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Monitoring Methods
Phosphorus
Introduction
Forms of
Phosphorus
Soluble
Reactive P (SRP)
Soluble P (SP)
Particulate P
Total P (TP)
Collection
and Analysis
Sampling
and Preservation
Limits of Detection
Digestion Methods
Interferences
Recommendations
Literature
Cited
Contact the Dip-In at:
dipin@kent.edu
Web Site Updated:
June 09, 2008
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Portions of the following has been excerpted
with permission from copyrighted material. Material in this
section should not be reproduced without specific permission of the
North American Lake Management Society (www.NALMS.org).
The reader is encouraged to read the original material for more details
on phosphorus and phosphorus analytical methods.
Carlson,
R.E. and J. Simpson. 1996.
A Coordinator’s Guide to Volunteer Lake Monitoring Methods.
North American Lake Management Society.
96 pp.
Phosphorus
Introduction
Phosphorus is probably the most studied plant nutrient in freshwater
aquatic sciences. It is often found to be (and more often inferred as)
the nutrient that limits the growth and biomass of algae in lakes and
reservoirs. Whether this nutrient is as universally limiting as once
believed is debatable, but certainly there is substantial evidence of
its importance in many lakes. Numerous correlations and regressions have
been constructed linking phosphorus, especially total phosphorus, with
variables such as algal chlorophyll, algal weight, and productivity.
Because of its possible importance in limiting the growth and biomass of
algae and because of the numerous empirical models available for
phosphorus, it is an important addition to the list of variables to be
measured in volunteer programs
Forms of Phosphorus
Phosphorus in natural waters is divided into three component parts:
soluble reactive phosphorus (SRP), soluble unreactive or soluble organic
phosphorus (SUP) and particulate phosphorus (PP) (Rigler 1973). The sum
of SRP and SUP is called soluble phosphorus (SP), and the sum of all
phosphorus components is termed total phosphorus (TP). Soluble and
particulate phosphorus are differentiated by whether or not they pass
through a 0.45 micron membrane filter. The phosphorus fractions are
illustrated below:
Soluble Reactive Phosphorus (SRP)
This phosphorus fraction should consist largely of the inorganic
orthophosphate (PO4) form of phosphorus. Orthophosphate is
the phosphorus form that is directly taken up by algae, and the
concentration of this fraction constitutes an index of the amount of
phosphorus immediately available for algal growth.
In phosphorus limited situations, the concentration of this form should
be very low to undetectable (<5 ug/L). As concentrations of
orthophosphate (as reflected in the SRP fraction) increase, it can be
inferred that phosphorus is either not needed by the algae or that it is
being supplied at rates faster than it can be taken up by the biota.
Measurement of SRP can be used as an indicator, albeit a potentially
inaccurate one, of the degree of phosphorus limitation of the algae.
At one time SRP was called "dissolved inorganic phosphorus."
This terminology was changed to "soluble reactive phosphorus"
(Rigler 1964; Strickland and Parsons 1965) to reflect a more realistic
interpretation of what forms of phosphorus were found in this fraction.
The terms "soluble" and "reactive" were chosen
instead because this form of filtered phosphorus was neither necessarily
dissolved nor necessarily inorganic.
The term "reactive" is used to indicate that the phosphorus in
the SRP fraction is not solely inorganic phosphorus, but could include
any form of phosphorus, including some organic forms, that react with
the reagents. Some organic forms apparently do hydrolyze and react under
the conditions of this test, while some forms of inorganic phosphorus
(polyphosphates), in fact, do not react. There is a continuing debate as
to what extent SRP represents solely the ortho form of phosphorus or is
biologically available (Nürnberg and Peters 1984). The actual
composition of SRP probably varies with the nature of the water body.
The "soluble" fraction does not necessarily contain only
dissolved phosphorus forms: the phosphorus containing material in the
soluble fraction is dependent on the porosity and characteristics of the
filter used. Typically, a 0.45 micron cellulose (Millipore) filter is
used as a standard. This filter excludes most particulates, but
colloidal phosphorus may be present in the filtered fraction.
Some analytical protocols use glass fiber filters instead of a membrane
filter. Using a glass filter increases the amount of particulate
material that passes through the filter and therefore increases the
amount in the "soluble" fraction. Small particulates,
including very small algae and bacteria will be present in the filtered
sample. Whether or not they become represented as SRP will depend on the
extent that they react with the reagents. Glass fiber filters are less
costly than the membrane filters, and, more important, are used in a
number of other tests such as chlorophyll, particulate carbon, and
suspended solids. There is no consensus as to whether the necessity of
separating the soluble fraction from all possible particulate forms is
more important than analytical consistency between variables.
