WATER POLLUTION CONTROL RESEARCH SERIES • 16010 EHC 12/71
Eutrophication In Coastal Waters:
Nitrogen As A Controlling Factor
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
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results and progress in the control and abatement of pollution
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(Water), Research Information Division, R&M, Environmental
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EUTROPHICATION IN COASTAL WATERS: NITROGEN AS A CONTROLLING FACTOR
by
Institute of Marine Resources
Scripps Institution of Oceanography
University of California, San Diego
La Jolla, California 92037
for the
ENVIRONMENTAL PROTECTION AGENCY
Project #16010 EHC
December 1971
For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C. 20402 - Price 70 cents
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EPA Review Notice
This report has been reviewed by the Environmental Protection
Agency and approved for publication. Approval does not
signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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ABSTRACT
The role of southern California coastal sewage outfalls in
the eutrophication of local seawater was investigated. The
outfall effluents have a measureable influence on standing
stocks of phytoplankton, and on primary production. Two
cruises were undertaken, in July, 1970 and June, 1971. Kine-
tic parameters for the assimilation of ammonium, nitrate and
urea were determined at the outfall sites using ^%-labelled
substrates. These parameters will be useful for simulation
models of phytoplankton growth as influenced by local sewage
effluents.
The utilization of various forms of nitrogen by phytoplankton,
mechanisms and rates of nitrogen assimilation, and enzymes of
nitrogen assimilation were investigated in laboratory cultures.
Ammonium and nitrate assimilation were found to vary from day
to night as does the capacity for photosynthesis when cultures
were grown on light-dark cycles simulating natural illumination.
In fitting data on rates of nitrogen assimilation vs. concentra-
tion of nitrogen to the Michaelis-Menten equation, modified to
describe nutrient uptake, it was found that the maximum growth
rate was a variable while the saturation constant was uniform
over a range of dilution rates of N-limited chemostat cultures.
The chemical composition of phytoplankton, particularly ratios
of carbon/chlorophyll and carbon/nitrogen, varied with dilution
rate in reproducible ways. By varying the dilution rate of such
cultures one seems to regulate the degree of nitrogen-deficiency
of the phytoplankton.
This report was submitted in fulfillment of Project Number
16010 EHC under the sponsorship of the Water Quality Office,
Environmental Protection Agency.
iii
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CONTENTS
Section
I Conclusions
II Recommendations
III Introduction
IV Methods
V Shipboard Studies of Eutrophication around
Southern California Coastal Sewage Outfalls,
July, 1970 and June, 1971.
VI Diel Periodicity in Nitrogen Assimilation
VII Kinetics of Nitrogen Assimilation
VIII Influence of the Rate of Nitrogen Input on
The Chemical Composition of Phytoplankton
IX Comparison of Methods of Measuring Nitrogen
Assimilation Rate
X Discussion
XI Acknowledgements
XII References
XIII Publications
Page
1
3
5
9
13
25
35
47
55
59
61
67
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FIGURES
PAGE
Map showing the location of the White Point 14
(station number 19) and Point Loma (station
numbers 12, 18) sewage outfalls and the control
station of La Jolla, California (station numbers
1, 6, 10). The station numbers refer to the
1970 cruise and are not used further in this
report.
The rate of ammonium assimilation by natural 23
marine phytoplankton vs. concentration of am-
monium in the water. Circles represent measure-
ments at the Pt. Loma outfall, triangles are
for White Pt. data and squares are for a control
station off La Jolla, California.
The rate of nitrate assimilation by natural 24
marine phytoplankton vs. nitrate concentration
in the water. Circles: Pt. Loma; triangles:
White Pt.; square; off La Jolla, California;
diamonds: a second station at Pt. Loma.
Chlorophyll ^ concentration in shipboard cul- 26
tures of natural marine phytoplankton during
growth with nitrate, ammonium, or urea as the
nitrogen source. Abrupt changes in slope
represent depletion of vitamin B..2 from the
culture media.
Concentration of diatom cells in the shipboard 27
cultures. Cell division appears to take place
in the late afternoon and early evening.
Diel periodicity in the rate of nitrate (upper), 28
ammonium (middle), and urea assimilation of the
shipboard cultures. The capacity for photosyn-
thesis (measured under artificial light) is
also plotted (solid symbols).
Diel periodicity in the rate of phosphate 29
assimilation in the shipboard cultures.
Diel periodicity in the activity of three enzymes 31
of nitrogen assimilation in an N-limited chemostat
culture of Coccolithus huxleyi grown on a light-
dark cycle. Upper curve: nitrate reductase;
VI
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PAGE
middle: glutamic dehydrogenase measured with
NADH; lower: nitrite reductase. Activity of
the latter appears to be out of phase with
nitrate reductase and glutamic dehydrogenase
activity, but in phase with photosynthetic
capacity (see Table 6).
9 Diel periodicity in ambient concentration of 34
nitrate and in the velocity of nitrate assimi-
lation (upper curves) and in the concentration
of ammonium and rate of ammonium assimilation
in an N-limited chemostat culture of Skeletonema
costatum grown on light-dark cycles (lower curves).
10 Variation in the apparent value of y with dilu- 41
tion rate, y, based on phytoplankton growth in
N-limited chemostat cultures. The value of
ym' (the apparent v^) was calculated from equa-
tion 1 (see text). Circles: Gymnodinium
splendens; triangles: Coccolithus huxleyi;
squares: mixed culture of _C. huxleyi with
Skeletonema costatum; diamonds: Leptocylindrus
danicus.
11 Ratios of carbon to nitrogen in phytoplankton 44
grown j.n N-limited chemostat cultures at dif-
fe rat dilution rates. The abscissa represents
the Dilution rate, y, as a fraction of the dilu-
tion rate at washout of the culture, ym. Circles:
Gymnodinium splendens; triangles: a mixed cul-
ture of Coccolithus huxlevi with Skeletonema
costatum; squares: Leptocylindrus danicus.
12 Ratios of carbon to chlorophyll ji of phytoplankton 45
grown in N-limited chemostat cultures. Symbols
as in Fig. 10.
vii
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TABLES
°^ Page
1 Depth profiles of ammonium concentration off south- 15
ern California in June, 1971.
2 Photosynthetic carbon assimilation, nitrogen assimi- 16
lation and chlorophyll a_ at coastal seawater stations
off southern California.
3 Variation in the chlorophyll a_ content of the upper 17
50 meters of water about the Pt. Loma and White Pt.
outfalls and about a control station off La Jolla,
California.
4 Relative importance of urea as a source of nitrogen 19
for phytoplankton growth in southern California
coastal water.
5a Nitrogen productivity in southern California coastal 21
waters in July, 1970.
b Nitrogen productivity in southern California coastal 22
waters in June, 1971.
6 N-limited chemostat culture of Coccolithus huxleyi. 30
7 N-limited chemostat culture of Skeletonema costatum. 32
8 Kinetics of short-term ammonium uptake by Coccolithus 36
huxleyi.
9 Variation with dilution rate of the apparent half- 37
saturation constant (Ks') for the uptake of nitrate
and ammonium by phytoplankton grown in nitrogen-
limited chemostat cultures.
10 Comparison of half-saturation values determined by 38
two independent methods.
11 Ratios of Skeletonema costatum cell concentration to 40
that of Coccolithus huxleyi when the two phytoplank-
ters were grown together in the same culture, as a
function of the dilution rate of the culture.
Vlll
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No.
12 Changes in particulate nitrogen; nitrate, ammonium 49
and urea concentrations; utilization of nitrogen;
and nitrite reductase activity in shipboard cultures.
13 The mean rate of uptake for nitrate, ammonium, and 52
urea and the nitrite reductase and glutamic dehydro-
genase activities in shipboard cultures.
14 Ratios of enzymatic activity to chlorophyll,a_ and 54
to ATP.
ix
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SECTION I
CONCLUSIONS
1. Southern California coastal sewage outfalls have measureable
effects on the concentration of nutrients (ammonium) available
for phytoplankton growth in the immediate vicinity of the out-
falls. Phytoplankton standing stocks are also elevated in these
areas.
2. Nitrogen appears to be limiting for the growth of phyto-
plankton stocks in Southern California coastal waters, and
ammonium and urea exceed nitrate in importance as nitrogen
sources for phytoplankton growth except during upwelling periods.
Increased ambient nitrate concentrations during upwelling permit
increases in the phytoplankton standing stocks along the coast
to levels observed about the outfalls.
3. Laboratory studies of phytoplankton growth and physiology
complement the work at sea by providing data and concepts neces-
sary to an understanding of mechanisms and rates of processes to
be expected in nature.
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SECTION II
RECOMMENDATIONS
The study of phytoplankton standing stocks and nutrient levels
in waters about Southern California sewage outfalls is being
continued under the auspices of the Southern California Coastal
Water Research Project. The increases in phytoplankton stocks
attending sewage inputs are not in themselves detrimental for
present uses of coastal waters, although blooms of certain
species such as in dinoflagellate red tides may present problems,
largely in enclosed embayments. Bottom-living organisms are more
readily perturbed by the outfalls, and it is gratifying that studies
of these benthic communities are also in progress under SCCWRP.
It is not clear at present why nutrient enrichment of our coastal
waters sometimes results in a preponderance of diatoms and at
other times of dinoflagellates in the phytoplankton crop. Solu-
tion of this problem would be significant for the eventual control
of dinoflagellate blooms. Laboratory work on the problem seems
in order.
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SECTION III
INTRODUCTION
The Marine Food Chain Research Group of the Institute of Marine
Resources, University of California, San Diego, is the site of
an interdisciplinary program of research into factors which con-
trol the food web in the plankton and is concerned not only with
the kinetics of phytoplankton growth, but with the more subtle
effects that a change in the nature (as well as the biomass) of
the flora may have on the whole food chain leading to the produc-
tion of harvestable marine life. These aspects must be considered,
as the undesirable feature of eutrophication is often not so much
the high productivity but the production of the wrong organisms
at the wrong time for a food web to be established which is favor-
able to man.
It is impossible to predict the course of eutrophication in the
marine environment with any degree of sophistication without an
understanding of how the phytoplankton respond to an added load
of nitrogen compounds, in particular ammonia and nitrate. These
compounds are nearly always "limiting" nutrients in the sea and
affect the standing stock and nature of the plant crop (ref. e.g.
Strickland, 1965; Ryther and Dunstan, 1971). A shortage of sili-
cate and organic trace factors may influence the nature of the
flora but probably has little effect on the total biomass.
The near-shore coastal area of Southern California, where pollu-
tion now occurs and will increase in the future, has been surpri-
singly little studied with respect to phytoplankton ecology, des-
pite the fact that the California Current is one of the most ex-
tensively investigated regions with respect to physical oceanography
and certain aspects of the zooplankton, in particular fish larvae.
