 436 914 3521005 412 955 515 850 623 724 729 559 842 343 9 3 9 ,106
14 9 36 4 40 8 34 5 52 7 10 6 59 5 5 0 8 09 4 34 9 17 3 52 1 0 06 4 14 9 53 5 17 8 47 6 25 7 21 7 32 5 56 8 44 4 41 9 40 4 06
15 9 34 4 42 8 31 5 55 7 15 7 01 5 47 8 11 4 32 9 19 3 5 1 1 0 06 4 16 9 52 5 19 8 45 6 27 7 18 7 34 j 53 8 47 4 39 9 ill .: 06
16 9 33 4 44 8 29 5 57 7 12 7 03 5 44 8 13 4 30 9 21 3 5 1 1 0 07 4 17 9 50 5 2 2 8 42 6 29 7 15 7 36 5 51 8 49 4 37 9 02 3 26
17 9 32 4 46 R 26 6 00 7 09 7 05 5 42 8 16 4 20 9 23 3 5 1 10 00 4 19 9 40 5 24 8 39 6 32 7 13 7 38 5 48 8 51 4 35 9 43 '1 Oh
18 9 30 4 48 8 24 6 02 7 06 7 08 5 39 8 18 4 26 9 25 3 5 1 1 0 08 4 21 9 47 5 26 8 37 6 34 7 10 7 41 5 45 0 53 4 33 9 44 ;i 06
19 9 29 4 50 8 21 6 05 7 04 7 10 5 36 8 20 4 24 9 27 3 5 1 1 0 08 4 23 9 45 5 28 8 34 6 36 7 07 7 43 5 43 8 56 4 31 9 45 3 06
20 9 27 4 53 8 18 6 07 7 01 7 12 5 33 8 23 4 22 9 29 3 5 1 1 0 09 4 25 9 43 5 3 0 8 31 6 38 7 04 7 45 5 40 8 58 4 29 9 45 4 07
21 9 25 4 55 8 16 6 09 6 58 7 15 5 31 8 25 4 20 9 31 3 5 1 1 0 09 4 26 9 42 5 33 8 29 6 40 7 01 7 48 5 37 9 00 4 20 9 46 4 07
22 9 24 4 57 8 13 6 12 6 55 7 17 5 28 8 27 4 18 9 33 3 5 1 1 0 09 4 28 9 40 5 3 5 8 26 6 43 6 58 7 50 5 35 9 02 4 26 9 46 4 08
23 922 459 810 614 652 719 525 829 416 935 3511009 430 938 537 823 645 655 752 532 904 425 947 408
24 9 20 5 02 8 08 6 17 6 49 7 21 5 23 8 32 4 15 9 37 3 52 1 0 09 4 32 9 36 5 39 8 21 6 47 6 52 7 55 5 29 9 07 4 23 9 47 4 09
25 9 18 5 04 8 05 6 19 6 46 7 24 5 20 8 34 4 13 9 39 3 52 1 0 09 4 34 9 34 5 4 1 8 18 6 49 6 50 7 57 5 27 9 09 4 21 9 47 4 10
26 9 16 5 06 8 02 6 21 6 44 7 2 6 5 17 8 3 6 4 11 9 41 3 53 1 0 09 4 36 9 32 5 44 8 15 6 5 1 6 47 7 59 5 24 9 11 4 20 9 48 4 11
27 9 14 5 09 8 00 6 24 6 41 7 28 5 15 8 3 9 4 10 9 42 3 53 1 0 09 4 38 9 3 0 5 46 8 12 6 53 6 44 8 02 5 22 9 13 4 19 9 48 4 11
28 9 12 5 11 7 57 6 26 6 38 7 3 0 5 12 8 4 1 4 08 9 44 3 54 1 0 08 4 40 9 28 5 48 8 09 6 5 6 6 41 8 04 5 1 9 9 15 4 17 9 48 4 12
29 9 10 5 13 7 56 6 27 6 35 7 33 5 1 0 8 43 4 07 9 46 3 55 1 0 08 4 42 8 07 6 58
9 25 5 5 0 6 38 8 06 5 17 9 17 4 16 9 48 4 13
30 9 08 5 16 6 32 7 3 5 5 07 8 45 4 05 9 47 3 56 1 0 07 4 45 8 04 7 0 0
9 23 5 52 6 35 8 09 5 1 4 9 19 4 15 9 47 4 15
31 906 518 629 737 4 04 9 49 4 47 9 2 1 5 55 8 0 1 8 11 5 1 2 9 47 4 16

Add one hour for Dayliql~tSaving Time if and when in use. I certify that the above data are the result of an accurate and true corn.
putotion by the Nautical Almanac Office, United States Naval Obscrvatory.
a n agency cl~argedby Federal Statute (9 Stat. L 374, 375) w11l1~ h cduly
of mokinq such computations and publishrnq the results

E. W. WOOLAKD
Director Nautical Allntsnac
U. S. NOVCI~Obscivotory
 SUNRISE AND SUNSET AT KING SALMON, ALASKA NO. 1014
STANDARD TIME -OFTHE 150th MERIDIAN WEST.