Some researchers in phosphorus dynamics use Nucleopore™ filters
instead of either membrane or glass fiber filters. These filters
partition the water into size fractions more accurately than do membrane
or glass fiber filters, and therefore allow much better separation of
size related phosphorus forms. Nucleopore filters also do not contain
phosphorus, which is a contaminant in membrane filters (Peters, personal
communication). However, these filters are more expensive than either
membrane or the glass fiber filters, and the increased control of size
may not be important to a general monitoring effort.
Soluble Unreactive Phosphorus (SUP)
This phosphorus fraction contains filterable phosphorus forms
that do not react with the phosphorus reagents under the time and
conditions of the test. It is measured as the difference between soluble
phosphorus (SP) and SRP. The compounds in the SUP fraction are organic
forms of phosphorus and chains of inorganic phosphorus molecules termed
polyphosphates. The size of this fraction relative to the other
phosphorus fractions is highly dependent on the type of filter used to
separate the soluble from particulate fractions.
A number of organic phosphorus molecules have been identified, but two
main classes seem to predominate in natural waters. The first are low
molecular weight compounds, apparently derived from algal and bacterial
metabolism, which release orthophosphate upon treatment with alkaline
phosphatase. These compounds do not react with the phosphorus reagents
without prior digestion (Franko and Heath 1979). The second are colored,
large molecular weight compounds, perhaps phosphorus bound in humic
complexes, which release orthophosphate in the presence of ultraviolet
light.
These compounds may (Downes and Paerl 1978) or may not (Franko and Heath
1979) react without prior digestion and could be measured as SRP. These
organic forms apparently form a pool of phosphorus for algal and
bacterial growth, but they must be first converted to orthophosphate,
either by enzymes or by UV light, before they are available for uptake
by the biota. Although the phosphorus in the pool appears to be highly
dynamic, the total amount of phosphorus in this fraction seems to be
fairly stable seasonally in lakes with long residence times (Rigler
1964). However, changes in the pool might signal shifts in either the
availability of this phosphorus form to algae or bacteria or shifts in
the biotic community's ability to use this form, or more likely, runoff
variability into the lake.
Soluble Phosphorus (SP)
This form is measured after the digestion of the filtrate and
should contain all filterable forms of phosphorus, both organic and
inorganic that are converted to orthophosphate by the digestion process.
However, the amount of phosphorus in this filterable pool is highly
dependent on the filter used. The larger the effective pore size of the
filter, the more particulate material that will pass through the filter,
be digested, and be considered "soluble."
Rigler (1964) estimated the percent of phosphorus that would be
considered part of the soluble phosphorus pool of three lakes, using 7
different separation techniques and filter sizes. Particulates were
removed using a Foerst centrifuge, or by passing the water through 3
layers of Whatman #44 paper filters, or through 5.0, 1.2, 0.45, 0.22,
and 0.1 micron Millipore filters. Considerable differences in the
percent soluble phosphorus were found as the pore size decreased.
 |
The effect of particle separation method or filter pore
size on the estimate amount of soluble phosphorus in lakewater
(mean plus or minus standard error). Data from Rigler 1964. |
Deviation from the 0.45 micron membrane standard will have a significant
effect on the soluble phosphorus concentration, and, because it is
calculated as the difference between SP and SRP, soluble organic
phosphorus will be affected as well. SRP may or may not be affected,
depending on the extent to which phosphorus in these particulates are
released during the analytical procedure. Particulate Phosphorus
This fraction of phosphorus contains all material, inorganic and
organic, particulate and colloidal, that was captured on the filter.
Typically, particulate forms will contain bacteria, algae, detritus, and
inorganic particulates such as clays, smaller zooplankton, and
occasionally, larger zooplankton, sediments, or large plant material.
Particulate phosphorus can be measured either by filtering a known
volume of water through a membrane filter and then digesting the filter,
or it can be obtained by subtraction of total soluble phosphorus (SP) from the total phosphorus
concentration (TP). The filtration method allows the analyst to concentrate
samples from low particulate waters, thereby increasing the sensitivity
of the test, but it also increases the probability that large particles,
such as zooplankton will be captured as well. Caution must be taken so
that adequate oxidant is present to completely digest the sample.
