BULLETIN OF MARINE SCIENCE, 27(1):
44-57, 1977
PHYTOPLANKTON NITROGEN METABOLISM, NITROGEN
BUDGETS, AND OBSERVATIONS ON COPPER
TOXICITY: CONTROLLED ECOSYSTEM
POLLUTION EXPERIMENT
W. G. Harrison, R. W. Eppley, and E. H. Renger
ABSTRACT
Phytoplankton
growth and nitrogen metabolism seemed typical of coastal temperate
plankton in the 0.25-scale CEPEX enclosures used at Saanich Inlet, B.C., in the summer
of 1974. Maximum rates of nitrogen assimilation corresponded
to a growth rate of
about 1.0 day" and half-saturation values for nitrate and ammonium uptake were about
1 MM. Although assimilation rates on a day-to-day basis were regulated primarily by
ambient nitrate and ammonium levels, overall phytoplankton levels and settlement rates
were related to the rate of nutrient loading. Remineralization
of nitrogen to ammonium
was rapid and important.
Although only nitrate (along with phosphate and silicate nutrients) were added to the enclosures, rates of ammonium assimilation by the phytoplankton were similar to those for nitrate.
Several acute effects of copper on phytoplankton were observed, including inhibition of
nitrate uptake, photosynthetic
carbon assimilation, synthesis of nitrate reductase, and
cell disruption and loss of accumulated ammonium in Noctiluca sp. Evidence suggests
that the addition of copper to the enclosures resulted in acute inhibition of phytoplankton
growth and a replacement of the initial phytoplankton by a copper-tolerant
assemblage.
Bioassay experiments indicated that even after the shift to copper-tolerant
forms, (1)
the copper in the enclosures remained in a chemical form still toxic to phytoplankton
from control enclosures, and (2) the degree of copper-tolerance
of the phytoplankton
was related to ambient copper concentrations.
Copper is one of the more interesting
heavy metals to aquatic ecologists because
naturally-occurring concentrations may reach
inhibitory levels for some species in some
environments and during some seasons. For
example, Riley (1939) studied the copper
cycle in three Connecticut lakes and found
that natural copper concentrations in the
faIl, and in the hypolimnion in summer,
reached inhibitory levels for Hydra sp.,
Daphnia pulex, and perhaps other species.
This was judged from laboratory experiments on isolated animals and chemical
measurements of copper in the lakes. Other
events taking place in the lakes precluded
any conclusions as to whether copper actually influenced the seasonal or depth distribution of the animals, however.
Species differ in their sensitivity to copper
poisoning. Riley (1939) presented some
toxicity curves for freshwater invertebrates,
discussed much of the early work, and provided a historical review of copper toxicity
studies going back to 1881. For phytoplankton the studies of Moore and Kellerman
(1904, 1905) and Hale (1922), relative to
removing undesirable microorganisms from
reservoirs with copper sulfate, are significant. Palmer and Maloney (1955) and
Maloney and Palmer (1956) provided more
recent evidence for species differences in
copper sensitivity among freshwater phytoplankton. Saward et aI. (1975) also provided a historical review, citing quite a different suite of references than Riley (1939)
and including references on the different
sensitivities to copper among various taxonomic groups of algae (diatoms seem most
sensitive, green algae least sensitive, with
dinoflagellates and blue-green algae inter44
HARRISON
ET AL.:
PHYTOPLANKTON
METABOLISM
mediate). They also cited earlier observations on the development (or selection) of
copper-resistant strains and a case of copper
pollution along the Dutch shore.
The purpose of the CEPEX (Controlled
Ecosystem Pollution Experiment) program
at Saanich Inlet, B.C., Canada, and a description of the enclosures are given in detail by Menzel and Case (1977). The present authors participated in three CEPEX
experiments in the summer of 1974: two on
effects of added copper (June-July and September), and one on effects of an aqueous
extract of petroleum (August, referred to
later as the hydrocarbon experiment).
In addition to documenting the course of
phytoplankton nitrogen assimilation in the
CEPEX enclosures, and effects of pollutants
thereon, we viewed our role as one of helping to delineate the effects of efforts to
manage the experimental plankton crops in
the enclosures and to identify and segregate
these effects from those due to added pollutants. Differences in rates of nutrient addition in the various experiments provide an
example of such effects in the present study.
That is, the 1974 summer experiments inadvertently constituted a study of the effects
of nutrient loading rate on plankton stocks,
settlement rates, and nutrient recycling; in
short, a eutrophication experiment.
The experimental plankton assemblages
provided opportunity for testing methods of
assessing pollutant effects, and for developing bioassay techniques appropriate to specific pollutants. For example, HC photosynthesis measurements used to assess
copper inhibition of phytoplankton growth
affirmed that copper inhibition was not immediate but required several hours' exposure for full development (Steemann
Nielsen and Wium-Andersen, 1971). They
also suggested that threshold concentrations
for copper inhibition exist and that they vary
with the ambient copper concentration that
prevailed during previous growth of the phytoplankton. Acute effects of copper were
noted at the 5-10 p.gjllevel as in other recent
studies of both freshwater (Steemann Niel-
AND COPPER
TOXICITY
45
sen et aI., 1969) and marine phytoplankton
(Erickson, 1972; Krock and Mason, 1971;
Saward et aI., 1975).
METHODS
Nitrogen Assimilation
Nitrate and ammonium assimilation rates
were measured isotopically with 15N-Iabelled
nitrate and ammonium using water samples
from the CEEs. Four-liter samples were enriched with 0.1 p.M liter1 of the 15N-Iabelled
ion and incubated in pyrex bottles. One set
of bottles was covered with neutral density
screens to give a range of light intensity approximately 1 to 90% of ambient light. These
were used to obtain depth profiles of N-assimilation in simulated in situ incubations.
A set of clear bottles was used to provide
uniform light for measuring assimilation rate
as a function of nitrogen concentration
added. Samples for the depth profiles were
taken from 1-, 5-, and 9-m depths in the
CEEs. Samples for uptake kinetics measurements were taken from I-m depth. All samples were incubated in natural light in lucite
boxes cooled by running water. In the first
copper experiment (June-July)
samples
were incubated 24 h, in the second experiment (Sept.-Oct.) for approximately 4 h.