JAN. FEB. MAR. APR. MAY JUNE JULY AUG. ; SEPT. OCT. NOV. DEC.
DAY 'Rise Set Rise Set Rise Set Rise Set ~ i s e Set Rise Set Rise Set R ~ s e Set Rise Set Rise Set ~ i s i Set Rise Set
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.
1 9 18 3 43 8 34 4 48 7 23 5 56 5 54 7 08 4 31 8 18 3 28 9 22 3 22 9 38 4 15 8 49 5 24 7 27 6 3 1 6 00 7 43 4 36 8 51 3 40
2 9 17 3 45 8 32 4 50 7 20 5 59 5 51 7 11 4 29 8 20 3 27 9 23 3 23 9 37 4 17 8 47 5 27 7 24 6 33 5 57 7 46 4 34 8 53 3 39
3 9 17 3 46 8 29 4 53 7 17 6 0 1 5 48 7 13 4 26 8 22 3 25 9 25 3 24 9 37 4 20 8 44 5 29 7 21 6 35 5 55 7 48 4 31 8 55 3 38
4 9 16 3 48 8 27 4 55 7 14 6 03 5 45 7 15 4 24 8 25 3 24 9 26 3 25 9 36 4 22 8 42 5 31 7 19 6 38 5 52 7 50 4 29 8 57 3 37
5 9 15 3 49 8 25 4 57 7 12 6 06 5 43 7 17 4 21 8 27 3 23 9 28 3 26 9 35 4 24 8 39 5 33 7 16 6 40 5 49 7 53 4 27 8 58 3 36
6 9 15 3 51 8 22 5 00 7 09 6 08 5 40 7 20 4 19 8 29 3 22 9 29 3 28 9 34 4 26 8 37 5 36 7 13 6 42 5 46 7 55 4 24 9 00 3 35
7 9 14 3 53 8 20 5 02 7 06 6 11 5 37 7 22 4 16 8 31 3 21 9 30 3 29 9 33 4 28 8 34 5 38 7 10 6 44 5 43 7 58 4 22 9 02 3 34
8 9 13 3 54 8 18 5 05 7 03 6 13 5 34 7 24 4 14 8 34 3 20 9 3 1 3 30 9 32 4 3 1 8 32 5 40 7 07 6 47 5 40 8 00 4 20 9 07 3 34
9 9 12 3 56 8 15 5 07 7 00 6 15 5 31 7 27 4 12 8 36 3 20 9 33 3 32 9 3 1 4 33 8 30 5 42 7 04 6 49 5 38 8 03 4 18 9 05 3 33
10 9 11 3 58 8 13 5 10 6 57 6 18 5 28 7 29 4 09 8 38 3 19 9 34 3 33 9 29 4 35 8 27 5 44 7 0 1 6 5 1 5 35 8 05 4 15 9 06 3 33
11 9 10 4 00 8 10 5 12 6 55 6 20 5 25 7 31 4 07 8 40 3 18 9 35 3 35 9 28 4 37 8 24 5 47 6 58 6 54 5 32 8 07 4 13 9 07 3 32
12 9 09 4 02 8 08 5 15 6 52 6 22 5 23 7 34 4 05 8 43 3 18 9 36 3 36 9 27 4 40 8 22 5 49 6 55 6 56 5 29 8 10 4 11 9 09 3 32
13 9 07 4 04 8 05 5 17 6 49 6 25 5 20 7 36 4 02 8 45 3 17 9 36 3 38 9 25 4 42 8 19 5 51 6 53 6 58 5 26 8 12 4 09 9 10 3 32
14 9 06 4 06 8 03 5 20 6 46 6 27 5 17 7 38 4 00 8 47 3 17 9 37 3 40 9 24 4 44 8 17 5 53 6 50 7 0 1 5 24 8 14 4 07 9 11 3 32
15 9 05 4 08 8 00 5 22 6 43 6 29 5 14 7 41 3 58 8 49 3 16 9 38 3 4 1 9 22 4 46 8 14 5 55 6 47 7 03 5 21 8 17 4 05 9 12 3 31