Large zooplankton, suspended sediments, or large plant remains may be
captured on the filter. These latter particulates are generally
considered to be contaminants rather than normally occurring portions of
the fraction. Although suspended sediments, especially if stirred up
during the sampling process or stray leaf or plant remains should be
considered a contaminant, zooplankton in the sample present an
interpretational problem, especially in low phosphorus situations.
Zooplankton can significantly increase the phosphorus concentration in
the water sample. Chamberlin (1968), as cited in Rigler (1973), found
that a single individual of the zooplankter, Daphnia, containing
0.19 g P, would increase the total P in a 50 ml water sample by 4 ug/L.
Prepas and Rigler (1982) found that one phantom midge larva (Chaoborus)
can add 100 ug/L to a 50 ml sample.
Zooplankton and other large particulates cause a problem to particulate
and total phosphorus analyses because they are probably inadequately
sampled, and therefore give an inaccurate phosphorus estimation. If
particulate P is determined by subtraction, zooplankton are probably not
sampled accurately or randomly in a 10 or 50 ml aliquot used in the
total phosphorus determination. The presence or absence of the few
zooplankton that may be captured would cause a significant overestimate
or underestimate of the limnetic phosphorus pool if zooplankton are
considered part of that pool. Although it might seem that filtering and
digesting the filter would increase the precision of the particulate
measurement, its accuracy is still in doubt because the tools used for
collecting total phosphorus from the lake (water samplers or tube
samplers) will generally under sample zooplankton populations. By either
method, the limnetic phosphorus pool will be inaccurately measured.
Certainly, the larger size fraction of the limnetic phosphorus pool
(zooplankton and fish) are generally ignored in most studies, although
they may make up a sizeable fraction of the total phosphorus in the
water. For example, Prepas and Rigler (1982) found that the phosphorus
fraction greater than 250 microns (which might contain larger algae as
well as zooplankton) constituted between 14 and 28% of the total
phosphorus in the epilimnion on an Ontario lake. Fish can also
constitute a sizable proportion of the total phosphorus in a lake.
Kitchell et al. (1975) reported that zooplankton constituted 18% and
fish, 74%, of the total pelagic zone phosphorus in Lake Wingra,
Wisconsin. Generally these larger size fractions do not interfere with
the use of total phosphorus in empirical models because the data used in
model construction probably ignored these size fractions as well.
However, if the data were used to estimate lake phosphorus content for a
nutrient budget, then it is possible that the content could be seriously
underestimated without consideration of the larger fractions.
The ideal would be to have a size fractionated phosphorus, where
phosphorus would be sampled by methods appropriate for each size
fraction. In the absence of the ideal, which is probably the case of
volunteer programs, particulate phosphorus is probably best measured by
subtraction from total phosphorus rather than filtration. In this case,
the particulate value will be influenced by the pore size of the filter
used. Volunteers could be cautioned to try to exclude large particles
such as leaves. Total Phosphorus
This form incorporates the total of all filterable and particulate
phosphorus forms mentioned above. It is probably the most often analyzed
fraction of phosphorus because it is used in a wide variety of empirical
models relating phosphorus to a wide variety of limnological variables
(Peters 1986); and the link between phosphorus loading estimates and
phosphorus content in the lake.
Considering the wide variety of materials that might constitute
"total phosphorus," it is remarkable that total phosphorus
correlates well with any other single variable, especially an algal
variable such as chlorophyll. The chlorophyll molecule itself does not
even contain phosphorus. For such correlation to be strong, chlorophyll
must be related to phosphorus containing compounds in algal cells.
Variations in TP chlorophyll relationships come, in part, from
variations in the amount of chlorophyll generated per unit of total
phosphorus in algal cells. As cells vary in their phosphorus or
chlorophyll content, the TP/chlorophyll ratio will vary. All other
phosphorus forms found in the water must be constant, negligible, or at
least change as a function of chlorophyll.
For example, SRP is generally low in phosphorus limited situations while
SUP may be relatively constant. Increases in SRP or variations in SUP
would adversely affect chlorophyll phosphorus relationships. Forms of
particulate phosphorus, other than algal phosphorus, must also be
negligible, constant, or change as a function of chlorophyll.