Following incubation, the particulate matter
was removed by filtration using Reeve Angel
984H glass fiber filters. These were rinsed,
placed in waxed paper envelopes, and dried
in a vacuum desiccator. Analyses were
carried out by mass spectrometry following
conversion of the particulate nitrogen to
nitrogen gas by the Dumas procedure (Dugdale and Goering 1967). Assimilation rates
were calculated as specific rates per weight
of particulate nitrogen (V, day-1 or h-1) or
as moles 1-1 time-1 by multiplying the V
values by the particulate N content of the
samples. The latter were measured with a
Hewlett-Packard Model 185B CHN Analyzer (Sharp, 1974). Although nutrient
analyses of CEE water were carried out as
part of the routine monitoring of the experiments, separate samples for nitrate
46
BULLETIN OF MARINE SCIENCE, VOL. 27, NO.1,
(Wood et at, 1967) and ammonia (SoI6rzano, 1969) were analyzed at the initiation
of each 15N experiment.
Nitrate Reductase Assays
Particulate matter concentrated on glass
fiber filters from 2- to 4-1 water samples
from the CEEs was homogenized in phosphate buffer containing dithiothreitot The
centrifuged extracts were used as a source
of crude enzyme and aliquots were added
to a reaction mixture containing buffer,
KN03, MgS04, and NADH (Eppley et at,
1969a). After 30 min incubation the reac-
tion was stopped by adding zinc acetate and
ethanol. The mixture was centrifuged and
nitrite, the product of the reaction, was measured spectrophotometric ally (Strickland and
Parsons, 1972), Chlorophyll a was determined on the same samples by a fluorometric
method also described in Strickland and Parsons (1972). Results were calculated as
moles of nitrite formed per weight of chlorophyll a per time. The limits of detection
will depend upon the amount of material
extracted and in the present work would be
about 0.05 X 10-9 moles of nitrite formed
(p.g chlorophyll a)-l h-1.
Nitrogen Budgets
The amount of nitrogen in various fonns
(soluble inorganic-N, particulate-N, etc.) on
any particular day was determined by summing the products of the measured concentrations at the depths used and a volumesection corresponding to the particular
depth. For example, particulate-N concentrations (measured at 1, 5, and 9 m), were
multiplied by volumes corresponding to
depth intervals of 0-3 m, 3-7 m, and 7-15 m,
respectively. Nitrate was measured at 0, 1,
3, 5, 7, 9, 11, and 13 m, ammonium at 0,
7, and 13 m. An enclosure diameter of 2.5
m was used in the volume calculations and
the total volume was assumed to be 68 m3•
Values were expressed as grams nitrogen per
enclosure. Day-to-day changes are indicated
below as .6N.
1977
In addition, the total amount of nitrogen
(as nitrate) added to each enclosure over the
experimental period was noted. Finally,
from these derived parameters, the gross
amount of material settling out of the enclosures (expressed as grams N) was estimated from the equation
Gross settlement =
nitrate added - 6. total inorganic-N
- .6 particulate-N
A single pathway of nitrogen flow (inorganic-N ~ particulate-N ~ settlement-N)
is assumed in this equation.
Average net flux rates of nitrogen to phytoplankton were estimated from the budget
data by dividing the overall decrease in inorganic-N (sum of measured inorganic-N
decrease and total N added) by the duration
of the experimental periods. Likewise, nitrogen remineralization rates were calculated
by summing the overall net increase in ammonium concentrations with the ammonium
assimilated (measured in the 15N productivity experiments) and dividing by the duration of the experiments. From this information, a figure for net settlement production
could be calculated by subtracting the remineralization value from the gross settlement value previously obtained. These "remineralization" values include not only
direct ammonification of the settling particulate matter, but other sources such as zooplankton excretion, leakage from phytoplankton, and so forth.
Nutrient Additions to the CEEs
A schedule of nutrient additions was
carried out based upon ambient nitrate concentrations. When nitrate fell below 1 p.gat Nil at any depth a nutrient addition was
made at those depths (usually the upper 510 m) to bring the final concentration of
nitrate either to 3 p.g-at Nil (first copper
experiment) or 1p.g-at Nil (second copper
experiment). The added nutrient mixture
contained equimolar amounts of nitrate and
HARRISON
ET AL.:
PHYTOPLANKTON
METABOLISM
Table 1. Nitrate and ammonium assimilation
rates from "simulated ill situ" incubations. Values
are averages of the number of measurements given
in parentheses. Copper analysis for these experiments are described by Topping and Windom
( 1977).
Depth
m
Treatment
47
TOXICITY
Table 2. Nitrate and ammonium specific assimilation rates measured by simulated ill situ techniques 2-3 days after adding copper to the CEEs
to show copper-inhibition.
Values are averages
± S.D. for samples from 1-, 5-, and 9-01 depths.
Specific
Assimilation
Rate
CEE
Assimilation Rates as
'" moles N 1-1h-1
NO,
AND COPPER
Date
(",gil ClI)
(h-1 X 10-')
Experiment 1. Copper added, 17 June 1974
NH.
20 June
6.2 ± 0.4
0.54 ± 0.12
Nitrate
Nitrate
June-July,
controls
1
5
9
0.035(5)
0.012(5 )
0.014(5)
0.011 (5)
0.010(5)
0.0096(5)
Experiment 2. Copper added, 10 September 1974
12 Sept.
1
5
9
0.018(3 )
0.017(3)
0.0058(3 )
0.0088(3 )
0.0079(3)
0.013(3)
R
S (5)
13 Sept.
R
S (5)
1
5
9
0.0022(2)
0.0026(2)
0.0014(2)
0.010(2)
0.0058(2)
0.0042(2)
10 I'g/1 Cu
50 I'g/l Cu
Second Cu Experiment.
1974
J
M (50)
First Cu Experiment.
September-October,
1974
Patricia Bay
1
5
9
0.021(3)
0.094(3 )
0.040(3 )
0.055 (2)
0.026(2)
0.0074(2)
5 I'g/1 Cu
1
5
9
0.033(3)
0.0085(3 )
0.0026(3 )
0.019(2)
0.033 (2)
0.028(2)
0.1 that
amount
silicate
and
of phosphate.