Sverdrup and Allen (1939) and Sargent and Walker (1948) related
diatom populations to the large-scale eddies and areas of upwelling
off the coast. In addition, there have been several investigations
arising from projects to study the disposal of raw and treated
sewage (e.g. Stevenson and Grady, 1956, Water Resources Engineers,
1967), but these and numerous published and unpublished floristic
studies have produced no basic understanding of the phytoplankton
ecology or conclusive evidence for the effect of pollution on the
planktonic flora, even adjacent to a sewage outfall. In this respect
the area is more difficult to study than an estuary to marshy bay
where there is poor circulation. In such contained areas abnormal
blooms of organisms are clearly associated with pollution. Such
situations have been documented and the general ecology worked out
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(e.g. coccolithophores in the Oslo Fjord; Berge, 1962; or green
algae in parts of Long Island Sound exposed to the run-off from
duck ponds, Ryther, 1954).
Much of the year surface waters off southern California are de-
pleted of plant nutrients, especially nitrogen, with nitrate
undetectable at the surface and ammonium concentrations less
than 1 yM (cf. Strickland, ed., 1970, and earlier references
cited therein). Urea concentrations are likewise less than
1 yM (McCarthy, 1971). Enrichment takes place periodically,
especially in spring and summer, when the upwelling of nutrient-
rich water markedly increases the concentrations of nitrate,
phosphate, and silicate. A detailed study carried out April
through September, 1967, off La Jolla, California, provided com-
parative data on phytoplankton crop size and nutrient concentra-
tions in both quiescent and upwelling periods (Strickland, ed.,
1970). The physical oceanography of upwelling has been reviewed
recently (Smith, 1968) and the importance of the processes for
local phytoplankton production has been recognized for many years
(Moberg, 1928). Nutrient enrichment during upwelling tends to
increase the size of the phytoplankton crop in local waters. In
some cases, especially offshore, diatoms are the principal com-
ponents of the resulting blooms (Sverdrup and Allen, 1939; Sargent
and Walker, 1948) whereas dinoflagellates often form blooms (red
tides) within a few miles of shore (Allen, 1946; Holmes et al.
1967). At present we cannot predict whether dinoflagellates or
diatoms will increase in response to nutrient enrichment nearshore
(Strickland, ed., 1970) and further research on the character and
mechanisms of species succession is needed.
Enrichment of surface waters also results from sewage disposal
off southern California in outfalls serving Ventura, Los Angeles,
Orange and San Diego counties. At present we lack sufficient
data to compare the nutrient contributions from upwelling and
sewage disposal to local surface waters. Very preliminary and
approximate estimates suggest that natural upwelling may exceed
sewage by an order of magnitude as a source of nitrogen for
phytoplankton growth over a year. Fairly accurate estimates of
the nitrogen input from sewage can be made, but determining the
contribution from upwelling would be very costly of ship time and
no doubt variable from year to year.
Upwelling provides nitrogen as nitrate while sewage would be
expected to supply ammonium as the principle form of nitrogen.
Phytoplankton appear to utilize both forms equally well although
their chemical composition, especially C/N and C/chlorophyll £
ratios, may vary somewhat with the nitrogen source used for
growth (Eppley £t al., 1971a). Since upwelling is seasonal and
intermittent a survey of the region during a quiescent period
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(no upwelling) would be expected to show the outfall areas as
as points of nutrient-rich water, with high phytoplankton crops,
against a low nutrient, low crop background. This was the case,
in part, in July 1-15, 1970, for coastal waters between Los
Angeles and San Diego: phytoplankton crops were high only at
the outfalls but surface nutrient concentrations were low every-
where. In June, 1971, crops were high at all stations and the
effects of sewage effluents were less obvious.
Grigg and Kiwala (1970) and Turner, Ebert and Given (1968)
provide maps of the White Point and Point Loma outfall areas,
respectively, the two outfalls we studied, and report studies
on benthic organisms.
Laboratory studies were carried out to aid in the interpretation
of the results from cruise work. Intensive studies of diel
periodicity in growth (cell division), photosynthetic rate,
and nitrogen assimilation were carried out with single phyto-
plankton species in the laboratory and with cultures of natural
phytoplankton aboard ship. Continuous cultures, operated as
nitrogen-limited chemostats, were also studied in the laboratory.
Such cultures are particularly amenable to studies of nitrogen
assimilation rates and the kinetics of assimilation.
Results of the culture studies fall into four categories: (1)
periodicity in nitrogen assimilation, and in cell division,
chlorophyll synthesis, and photosynthetic carbon assimilation
rate; (2) kinetic studies of nitrogen assimilation; (3) the
chemical composition of phytoplankton in N-limited chemostat
cultures and the influence of rate of nitrogen input on carbon/
nitrogen, carbon/chlorophyll, and nitrogen/chlorophyll ratios
in the cells; (4) comparison of results of measuring nitrogen
assimilation by direct chemical, isotopic "N, and enzymatic
methods.
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SECTION IV
METHODS
Nutrient and Particulate Analyses
The determination of chlorophyll a_ by the fluorometric technique
(Holm-Hansen et al. 1965), nitrate by the cadmium-copper reduction
and subsequent determination of nitrite (Wood, Armstrong, and
Richards, 1967), and particulate nitrogen by the Kjeldhal method
with a ninhydrin finish (Holm-Hansen, 1968) and particulate adeno-
sine triphosphate by the luciferin luciferase method (Holm-Hansen
and Booth, 1966) followed the procedures outlined in Strickland
and Parsons (1968). Ammonium was determined by the phenolhypo-
chlorite method (Solorzano, 1969) and urea by the urease method
(McCarthy, 1970), and both incorporated the modifications of tech-
nique, sample preparation, and sample storage reported elsewhere
(McCarthy, 1971; McCarthy and Kamykowski, unpublished results).
Reactive phosphate and silicate were analyzed according to methods
in Strickland and Parsons (1968).
Primary Production
Samples were taken at depths corresponding to sunlight irradiances
of 100, 45, 20, 8, 4, and 1% of surface irradiance for measurement
of both carbon and nitrogen productivity. (These depths were
chosen to match the transmission of neutral density filters in the
deck incubators.) The light depths for sampling were based on
three times the Secchi disc depth as the 1% light level and the
further assumption of a constant attenuation coefficient with depth.
The water samples were passed through 183-u netting and placed in
300-ml glass-stoppered bottles. Radiocarbonate solution (5 or 20
yCi in one ml) was added with rinsing, the contents of the bottles
mixed and the bottles placed in incubators on deck in unobstructed
sunlight. The incubators provided cooling water at sea surface
temperatures. Samples were incubated for 24 hours. They were
then passed through 0.45-y membrane filters and the filters were
dried immediately in a vacuum desiccator over silica gel. Finally,
the radiocarbon of the filtered particulate matter was assayed
with a scintillation counter and the counts were corrected for
counter efficiency, background radiation and^coincidence effects.
Carbon assimilation was calculated as mg C/m /day. These values
were integrated over depth to express production as g C/m /day.
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Phytoplankton Uptake of Nitrogen Using the N Isotope Technique
The N analyses of phytoplankton nitrogen uptake followed the
procedure outlined by Dugdale and Goering (1967). After incuba-
tion (24 hours unless noted) with the 15N isotopes [99 at % K15NO_,
95 at % C115NH4, and 97 at % (15NH2)2CO] the particulate material
was collected on a Reeve Angel 984H glass fiber filter, desiccated
under a partial vacuum over silica gel, and converted to gaseous
nitrogen by an automated Dumas method. A Coleman nitrogen ana-
lyzer was used to combust the particulate sample and filter and
to sweep it into a glass vacuum system which trapped the C0_ and
reduced the volume of the gaseous nitrogen sample (Barsdate and
Dugdale, 1965). The sample then entered a single beam Nier sector-
type mass spectrometer for determination of the ^N/ ^N ratio.
The resultant ratio was compared with standards to determine the
quantity of N isotope incorporated by the algae during the in-
cubation. The precision of the mass spectrometer was examined
using 13 replicate samples of low enrichment (0.448 at % -*-^N);
and one SD was 0.0124 at % 15N.
From the mass spectrographic analysis the variable V was deter-
mined. mass of N taken up and hence has only
mass of particulate N X time
dimensions of (time)~l and can be considered a specific growth
rate in terms of nitrogen (Dugdale and Goering, 1967).
When detrital particulate nitrogen is present in a sample, V
will underestimate growth rate since the biologically active
particulate nitrogen is diluted by the inert fraction in the
mass spectrographic analysis. For laboratory cultures and en-
riched shipboard cultures, where there is proportionately little
particulate detrital nitrogen, the consequences are negligible.
The product of V and the particulate nitrogen concentration for
the same sample is an assimilation rate (p), which has dimensions
of mass/volume-time and units of ymole N/l-hour or ymole N/l-day
will be used. Dugdale and Goering (1967) have shown that this
parameter is not affected by detrital particulate nitrogen, hence
assimilation rates may be compared both within and between samples.
Enzyme Assays
All cell-free extracts for enzyme assay were prepared by filtering
cells on a glass fiber filter paper, homogenizing filter and cells
in 0.2 M phosphate buffer, pH 7.9, and centrifuging 1-2 min at
about 2000 RCF to obtain a clear extract (Eppley, Coatsworth and
Solorzano, 1969).
10
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Nitrite reductase (NiR). The enzyme was assayed essentially
as described by Joy and Hageman (1966) with reduced (by dithio-
nite) methyl viologen as reductant. The 1.0 ml methyl viologen
solution (40 mg/10 ml water), 0.1 ml NaNO- solution (9 mM in
water), and 0.1 ml sodium dithionite solution (75 mM in buffer,
freshly prepared). Reaction tubes were fitted with serum stop-
pers and evacuated through a hypodermic syringe needle plumbed
to a vacuum pump. Incubations were carried out at room tempera-
ture (20-25 C) usually for 60 min. Activity was calculated as
the difference in IMK" remaining in control tubes (reaction tubes
lacking enzyme, with boiled enzyme, or lacking MV)'and that in
experimental tubes. Nitrite in the reaction tubes was determined
on 1-ml samples (in duplicate or triplicate) after 1/10 or other
appropriate dilution, by adding 1 ml sulfanilamide solution and
and 1 ml N-(l-naphthyl)-ethylenediamine dihydrochloride solution,
and absorbance was read at 543 nm with a spectrophotometer.
Glutamic dehydrogenase (GDH). The ammonium-dependent oxidation
of pyridine nucleotide in the presence of a-ketoglutarate was
taken as GDH activity. To each reaction tube was added 0.6 ml
cell extract in 0.2 M phosphate buffer, pH 7.9, 0.1 ml a-keto-glutarate
solution (0.2 M), 0.1 ml (NH^)2 SO^ solution (1.5 m), and 0.2 ml
NAD(P)H solution (1 mM). After an appropriate incubation time
(usually 10 min) the reaction was stopped by adding 0.3 ml 3 N
HCL followed by 1.0 ml 30% NaOH solution. The reaction mixture
was then placed in a boiling water bath for 5 min if NADH was
included, but not if NADPH was used. Five ml water was then
added and the fluorescence of oxidized pyridine nucleotide,
formed in the reaction, was determined (Turner and Associates,
1968). Activity was calculated from the difference in fluores-
cence between tubes with and without added (NH,)2 SO^, i.e.
from the ammonium-dependent fluorescence. Standard curves were
prepared using NAD(P) dissolved in buffer and treated as the
samples.