16 9 03 4 10 7 58 5 25 6 40 6 32 5 11 7 43 3 56 8 5 1 3 16 9 38 3 43 9 2 1 4 49 8 11 5 58 6 44 7 05 5 18 8 19 4 03 9 13 3 31
17 9 02 4 12 7 55 5 27 6 37 6 34 5 09 7 45 3 54 8 53 3 16 9 39 3 45 9 19 4 5 1 8 09 6 00 6 4 1 7 08 5 15 8 21 4 01 9 14 3 32
18 9 00 4 15 7 53 5 30 6 35 6 36 5 06 7 47 3 52 8 56 3 16 9 39 3 47 9 17 4 53 8 06 6 02 6 38 7 10 5 13 8 24 3 59 9 15 3 32
19 8 59 4 17 7 50 5 32 6 32 6 38 5 03 7 50 3 50 8 58 3 16 9 40 3 49 9 16 4 55 8 03 6 04 6 35 7 12 5 10 8 26 3 57 9 16 3 32
20 8 57 4 19 7 47 5 35 6 29 6 4 1 5 00 7 52 3 48 9 00 3 16 9 40 3 5 1 9 14 4 58 8 0 1 6 06 6 32 7 15 5 07 8 28 3 56 9 16 3 32
21 8 55 4 21 7 45 5 37 6 26 6 43 4 58 7 54 3 46 9 02 3 16 9 40 3 52 9 12 5 00 7 58 6 09 6 29 7 17 5 05 8 31 3 54 9 17 3 33
22 8 54 4 24 7 42 5 39 6 23 6 45 4 55 7 57 3 44 9 04 3 16 9 4 1 3 54 9 10 5 02 7 55 6 11 6 26 7 19 5 02 8 33 3 52 9 17 3 33
23 8 52 4 26 7 39 5 42 6 20 6 48 4 52 7 59 3 42 9 06 3 17 9 4 1 3 56 9 08 5 04 7 52 6 13 6 23 7 22 4 59 8 35 3 51 9 18 3 34
24 8 50 4 28 7 37 5 44 6 17 6 50 4 50 8 0 1 3 40 9 08 3 17 9 4 1 3 58 9 06 5 07 7 50 6 15 6 2 1 7 24 4 57 8 37 3 49 9 18 3 35
25 8 48 4 31 7 34 5 47 6 14 6 52 4 47 8 04 3 39 9 10 3 18 9 40 4 00 9 04 5 09 7 47 6 18 6 18 7 26 4 54 8 39 3 48 9 18 3 35
26 8 46 4 33 7 31 5 49 6 11 6 55 4 44 8 06 3 37 9 11 3 18 9 40 4 03 9 02 5 11 7 44 6 20 6 15 7 29 4 5 1 8 41 3 46 9 18 3 36
27 8 44 4 35 7 28 5 51 6 09 6 57 4 42 8 08 3 35 9 13 3 19 9 40 4 05 9 00 5 13 7 41 6 22 6 12 7 3 1 4 49 8 43 3 45 9 19 3 37
28 8 42 4 38 7 26 5 54 6 06 6 59 4 39 8 11 3 34 9 15 3 19 - 9 40 4 07 8 58 5 16 7 38 6 24 6 09 7 34 4 46 8 45 3 43 9 19 3 38
29 8 40 4 40 7 25 5 55 6 03 7 01 4 36 8 13 3 32 9 17 3 20 9 39 4 09 8 56 5 18 7 36 6 26 6 06 7 36 4 44 8 47 3 42 9 18 3 39
30 8 38 4 43 6 00 7 04 4 34 8 15 3 3 1 9 19 3 2 1 9 39 4 11 8 53 5 20 7 33 6 29 6 03 7 38 4 4 1 8 49 3 4 1 9 18 3 40
3 1 8 36 4 45 5 57 7 06 329 920 4 13 8 51 5 22 7 30 7 41 439 9 18 3 42
A d d one hour for Daylight Saving Time if a n d when in use.