Chlorophyll would be expected to relate best to total phosphorus in
situations where there are negligible amounts of clays, suspended
sediments, and detritus, where phosphorus limits algal biomass, and in
natural lakes that have sufficient depth that bottom sediments are not
resuspended.
"Total" phosphorus is largely defined on the basis of how much
phosphorus in its various forms will be oxidized into orthophosphate by
a specific oxidant. Different analytical tests used for the digestion
and analysis of phosphorus may change the amount of phosphorus reported.
It is important to remember that all these phosphorus designations are
functionally defined, and the coordinator must be very cautious about
modifying phosphorus analysis procedures. It is possible that some
modifications would alter the results significantly and therefore limit
the use of the data. It is also important to remember that all of the
empirical equations are based on certain methodologies (hopefully, the
same ones). Deviations from these methods could introduce error in these
empirical relationships (Griesbach and Peters 1991). Phosphorus
Collection and Analysis
Sampling and Preservation
Although field kits that measure phosphorus potentially could be used by
volunteers, the limits of detection of the kits that use visual
comparators are too high to be useful in lake monitoring programs. A
sensitive spectrophotometer is necessary to measure the phosphorus
levels found in most lake situations. Because of this, volunteers will
most often be asked to collect samples for later analysis in a
laboratory. Prime concerns should be that the volunteer collects the
sample correctly and that the sample is stored in a manner that will
allow subsequent analysis with minimal changes in either the absolute
concentration or phosphorus form.
Collection of the water sample for subsequent analysis can be done with
any of the sampling techniques discussed in the earlier chapter on
sampling techniques. Surface (0.5 m) samples are usually used in trophic
state determinations, but see the chapter on sampling for a discussion
of alternative sampling procedures. Whatever the technique chosen, the
phosphorus sample should be taken from the same water sample that is
used for chlorophyll. Hypolimnetic phosphorus samples can also be taken,
but as the Wisconsin program discovered, it is difficult to obtain a
near sediment sample without disturbing the sediments. Care should be
taken that the sampler is clean, without any dirt or material from
earlier excursions clinging to the inside surface. Several rinsings with
lake water prior to use may be beneficial.
Once collected, the water should be poured into a pre-cleaned sample
container. It would be best that this sample container be cleaned and
supplied by the program rather than relying on the volunteer to supply
or to clean the container. Cleaning should consist of washing the
containers in phosphorus free detergent. (usually dish washing
detergent, not dishwasher detergent, is phosphorus free), several tap
water rinses, a rinse in dilute HCl (Standard Methods APHA 1989,
recommends hot dilute HCl) and then several rinses in distilled water.
If care is not taken in the cleaning of the containers, subsequent care
becomes meaningless. Standard Methods (APHA 1989) also recommends that
plastic jars not be used unless the sample is frozen because the
phosphorus may be adsorbed onto the walls of the plastic container. If
the sample is to be poured from the container in the laboratory prior to
analysis, the sample should be preserved prior to storage to prevent any
transformations (uptake by algae or bacteria) or sorption to the walls
of the container during storage. If only total phosphorus will be
measured, then the sample can be preserved with one ml of HCl per liter
or by freezing the sample.
An alternative method used in several Midwest program is to measure
and pour an exact amount of the sample into a pre-cleaned borosilicate
bottle. Later, digestion is done in this same container. The assumption
is that preservation is not necessary because any material adsorbed onto
the side of the container will be stripped off during the digestion
process.
Griesbach and Peters (1991) found that TP in unpreserved, unfiltered
samples stored frozen was stable for up to 12 months, if stored in the
tubes in which they would eventually be analyzed. At room temperature,
samples could safely be stored for a month, again, if stored in the
tubes in which they would be analyzed. If other forms are to be
analyzed, filtration prior to preservation seems to yield the best
results. If clean filtration equipment can be assured, there is no
reason that the volunteers could not do the filtration to differentiate
between soluble and particulate phosphorus. If glass fiber filters were
used, then the filtrate obtained from the chlorophyll filtration could
be used for the soluble phosphorus fraction. The problem of volunteer
obtained filtered samples is that of preservation. Standard Methods
(AHPA 1989) recommends that the sample should be immediately frozen at
or below 10 °C or preserved with HgCl2. Mercuric chloride is
extremely hazardous and should never be given to volunteers.
Griesbach and Peters (1991) found that filtered samples kept at room
temperature could be analyzed for SRP and SP within a week, if stored in
the tubes in which they would be eventually analyzed. They did not
recommend freezing samples for analysis of the phosphorus fractions.