Viologens were purchased from Mann Research Laboratories, Inc.;
pyridine nucleotides from Sigma Chemical Co.
Shipboard Culture Methods
Polyethylene culture vessels on an exposed deck of the ship
were filled with 200 1 of low-nutrient, near-surface seawater
which had been passed through 183-y nylon mesh to remove the
larger zooplankton. The vessels were covered with translucent
lids, wrapped with cheesecloth, and cooled by a continuous spray
of surface seawater. A full complemement of nutrients in the
proportions recommended by Eppley, Holmes, and Strickland (1967),
except for nitrogen, were introduced at the initiation. Nitro-
gen as the limiting nutrient was added as nitrate to one culture,
11
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ammonium to another, urea to the third (hereafter referred to
as the nitrate, ammonium, and urea cultures) and a fourth
culture received no nitrogen. Additional quantities of nu-
trients were added when necessary to prevent depletion during
the experimental period. The cultures were sampled at 6
hour intervals through 24 hours of the stationary phase of
growth in one experiment and at 6 hour intervals through 42
hours of the logarithmic phase of growth in another. Every
6 hours analyses were made for nitrate, ammonium, urea, par-
ticulate nitrogen, chlorophyll £, enzyme activities, and
photosynthetic rate (samples incubated 1 hr with ^C under
artificial light). At the same time each of three 200 ml
samples from each culture was enriched with 5.0 yg-at N/l
l-^N labeled nitrate, ammonium, or urea for determining N-
assimilation rates. The bottled samples were suspended in
the culture vessels and incubated for one hour.
Laboratory Culture Methods
Organisms cultured were the coccolithophorid, Coccolithus
huxleyi, clone BT-6, isolated by Dr. R. R. L. Guillard in
1960 from the Sargasso Sea, two coastal diatoms: Skeletonema
costatum, isolated from Long Island Sound in 1956 by Dr.
Guillard, and Leptocylindrus danicus, isolated by Mr. J. B.
Jordan off Pt. Loma, California, in 1970, and a large-celled
dinoflagellate, Gymnodinium splendens, isolated off the Scripps
Institution pier by Mr. Jordan in 1969. The latter is often
a minor species component in local dinoflagellate blooms.
Enriched sea water media were used both for maintenance of
cultures and for the experiments (half-strength IMR medium
described by Eppley, Holmes, and Strickland, 1967). Nitrogen
levels were reduced for N-limited chemostat experiments and
are indicated in the appropriate data tabulations. All ex-
periments were carried out at 18°C. Illumination was provided
by tungsten lamps with the light filtered through copper sul-
fate solutions. Details of the light source are given in Eppley
and Coatsworth (1966). Irradiance during the experiments, and
daylength are given in the data tables. Dilution rate of the
continuous cultures was regulated by a Technicon peristaltic
pump. Cell concentration was measured in the laboratory cul-
tures with a CelloscopeR model 101 electronic particle counter.
12
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SECTION V
SHIPBOARD STUDIES OF EUTROPHICATION AROUND SOUTHERN CALIFORNIA
COASTAL SEWAGE OUTFALLS, JULY, 1970, AND JUNE, 1971
The White Point (Los Angeles County) and Point Loma (San Diego)
coastal sewage outfalls were visited on cruises of the R/V
ALPHA HELIX and ALEXANDER AGASSIZ in July, 1970, and June, 1971,
respectively (Fig. 1). The outfall diffuser piping lies at ap-
proximately 70 meters depth at each site allowing the ship to
move directly over the pipes for taking samples. The outfall
diffusers are said to be designed for an initial dilution of
sewage with sea water of the order 200-fold, hence one would see
only small salinity changes resulting from fresh water dilution,
even at depth. We have found the best "tracer" for the sewage
to be ammonium ion. Ammonia in sewage is probably of the order
1-2 millimolar in concentration and is generally present at <1
micromolar in sea water. The 200-fold dilution would be expected
to produce an ammonia concentration 5 to 10 micromolar directly
above the outfall, a concentration easily measured with present
methods. We have found concentrations up to 30 micromolar at
White Pt. (Table 1) but concentrations were usually lower.
The depth of the euphotic zone at the outfalls is roughly 20 m
or about 1/4 the water depth (Table 2). Ammonia concentration
in the euphotic zone is low, presumably because the abundant
phytoplankton rapidly absorb the nutrient in the presence of
light. Other nutrient substances measured include phosphate,
silicate and nitrate, none of which were different, either
elevated or depressed, at the outfalls in comparison with con-
centrations measured at control stations (Eppley et al. 1971b).
Phytoplankton crops were high at the outfalls at each visit. In
July, 1970, phytoplankton concentrations were fairly low along
the southern California coast and the outfall crops stood out
clearly above background levels. In June, 1971, crops were ele-
vated everywhere measurements were made and the outfalls were not
conspicuous by their phytoplankton crops (Table 2). Limited areal
coverage was achieved in 1971 by recording several depth profiles
of chlorophyll fluorescence (Lorenzen, 1966) about the outfalls
with a hose, pump and fluorometer (Table 3). However, the above
conclusions based upon chlorophyll concentration were supported
by other parameters related to the standing stock of phytoplankton:
primary productivity, particulate carbon, particulate nitrogen, and
adenosine triphosphate (Eppley £t_ al. 1971b). The phytoplankton
crop off southern California has shown marked temporal fluctuations
13
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34°
Stations 12, 18 XX
Fig. 1. Map showing the location of the White Point (station number 19) and Point Loma
(station numbers 12, 18) sewage outfalls and the control station of La Jolla,
California (station numbers 1, 6, 10). The station numbers refer to the 1970
cruise and are not used further in this report.
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Table 1. Depth profiles of ammonium concentration (ymoles/liter) off southern
California in June, 1971.
Pt. Loma Outfall
5 June 1971
8 June 1971
White Pt. Outfall
6 June 1971
Depth
m
5
15
25
40
45
50
+
yM
0.16
0.20
0.31
2.72
0.07
0.24
Depth
m
0
5
10
25
45
60
+
yM
0.83
1.04
0.79
0.91
2.19
0.35
Depth
m
0
5
10
25
35
48
+
yM
0.40
1.30
18.3
26.0
30.1
27.6
Control Station
off La Jolla, Calif.
7 June, 1971
Depth NH,
m yM
0
5
10
25
35
50
0.08
0.19
0.17
0.23
0.19
0*13
-------
Table 2. Photosynthetic carbon assimilation, nitrogen assimilation, and chlorophyll a.
at coastal seawater stations off southern California. Nitrogen assimilation
values are the sum of nitrate, ammonium, and urea assimilation. Values were
integrated over the depth of the euphotic zone.
Location
Pt. Loma Outfall
White Pt. Outfall
Control station off
La Jolla
Pt. Loma Outfall
White Pt. Outfall
Control station off
La Jolla
Photosynthetic Nitrogen
Rate Assimilation
Date g C/nr-day m moles N/m^-day
9 July
13 July
1 July
3 July
8 July
5 June
8 June
6 June
7 June
July, 1970
2.64
1.76
1.37
1.10
0.36
June, 1971
2.07
0.96
2.54
2.46
ND
10.0
10.4
9.4
ND
14.0
12.8*
10.5
18.5*
Euphotic
Chlorophyll a Depth
/ 2
mg/m m
163
83
33
18
14
21
50
66
53
21
17
33
33
46
20
23
16
28
*Based on three depths only, instead of the usual six depths.
NB: Ratios of the rate of carbon assimilation to nitrogen assimilation in these
experiments averaged 9.7+2.5 (s.d.) g C/g N.
-------
Table 3. Variation in the chlorophyll a_ content of the upper
50 meters of water about the Pt. Loma and White Pt.
outfalls and about a control station off La Jolla,
California.
Station
Time
Position
N. Latitude W. Longitude
Chlorophyll a_
.g/n
mg/nr
1*
2
3
4
5
6
7
8
9
10
U*
12
13
14
15
17
18
19
20
21*
Point
0930
1406
1500
1545
1648
1828
1912
White
0800
0906
0952
1030
1130
1500
1539
1636
Control Station
0850
0940
1106
1200
1236
Loma Outfall
32°40.3'
41.3'
43.5'
46.3'
40.3'
39.1'
37.0'
Point Outfall
33°42.2'
45.0'
43.4'
42.6'
42.1'
41.6'
40.8'
40.5'
Off La Jolla,
32°55.9'
54.0'
49.4'
50.8'
52.2'
5 June, 1971
117°17.0'
17.3'
17.3'
17.2'
22.8'
17.2'
17.2'
6 June, 1971
118°20.3'
26.6'
23.4'
21.4'
19.9'
19.1'
16.9'
21.6'
California 7 June,
117°19.0'
18.0'
21.3'
19.7'
18.0'
33
83
32
18
115
36
50
292
250
247
168
206
201
85
141
1971
53
55
79
78
72
(40 m)
(25 m)
(40 m)
(45 m)
(40 m)
(30 m)
(45 m)
Stations for productivity and chemical measurements.
17
-------
in the past and much of this variation is attributable to inter-
mittent coastal upwelling (Strickland, 1970). Nutrient inputs
from upwelling could account for the year to year differences we
observed.
Primary production, as nitrogen assimilation, was measured as
well as the customary carbon-14 measure of photosynthetic carbon
assimilation. Nitrate and ammonium uptake rates were measured
with the N-labeled compounds following procedures of Dugdale
and Goering (1967). Uptake of 15N-urea was also measured (ap-
parently for the first time at sea) with rather surprising results.
Urea appeared to provide as much as one-half the nitrogen assimi-
lated by coastal phytoplankton (Table 4). Urea concentrations
in seawater are of the same order as those of ammonium (McCarthy,
1971) but were not elevated at the outfall stations even when
high ammonia concentrations were noted. The source of urea in
seawater is thought to be the excretory metabolism of animals,
including zooplankton, bony fishes and sharks (McCarthy, 1971).
