contrasting nitrogen uptake by diatom and phaeocystis-dominated phytoplankton assemblages in the...

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Journal of Experimental Marine Biology and Ecology

292 (2003) 19–41

Contrasting nitrogen uptake by diatom and

Phaeocystis-dominated phytoplankton

assemblages in the North Sea

Clavery Tungarazaa,*, Veronique Rousseaub, Natacha Briona,Christiane Lancelotb, John Gichukia, Willy Baeyensa, Leo Goeyensa

aVrije Universiteit Brussel, Laboratorium Analytische Chemie, Pleinlaan 2, 1050 Brussel, BelgiumbUniversite Libre de Bruxelles, Groupe de Microbiologie des Milieux Aquatiques, C.P. 221,

1050 Bruxelles, Belgium

Received 29 May 2002; received in revised form 2 March 2003; accepted 11 March 2003

Abstract

This paper documents ambient concentrations of nutrients in the Belgian coastal waters of the

North Sea during the spring of 1996 and 1997. The paper elaborates the differences of uptake rates of

oxidised nitrogen (NO3�) and reduced nitrogen (NH4 and urea) by Phaeocystis and diatoms. The

nitrogen concentrations were dominated by NO3� with a maximum concentration of 30 AM (January

1997) and 40 AM (March 1996). In 1996, Phaeocystis dominated the spring biomass with a

maximum of 521 Ag C l� 1, while maximum diatom biomass was 174 Ag C l� 1. In 1997, the

maximum Phaeocystis spring biomass was 1600 Ag C l� 1 and diatom maximum biomass was below

100 Ag C l� 1. A maximum bacteria biomass of about 55 Ag C l� 1 was observed in mid-May 1996.

The maximum nitrogen uptake rates were recorded during spring and were dominated by NO3�

(0.005 h� 1 in 1996 and 0.032 h� 1 in 1997). Maximum specific NH4 uptake rates were between

0.005 h� 1 in May 1996 and 0.006 h� 1 in April 1997. The NO3� uptake rates displayed exponential

decrease versus increasing ambient reduced nitrogen concentrations (ammonium and urea), whereas

the reduced nitrogen uptake increased but never compensated the decreased nitrate uptake. The NH4

uptake kinetics of diatoms displayed lower vmax compared to Phaeocystis. Consequently,

Phaeocystis showed ability to increase their NH4 uptake capacity when more NH4 became available

while diatoms failed to do so, after ammonium had exceeded their saturation concentration (>1 AM).

Although reduced nitrogen has a negative effect on the uptake of NO3�, Phaeocystis have more

0022-0981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0022-0981(03)00145-X

* Corresponding author. Faculty of Science, Sokoine University of Agriculture (SMC), P.O. Box 3038,

Morogoro, Tanzania. Fax: +255-23-2603404.

E-mail address: [email protected] (C. Tungaraza).

C. Tungaraza et al. / J. Exp. Mar. Biol. Ecol. 292 (2003) 19–4120

advantage than diatoms on the uptake of ammonium. This might be contributing to the biomass

domination shown by Phaeocystis over extended periods in spring.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Ammonium uptake rates; Diatoms; Inhibition effects; Nitrate uptake rates; Phaeocystis

1. Introduction

The Southern Bight of the North Sea is a prominent example of marine coastal systems

submitted to discharges of domestic, industrial and agricultural wastewater, which cause a

major concern for the sustainable management of the environmental quality. Rivers as well

as the English Channel have a significant contribution to the nutrient load of this area

(North Sea Task Force, 1993). The pollution problems in this area include, among others,

excess nutrient input. This has resulted to subsequent high primary production (Baeyens et

al., 1984; Hesse, 1988), and increasing frequency of phytoplankton blooms (Cadee, 1986),

dominated by flagellates (Owens et al., 1990). In general, accelerated primary production

due to over enrichment of nutrients has occasionally led to oxygen depletion problem in

some areas, as was observed in the German Bight (Gerlach, 1984).