Nutrient additions were required once or
twice each week.
RESUL TS AND DISCUSSION
Nitrogen Assimilation Rates
In spite of the fact that only nitrate was
added as a source of nitrogen when the
CEEs were enriched with nutrients, assimilation rates of ammonium were about equal
to those of nitrate throughout the experiments. Average rates per liter are given in
Table 1. Assimilation rates of both nitrate
(Fig. 1) and ammonium (Fig. 2) were regulated on a day-to-day basis primarily by the
ambient concentrations of nitrate and ammonium in the CEEs and overall by the rate
of nutrient loading. Differences between
the two copper experiments resulted from
different incubation periods (24 h in ex-
13
±11
0.57 ± 0.67
8.3 ±
5.2 ±
2.4
0.8
Nitrate
Nitrate
Ammonium
Ammonium
periment 1, and about 4 h in experiment 2).
Rates in experiment 1, calculated per hour,
are thus averaged over a 24-h day-night
cycle and night-time rates are expected to
be lower than day rates. An exception to
the regulation of the rates by ambient concentration is apparent (Fig. I and Table 2)
in the 2-3 days just following addition of
copper to the CEEs. In this period the
rates were low, particularlly for nitrate assimilation,
suggesting
acute
inhibition
by
copper. Ammonium assimilation was barely
affected (Table 2, 13 Sept. samples). Rates
returned to normal a few days after the
copper additions, as would be implied by the
population assessments of Thomas et al.
(1977) and Thomas and Seibert (1977).
Kinetic Parameters of N Assimilation
Uptake kinetics measurements for nitrate
and ammonium ions were carried out several times during each of the experiments
(Table 3). Uptake was a hyperbolic function of added concentration allowing calculation of the maximum uptake velocity
(Vm) and the half-saturation constant (Ks,
the concentration resulting in one-half the
maximum rate). Mostly, these experiments
were carried out several days after the copper additions and after the period when
BULLETIN OF MARINE SCIENCE, VOL. 27, NO.1,
48
1977
.~
'"Q
~
T
10
.<=
'"
g
25
W
~
0:: 20
•
Z
a
~
...J
~
o
15
ii5
If)
<l
W
~
0::
10
'=Z
~
lJ..
U
AMBIENT AMMONIUM IN CEEs fLM
5
W
a.
If)
o
AMBIENT
,
I
5
10
NITRATE
CONC IN
CEEs fJ-M
Figure 1. Variation in nitrate assimilation rate
with ambient nitrate concentration in the CEEs.
Open circles: control CEEs, 1 and 5 m (1st expt.);
solid circles: control CEEs, 1 and 5 m (2nd
expt.); triangles: 50 p,g/l copper CEE, 1, 5, and
9 m (1st expt. 3 days after adding copper);
diamonds:
5 p,g/l copper CEE, 1, 5, and 9 m
(2nd expt. 2 days after adding copper). The lines
were drawn by inspection and are not based on
calculation of VIll and K•.
copper inhibition of N-assimilation was evident. However, the nitrate experiment of
16 Sept. (6 days after copper addition)
shows a low Vm for nitrate assimilation compared to a sample of Patricia Bay water
(l-m depth) used as the control. However,
the 16 September sample with copper gave
a rate not different from other samples from
the CEEs suggesting that the Patricia Bay
sample gave an atypically high rate for that
time. The variability in Vm values is fairly
high and typical of our unpublished data
for Southern California coastal waters.
Average Vm values were 0.027 h-1 for nitrate
and 0.039 h-1 for ammonium. The specific
Figure 2. Variation in ammonium assimilation
rate with ambient ammonium concentration in the
CEEs. Data refer to incubation of 1 and 5 m
water. Open circles: control CEEs (1st expt.);
solid circles: control CEEs (2nd expt.); triangles:
10 pg/l copper CEE (1st expt.); diamonds: 5
p,g/l copper (2nd expt.).
rates would be equivalent to maximum specific growth rates, if all the particulate nitrogen were indeed phytoplankton. The average
Vm values are equivalent to 0.93 doublings
of particulate N day-l for nitrate and 1.3 for
ammonium. Such specific growth rates are
also typical of coastal marine phytoplankton
in temperate surface waters (Eppley et aI.,
1969b).
The half-saturation values show considerable variability, but again they are in
the expected range for coastal marine phytoplankton. Average values were 1.1 p.M
for nitrate and 1.2 for ammonium. The 3
October sample for nitrate from CEE S gave
a negative value (Table 3). This usually results from inhibition of nitrate uptake by high
ammonium levels (> about 2 p.M), and was
a common feature of the 1974 hydrocarbon
experiment during periods when ammonium
levels were elevated (Table 4). The very
high value for the 30 June ammonium ex-
HARRISON ET AL.: PHYTOPLANKTON METABOLISM AND COPPER TOXICITY
49
Table 3. Nitrate and ammonium uptake kinetics experiments in the two copper experiments, summer 1974. All samples from CEEs were taken at 1-01 depth. In the second experiment, some measurements were made with Patricia Bay water adjacent to the CEEs at I-m depth. ND
not determined.
=
CEE
(pg/I Cu)
Date
Chlorophyll a
pg/I
Nitrate
Experiment
28 June
4 July
3 Oct.
7.49
7.59
3.90
7.28
10.9
9.13
5.2
2.4
0.53
0.39
0.26
0.08
0.025
0.012
0.10
0.0090
0.013
0.017
0.67
0.Q1
0.84
0.0
0.0
0.06
Bay
S (5)
R
S (5)
Bay
S (5)
T (10)
7.7
2.5
4.0
4.3
4.4
5.4
5.9
3.7
0.49
0.26
0.43
0.13
-0.37
0.22
0.080
0.013
0.0088
0.020
0.022
0.Q18
0.019
ND
ND
0.09
0.Q2
ND
ND
ND
4 Oct.
Assimilation
Kinetics
1.