The distribution of ammonia and of phytoplankton was patchy
about the outfalls. This would be expected if one visualized
the effluent leaving the diffusing mechanism of the outfall
and maintaining a certain integrity over time and space before
intermixing completely with the ambient seawater. Depth profiles
in several instances showed an elevated ammonia concentration at
a single depth, as if that sample bottle had filled in such a
discrete "patch" or "plume" of sewage-seawater mixture (Table 1,
see for example, Pt. Loma: depths 40-50 m). Phytoplankton
patchiness was evident in continuous depth profiles of chloro-
phyll fluorescence and in the total quantity of chlorophyll
integrated over the upper 50 meters (Table 3). This kind and
scale of patchiness in chlorophyll distribution was seen earlier
off La Jolla (Strickland, 1970) where its origin was presumably
the temporal and spatial irregularity in natural upwelling off
headlands and submarine canyons. The apparent patchiness in
the properties measured about the outfalls makes one wonder how
typical or representative our results may be. Finding high
ammonia concentrations may depend largely upon fortune, depending
on the ships drift rate over the outfall piping and the geometry
of the plumed "high ammonia water." Chlorophyll distribution
would be expected to be less patchy since chlorophyll synthesis,
like other aspects of plant growth, is not instantaneous but
occurs on a time scale of days and mixing could be more extensive
on this time scale.
Our chlorophyll profiles, made at intervals about the outfalls,
gave no evidence of a discrete phytoplankton bloom developing
"downstream" of the outfall, as would be predicted if a steady
longshore current were present (Dugdale and Whitledge, 1970).
18
-------
Table 4. Relative importance of urea as a source of nitrogen for
phytoplankton growth in southern California coastal waters.
Urea Assimilation as % of
Urea + Nitrate + Ammonium Assimilation
Light Depth
as % of Surface Irradiance
Mean Values of 1970 and 1971
Control Stations
Pt. Loma White Pt. off La Jolla, Calif.
87
43
20
8
4
1
40
60
30
44
17
3
62
47
43
38
36
12
41
37
38
49
16
15
19
-------
However, it is quite possible that such a bloom could occur at
those times when a persistent longshore current is present. One
wonders if the frequent dinoflagellate blooms observed off Newport.
California are caused by longshore transport of sewage nutrients
from outfalls "up" the coast toward Los Angeles.
An interesting parameter which can be calculated from the nitro-
gen assimilation data and the concentrations of nitrogenous sub-
stances in the water is the time required for the phytoplankton
crop to deplete the available nutrient, assuming no new input.
Such turnover times ranged about 2-3 days in July, 1970, and
1-2 days in June, 1971 for the upper ten meters. Values tended
to increase with depth either because of decreased crop, de-
creased rate of uptake per unit crop, or increased ambient nu-
trient (Table 5). Turnover times as low as those in the upper
ten meters must be regarded as unusual for waters with high
rates of nutrient input as one would intuitively consider near
shore coastal waters to be. They imply rather close coupling
between nutrient input rate and phytoplankton growth as in con-
tinuous phytoplankton cultures of the chemostat variety. The
waters sampled for measurement of nitrogen assimilation fortui-
tously included a range of nitrate and ammonium concentrations
so that the dependence of assimilation rate upon the concentra-
tion of these nitrogen compounds could be assessed and the
maximum assimilation rate, Vm, and the half-saturation constant,
Ks, could be evaluated in the Michaelis-Menten equation:
V = VS/K+S (1)
m s
-3 -1
Values of V were 5 to 6 X 10 day for nitrate and ammonium
and the corresponding K values were 0.3 to 0.5 micromolar (Figs.
2 & 3). The K values Igree well with earlier measurements
with natural eutrophic phytoplankton (Maclsaac and Dugdale, 1968)
and with cultures of neritic phytoplankton species (Eppley,
Rogers, and McCarthy, 1969; Eppley and Thomas, 1969; Carpenter
and Guillard, 1971). The V values are typical of oligotrophic
waters of the Pacific Ocean (Maclsaac and Dugdale, 1968; Goering,
Wallen and Naumann, 1970) rather than eutrophic waters of rich
coastal regions. Assimilation of nitrate and urea decline with
depth in a way which suggests a dependence upon light intensity
(Tables 4 & 5). However, ammonium assimilation showed no light
dependence in these ocean profiles (Table 5).
20
-------
Table 5a. Nitrogen productivity in southern California coastal waters
in July, 1970. Assimilation rates of nitrate, ammonium, and
urea (pinoles/liter-day) were determined with ^%-lahelled
substrates. Turnover times (days) were calculated for the
nutrients dissolved in the water as (ambient concentration of
nutrient + 1%-nutrient added)/assimilation rate.
, . , ,_ Assimilation Rate Turnover Time
Light
Level1" NO, NH, Urea NO, NH, Urea
White Point Outfall, 13 July, 1970
1 100 0.444 0.125 0.338 2.4 1.9 1.5
3 45 0.526 0.139 0.125 2.0 1.6 1.6
6 20 0.422 0.166 0.154 2.5 1.5 1.6
9 8.1 0.377 0.187 0.074 3.1 1.3 1.3
12 4.9 0.163 0.120 0.091 3.6 1.9 2.8
15 1.4 0.259 0.216 0.122 2.3 1.8 2.5
Control Station off La Jolla, Calif., 3 July, 1970
2 100 0.380 0.038 0.115 3.0 2.4 2.8
7 45 0.255 0.079 0.230 1.8 1.9 3.0
13 20 0.144 0.106 0.134 15* 2.5 3.0
16 8 0.029 0.085 0.149 100 2.6 4.0
23 4.9 0.010 0.060 0.017 >100 3.0 5.8
30 1.4 0.010 0.043 0.017 >100 3.8 7.5
*Sudden increase in nitrate turnover time represents increase in
nitrate concentration with depth. Concentrations were 0.0, 0.0, 5.50,
7.30, 8.70, and 12.0 ymolar for depths shown. This effect is the rule
rather than the exception. Ammonium and urea concentration show little
depth variation and are usually <1 yM.
^As 7, surface irradiance.
21
-------
Table 5b. Data for June, 1971. Values and units as in Table 5a.
Depth
(m)
T.I. ii£>S.J.lUJ.J. CILJ.UU
Light
Level7 N03 NH4
jxate
Urea
±u:
N°3
mover .
NH4
nme
Urea
Pt. Loma Outfall, 5 June, 1971
1
3
7
11
15
20
1
3
6
9
12
16
1
10
21
5
15
25
100
45
20
8.1
4.9
1.4
100
45
20
8.1
4.9
1.4
Control
100
20
4.9
45
8.1
1.4
0.389
0.140
0.382
0.178
0.171
0.187
White Point
0.083
0.076
0.077
0.066
0.056
0.028
0.158
0.190
0.332
0.209
0.102
0.266
Outfall
0.180
0.150
0.139
0.114
0.260
0.738
Station off La Jolla,
0.223
0.272
0.169
Point Loma
0.202
0.016
0.028
0.173
0.421
0.133
Outfall,
0.130
0.136
0.290
0.613
0.565
0.303
0.178
0.054
0.0083
, 6 June,
0.529
0.740
0.389
0.305
0.290
0.038
Calif. ,
0.350
0.322
0.037
8 June,
0.437
0.238
0.014
1.5
1.4
18
67
94
96
1971
1.2
1.3
1.3
1.5
1.8
12
7 June ,
1.8
1.5
69
1971
1.7
1.5
43
1.8
1.5
2.1
4.4
6.2
23
1.3
1.5
1.4
1.5
1.2
1.8
1971
1.9
1.5
3.5
1.8
1.7
1.1
1.4
1.5
3.2
7.2
18
140
1.2
1.5
1.3
1.4
1.3
2.6
1.7
1.5
8.0
1.9
1.9
1.7
Nitrate hardly detectable above 16 m.
"""As % surface irradiance
22
-------
5.0-
4.0-
ro
oo
o
X
»
X
1.0-
1.0 2.0 3.0 4.0
AMMONIUM CONCENTRATION juM
5.0
6.0
Fig. 2. The rate of ammonium assimilation by natural marine phytoplankton vs. concentration
of ammonium in the water. Circles represent measurements at the Pt. Loma outfall,
triangles are for White Pt. data and squares are for a control station off La Jolla,
California.
-------
6.0-
ro
1.0
2.0 3.0 4.0
NITRATE CONCENTRATION
5.0
6.0
7.0
Fig. 3. The rate of nitrate assimilation by natural marine phytoplankton vs. nitrate concentration
in the water. Circles: Pt. Loma; triangles: White Pt.; square; off La Jolla, California;
diamonds: a second station at Pt. Loma.
-------
SECTION VI
DIEL PERIODICITY IN NITROGEN ASSIMILATION
Three experiments were carried out for the purpose of studying
day-night differences in nitrogen assimilation rate of marine
phytoplankton: (1) a shipboard batch culture series in which
200 liters of seawater containing its natural phytoplankton
flora were enriched with phosphate, silicate, chelated trace
metals and vitamins and with either no added nitrogen, with
nitrate, with ammonium, or with urea as the nitrogen source for
growth. Details of the experimental procedures are described
in Eppley et al. (1971a). There was essentially no growth in
the culture with no added nitrogen as is typical of local waters
except during upwelling (Eppley, 1968; W. H. Thomas, unpublished
results). In the other cultures chlorophyll a_ synthesis was
exponential over time with no evidence of diel periodicity (Fig.
4). The sudden change in slope for the nitrate and ammonium
cultures represents depletion of vitamin B..^. Cell division was
periodic (Fig. 5) and was most rapid in trie afternoon and early
evening. Nitrate and ammonium assimilation were also periodic
(Fig. 6) and photosynthetic capacity showed its usual diel
periodicity in the nitrate, ammonium, and urea cultures. Phos-
phate assimilation rate likewise showed diel periodicity irres-
pective of the form of nitrogen added (Fig. 7).
In a second experiment Coccolithus huxleyi, a small 5 y diameter
phytoplankter of cosmopolitan distribution in the oceans, was
studied in a nitrogen-limited chemostat culture in the laboratory.
Measurements included cell concentration, nitrate and ammonium
in the culture (both forms of nitrogen were offered in the feed
medium), cell nitrogen content, photosynthetic rate, and enzyma-
tic activity of nitrate reductase, nitrite reductase, and glutamic
dehydrogenase. Further details are given in Eppley, Rogers,
McCarthy and Sournia (1971). Results are presented in Table 6
and Figure 8. Diel periodicity was noted in cell division and
photosynthetic rate (Table 6) and in the activity of all three
enzymes assayed (Fig. 8). However, nitrogen assimilation rates
sufficed to maintain low ambient concentrations of nitrate and
ammonium in the culture and no periodicity in assimilation rate
of nitrogen could be observed.
In the third experiment Skeletonema costatum, a common coastal
diatom, was studied in an N-limited chemostat culture grown,
as was C_. huxleyi, with illumination provided in light-dark
cycles. Cell division and photosynthetic rate again showed diel
periodicity (Table 7) and the ambient nitrate and ammonium con-
centration within the culture fluctuated between day and night
25
-------
LOG
Scale
10
0|
.C
o
1.0
.9
£
.7
.6
.5
.4
.2
O.I
NH4
UREA
NO,
A NH4
B UREA
1030
JULY II
1100
I JULY 12
1200
JULY 13
0000
I
1200
JULY 14
0000
I JULY 15
Fig. 4. Chlorophyll a_ concentration in shipboard cultures of natural marine
phytoplankton during growth with nitrate, ammonium, or urea as the
nitrogen source. Abrupt changes in slope represent depletion of
vitamin BI_ from the culture media.