NAUTICAL ALMANAC OFFICE

IJNlTED
-- STATES NAVAL OBSERVATORY
WASHINGTON D C 30?9n
 SUNRISE AND SUNSET AT YAKUTAT, ALASKA NO. 1019
STANDARD TIME OF THE 135th MERIDIAN WEST

JAN. FEB. MAR. MR.- MAY JUNE JULY AUG. SEPT. OCT. "rN0V. DEC. .
DAY'Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Rise Set Riae Set
A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M. A.M. P.M.
1 9 17 3 28 8 30 4 35 7 17 5 475 45 7 01 4 19 8 14 3 13 9 2 1 3 06 9 38 4 02 8 46 5 14 7 2 1 6 23 5 52 7 39 4 25 8 50 3 25
2 9 16 3 30 8 28 4 38 7 14 5 495 42 7 04 4 17 8 16 3 11 9 23 3 07 9 38 4 04 8 44 5 17 7 18 6 26 5 49 7 4 1 4 23 8 52 3 24
3 9 16 3 3 1 8 26 4 40 7 11 5 525 39 7 06 4 14 8 19 3 10 9 25 3 08 9 37 4 07 8 4 1 5 19 7 15 6 28 5 46 7 44 4 20 8 53 3 23
4 915 333 823 443 708 554 536 709 412 8 2 1 309 926 309 936 409 839 5 2 1 713 630 543 746 418 855 322
5 914 334 8 2 1 446 705 557 533 711 409 823 3 0 8 928 310 935 411 836 524 710 633 540 749 415 857 3 2 1
6 9 13 3 36 8 18 4 48 7 02 5 595 30 7 13 4 06 8 26 3 06 9 29 3 12 9 34 4 14 8 34 5 26 7 07 6 35 5 37 7 5 1 4 13 8 59 3 20
7 9 13 3 38 8 16 4 51 6 59 6 015 27 7 16 4 04 8 28 3 05 9 30 3 13 9 33 4 16 8 3 1 5 28 7 04 6 37 5 34 7 54 4 10 9 0 1 3 20
8 9 12 3 40 8 13 4 53 6 56 6 045 24 7 18 4 01 8 3 1 3 04 9 32 3 14 9 32 4 18 8 29 5 3 0 7 01 6 40 5 31 7 56 4 08 9 02 3 19
9 9 11 3 42 8 11 4 56 6 53 6 065 21 7 21 3 59 8 33 3 04 9 33 3 16 9 31 4 21 8 26 5 33 6 58 6 42 5 28 7 59 4 06 9 04 3 18
10 9 09 3 44 8 08 4 59 6 50 6 095 19 7 23 3 56 8 35 3 03 9 34 3 18 9 29 4 23 8 23 5 35 6 55 6 45 5 26 8 01 4 03 9 05 3 18
11 9 08 3 46 8 06 5 01 6 48 6 11 5 16 7 25 3 54 8 38 3 02 9 35 3 19 9 28 4 25 8 2 1 5 37 6 52 6 47 5 23 8 04 4 01 9 07 3 17
12 9 07 3 48 8 03 5 04 6 45 6 14 5 13 7 28 3 52 8 40 3 0 1 9 36 3 21 9 26 4 28 8 18 5 40 6 49 6 49 5 20 8 06 3 59 9 08 3 17
13 9 06 3 50 8 0 1 5 06 6 42 6 16 5 10 7 30 3 49 8 42 3 0 1 9 37 3 23 9 25 4 30 8 15 5 42 6 46 6 52 5 17 8 09 3 57 9 09 3 17
14 9 04 3 52 7 58 5 09 6 39 6 18 5 07 7 33 3 47 8 45 3 00 9 38 3 24 9 23 4 32 8 13 5 44 6 43 6 54 5 14 8 11 3 54 9 10 3 16
15 9 03 3 54 7 55 5 11 6 36 6 21 5 04 7 35 3 45 8 47 3 00 9 38 3 26 9 22 4 35 8 10 5 47 6 40 6 57 5 11 8 14 3 52 9 11 3 16
16 9 01 3 56 7 53 5 14 6 33 6 23 5 0 1 7 37 3 42 8 49 3 00 9 39 3 28 9 20 4 37 8 07 5 49 6 37 6 59 5 08 8 16 3 50 9 12 3 16
17 9 00 3 59 7 50 5 16 6 30 6 26 4 58 7 40 3 40 8 51 3 00 9 40 3 30 9 18 4 39 8 04 5 5 1 6 34 7 01 5 06 8 18 3 48 9 13 3 16
18 8 58 4 0 1 7 47 5 19 6 27 6 28 4 55 7 42 3 38 8 54 2 59 9 40 3 32 9 16 4 42 8 02 5 53 6 31 7 04 5 03 8 2 1 3 46 9 14 3 16
19 8 56 4 03 7 45 5 22 6 24 6 30 4 53 7 45 3 36 8 56 2 59 9 41 3 34 9 14 4 44 7 59 5 56 6 28 7 06 5 00 8 23 3 44 9 15 3 17
20 8 55 4 05 7 42 5 24 6 2 1 6 33 4 50 7 47 3 34 8 58 2 59 9 41 3 36 9 13 4 46 7 56 5 58 6 25 7 09 4 57 8 26 3 42 9 16 3 17