J
L (10)
L (10)
L (50)
6 July
8 July
20 Sept.
Kinetics
J
L (10)
J
L (10)
K
M (50)
Ammonium
Experiment
17 Sept.
AmbientNH.
cone. pM
2.
19 Sept.
Experiment
30 June
Assimilation
h-1
1.
2 July
Experiment
16 Sept.
Vm
K.
pM/I
3.4
8.00
8.00
11.9
1.8
20
0.45
0.98
0.013
0.050
0.067
0.067
2.5
3.5
3.5
5.8
8.2
8.2
8.0
0.82
1.1
0.61
1.8
1.4
1.3
1.1
0.027
0.026
0.016
0.063
0.037
0.035
0.027
2.
Bay
S (5)
R
S (5)
Bay
S (5)
T (10)
periment, CEE L, resulted from assimilation
rate being essentially linear with concentration and is anomalous. This value was excluded in the average above. Occasionally
similar high values have been previously observed in coastal waters but no explanation
has been afforded for them. In general, the
present results seem typical of eutrophic
coastal sea-waters and none of the measurements suggest a marked copper inhibition,
except for the days immediately following
copper addition.
Synthesis of Nitrate Reductase
The extractable nitrate reductase activity
of natural marine phytoplankton appears to
be a function of the ambient concentration
of nitrate (Eppley et a1., 1970) when not
repressed by ammonium. The enzyme appears to be synthesized within a few hours
when nitrate concentrations are increased
in the medium. Therefore, to achieve enzyme synthesis simultaneously we added
copper and nitrate to water samples from
the CEEs and from Patricia Bay, incubated
BULLETIN
50
OF MARINE
SCIENCE,
Table 4. Reduction in the maximum velocity of
nitrate assimilation by ambient ammonium.
Data
are from the hydrocarbon experiment of August,
1974, control enclosure N.
VIll for
Nitrate
Assimi~
lation
(h-l X I(}')
Date
8
28
19
27
Aug.
Aug.
Aug.
Aug.
74
74
74
74
8.8
9.8
0.Q7
1.5
Ammonium
Coneentration
CuM)
<0.2
1.6
4.5
9.2
VOL. 27, NO.1,
Table 5. Synthesis of nitrate reductase
plankton from CEEs
+
+
+
+
Experiment
the
samples,
and
measured
extractable
ni-
trate reductase activity. Copper inhibited
the apparent synthesis of enzyme when
added to water samples from the CEEs or
from Patricia Bay (Table 5). On the other
hand, the 10 July experiment, with copperresistant phytoplankton from the 50 fLg/1
copper CEE, showed enzyme activity as
high as the control CEE and a greater tolerance to added copper.
Nutrient Loading Effects on
Phytoplankton Standing Stocks and
Apparent Settlement Rates
Since particulate material settling out of
the CEEs was routinely removed in the
management of the experiments, nutrients
(NOa, P04, Si) were added at periodic intervals to compensate for the loss by settlement. The total addition of nitrogen per
enclosure ranged from 7 to 24 g N during
the three experiments carried out between
June and October, 1974 (Table 6). Despite considerable variation in particulate
nitrogen (PN) with time and depth during
the experiments, a positive, linear relationship was found between the mean daily nitrate-N addition rate and the average standing stock of PN present (Fig. 3). Accumulation of the added nitrate was apparent
during early stages of the experiments when
PN values were generally low (Figs. 4, 5).
Later on, as PN increased, inorganic nitrogen (IN) decreased to almost undetectable
levels. The accumulation of ammonium was
by
1. 10 July 1974"
Control CEE
Control CEE
10 }Lg/ICu
Control CEE
20 ,Ltg/ICu
50 ,Ltg/ICu - CEE
50 JLg/1 Cu - CEE
10 JLg/1 Cu
50 ,Ltg!lCu - CEE
20 !<g!l Cu
0.10
-0.57
-8.2
-0.9
(NR)
NR as 10-·
moles NO,,(/1g Chi a)-l"h-l
Experiment
Apparent
K.
CuM)
1977
2. 23 September
1974"t
ControlCEE
5 !<g/I CEE
Experiment
5.9
3.9
3. 3 October
Control CEE
5 ,Ltg/I Cu
10 JLg/1 Cu
+
+
0.5
0.0
0.0
1.3
1.9
0.8
1974t
1.2
0.9
0.6
• Water from control and 50 /Lg/I CEEs, 2-m depth. Four
I samples were spiked with 50 /1M KNOn•
NR analysis
after 22 h of preincubation
with and without
added
CuSO •. 5Hp.
t Samples from control and 5 /1g/1 copper CEEs (1, 5,
9 m).
Spiked with 5 11M KNOn• incubated
7 h in light
prior to assay.
t Patricia Bay water (I-m depth) incubated 24 h with 0,
5, 10 /1g/1 copper
(as CuSO •. 5H20), then 11M KNO.
added, incubation
continued
6.5 h before assay.
observed following PN declines and near the
end of the experiments. Overall, there was
a net accumulation of IN (relative to initial
concentrations) in the CEEs receiving the
higher nutrient additions and a net decrease
in those receiving the lower additions (Table
6). Stated another way (Table 7), the rate
of nutrient addition surpassed the budget
estimated net nitrogen flux rate to phytoplankton in the first copper experiment (16
June-12 July) while flux rates surpassed
addition rate in the second copper experiment (3-30 Sept.). The overall average uptake rate (from the budget calculations)
was more closely related to the input rate
than to the ambient concentration of nitrate
and ammonium.
The average concentration of IN in all
enclosures was strikingly similar, regardless
HARRISON
ET AL.:
PHYTOPLANKTON
METABOLISM
AND COPPER TOXICITY
51
/5
••~
::J
'"
0
u
c:
W
•
:2
c..
'"E
~
,.,:
10
C>
u
12
Y; 10.67x+0.87
0.88
<f)
I~;
C>
:2
6
:2
5
t:!
<f)
w
~
a::
w
o
:a:
o
10
\
,
..••...