-------
LOG
Scale
10
E
in
d 3
^
to
LJ
o
10'
o N03-
A NH4*
a UREA
1200 1800
JULY 13
0000 0600 1200
I JULY 14
1800 0000
0600
JULY 15
Fig. 5. Concentration of diatom cells in the shipboard cultures. Cell divi-
sion appears to take place in the late afternoon and early evening.
-------
15
-------
1.2 -
1.0 -
-------
Table 6. N-limited chemostat culture of Coccolithus huxleyi, clone BT-6, with 16 hr. light/8 hr.
dark illumination cycle: cell concentration, nitrate and ammonium concentration in the
culture. Dilution rate was 0.78/day.
(1)
(2)
(2) - (1)
Cell
Cell (1) Expected Concentration NO,
Concentration From Dilution Due to Cell
Day
Hour
(millions/I)
Rate'
Division
yM
N/cell
(picograms)
16 Feb.
17 Feb.
18 Feb.
19 Feb.
0800
1500
0000
0400
0800
1500
0000
0500
0930
1500
0000
0800
L
L
D
D
L
L
D
D
L
L
D
L
920
780
660
790
920
800
630
750
980
680
660
900
920*
760
570
500
440/920*
760
570
470
420/980
840
630
490
0
20
90
290
480/0
40
60
280
560/0
-160
30
410
.12
.14
.19
.13
.13
-
-
-
-
-
—
"
.02
.05
.09
.14
.08
0.3
0.15
0.1
0.02
0.25
0.1
0.1
3.
6.
1.
0.
5.
6.
1.
0.
6.
6.
0.
2.
4
0
0
38
6
1
4
67
8
4
45
5
0.
0.
1.
0.
0.
0.
1.
0.
0.
0.
0.
0.
73
86
0
85
73
84
1
89
68
98
83
74
"•A picogram is
grams.
JL
_Q 7gf-
Assuming no cell division. N = NQe ' where N is taken as the cell concentration at 0800
on Feb. 16 and 17 and 0930 on Feb. 18; t is in days. °
-------
200
Coccolithus
huxleyi BT-6
Nitrate Reductase
(NADH)
Glutamic
Dehydrogenase
(NADH)
Nitrite
Reductase
0800 0000 0800 0000 0800 0000 0800
1600 1600 1600
TIME OF DAY
Fig. 8. Diel periodicity in the activity of three enzymes of nitrogen
assimilation in an N-limited chemostat culture of Coccolithus
huxleyi grown on a light-dark cycle. Upper curve: nitrate
reductase; middle: glutamic dehydrogenase measured with NADH;
lower: nitrite reductase. Activity of the latter appears to
be out of phase with nitrate reductase and glutamic dehydro-
genase activity, but in phase with photosynthetic capacity
(see Table 6).
-------
Table 7. N-limited chemostat culture of Skeletonema costatum, clone Skel. with 12 hr. light/12 hr.
NJ
dark illumination cycle: cell concentration, chlorophyl!
photosynthetic rate.
Day
13 May
14 May
15 May
Cell
Concentration
Hour (105/liter) Cells/chain
1400
2000
0200
0800
1400
2000
0200
0800
1400
2000
L
D
D
L
L
D
D
L
L
D
455 5.6
272 3.5
292 4.8
342 6.6
230 5.2
193 5.4
157 4.5
254 7.2
220 5.0
167
Chlorophyll a
per cell
(picograms)
3.4
5.7
3.4
2.6
4.8
6.9
5.7
2.5
2.9
—
Nitrogen/cell
(picograms)
15
25
20
15
30
36
31
14
17
Photosynthetic
Rate
g C/g Chi, a'hr.
3.00
2.25
1.87
3.01
3.65
2.35
2.14
2.72
3.11
—12
A picogram is 10 grams
-------
such that assimilation rate of these ions could be evaluated
(Fig. 9). Rates in the figure are expressed as V with units
hr and were calculated as the uptake per hour per cell nitr-
gen content. Units so expressed are equivalent to a specific
growth rate and can be compared with the data from ocean profiles
based upon the N method of measuring nitrogen assimilation.
Values of V in the culture experiments are nearly an order of
magnitude greater than in the ocean profiles. This may result
from the dilution effect of detrital nitrogen on the calcula-
tion of V in natural samples, as well as from the higher ambient
nutrient concentrations in the cultures and higher specific
growth rates.
33
-------
0.15
0.10 -
'*>
o
0.05 -
0.0
30
Y/////////A
Skeletonema costatum
0.03
~ 0.02 -
0.01
0.0
V/////////A
1400
TIME OF DAY
Fig. 9. Diel periodicity in ambient concentration of nitrate and in the velocity
of nitrate assimilation (upper curves) and in the concentration of am-
monium and rate of ammonium assimilation in an N-limited chemostat cul-
ture of Skeletonema costatum grown on light-dark cycles.
-------
SECTION VII
KINETICS OF NITROGEN ASSIMILATION
Coccolithus huxleyi was grown in a nitrogen-limited chemostat
culture until a steady-state was reached (constant cell concen-
tration from day to day) at each of two dilution rates. A
large volume of the culture was withdrawn and divided into
aliquots. To these was added ammonium sulfate solution to
achieve a range of ammonium concentrations 0.1 to about 10 yM.
The aliquots of cell suspension were incubated 10 to 30 minutes,
as appropriate, the cells were removed by filtration and the
remaining ammonium measured. Uptake rate was computed from the
difference between initial and final concentrations and the half-
saturation constant and maximum uptake rate were evaluated using
Wilkinson's method (Wilkinson, 1961). The values could be com-
pared with results from a stationary phase batch culture obtained
earlier (Eppley, Rogers, and McCarthy, 1969). The half-saturation
constant (K ) was found to be invariant with dilution rate but
large differences were noted in the maximum velocity of uptake
(V ) as shown in Table 8. The V values increased as the dilution
rate decreased, that is, as the cultures became progressively
more nitrogen-deficient.
The apparent half-saturation constant (Ks') can also be computed
in another way, from the dilution rate and the concentration of
ammonium in the culture, if ym is evaluated and if data are com-
piled from several steady state cultures at different dilution
rates (cf. Droop, 1970) using the equation
y = ym S/K + S (1)
s
The value of y is taken as the dilution rate required to
washout the culture, S is the ammonium of nitrate concentration
in the vessel and y is the dilution rate. This technique gives
apparent K' values which decrease systematically with increasing
dilution rite (Table 9). Droop (1970) regards the "true" value
of K to be that minimal value obtained as y approaches y and
this agrees with our data (Table 10).
A culture of £. huxleyi and j^. costatum, together was studied
in an attempt to observe species succession resulting from dif-
ferent kinetic parameters of nitrogen uptake between the two
species. The cosmopolitan _C. huxleyi has a lower K for uptake
of both ammonium and nitrate than does the neritic diatom, _§_.
costatum. However, the latter has a greater maximum growth rate,
y . Hence if one graphs expected growth rate against concentration
35
-------
Table 8. Kinetics of short-term ammonium uptake by Coccolithus
huxleyi, clone BT-6.
Dilution Rate
as doublings /day
(actual doublings /day
of cell-N)
0*
0.32
0.63
K
s
yM + S.E.
0.35 + 0.10
0.28 + 0.07
0.33 + 0.10
V
max
as doublings /day
of cell-N
7.3
4.8
2.4
*N-depleted batch culture in stationary phase of growth.
36
-------
Table 9. Variation with dilution rate of the apparent half-
saturation constant (K ) for the uptake of nitrate
and ammonium by phytoplankton grown in nitrogen-
limited chemostat cultures. 18°C. Values in paren-
thesis are extrapolated to the maximum dilution rate
by graphing apparent K' vs y.
Dilution Rate
as doublings/day
(y)
Apparent Half-Saturation,Constant
in moles/liter (K )
Nitrate Ammonium
0.24
0.37
0.51
0.57
0.23
0.78
1.43
1.85
2.35
= U
m
= y
m
Gymnodinium splendens
0.66
0.75
2.4
72.4
Leptocylindrus danicus
4.9
1.8
0.57
0.62
(0.6)
0.62
1.0
1.2
(1.2)
4.3
1.2
0.44
0.85
(0.5)
0.32
0.56
0.76
1.08
1.41
1.7
= H
m
Coccolithus huxleyi BT-6
1.08 0.73
0.51 0.42
0.36 0.16
0.14 0.10
0.05 0.03
(0.1) (<0.1)
37
-------
Table 10. Comparison of half-saturation values determined by two
independent methods. In column (a) are values based on
short-term uptake experiments in which uptake was mea-
sured vs. concentration with N-depleted batch cultures.
In column (b) values are obtained from extrapolating
the apparent Ks to y = y , as in Table 9. Values in
column (a) from Eppley, Rogers, and McCarthy, 1969.
Half-Saturation Constant, Ks,
ymoles/liter
Species For Nitrate For Ammonium
(a) (b) (a) (b)
Coccolithus huxleyi 0.1 0.1 0.1 <0.1
Leptocylindrus danicus 1.2 0.6 0.5 0.5
Gymnodinium splendens 3.8 >2.4 1.1 1.2
38
-------
of nitrate or ammonium, based on eq (1) and with measured values
of K and V , the curves cross. The coccolithophorid would be
expected to grow fastest, i.e. to eventually win the competition,
at low nutrient concentrations and low dilution rates while the
diatom would do better at higher levels. However, both species
maintained themselves at the dilution rates employed although the
ratio of their concentrations did follow the expected trend
(Table 11). The experiment was not entirely satisfactory for
two reasons. At the lowest dilution rate S_. costatum lost its
buoyancy and appeared to become sticky. Even vigorous aeration
did not keep the cells suspended and they tended to settle out
and stick to the walls of the vessel. Furthermore C_. huxleyi
showed a high percentage of motile cells at the low dilution
rates and this may represent a significant alteration of its
life cycle with possible physiological differences from the
usual non-motile cells. The ability to become motile under
conditions of nutrient stress has obvious survival value (Munk
and Riley, 1952).