Others report maximum holding times for SRP analysis from less than 24
hours to a week.
Limits of Detection
Although a number of analytical tests exist for the measurement
of phosphorus, the ascorbic acid method described in Standard Methods
(AHPA 1989) is probably the most commonly used test. In this test, the
molybdate reagent reacts with orthophosphate producing phosphomolybdic
acid, which forms the colored molybdenum blue upon reduction with
ascorbic acid. While the compound appears blue, the peak absorbance at
885 nm is in the infrared region. Absorbance is linearly related to
concentrations by Beers Law, and this test detects phosphate
concentrations of 5 to 1300 µg/L with a cuvette pathlength of l cm.
It is important to have an appropriately defined phosphorus detection
limit. For example, a TP detection limit of 50 ug/L will not be adequate
for a great deal of limnological efforts. Some phosphorus tests are
designed for soils or sewage analysis and their minimum detection limits
are inappropriate for limnological phosphorus analysis. As a
matter of protocol, detection limits of 10 or 5 ug/L (or lower) will
likely be necessary, to have adequate resolution for typical stream and
lake monitoring. Using a rule of thumb that the detection level is about
two times the standard deviation of replicate blanks, the lowest level
of quantification is about five times this standard deviation. Hence,
the level of quantification better defines the lower levels of
identifiable TP concentrations, which occur with better (lower)
detection levels.
Lower limits of detection can be obtained with a longer pathlength
cell. Griesbach and Peters (1991) found that TP concentrations (after
digestion) were relatively insensitive to even substantial changes in
the concentration of the phosphorus reagents, and high precision was
unnecessary when preparing the reagents. Results should be reported as
the concentration of phosphorus, not phosphate, present in the water.
Since orthophosphate is the form measured in the test, the results can
be presented as PO4P.
Digestion Methods
As mentioned earlier, total phosphorus and total soluble phosphorus
require a digestion step prior to the measurement of the orthophosphate
form. There is no one perfect oxidant. The acid persulfate test that is
often used is a compromise method that extracts most but not all
phosphorus, but is a safer technique than others. Perchloric acid
digestion, for example, will extract phosphorus from soils, but is a
more complicated procedure and requires special facilities and safety
precautions. Harwood et al. (1969) compared three different digestion
methods (magnesium nitrate fusion, persulfate digestion, and peroxide
digestion) on five types of liquid samples but found no significant
differences in the resulting phosphorus concentrations.
Prepas and Rigler (1982) found that using persulfate alone produced
less variable results than did perchloric acid or potassium persulfate
with sulfuric acid (Jeffries et al. 1979). Standard Methods (1989)
recommends the use of acid together with persulfate. It may be that the
acid aids in the digestion of phosphorus from clays and other soil
particles. Griesbach and Peters (1991), in an extensive examination of
phosphorus techniques, found that the total phosphorus values resulting
from varying the amount of persulfate added was not important within a
range of 0.5 to 0.8 g persulfate per 40 ml sample. They suggested that
weighing of each sample was not necessary; using a simple, volumetric
scoop of persulfate was adequate. They also found that digestion time
and whether the samples were autoclaved or boiled had no significant
effect on the final phosphorus concentration. They suggest that boiling
is more time-consuming than autoclaving but can be used if no autoclave
is available.
Interferences
Several substances can interfere with the phosphorus analysis. Arsenate,
which has been used to control algae and aquatic macrophytes, and high
concentrations of silica can interfere. Where there is considerable
humic color or clay turbidity, it may be necessary to run
color/turbidity blanks for each sample. Keep a close watch on the
physical characteristics of water samples. The phosphorus test is
sensitive to trace amounts of contamination as is normally present in
tap water, on fingers, in soap and some detergents, and in buffers or
other reagents; be sure to clean glassware thoroughly, and observe
maximum cleanliness in the field collection and in the laboratory. It is
very important to run distilled water blanks and standards, keeping
records of the absorbance in order to detect changes in distilled water
quality or reagents.
Recommendations
Based on the theory discussed above and on the methods, observations,
and comments by existing programs, the following recommendations are
made.
1. Based on ease of sampling, questions about storage, and utility of
the information, total phosphorus should be the primary form of
phosphorus measured in volunteer programs.