0.2
0.4
0.6
0.8
1.0
AVERAGE NUTRIENT ADDITION (grams N Enclosure-I doll)
o
20
Figure 3. Relationship between nitrate addition
rate and average particulate nitrogen standing Figure 4. Daily variationsin the forms of nitrostock. Squares: first copperexperiment(16 June- gen in the CEEs,first copperexperiment: J (con12 July); triangles:
hydrocarbon experiment (2- trol); K (control); L (10 /Lgjlcopper); M (50
26 Aug.); circles: second copper experiment (3- /Lgjl copper); (0)
PN; (e)
N0 -N; (X)
30 Sept.). Open symbols are controls; solid
NH.-N.
symbolsare treatments.
=
of nitrogen additions. As with the standing
stock and N-uptake rate, settlement production also appeared to be primarily a
function of nutrient additions (Tables 6,
8). This is reminiscent of the relation between algal stocks and phosphorus loading
in lakes (Dillon and Rigler, 1974).
Analysis of the settled material from the
second copper experiment revealed a composition almost exclusively of intact or fragments of phytoplankton (J. Davies, personal communication), even in the control
enclosure which contained a healthy population of herbivorous zooplankton. Apparently, the same was true in the first
copper experiment since even lower zooplankton stocks were present (Gibson and
Grice, 1977). This may help explain the
close relationship between nutrient uptake,
standing stock and settlement production.
Apparently, the nutrient enrichment was reflected in corresponding increases in the
=
3
=
phytoplankton stocks which then settled out
of the enclosures without being significantly
cropped. These results contrast with the
findings of the Great Central Lake eutrophication study of Parsons et al. (1972), LeBrasseur and Kennedy (1972) and Barraclough and Robinson (1972) in which
primary productivity increases from nutrient
loading were passed on to the herbivorous
zooplankton,
while the phytoplankton
standing stocks increased only slightly.
Copper Effects as Seen in the
Nitrogen Budgets
Although nutrient additions had a more
pronounced influence on the budget parameters investigated than did copper, some
copper effects were apparent. There was a
greater retention of PN in the water column
of the copper-treated enclosures relative to
that in the controls, based upon values of
Table 6. This may be related to the lowered
BULLETIN
52
OF MARINE
SCIENCE,
VOL. 27, NO. I, 1977
of the experiments (Thomas et al., 1977;
Thomas and Seibert, 1977) and the virtural
elimination of herbivorous zooplankton in
the copper-treated enclosures (Gibson and
Grice, 1977) contributed to these differences.
Remineralization
and Nutrient Loading
The ammonium assimilation by the phytoplankton resulted from nitrogen recycling.
The pathways expected to be important were
microbial decomposition of the settling material, of dissolved organic nitrogen, and
zooplankton excretion. It is interesting to
Figure 5. Daily variations in the forms of nitrogen in the eBBs, second copper experiment. R
(control); S (5 p.g/l copper); T (10 p.g/l copper).
Symbols as in Fig. 4.
settlement production rate (Table 8). Probably cell size differences in phytoplankton
assemblages (primarily micro-flagellates in
the copper, and chain-forming diatoms in
the control enclosures) in the latter stages
note that ammonium assimilation was about
equal to that of nitrate in two systems with
heavy nitrate inputs: the Peru upwelling
(Walsh, in press) and the CEPEX enclosures. In the Peru upwelling the ammonium
is thought to arise largely from anchovy
excretion.
Earlier studies of remineralization have
concerned ammonium release from decaying phytoplankton (Grill and Richards,
1964; Otsuki and Hanya, 1972, as well as
the classic studies of von Brand and Rakestraw, 1937-41; see review of Vaccaro,
1965). Typical rates of ammonia formation
Table 6. Nitrogen budget parameters for the first copper experiment (enclosures J-M), hydrocarbon
experiment (enclosures N-Q), and second copper experiment (enclosures R-T), summer 1974. All
values are expressed as grams of nitrogen per enclosure.
Change in·
inorganic
nitrogen
(I1IN)
Change int
particulate
organic
nitrogen
(I1PN)
J
K
L
M
+2.95
+3.29
+2.07
+0.68
-6.58
-2.82
+4.19
- 1.24
21.84
23.80
17.22
13.30
25.47
23.33
10.96
13.86
2 Aug.-27 Aug.
N
0
P
Q
+6.50
+4.08
+ 1.16
+1.32
-2.73
- 3.25
- 1.35
-2.80
13.72
13.72
13.72
13.72
9.95
12.89
13.91
15.20
3 Sept.-30 Sept.
R
-1.47
-3.90
- 2.55
-4.25
+0.28
- 0.51
7.42
7.14
8.12
13.14
10.76
11.18
Enclosure
Date
16 June-12
July
S
T
• Overall net change in nitrogen content (grams N). (IN) inorganic-N, (PN)
net decrease.
t Total settlement material produced (uncorrected for remineralization loss)
Nitrogen-added
(as nitrate)
particulate-N,
=
(+)
Gross
Settlcmentt
net increase,
(N added - I1IN) - I1PN.
H
HARRISON ET AL.:
PHYTOPLANKTON METABOLISM AND COPPER TOXICITY
53
Table 7. Budget estimates of nitrogen assimilation rates in the first and second copper experiments.
Values are averages over the duration of each experiment.
Average
Average
rate of N
addition
(g N day-l)
Nitrogen
uptake rate
(g N day-l)
0.81
0.88
0.64
0.73
0.79
0.58
M
2.33
2.38
2.86
3.00
0.49
0.49
R
S
T
2.68
2.48
2.47
0.27
0.26
0.29
0.33
0.41
0.40
inorgank-N
concentration
Date
16 June-12
3 Sept.-30
(g N per
enclosure)
Enclosure
July
J
K
L
Sept.
=
• Average nitrogen uptake rate (grams N day-')
(measured net decrease in inorganic-N
experiment. This actually represents net flux into the phytoplankton.
were of the order of 5% of the particulate
nitrogen per day and J. Davies (personal
communication) directly measured a similar
rate in settlement material from the CEPEX
enclosures. Ammonification rates were high
compared to gross settlement rates (Table
8). These no doubt reflect excretion by zooplankton as well as bacterial decomposition
of settled phytoplankton. The overestimation of ammonium assimilation rate by the
15N method,
because the added 15N ammonium would often approach ambient
ammonium levels already present, would
also lead to high values. A more detailed
analysis of nitrogen flow in enclosures en-
Table 8. Budget estimates
the two copper experiments
of nitrogen
+
Average·
N-added)
-+- duration of
riched with 15N nitrate and ammonium is in
progress and will be reported separately
(Harrison and Davies, unpublished). Vaccaro et al. (1977) have reported on bacterial activity in the enclosures.