Data from N-limited chemostat cultures of Leptocylindrus danicus,
a coastal, chain-forming diatom, Gymnodinium splendens, a 50
micrometer diameter dinoflagellate often present as a minor
species component of local red water blooms, and C^. huxleyi
were collected for evaluation of the apparent K value for
ammonium and nitrate uptake using methods analogous to those of
Droop. K values obtained earlier from short-term uptake
measurements of N-depleted batch cultures were available for
comparison. Results were as noted by Droop: i.e. the apparent
Ks value obtained as y approached y agreed with the earlier values
reported (Tables 10 & 11). m
The implication of these comparisons is that y in eq (1) varies
with dilution rate while K remains constant. If this is so then
this variation in apparent y can be calculated from eq (1) taking
a constant K (the minimum value as y approaches y or the value
determined in short-term uptake experiments). When this is done
for the three species studied the value of y was found to be a
linear function of the dilution rate (y) and^ad a value about
twice the value of y (Fig. 10). Recent results of Thomas and
Dodson (1971) show that the rate of photosynthesis at light
saturation varied with dilution rate in N-limited chemostat cul-
tures of marine phytoplankton in the same direction as the varia-
tion in y . Since one would expect the two parameters to be
closely related Thomas' observations seem to confirm variation
in apparent y we calculated.
39
-------
Table 11. Ratios of Skeletonema costatum cell concentration
to that of Coccolithus huxleyi BT-6, when the two
phytoplankters were grown together in the same cul-
ture, as a function of the dilution rate of the cul-
ture. Dilution rate is expressed as doublings/day.
Dilution Rate Skeletonema/Coccolithus
0.26 No steady state reached*
0.79 2.60
0.84 3.07
1.49 5.97
1.52 7.10
See text for explanation.
40
-------
5.0-
4.0
J.O
UJ
(E
<
O.
3[
1.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Fig. 10. Variation in the apparent value of v^ with dilution rate, y, based on
phytoplankton growth in N-limited chemostat cultures. The value of ym
(the apparent ym) was calculated from equation 1 (see text). Circles:
Gymnodinium splendens; triangles: Coccolithus huxleyi; squares: mixed
culture of Q. huxleyi with Skeletonema costatum; diamonds: Leptocylindrus
danicus.
-------
SECTION VIII
INFLUENCE OF THE RATE OF NITROGEN INPUT
ON THE CHEMICAL COMPOSITION OF PHYTOPLANKTON
The carbon, nitrogen, and chlorophyll a_ content of the phyto-
plankton cultures grown as N-limited chemostats were measured
at steady-state with several dilution rates. The carbon/nitro-
gen and carbon/chlorophyll a_ ratios varied systematically with
dilution rate as would be expected if the dilution rate of an
N-limited cultures were a quantitative measure of the degree
of N-deficiency of the cells in the culture (Thomas and Dodson,
1971). Certain similarities between the species can be seen if
the composition ratios are plotted not against dilution rate,
y, but against the ratio y/y as Thomas has done. The C/N
ratio then shows little variation among the species (Fig. 11).
Species differences are still noted, however, in the C/Chloro-
phyll a. ratio (Fig. 12).
43
-------
30-
20
>5*
o
o
or
\
o
10
O.I
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fig. 11. Ratios of carbon to nitrogen in phytoplankton grown in N-limited chemostat cultures at
different dilution rates. The abscissa represents the dilution rate, y, as a fraction
of the dilution rate at washout of the culture, ym. Circles: Gymnodinium splendens;
triangles: a mixed culture of Coccolithus huxleyi with Skeletonema costatum; squares:
Leptocylindrus danicus.
-------
300
o>
o|
200
Q.
O
-------
SECTION IX
COMPARISON OF METHODS OF MEASURING NITROGEN ASSIMILATION RATE
Direct chemical measurements lack sufficient sensitivity and
precision for measuring rates of phytoplankton assimilation of
nitrate, ammonium and urea or of the increase of phytoplankton
particulate nitrogen in natural seawater samples. Typical
rates are of the order nanomoles of N assimilated per liter
and hour and depend upon the phytoplankton and substrate con-
centrations in the water, as well as upon the metabolic rate of
the organisms, as influenced by irradiance, temperature, and
the species composition of the phytoplankton assemblage.
The introduction of N methodology (Dugdale and Goering, 1967)
for measuring nitrogen productivity of seawater samples incu-
bated on shipboard provided a significant addition to the
available methods for studying phytoplankton growth in the sea.
The technique has broad appeal for use in field phytoplankton
studies. It allows one to measure nitrogen productivity and
to distinguish between the fractions due to different forms
of nitrogen utilized (Dugdale and Goering, 1967; Goering,
Wallen and Naumann, 1970). Assay of phytoplankton enzymes
involved in nitrogen assimilation, such as nitrate reductase,
is also feasible at sea (Eppley, Coatsworth and Solorzano,
1969; Eppley, Packard and Maclsaac, 1970) and may indicate
qualitatively the utilization of nitrate or its suppression by
ammonium.
The following study used shipboard culture experiments to compare
the ^N-method and direct chemical analyses of nitrogenous
nutrients and particulate nitrogen for estimating the phytoplank-
ton uptake of nitrate, ammonium, and urea. At the same time
enzyme assays of nitrite reductase and glutamic dehydrogenase
were carried out with the heuristic purpose of assessing their
utility in studies of phytoplankton nitrogen assimilation. More-
over, the shipboard cultures provided an opportunity to observe
interactions between phytoplankton uptake of nitrate, ammonium,
and urea when more than one of these forms of nitrogen was added.
Results of the N- and enzyme methods are also compared for
several stations off the California coast, between San Diego
and Los Angeles, where assessment of phytoplankton uptake of
nitrogen by direct chemical measurement was not possible. Other
aspects of this work are described in Eppley ^t aJ. (1971a, b).
47
-------
Shipboard Cultures
The N technique gave good results in comparison x^ith the direct
chemical methods of measuring N-assimilation, i.e. loss of nitrate,
ammonium, or urea from the culture medium and increase in particu-
late nitrogen in the cultures (Table 12). The isotope technique
overestimated nitrogen utilization over a 36 hour period by 9.2%
in the nitrate cultures and by 22.8% in the ammonium culture and
underestimated it by 0.5% in the urea culture, when compared to
an average value of particulate nitrogen increase and nutrient
loss. Furthermore, specific growth rates computed from nitro-
gen uptake rates compared well with those computed from increases
in cell concentration.
Nitrite reductase (NiR) activity would be expected to follow the
capacity for nitrate uptake and it showed a fair correspondence
to the measured rate of nitrate assimilation (Tables 12, 13).
Rates of nitrate uptake capacity and NiR activity were highest in the
nitrate culture, intermediate in urea cultures and lowest when
ammonium was the nitrogen source. A linear regression of nitrite
reductase specific activity vs ^NO-j gave
NiR/PN = 0.95 VXT. - 0.09
NO,
with 95% confidence limits on the slope of 0.55 with 17 pairs
of measurements.
Glutamic dehydrogenase activity would be expected to parallel
the capacity for ammonium assimilation but GDH assays proved to
be a poor measure of the latter, either when enzymatic activity
was measured with NADH, NADPH or as the sum of that with both
pyridine nucleotides (Table 13). The ratio of the total GDH
activity to the rate of ammonium assimilation measured with
in the cultures was 0.73 + 0.56 (2a) for samples taken during
the day when the two measures would be expected to most closely
correspond to one another.
The close relation between nitrite reductase activity and rate of
nitrate assimilation noted in the culture experiments was not ob-
served in the natural seawater samples^ Nitrite reductase activity
was always higher than the rate of N0» assimilation measured with
•'•%03~, often by an order of magnitude. Since NiR also occurs in
micro-organisms other than phytoplankton, heterotrophic NiR offered
a potential explanation. Hence, we compared NiR activity per weight
of chlorophyll a_ (present only in photosynthetic organisms) and
per weight of ATP (present in all organisms) in the filtered par-
ticulate matter, for both seawater samples and culture samples
(containing few heterotrophs) without finding any significant
48
-------
Table 12. Changes in particulate nitrogen; nitrate, ammonium, and urea concentrations; utilization
of nitrogen; and nitrite reductase activity for each six-hour period by shipboard cultures.
NITRATE CULTURE
VO
Utilization of Nitrogen Nitrite Reductase
Date
13 July
1200
13 July
1800
14 July
0000
14 July
0600
14 July
1200
14 July
1800
15 July
0000
15 July
0600
I
13-15 July
1800 0600
A Particulate Nitrogen A N03~
(yg at N/l) (yg at N/l)
4.5 7.6
5.1 1.5
-0.7 2.2
13.1 8.0
10.4 6.0
3.7 0.6
0.0 4.2
31.6 22.5
( P N03)
(yg at N/l)
2.6
2.5
3.2
4.8
4.5
5.7
8.9
29.6
Activity
(yg at N/l)
5.1
5.1
3.9
4.8
4.8
6.5
6.2
31.3
-------
Ui
o
Table 12, Cont'd.
AMMONIUM CULTURE
Utilization of Nitrogen
Date
13 July
1200
13 July
1800
14 July
0000
14 July
0600
14 July
1200
14 July
1800
15 July
0000
15 July
0600
E
13-15 July
1800 0600
A Particulate Nitrogen A NH^4" ( P NH4+)
(Ug at N/l) (yg at N/l) (ug at N/l)
13.8 8.2 3.5
4.3 2.0 5.8
15.9 7.9 7.9
12.1 10.8 13.1
4.5 14.7 12.4
9.8 6.3 8.3
1.2 1.7 9.0
48.8 43.4 56.5
-------
Table 12. ContM.
Date
13 July
1200
13 July
1800
14 July
0000
14 July
0600
14 July
1200
14 July
1800
15 July
0000
15 July
0600
A Particulate Nitrogen
(yg at N/l)
1.1
1.0
4.7
8.4
2.0
UREA CULTURE
A Urea
(yg at N/l)
2.3
5.3
6.4
6.2
2.6
Utilization of Nitrogen
(Purea)
(yg at N/l)
2.9
2.5
7.1
3.2
4.2
13-15 July
1800 0600
17.2
22.8
19.9
The uptake rate of N-labelled nitrate, ammonium, or urea.
-------
Table 13. The mean rate of uptake (V) for nitrate, ammonium, and
urea and nitrite reductase (NiR) and glutamic dehydroge-
nase (GDH) activities per weight of particulate nitrogen
in each culture averaged over the duration of the experi-
ment. All units are hrs.~ . GDH activity is the sum of
that with NADH and NADPH measured separately.
Culture
VNO~
Experiment I
urea
NiR
PN
GDH
PN
Nitrate
Ammonium
Urea
.0236
,0034
,0080'
.0304
.0309
.0367*
.0087
.0052
.0237
.017
.004
ND
.025
.025
.022
Experiment II
Nitrate
Ammonium
Urea
.0278
.0014
.0164*
.0245
.0318
.0417*
.0105
.0039*
.0418
.025
.008
.012
ND
ND
ND
*Potential uptake. Phytoplankton were exposed to this form of
nitrogen only during measurement period.
52
-------
differences: NiR/Chl. a_ and NiR/ATP were similar in the nitrate
culture and in the seawater samples. Furthermore, for natural
seawater samples, a graph of the ratio NiR/ATP vs Chi. a/ATP
had a positive slope, with 95% confidence limits not including
a slope of zero, and the intercept was not significantly dif-
ferent from zero (p<.05). This seems to imply that the nitrite
reductase activity observed is primarily in chlorophyll-contain-
ing cells. Differences between NiR activity and rate of nitrate
uptake would be due to low nitrate uptake rates rather than lack
of assimilatory enzymes. Nitrite reductase activity >ras found
even in surface sea water samples lacking nitrate and with low
rates of nitrate assimilation.