In the Southern Bight of the North Sea, the undesirable developments of Phaeocystis is

known to have been generated by the perturbed nutrient patterns, which have changed the

relative availability of Si in relation to N (Lancelot et al., 1987; Billen et al., 1991;

Lancelot and Rousseau, 1994) resulting into the decrease of the food quality for the marine

herbivores (Hansen and Van Boekel, 1991). In addition, two to threefold increase in

phytoplankton biomass, following upon excess input of nitrogen in the southeastern North

Sea were observed by Bauerfeind et al. (1990).

The Belgian coast is greatly under the influence of the Scheldt river. In addition to

considerable riverine supply, an elaborated monitoring program in the Belgian coastal area

revealed predominance of DIN advection through the English Channel. Seasonal concen-

tration fluctuations were largely related to variable autotrophic removal of nutrients and to

significant pelagic and benthic remineralization (Van Bennekom et al., 1975; Baeyens et

al., 1984). In spring and summer, the Belgian coastal waters are generally characterized by

two distinct peaks of diatom abundance, which are separated by an intensive Phaeocystis

development (Rousseau et al., 1996).

The trend of major nutrients (nitrate and silicate) in the Belgian coast and their

corresponding uptake regime are almost completely governed by the two major phyto-

plankton communities, the diatoms and Phaeocystis, which bloom during spring. Appa-

rently, the Phaeocystis biomass dominates the season through an extended bloom period.

However, there is a remarkable interannual variability in peak intensities and timing.

Higher phytoplankton biomass in Belgium coastal water at the end of April was also

reported by Joint and Pomroy (1993), which was associated with an extensive bloom of

the Phaeocystis sp.

The parameters governing the diatoms–Phaeocystis succession and the possible

reason for the dominance of Phaeocystis blooms are insufficiently understood. Reid

et al. (1990) explained an early appearance of diatoms to be due to the presence of

C. Tungaraza et al. / J. Exp. Mar. Biol. Ecol. 292 (2003) 19–41 21

Si and when depleted, Phaeocystis starts blooming. This is not always observed and

early appearance of Phaeocystis has been reported while Si is still available (Bakker

et al., 1990; Veldhuis et al., 1986). A work by Cadee and Hegeman (1986) excluded

water temperature as a triggering factor of Phaeocystis bloom in spring. Peperzak et

al. (1998) investigated the relationship of Si depletion and diatoms-Phaeocystis

spring bloom succession by testing two theories related to spring bloom. They

followed light threshold at which Phaeocystis starts blooming under nitrogen and

phosphorus replete condition. Their results did not support the Si depletion theory,

but a light threshold of 100 W h m� 2 day� 1 was found to be a triggering factor

for Phaeocystis bloom.

The study area was on the Belgian coastal zone in the Southern Bight of the North Sea.

This area is characterised by strong along shore tidal currents, resulting in well mixed

water masses (Creutzberg and Postma, 1979). The area is under the influence of the

Scheldt river (Van Bennekom and Wetsteijn, 1990) and has a bathymetric depth of about

17 m. The water discharge of river Scheldt is estimated to be at an average of 129 m3 s� 1

and maximum discharge can be as high as 753 m3 s� 1 (Lenhart et al., 1996).

Average total nutrient input supplied by river Scheldt to the Belgian coast was compiled

by Lenhart et al. (1996) for the period between 1977 and 1993. Between 1990 and 1993,

the maximum annual total nitrogen load was reported to be 34 kt (in 1993) and the

maximum load of NO3� was 20 kt (in 1992), whereas NH4 was 5 kt in 1991. The

phosphate annual load from 1990 to 1993 remained at an average of 1 kt, although in the

1980s about 2 kt were observed to be the average input. Silicate maximum contribution of

about 14 kt was observed in 1992.