Experiments to Test Whether the
Copper in the CEEs was Toxic
for Phytoplankton
Since inhibition of carbon and nitrate assimilation rates was observed only briefly
after adding copper to the CEEs we were
curious as to whether the copper remained
in its original chemical form and whether
it retained its toxicity. If so, it must be con-
remineralization
and net production
of settlement
material
in
Enclosure
Gross·
Settlement
Rate
Remineralizationt
Rate
Net:l:
Settlement
Rate
Remineralization
as % of Gross
Settlement
Rate
J
K
L
M
0.98
0.90
0.42
0.53
0.29
0.29
0.26
0.16
0.69
0.61
0.16
0.37
29.6
32.2
61.9
30.2
R
0.49
0.40
0.41
0.25
0.20
0.21
0.24
0.20
0.20
51.0
50.0
51.2
S
T
• Uncorrected settlement production rate (g N day-l) from Table 6.
t Remineralization rate (g N day-l) from average ammonium assimilation rates measured with 15NH,Cl
increase in ammonium in the enclosures
:I: Settlement production rate, corrected for remineralization (g N day-l), i.e. column 2 less column 3.
+ overall
net
BULLETIN OF MARINE SCIENCE, VOL. 27, NO.1,
54
1977
Table 9. Experiments to determine if copper in the CEEs was inhibitory for photosynthesis of phytoplankton from control CEEs. Water from 1-2 m depth with phytoplankton
from control CEEs was
mixed with filtered water (phytoplankton
removed) from control CEEs or copper containing CEEs
followed by photosynthesis measurement with "C.
Source of
filtered
Water
Approx. Final
Cu Cone. (,"g/l)
Experiment
Control CEE
10 ,ug/I Cu CEE
50 ,ug/I Cu CEE
Experiment
Control
Photosynthetic Rate mg C
(mg Chi a)-l h-1and (% control)
Phytoplankton from Control CEE:
Experiment
Control CEE
10 ,ug/l Cu CEE
50 ,ug/I Cu CEB
Experiment
K
1.74(100)
1.76(101)
1.74 ( 100)
2.20(100)
1.97(90)
1.75 (80)
2.26(100)
2.37(105)
1.55(69)
1.66(100)
1.81(109)
1.38(83 )
1. 8 July 1974. 3-h incubation
<1
5
17
2. 9 July 1974. 4-h incubation
<1
CEE
10 ,ug/l Cu CBE
50 ,ug/l Cu CBB
J
5
17
3. 10 July 1974. 24-h preincubation,
<1
5
17
1.46(100)
1.41 (97)
0.26(18)
4. 17 July 1974. 24-h preincubation,
o
5
7.5
9.0
9.5
4-h HC incubation
3.11(100)
2.46(79 )
0.79(25)
3-h HC incubation*
100
87
100
75
75
51
32
32
87
44
• Dilution series of CEE J plankton with different volumes of water from 10 ,ug/I Cu eEE to give a series of Cu con·
centrations. Values as % control.
cluded that the phytoplankton growing in
the CEEs several days after the copper additions were somehow tolerant to the copper
present, as were the heterotrophic bacteria
(Vaccaro et aI., 1977). To test these alternatives some experiments of a bioassay
character were carried out in which phytoplankton from control CEEs were incubated
in filtered water from copper-containing
CEEs (Table 9). In the first of these,
volumes of unfiltered water (with phytoplankton) from control CEEs were mixed
with an equal volume of filtered water from
copper-containing CEEs, HC was added and
photosynthetic carbon assimilation was measured for 3 h. Only a slight inhibition was
seen with phytoplankton from control CEE
K and filtered water from the 50 f-tg/lcopper
CEE.
The second experiment was essentially a
repeat of the first, with similar results. In
the third experiment the filtered and unfiltered samples were mixed and pre-incubated 24 h before adding HC for a 4-h
photosynthesis measurement. Marked inhibition was seen with filtered water from
the 50 f-tg/l copper CEE. In the fourth experiment, a dilution series of unfiltered water
from control CEEs was added to filtered
water from the 10 f-tg/l copper CEE, preincubated 24 h, then photosynthesis was
measured for 3 h. Inhibition of 50% or
greater, was observed at a dilution corresponding to 1 part unfiltered water to 9 parts
filtered water and 9 f-tg/l copper. We concluded that the copper in the CEEs was still
in a form inhibitory to phytoplankton from
the control CEEs.
HARRISON
ET AL.:
PHYTOPLANKTON
METABOLISM
•
...J
100
Z
0
U
lI..
0
~
Vi
w
TOXICITY
55
and 4+ p.g/l for the control CEEs and 10
and 35 for the nominally 10 and 50 (actually 35; Topping and Windom, 1977) p.g/l
copper-containing CBBs. These results imply that the phytoplankton in all the CBBs
were still subject to inhibition by copper,
but that plants in the copper-containing
CEEs were resistant to copper concentrations equal to those in their environment.
~
•....
en
AND COPPER
50
:z:
•....
z
>en
Experiments with Noctiluca sp.
0
•....
0
:z:
a..
0
1000
COPPER
CONCENTRATION
Figure 6. Inhibition of photosynthesis of phytoplankton from CEEs by added copper sulfate.
Samples were incubated 24 h after adding copper,
then photosynthetic carbon assimilation was measured in a 4-h incubation. Copper concentrations
on the abscissa include ambient plus added copper.
9 July 1974. Open circles: control CEE J; solid
circles: control CEE K; triangles: 10 p.g/I copper
CEE; diamonds: 50 p.g/l (actually 35 /-Lg/I) copper
CEE.