Results with glutamic dehydrogenase assays of seawater samples
were even less satisfactory. Control samples, containing a
complete reaction mixture except ammonium sulfate, showed con-
siderable oxidation of pyridine nucleotide not seen in work with
cultures. This suggested that high levels of ammonium were somehow
introduced in the enzyme preparation from the filtered particulate
matter and this suggestion was later confirmed by ammonium analysis
of homogenated particulate matter from sea water collected at
the Scripps Institution Pier. It is apparent that we were, in
fact, measuring the rate of oxidation of reduced pyridine nucleo-
tides in the presence of a-ketoglutarate and ammonium and that
actual GDH levels could not be calculated for lack of adequate
experimental controls, lacking ammonium.
Since even this information may be of value in assessing metabolic
activity, the data for culture and sea water samples were re-
calculated as rate of pyridine nucleotide oxidation. The sum of
the activities with both nucleotides was a constant fraction of
the ATP content of the samples (Table 14) in both culture and
seawater samples over 3 orders of magnitude variation in ATP
content. The summed activities (i.e. that with both pyridine
nucleotides) were also proportional to the chlorophyll content
of the sample, although with greater scatter in the seawater
samples. In contrast to results with NiR a graph of the summed
activity/weight ATP vs chlorophyll a./ATP had a slope not signifi-
cantly different from zero (p>0.2). To the extent that the
Chi. a/ATP ratio represents the proportion of phytoplankton in
all living organisms in the sample, this result implies that the
activities were not restricted to phytoplankton.
53
-------
Table 14. Ratios of enzymatic activity to chlorophyll a^ and to
adenosine triphosphate (ATP). Units are ymoles/yg'hr.
(average + standard deviation).
Rate/Chlorophyll a Rate/ATP
Cultures
Nitrite reductase* 0.027 + .019 0.080 + 0.023
Pyridine nucleotide 0.12 + 0.04 0.29 + 0.07
oxidation
Seawater Samples
Nitrite reductase 0.020 + 0.017 0.065 + 0.058
Pyridine nucleotide 0.16 +0.10 0.22 +0.06
oxidation
*Nitrate cultures only
54
-------
SECTION X
DISCUSSION
The timing of the first cruise in July, 1970, was propitious for
showing the eutrophication around southern California sewage
outfalls where phytoplankton crops and primary production exceed
typical levels off this coast and approach values characteristic
of upwelling periods.
Concentrations of nutrients were not elevated in samples taken at
the outfall stations (except occasional ammonia values) even
when bottle casts were taken directly over outfalls. This would
seem to imply that the hydraulic diffusers were effective in
diluting the wastes prior to emission and that the phytoplankton
growth was sufficiently vigorous to keep up with the nutrient
input rate.
If a value for inorganic nitrogen (nitrate and ammonium) of 2 mM
is taken for sewage (Weibul, 1969), with a 200-fold dilution with
sea water during emission, the final nitrogen concentration would
be about 10 yM. This concentration of nitrogen would produce a
phytoplankton crop equivalent to about 10 yg/1 of chlorophyll a^
using conversion factors found in this laboratory. Maximum ob-
served chlorophyll a_ concentrations were 10 to 17 yg/1 at the two
outfalls, in reasonable agreement with the hypothesis that the
phytoplankton crop was assimilating the inorganic nitrogen of the
effluent as fast as it was released. This hypothesis was sup-
ported by the 1-3 days turnover times of nitrate, ammonium, and
urea in the upper 10 meters observed both in July, 1970 and June,
1971.
Nitrate and phosphate concentrations found in this study were
much lower than in eutrophic estuaries of the eastern coast
of the United States (Ketchum, 1969; Carpenter, Pritchard and
Whaley, 1969; Flemer ^t al. 1970; Ryther and Dunstan, 1971) and
fall within the range of values observed elsewhere along the
southern California coast (Strickland, ed., 1970). Chlorophyll
^ and phosphate concentrations resemble those from Long Island
Sound rather than polluted estuaries (see Fig. 4, in Ketchum,
1969; Flemer et _al. 1971; Ryther and Dunstan, 1971). The high
ammonium levels (up to 30 micromolar) observed below 15 meters
in June, 1971, at the White Point outfall constitute an exception
to this generalization.
55
-------
Earlier work has shown that while growth and uptake rates of
nitrogen by coastal marine phytoplankton are dependent upon
the ambient nutrient concentration, rates are essentially
saturated between one and five micromolar concentrations of
nitrate, ammonium (Maclsaac and Dugdale, 1968; Eppley, Rogers,
and McCarthy, 1969; Eppley and Thomas, 1969) and urea (McCarthy,
1971). Since concentrations were less than one micromolar in
the upper 10 to 15 meters the phytoplankton seem to be in a
favorable position to adjust rapidly to changes in ambient
nutrient levels. That is, their growth and uptake rate is on
a steep portion of the rate vs. concentration curve, and any
increase in nutrient can be accomodated by increasing uptake
rate of each algal cell. If nutrient concentrations were so
high as to be rate-saturating then the phytoplankton could
respond to increased nutrient concentration only by an increase
in crop size, certainly a slower and less sensitive mechanism
than that postulated, and one which requires the cooperation of
grazing animals as well. (I am indebted to Drs. John Caperon
and Alan Cattell, Univ. Hawaii, for this insight). This is
perhaps another way of saying that the input of nitrogen at
the outfalls, monumental as it is, is not yet great enough to
exceed the capacity of the local phytoplankton to assimilate
it. However, the elevated ammonium concentrations noted in 1971
at White Point below 15 meters depth seem to suggest that the
phytoplankton may not be able to keep up indefinitely. The
consequence of such a failure would be multifold: (a) increases
in phytoplankton crops about the outfalls; (b) increased concen-
trations of ammonium at the surface near the outfalls; and (c)
an increase in the spatial extent of the outfalls influence;
i.e. increased eutrophication of the southern California coastal
waters. Perhaps the most dangerous consequence for local food
webs would be a shift in the species composition of the phyto-
plankton flora to minute coccoid green algae as observed in bays
receiving duck farms wastes (Ryther, 1954; Hulburt, 1970).
Samples taken for determination of phytoplankton species will
not be counted by the time this report is due but will be avail-
able ultimately.
Some of the results of laboratory experiments suggest other
observations which might be made at the outfalls:
(1) The diel periodicity in assimilation rate of nitrate and
ammonium suggests that uptake rates might slow greatly at night
in natural phytoplankton populations. If so, this would lead
to increased ambient ammonia levels at night in the upper few
meters where the impact of phytoplankton nutrient assimilation
is greatest.
56
-------
(2) If one assumes that the phytoplankton crop about the outfalls
behaves as if in a nitrogen-limited chetnostat culture, albeit
of unknown volume, some further inferences can be made. From
the variation in the ratio of cell carbon to nitrogen (Fig. 10)
in the natural phytoplankton one can guess the specific growth
rate of the crop as a fraction of its maximum growth rate (i.e.
y/ym). In samples where large crops of phytoplankton allow such
estimates, relatively free of error due to particulate detritus
in the samples, ratios were in the range 7 to 9. This suggests
that y/ym is about 0.85. If v^ were about 0.7 doublings/day, as
measured in nutrient-rich waters off Peru (Strickland ^t al^. 1969;
Beers &t_ al. 1971) then y would be about 0.6 doublings/day. Other
estimates of y from rates of nitrogen and carbon assimilation
calculated as daily increments of the existing crops (Eppley
jit al. 1971; McCarthy, 1971) suggest lower values about 0.3 to
0.4 doublings/day, i.e. y/ym 0.4 - 0.6. This discrepancy may
be due to differences in irradiance between the cultures and
natural situations as irradiance has a marked influence on C/N
ratios of cultures.
(3) Perhaps the greater utility of N-limited chemostats as
models or natural phytoplankton growth about the outfalls
derives from the possibility of mathematical descriptions
permitted by chemostat theory.
However, with the present lack of measurements over the seasons
it cannot be assumed that our estimates of the phytoplankton crop
and growth rate represent a steady-state. Advective processes,
variation in the quantity and quality of wastes discharged, and
periodic upwelling would complicate mathematical simulation modeling
but efforts in this direction would be interesting and useful
(Dugdale and Whitledge, 1970).
While our data are too limited for good quality simulation
modeling because of infrequent sampling and poor areal coverage
certain of the measurements may prove useful, such as the kine-
tic parameters for nitrate and ammonium uptake. The short
turnover times for these nutrients and for urea in the upper few
meters imply a close coupling between nitrogen input and assimi-
lation by the phytoplankton. These turnover times are generally
less than the doubling times calculated for the phytoplankton.
The ambient nitrate and ammonium concentrations measured in the
upper 10-15 meters are so low that uptake rate is on the steep
portion of uptake rate vs. concentration curves for the crops.
These two observations strengthen the conclusion that the phyto-
plankton crops are able to keep up with the rate of nitrogen
input. They furthermore show the power of the 1% methods of
measuring nitrogen assimilation in southern California coastal
waters in which nitrogen is usually the growth rate-limiting
nutrient.
57
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SECTION XI
ACKNOWLEDGMENTS
This work was planned and carried out at the Scripps Institution
of Oceanography by members of the Food Chain Research Group,
Institute of Marine Resources, University of California at
San Diego. Mr. E. H. Renger, Mrs. Gail Hirota, Mrs. Jane Rogers,
Mrs. Melita Dee, Dr. James J. McCarthy (then a graduate student);
Dr. R. W. Eppley and Dr. 0. Holm-Hansen provided experimental or
analytical contributions. Dr. Eppley prepared this report.
Shiptime was funded by the National Science Foundation in 1970 via
the R/V Alpha Helix Eastern Pacific Program and in 1971 via a
grant to the Scripps Institution of Oceanography. The U.S.
Atomic Energy Commission, Contract AT(11-1)GEN 10, P.A. 20
shared in the support of the laboratory aspects of this program.
Drs. W. H. Thomas, A. F. Carlucci, P. M. Williams and J. R. Beers
provided advice and counsel. The late Dr. J. D. H. Strickland,
head of the Food Chain Research Group, helped the work in innumer-
able ways. We thank Dr. T. Enns for the use of his mass spectro-
meter for the •'••'N work.
59
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SECTION XII
REFERENCES
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61
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12. Eppley, R.W., and Thomas, W.H., "Comparison of half-saturation
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with nitrate, ammonium or urea as the nitrogen source."
Submitted to Limnol. Oceanog. (1971a).