Although the enhancement of nutrients is a key for the appearance of eutrophication, it

cannot be correctly understood neither can it be remedied without a sound understanding

of the processes behind it. In this study, the emphasis is on the seasonal patterns in diatoms

and Phaeocystis biomass in relation to their uptake rates of the major nitrogenous

nutrients, viz. NO3�, NH4 and CO(NH2)2.

2. Material and methods

Sampling was done at station 330 (51j26.05VN, 2j48.50VE; Fig. 1). Surface water

samples were collected between late winter and early summer (i.e., January to June) in

1996 and 1997. Measurements of uptake rates were performed during spring (March to

May), a period of high biological activities. The samples were collected from surface water

by using a plastic bucket aboard RV Belgica, then maintained at ambient water temperature

and transported to the laboratory for analyses. This technique warrants minimal disruption

of fragile Phaeocystis colonies.

2.1. Nutrient measurements

Samples for determination of Si, PO43�, NO3

� and NO2� concentrations were filtered

through Whatman glassfibre filters (GF/F), preserved in plastic bottles and frozen

immediately until analyses. Silicates samples were analysed by the silico-molybdic

Fig. 1. Reference sampling area, station 330. Rivers and their catchment area (broken line). Arrows indicate

directions of localised water currents.


complex formation method (detection limit 0.1 AM) according to Koroleff (1983b).

Phosphate concentrations were analysed by the phospho-molybdic complex formation

method (detection limit 0.08 AM) described by Koroleff (1983a). Nitrate and NO2�

concentrations were determined by an automated diazotation method (detection limit

0.1 AM; D’Elia, 1983; Elskens and Elskens, 1989). Ammonium and urea samples

were kept in glass bottles and fixed by immediate addition of the reagents. The

concentrations of ammonium were determined by the indophenol-blue method (detec-

tion limit 0.03 AM) according to Koroleff (1969) and urea concentrations were

determined by an adapted diacetylmonoxime method (detection limit 0.1 AM; Goeyens

et al., 1998). All sample concentrations were measured in duplicate. Reagents used in

the analyses and standards preparation were analytical grade. Solutions were prepared

in deionised water from a Millipore Milli-Q ion exchange unit.

2.2. Chlorophyll a and phytoplankton enumeration

The quantification of chlorophyll a was performed by the classical spectrophotometric

method of Lorenzen (1967). Seawater samples were filtered through Whatman glassfibre

filters (GF/F; 47 mm diameter) under low suction. Filters were immediately wrapped in a

clean aluminium foil and stored in a deep freezer for further laboratory analysis. The

filtered volumes of seawater were between 50 and 1000 ml, depending on the quantity of

particulate matter (PM) in the sample.

The extractions of chlorophyll a from the filters were done in 90% acetone at f 5 jCand for a minimum of 1 h. Samples were then centrifuged for 10 min at 300 rpm followed

by immediate recording of absorbance and determination of chlorophyll a concentrations,

as described by Strickland and Parsons (1972).

Phytoplankton samples for species composition analyses were preserved in a lugol-

glutaraldehyde solution (1%). The abundances of diatoms and Phaeocystis were deter-

mined by an inverted microscope (Leitz Fluovert) according to the Utermohl method

(Hasle, 1978). Counting was done on 10–100 ml subsamples after 12 h sedimentation.

Diatoms were identified and counted at � 100 or � 200 magnification; Phaeocystis cells

and colonies were enumerated according to the method described by Rousseau et al.

(1990). The carbon biomasses of both diatoms and Phaeocystis were calculated from

microscopic counts and biovolumes. The carbon biomass of Phaeocystis colonies was

defined by the equations recommended by Rousseau et al. (1990); for diatoms, we used a

conversion factor of 0.11 pg C Am� 3 of plasma volume (Edler, 1979).