Experiments to Test Whether
Phytoplankton Growing in the
Copper-Containing CBBs are
Resistant to Copper
Additions of copper, from a fresh solution of copper sulfate, were made late in the
first copper experiment to samples of water
from both control and copper-containing
CEBs to give a range of copper concentrations. Radiocarbonate was added and the
mixtures were incubated for 24 h for measurement of inhibition of photosynthetic
activity. Rates of photosynthesis were
graphed against the log of the copper concentration
(ambient plus that freshly
added) and these resulted in straight line
plots (Fig. 6). The copper concentration
for incipient inhibition of photosynthesis,
taken from the intersection of the straight
lines with the 100% activity line, were 3
Fairly dense aggregations of Noctiluca
sp. were noted at the surface of control
CEEs early in July. None were evident in
CEEs with added copper. Kesseler (1966)
has shown that Noctiluca miliaris accumulates high intracellular concentrations of
ammonium and that this aids in the observed
positive buoyancy of the organism. Since
one proposed mechanism of copper inhibition of algal growth is the alteration of membrane permeability and the resulting loss of
intracellular ions accumulated against the
electro-chemical gradient (such as potassium [McBrien and Hassall, 1965; Overnell,
1975]) loss of ammonium, and of buoyancy
would be expected to result on exposure of
Noctiluca sp. to copper.
To test this notion samples of water with
Noctiluca sp. from control CBBs were
either spiked with copper sulfate or mixed
with an equal volume of water from coppercontaining CEBs. The samples were placed
in 4-1 graduated cylinders in dim light and
were observed over a 48-h period. Replicate samples of the cells were then harvested
by netting, and placed in lO-ml volumes of
distilled water to burst the cells osmotically
for ammonium analysis.
Noctiluca sp. in the cylinders immediately
aggregated in the upper 2-3 em of water in
all treatments where they remained for the
48 h of observation. Sinking cells were not
observed. However, there was a slow buildup of cellular debris on the bottom of the
cylinders containing copper. Cellular ammonium concentrations after 48-h exposure
showed a small decline in the treatments
56
BULLETIN
OF MARINE
SCIENCE,
with copper. Values, as nanomoles per cell,
were: Controls-82
and 88; CEE L water
(10 p.g/l copper-83 and 58; CEE M water
(35 p.g!l copper) -60
and 51 ; 10 p.g/l
added copper-62
and 57; and 20 p.g/l
added copper-51 and 77. From Kesseler's
value of 0.06 M ammonium in the cell sap
of N. miliaris and a cell volume of 40 X
10-9 liter, we expected an ammonium content per cell of 2.4 nanomoles. Our values
seem high.
These observations confirm the high ammonium contents of Noctiluca sp. reported
by Kesseler (1966 and references therein),
and our values were even higher than his.
The visual observations, particularly the
slow build-up of debris in the cylinders with
copper, suggest that cell membrane disruption and cell lysis, with loss of intracellular
ammonium, took place slowly and progressively when Noctiluca sp. was exposed to
10-20 p.g/l copper. The high ammonium
contents of the cells that remained buoyant
suggests that ammonium loss by a cell may
be abrupt (Passow et al., 1961) rather than
slow and progressive (McBrien and HassaIl, 1965), and may take place largely on
cell lysis. The results further confirm that
one mode of copper-induced injury is to
impair the semi-permeable osmotic barriers
of cells.
ACKNOWLEDGMENTS
We are grateful to Dr. M. Takahashi for many
courtesies during oUI stay at the CEPEX site
near Sidney, B.C., Canada, and for his encouragement and collaboration on the copper toxicity experiments on phytoplankton in July, 1974. Don
Seibert provided the particulate nitrogen data
used in preparing the nitrogen budgets. Leslie
Watanabe, Janet Barwell-Clarke, and Bruce
Wright carried out the general purpose nutrient
and chlorophyll analyses. Peter Koeller and Frank
Whitney provided sampling and logistic support
at the CEPEX site. Zoe Eppley assisted in the
experimental work during the first copper experiment, July, 1974. Dr. John Davies provided
access to unpublished data on settlement material
in the second copper experiment, September, 1974.
Dr. T. Enns kindly provided his mass spectrometer
for our use in the 13Nanalyses at Scripps. Thanks
VOL. 27, NO.1,
1977
to each of these people and to Hanna Moshe for
typing the manuscript.
Support was provided by the National Science
Foundation, Oceanography Section, Grant No.
31167X, and NSF, International Decade of Ocean
Exploration, Grant No. GX-42579 (CEPEX).
LITERATURE
CITED
Barraclough, W. E., and D. Robinson. 1972.
The fertilization of Great Central Lake. III.
Effect on juvenile sockeye salmon. Fish. Bull.
70: 37-48.
Dillon, P. J., and F. H. Rigler. 1974. A test of a
simple nutrient budget model predicting the
phosphorus concentration in lake water. J.
Fish. Res. Bd. Can. 31: 1771-1778.
Dugdale,
R. C., and J. J. Goering.
1967.
Up-
take of new and regenerated forms of nitrogen
in primary productivity. Limnoi. Oceanogr.
12: 196-206.
Eppley, R. W., J. L. Coatsworth, and L. Sol6rzano.
1969a. Studies of nitrate reductase in marine
phytoplankton. Limnol. Oceanogr. 14: 194205.
---,
J. N. Rogers, and J. J. McCarthy. 1969b.
Half-saturation constants for uptake of nitrate and ammonium by marine phytoplankton. Limnoi. Oceanogr. 14: 912-920.
---,
T. T. Packard, and J. J. MacIsaac. 1970.
Nitrate reductase in Peru current phytoplankton. Mar. BioI. 6: 195-199.
Erickson, S. J. 1972. The toxicity of copper to
Thalassiosira pseudonana in unenriched inshore seawater. J. Phycoi. 8: 318-322.
Gibson, V. R., and G. D. Grice. 1977. Response
of macro-zooplankton populations to copper.
Controlled ecosystem pollution experiment.
Bull. Mar. Sci. 27: 85-91.
Grill, E. V., and F. A. Richards. 1964. Nutrient
regeneration from phytoplankton decomposing
in seawater. J. Mar. Res. 22: 51-69.
Kesseler, H. 1966. Beitrag zur Kenntnis der
chemischen und physikalishen Eigenschaften
des Zellaftes von Noctiluca miliaris. Veroff.