18. Eppley, R.W., Carlucci, A.F., Holm-Hansen, 0., Kiefer, D.,
McCarthy, J.J., and Williams, P.M., "Evidence of eutrophica-
tion in the sea near southern California coastal sewage
outfalls." Submitted to Calif. Cooperative Oceanic Fisheries
Investigations Reports (1971b).
19. Eppley, R.W., Rogers, J.N., McCarthy, J.J., and Sournia, A.,
"Light/dark periodicity in nitrogen assimilation of the
marine phytoplankters Skeletonema costatum and Coccolithus
huxleyi in N-limited chemostat culture," J. Phycol., 7.
pp 150-154 (1971c).
20. Flemer, D.A., Hamilton, D.H., Keefe, C.W., and Mihursky,
J.A., "The effects of thermal loading and water quality on
estuarine primary production," Natural Resources Inst.,
Univ. Maryland Ref. No. 71-6, 217 pp (1970).
21. Goering, J.J., Wallen, D.D., and Nauman, R.M., "Nitrogen
uptake by phytoplankton in the discontinuity layer of the
eastern subtropical Pacific Ocean," Limnol. Oceanogr., 15,
pp 789-796 (1970).
62
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22. Grigg, R.W., and Kiwala, R.S., "Some ecological effects of
discharged wastes on marine life," Calif. Fish and Game, 56,
pp 145-155 (1970).
23. Holmes, R.W., Williams, P.M., and Eppley, R.W., "Red water
in La Jolla Bay, 1964-1966," Limnol. Oceanogr., 12, pp 503-
512 (1967).
24. Holm-Hansen, 0., "Determination of particulate organic nitro-
gen," Limnol. Oceanogr., 13, pp 175-178 (1968).,
25. Holm-Hansen, 0., Lorenzen, C.J., Holmes, R.W., and Strickland,
J.D.H., "Fluorometric determination of chlorophyll," J. Cons.
Perm. Int. Explor. Her., 30. pp 3-15 (1965).
26. Joy, K.W., and Hageman, R.H., "The purification and properties
of nitrite reductase from higher plants and its dependence on
ferredoxin," Biochem. J., 100, pp 263-273 (1966).
27. Ketchum, B.H., "Eutrophication in estuaries," In: Eutrophication;
Causes, Consequences, Correctives, Publ. No. 1700, NAS-NRC
Washington, D.C. pp 197-209 (1969).
28. Lorenzen, C.J., "A method for the continuous measurement of
in vivo chlorophyll concentrations," Deep-Sea Res., 13, pp
223-227 (1966).
29. Maclsaac, J.J., and Dugdale, R.C., "The kinetics of nitrate
and ammonia uptake by natural populations of marine phyto-
plankton." Deep-Sea Res., 16, pp 415-422 (1968).
30. McCarthy, J.J., "A urease method for urea in seawater.
Limnol. Oceanogr., 15, pp 303-313 (1970).
31. McCarthy, J.J., "The role of urea in marine phytoplankton
ecology," Ph.D. Dissertation, Scripps Institution of
Oceanography, La Jolla, Calif. 165 p (1971).
32. Moberg, E.G., "The interrelationship between diatoms,
their chemical environment, and upwelling water in the
sea off the coast of southern California," Proc. Nat.
Acad. Sci. (U.S.). 14, pp 511-517 (1928).
33. Munk, W.H., and Riley, G.A., "Absorption of nutrients by
aquatic plants," J. Mar. Res.. 11, pp 215-240 (1952).
34. Ryther, J.H., "The ecology of phytoplankton blooms in
Moriches Bay and Great South Bay, Long Island, N.Y.,"
Biol. Bull.. 106, pp 198 (1954).
63
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35. Ryther, J.H., and Dunstan, W.M. , "Nitrogen, phosphorus, and
eutrophication in the coastal marine environment," Science,
171. pp 1008-1013 (1971).
36. Sargent, M.C., and Walker, T.J., "Diatom populations associated
with eddies off southern California in 1941," J. Mar. Res., 7,
PP 490-505 (1948).
37. Smith, R.L., "Upwelling," In: Oceanogr. Mar. Biol. Ann. Rev.
_6_, Allen and Unwin, London, pp 11-47 (1968).
38. Solorzano, L., "Determination of ammonium in natural waters
by the phenolhypochlorite method," Limnol. Oceanogr., 14,
pp 799-801 (1969).
39. Stevenson, R.E., and Grady, J.R., "Plankton and associated
nutrients in the waters surrounding three sewer outfalls in
southern California. A final report submitted to the Hyperion
Engineers, Inc. from the Geology Department, University of
Southern California (1956).
40. Strickland, J.D.H., "Production of organic matter in the
primary stages of the marine food-chain," In Chemical
Oceanography, Academic Press, London, pp 477-610 (1965).
41. Strickland, J.D.H. (ed.), "The ecology of the plankton off
La Jolla, California, in the period April through September,
1967." Bull. Scripps Inst. Oceanogr.. 17. pp 1-103 (1970).
42. Strickland, J.D.H., and Parsons, T.R., ''A Practical Handbook
of Seawater Analysis, Bull. Fish. Res. Bd., Canada, No. 167,
311 p (1968).
43. Strickland, J.D.H., Eppley, R.W., and Rojas de Mendiola, B. ,
"Phytoplankton population, nutrients and photosynthesis in
Peruvian coastal waters," Bol. Inst. del Mar del Peru, 2,
pp 1-45 (1969).
44. Sverdrup, H.U., and Allen, W.E., "Distribution of diatoms in
relation to the character of water masses and currents off
southern California in 1938," J. Mar. Res., 2, pp 131-144
(1939).
45. Thomas, W.H., and Dodson, A.N., "On nitrogen deficiency in
tropical oceanic phytoplankton. II. Photosynthetic and cel-
lular characteristics of a chemostat-grown diatom, (1971),
in press.
64
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46. Turner, C.H., Ebert, E.E., and Given, R.R., "The marine
environment offshore from Pt. Loma, San Diego County,"
Calif. Dept. Fish and Game, Fish. Bull., 140, pp 1-85
(1968).
47. Turner, G.K., and Associates, Inc., Manual of Fluorometric
Clinical Procedures, Palo Alto, Calif.
48. Water Resources Engineers, "Effects of ocean discharge of
waste water on the ocean environment near the city of San
Diego outfall," Water Resources Engineers, Inc. Walnut
Creek, Calif. Report - September 1967, 57 p (1967).
49. Weibul, S.R., "Urban drainage as a factor in eutrophication,"
In: Eutrophication: Causes, Consequences, Correctives, Publ.
No. 1700, NAS-NRC, Washington, D.C. pp 383-403 (1969).
50. Wilkinson, G.N., "Statistical estimations in enzyme kinetics,"
Biochem. J., 80, pp 324-332 (1961).
51. Wood, E.P., Armstrong, F.A.J., and Richards, F.A., "Determina-
tion of nitrate in sea water by cadmium-copper reduction to
nitrite," J. Mar. Biol. Ass. U.K.. 47, pp 23-31 (1967).
65
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SECTION XIII
PUBLICATIONS
The following research papers have resulted wholly or in part
from this research grant. Some of the papers are in press,
have been submitted for publication, or are in preparation.
Only those actually submitted are listed.
1. Eppley, R.W., and Rogers, J.N., "Inorganic nitrogen assimi-
lation of Ditylum brightwellii, a marine plankton diatom,"
J. Phycology, 6, pp 344-351 (1970).
2. Eppley, R.W., Rogers, J.N., McCarthy, J.J., and Sournia, A.,
"Light/dark periodicity in nitrogen assimilation of the marine
phytoplankters Skeletonema costatum and Coccolithus huxleyi in
N-limited chemostat culture," J. Phycology, 7, pp 150-154
(1971).
3. Eppley, R.W., Carlucci, A.F., Holm-Hansen, 0., Kiefer, D.,
McCarthy, J.J., and Williams, P.M., "Evidence for eutrophica-
tion in the sea near southern California coastal sewage out-
falls July, 1970," Rep. Calif. Coop. Oceanic Fisheries Invest.
(1971) In press.
4. Eppley, R.W., Carlucci, A.F., Holm-Hansen, 0., Kiefer, D.,
McCarthy, J.J., Venrick, E., and Williams, P.M., "Phyto-
plankton growth and composition in shipboard cultures sup-
plied with nitrate, ammonium or urea as the nitrogen source,"
Limnol. Oceanogr., (1971), In press.
5. McCarthy, J.J., "The role of urea in marine phytoplankton
ecology," Ph.D. Dissertation, Univ. Calif. San Diego. 165 p
(1971).
6. McCarthy, J.J., "Urea as a source of nitrogen for marine
phytoplankton," Submitted to J. Phycology (1971).
7. McCarthy, J.J., and Eppley, R.W., "A comparison of chemical,
isotopic, and enzymatic methods for measuring nitrogen as-
similation of marine phytoplankton," Submitted to Limnol.
Oceanogr., (1971).
67
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j I Accession .Yu/iiber
o i Subiect
* Field &. Croup
SELECTED WATER RESOURCES ABSTRACT
INPUT TRANSACTION FORM
_5j "*" '2B'°n
Institute of Marine Resources, Scripps Institution of Oceanography,
University, of California, San Diego
6 \Ti"f
Eutrophication in Coastal Waters: Nitrogen as a Controlling Factor,
,n | Authors)
Eppley, Richard W.
. . 1 Dale
' Dec., 1971
16
^2 Pages
67
Project Number
16010 EHC
.[• Contract Number
16010 EHC
2| Note
22
, Citation
23 Descriptors ("Starred Fiist)
25 ' Identifiers (Starred First)
'*Southern California Coastal Sewage Outfalls, Nitrogen Assimilation
in mar-ing phyt-npl anVfon . - _
27
Abstract
The role of southern California coastal sewage outfalls in the
eutrophication of local seawater was investigated. The outfall ef-
fluents have a measureable influence on standing stocks of phytoplank-
ton, and on primary production. Two cruises were undertaken, in July,
1970, and June, 1971. Kinetic parameters for the assimilation of
ammonium, ni-trate and'urea were determined at the outfall sites using
•*--%-labelled substrates. These parameters will be useful for simula-
tion models of phytoplankton growth as influenced by local sewage
effluents.
The utilization of various forms of nitrogen by phytoplankton,
mechanisms and rates of nitrogen assimilation and enzymes of nitr,ogen
assimilation were investigated in laboratory cultures. Ammonium and
nitrate assimilation were found to vary from day to night as does
the capacity for photosynthesis when cultures were grown on light-
dark cycles simulating natural illumination. (Eppley - UCSD).
Abstractor
R. W. Eppley
Institutton
San Diego
WO; 102 (REV. OCT. K9II
nnsic
University of Ca^^^:m•n^aJ San Pi
tCNO TO- WATER BESOURCES SClENTlFK
u 5. Oe"»RTME~T OF TH£ INT
WASHINGTON. O C. 2024C
FORM*'
ERIO«
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