2 1 8 53 4 08 7 39 5 27 6 18 6 35 4 47 7 50 3 32 9 00 3 00 9 41 3 38 9 11 4 49 7 53 6 00 6 22 7 11 4 54 8 28 3 4 1 9 16 3 17
22 8 51 4 10 7 36 5 29 6 15 6 38 4 44 7 52 3 30 9 02 3 00 9 41 3 40 9 09 4 51 7 50 6 03 6 19 7 14 4 52 8 30 3 39 9 17 3 18
23 8 49 4 13 7 34 5 32 6 12 6 40 4 4 1 7 54 3 28 9 04 3 00 9 41 3 42 9 06 4 53 7 47 6 05 6 16 7 16 4 49 8 33 3 37 9 17 3 19
24 8 47 4 15 7 3 1 5 34 6 09 6 42 4 38 7 57 3 26 9 06 3 01 9 41 3 44 9 04 4 56 7 45 6 07 6 13 7 19 4 46 8 35 3 35 9 17 3 19
25 8 45 4 18 7 28 5 37 6 06 6 45 4 36 7 59 3 24 9 08 3 01 9 41 3 46 9 02 4 58 7 42 6 10 6 10 7 21 4 43 8 37 3 34 9 18 3 20
26 8 43 4 20 7 25 5 39 6 03 6 47 4 33 8 02 3 22 9 10 3 02 9 41 3 49 9 00 5 00 7 39 6 12 6 07 7 24 4 41 8 39 3 32 9 18 3 2 1
27 8 41 4 23 7 22 5 42 6 00 6 50 4 30 8 04 3 20 9 12 3 02 9 41 3 51 8 58 5 03 7 36 6 14 6 04 7 26 4 38 8 41 3 31 9 18 3 22
28 8 39 4 25 7 19 5 44 5 57 6 52 4 27 8 06 3 19 9 14 3 03 9 40 3 53 8 56 5 05 7 33 6 16 6 01 7 29 4 35 8 44 3 29 9 18 3 23
29 8 37 4 28 7 18 5 46 5 54 6 54 4 25 8 09 3 17 9 16 3 04 9 40 3 55 8 53 5 07 7 30 6 19 5 58 7 31 4 33 8 46 3 28 9 18 3 24
30 8 35 4 30 5 5 1 6 57 4 22 8 11 3 16 9 18 3 05 9 39 3 57 8 51 5 10 7 27 6 2 1 5 55 7 34 4 30 8 48 3 27 9 18 3 25
31 832 4 33 5 48 6 59 3 14 9 20 4 00 8 49 5 12 7 24 7 36 4 28 9 17 3 26
Add one hour for Daylight Saving Time if a n d when in use. I certify that the above d a t a a r e the result of a n a c c u r a t e a n d true com-
putation by the Nautical A l m a n a c Office, United States Naval Observatory,
a n agency charged by Federal Statute (9 Stat. L 374, 375) with the duty
of making such computations a n d publishing the results.

E. W. WOOLARD
Director Nautical Almanac
U. S. Naval Observatory
The Alaska Department of Fish and Game administers all programs and activities free from discrimination
based on race, color, national origin, age, sex, religion, marital status, pregnancy, parenthood, or disability.
The department administers all programs and activities in compliance with Title VI of the Civil Rights Act
of 1964, Section 504 of the Rehabilitation Act of 1973, Title II of the Americans with Disabilities Act of
1990, the Age Discrimination Act of 1975, and Title IX of the Education Amendments of 1972.

If you believe you have been discriminated against in any program, activity, or facility, or if you desire
further information please write to ADF&G, P.O. Box 25526, Juneau, AK 99802-5526; U.S. Fish and
Wildlife Service, 4040 N. Fairfax Drive, Suite 300 Webb, Arlington, VA 22203 or O.E.O., U.S.
Department of the Interior, Washington DC 20240.

For information on alternative formats for this and other department publications, please contact the
department ADA Coordinator at (voice) 907-465-6077, (TDD) 907-465-3646, or (FAX) 907-465-6078.