Inst. Meeresforsch. Bremerh. Sonderbd. II:
357-368.
Krock, H. T., and D. T. Mason. 1971. Bioassays of lower trophic levels. San. Eng. Res.
Lab., Univ. Calif. Berkeley, Rept. No. 71-8.
A study of toxicity and biostimulation in San
Francisco Bay-Delta waters. 4: 123 pp.
LeBrasseur, R. J., and O. D. Kennedy. 1972.
The fertilization of Great Central Lake II.
Zooplankton standing stock. Fish. Bull. 70:
25-36.
McBrien, D. C. H., and K. A. Hassall. 1965.
Loss of cell potassium by Chlorella vulgaris
after contact with toxic amounts of copper.
Physiol. Plant. 18: 1059-1065.
HARRISON ET AL.:
PHYTOPLANKTON METABOLISM AND COPPER TOXICITY
Maloney, T. E., and C. M. Palmer.
1956. Toxicity of six chemical compounds to thirty
cultures of algae. Water and Sewage Works.
Nov., 1956, pages unnumbered.
Menzel, D. W., and J. Case. 1977. Concept
and design: Controlled ecosystem pollution
experiment.
Bull. Mar. Sci. 27: 1-7.
Otsuki, A., and T. Hanya.
1972. Production of
dissolved organic mater from dead green algae
cells. r. Aerobic microbial
decomposition.
Limnol. Oceanogr. 17: 248-257.
Overnell, J. 1975. The effect of heavy metals
on photosynthesis and loss of cell potassium
in two species of marine algae, Dunaliella
tertiolecta and Phaeodactylum
tricornutum.
Mar. BioI. 29: 99-103.
Palmer, C. E., and T. E. Maloney.
1955. Pre·
liminary
screening for potential
algicides.
Ohio J. Sci. 55: 1-8.
Parsons, T. R., K. Stephens, and M. Takahashi.
1972. The fertilization of Great Central Lake.
r. Effects of primary production. Fish. Bull.
70: 13-23.
Passow, H., A. Rothstein, and T. W. Clarkson.
1961. The general pharmacology
of heavy
metals. Pharmacol. Rev. 13: 185-224.
Riley, G. L. 1939. Limnological studies in Connecticut. Eco!' Mongr. 9: 55-94.
Saward, D., A. Stirling, and G. Topping.
1975.
Experimental studies on the effects of copper
on a marine food chain. Mar. BioI. 29: 351361.
Sharp, J. H. 1974. Improved analysis for "particulate organic carbon and nitrogen" from
seawater. Limnol. Oceanogr. 19: 984-989.
Sol6rzano, L. 1969. Determination of ammonia
in natural waters by the phenolhypochlorite
method. Limnol. Oceanogr. 14: 799-801.
Steemann Nielsen, E., and S. Wium-Andersen.
1971. The influence of Cu on photosynthesis and growth in diatoms. Physiol. Plant.
24: 480-484.
---,
L. Kamp-Nielsen, and S. Wium-Andersen.
1969. The effect of deleterious concentrations of copper on the photosynthesis of Chiarella pyrenoidosa.
Physio!. Plant. 22: 1121] 133.
Strickland, J. D. H., and T. R. Parsons.
1972.
A practical handbook of seawater analysis.
Bull. Fish. Res. Bd. Can. No. 167. 311 pp.
Thomas, W. H., and D. L. R. Seibert.
1977.
Effects of copper on the dominance and the
diversity of algae: Controlled ecosystem pollution experiment. Bull. Mar. Sci. 27: 23-33.
---,
O. Holm-Hansen,
D. L. R. Seibert, F.
Azam, R. Hodson, and M. Takahashi.
1977.
57
Effects of copper on phytoplankton
standing
crop and prOductivity: Controlled ecosystem
pollution experiment.
Bull. Mar. Sci. 27:
34-43.
Topping, G., and H. L. Windom.
1977. Biological transport of copper at Loch Ewe and
Saanich Inlet:
Controlled ecosystem pollution experiment. Bull. Mar. Sci. 27: 135-141.
Vaccaro,
R. F. 1965. Inorganic
nitrogen
in
seawater. Chern. Oceanogr. 1: 365-408.
---,
F. Azam, and R. E. Hodson.
1977.
Response of natural marine bacterial populations to copper: Controlled ecosystem pollution experiment.
Bull. Mar. Sci. 27: 17-22.
von Brand, T., and N. W. Rakestraw.
1940. Decomposition and regeneration of nitrogenous
organic matter in sea water. III. Influence of
temperature
and source and condition of
water. BioI. Bull. Woods Hole 79: 231-236.
---,
---,
and C. E. Renn.
1937. The
experimental decomposition and regeneration
of nitrogenous organic matter in sea-water.
BioI. Bull. Woods Hole 72: 165-175.
1939. Further experiments on the decomposition of nitrogenous organic matter in
sea water. BioI. Bull. Woods Hole 77: 285296.
Walsh, J. 1. (in press). Utility of systems models:
a consideration
of some possible feedback
mechanisms of the Peru upwelling ecosystem.
Proc. 2nd Internatl. Estuarine conf.
Wood, E. P., F. A. J. Armstrong,
and F. A.
Richards.
1967. Determination
of nitrate
in sea water by cadmium-copper reduction to
nitrite. J. Mar. BioI. Ass. U.K. 47: 23-31.
ADDENDUM
Historical references not seen by us
but cited by Riley (1939)
Hale, F. E. 1922. Tastes and odors in the New
York water supply. J. Am. Water Works
Assoc. 9: 829.
Moore, G. T., and K. F. Kellerman.
1904. A
method of destroying or preventing the growth
of algae and certain pathogenic bacteria in
water supplies. Bur. Plant Indust. U.S. Dept.
Agr. Bull. 64.
1905. Copper as an algicide and disinfectant in water supplies. Bur. Plant Indust.
U.S. Dept. Agr. Bull. 76.
DATE ACCEPTED: August 27, 1976.
ADDRESS:
Institute of Marine Resources, University of California, San Diego, La Jolla, California
92093.