2. Coordinators should first utilize surface (0.5 m) or an integrated
epilimnetic samples in their programs, especially if chlorophyll and
Secchi depth are to be measured at the same time. A second total
phosphorus sample taken in the hypolimnion would add another dimension
to the sampling program.
3. The sampling program should have samples taken during spring
turnover so that an estimate can be made of the total phosphorus content
of the lake. An alternative is to take a detailed vertical profile
during summer stratification.
4. Although preservation of total samples is recommended, there are
questions about the safety of having the volunteers handle strong acids.
Coordinators should consider freezing the samples or having the
digestion performed in the sample container itself.
5. Volunteers should be given pre-cleaned, acid washed sample
containers. Manipulation of the sample and the container by the
volunteer should be minimized. Phosphorus differences can rapidly set up
in a large container as particles settle. The volunteers should be
instructed to shake the sample well before pouring. It would be better
to pour directly from the water sampling device into the sample
container rather than into a second container so that settling in the
container and possible contamination is minimized.
6. Soluble reactive phosphorus can also provide valuable information,
but requires field filtration through a pre-cleaned membrane or glass
fiber filter under clean circumstances. There are also questions of its
stability with storage. If done, samples should be rapidly frozen and
brought to the laboratory for analysis. Thought should be given to using
a syringe filtration system so that the volunteers would not handle the
filters.
7. Research remains to be done as to the importance of the type and
porosity of the filter on SRP analysis.
Literature Cited
Betz, C.R., P.J. Howard, and P.J. Anderson. 1990. Wisconsin's expanded
self-help lake monitoring program: Results of the 1990 pilot program
expansion. Presented at the International Symposium of the North
American Lake Management Society.
Canfield, D.E. Jr. 1991. Assessment of water quality in the lakes of
north and central Florida. Project Completion Report.
Chamberlain, W.M. 1968. A preliminary investigation of the nature and
importance of soluble organic phosphorus in the phosphorus cycle of
lakes. Ph.D. Thesis, University of Toronto, Canada.
Downes, M.T. and H.W. Paerl. 1978. Separation of two dissolved
reactive phosphorus fractions in lakewater. J. Fish. Res. Board Can. 35:
1636-1639.
Franko, D.A., and R.T. Heath. 1979. Functionally distinct classes of
complex phosphorus compounds in lake water. Limnol. Oceanogr. 24:
463-473.
Griesbach, S.J. and R.H. Peters. 1991. The effects of analytical
variations on estimates of phosphorus concentration in surface waters.
Lake and Reservoir Management. 7: 97-106.
Harwood, J.E., R.A. Van Steederen, and A.L. Kahn. 1969. A comparison
of some methods for total phosphate analyses. Water Res. 3: 425-432.
Jeffries, D.S., F.P. Diken, and D.E. Jones. 1979. Performance of the
autoclave digestion method for total phosphorus analysis. Water Res. 13:
275-279.
Kitchell, Koonce, and Tennis. 1975. Phosphorus flux through fishes.
Verh. Internat. Verein Limnol. 10: 2478-2484.
Nürnberg, G. and R.H. Peters. 1984. Biological availability of
soluble reactive phosphorus in anoxic and oxic freshwaters. Can. J.
Fish. Aquat. Sci. 41: 757-765.
Peters, R.H. 1986. The role of prediction in limnology. Limnol.
Oceanogr. 31: 1143-1159.
Prepas, E.E. and F.H. Rigler. 1982. Improvements in quantifying the
phosphorus concentration in lake water. Can. J. Fish. Aquat. Sci. 39:
822-829.
Redfield, A.C. 1958. The biological control of chemical factors in
the environment. Amer. Sci. 46: 205-221.
Rigler, F.H. 1973. A dynamic view of the phosphorus cycle in lakes.
In: E.J. Griffith, A. Beeton, J.M. Spencer, and D.T. Mitchell (Eds),
Environmental Phosphorus Handbook. John Wiley & Sons.
Sakamoto, M. 1966. Primary production by phytoplankton community in
some Japanese lakes and its dependence on lake depth. Arch. Hydrobiol.
62: 486-489.
Strickland, J.D. H. and T.T. Parsons. 1965. A manual of sea water
analysis. Bull. 125. Fish. Res. Bd. Canada.
Vollenweider, R.A. 1976. Advances in defining critical loading levels
for phosphorus in lake eutrophication. Mem. Ist. Ital. Idrobiol. 33:
53-83.
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