2.3. Bacteria biomass

Bacteria abundance and biomass enumerations were only performed during the 1996

spring season. Samples were preserved in formalin (2% final concentration) and stored at 4

jC. The abundance and biomass of free living bacteria was determined by epifluorescence

microscopy (Leitz, Laborlux D). Before enumeration 1–5 ml of water sample were stained

with 4,6-diamidino-2-phenylindole (DAPI) to make the final concentration of 0.1 Ag l� 1

for 15 min (Porter and Feig, 1980). Stained bacteria were then collected by filtration on 0.2

Am pore-size black polycarbonate filters (Nuclepore), which were mounted on micro-

scopic slides and stored at � 20 jC until examination. Bacteria were enumerated on a

minimum of 20 different fields at � 1000 magnification. Their cell volume was calculated

from the measurement of the shortest and longest axes, considering rods and cocci as

cylinders and spheres, respectively. Between 300 and 600, bacteria were measured for

each sample and estimation of carbon biomass was done by a biovolume-dependent

carbon conversion factor of Simon and Azam (1989).

2.4. POC/PON analysis

The analyses of particulate organic nitrogen (PON) and carbon (POC) were

performed on particulate matter collected by filtration on precombusted (8 h at 450

jC) Whatmank glassfibre filters (GF/F; 47 mm diameter). PON and POC concen-

trations were analysed simultaneously by using a Carlo Erba NA 1500 CN Analyser.



The analysis combines conversion of PON and POC into N2 and CO2, separation on a

chromatographic solid phase column and detection by thermal conductivity (Pella,

1990). Concentrations of PON and POC were obtained by standardization with

acetanilide.

2.5. Nitrogen uptake experiments

The uptake rates of NO3�, NH4 and urea by phytoplankton were measured using the

15N tracer technique introduced by Dugdale and Goering (1967). Polycarbonate

incubation bottles (2 l) were filled with seawater and incubation experiments were

launched by the addition of labelled nutrients, i.e., NO3� (99.5% 15N), NH4 (99.8%

15N)

and urea (99% 15N). The tracer additions did not exceed 10% of the ambient

concentration. The samples were incubated at constant irradiance (340 A E m� 2 s� 1,

12:12-h light/dark cycle) and at their ambient sea water temperatures. At the end of each

experiment, the concentrations of each nutrient were measured for detection of possible

regeneration. PM, collected on pre-combusted Whatman glassfibre filters (GF/F) after

incubation, was converted to dinitrogen by a modified Duma’s method (Fiedler and

Proksch, 1975) and its 15N abundance was measured by emission spectrometry using

Jasco NIA-1 or N-151 15N Analysers. Calibration was done with certified standards

(Goeyens et al., 1985). The following uptake parameters were calculated: specific and

absolute uptake rates (Dugdale and Goering, 1967; Dugdale and Wilkerson, 1986).

Values for specific uptake rates were expressed as the amount of nitrogen taken up by

the phytoplankton per unit of particulate nitrogen, implying that the uptake rates

normalized to phytoplankton biomass (PON) and f-ratios (the ratio of NO3� uptake to

total nitrogen uptake; Eppley and Peterson, 1979).

3. Results

3.1. Seasonal changes of nutrient concentrations

The seasonal distributions of nutrient concentrations are presented in Fig. 2a–f.

Phosphate concentrations ranged from 0.1 to 3.4 AM and from 0.3 to 1.7 AM in 1996 and

1997, respectively. The concentration levels in both years displayed poor seasonal patterns

(Fig. 2a) with the highest concentration in mid-May 1996 and at the end of March in 1997.

The concentrations of Si showed a clear seasonal changes with high concentrations in

winter (with the maximum of f 30 AM at the end of February 1996 and in January 1997)

and early spring, and much lower concentrations were measured at the end of spring and

during summer (Fig. 2b). Minimum concentrations were f 3 AM in 1996, whereas in

1997 the concentrations were close to the detection limit (f 0.2 AM).

The seasonal variation of NO3� concentrations correlated that of Si. Following winter

replenishment, high concentrations occurred during late winter and early spring (f 40 AMin March 1996 and f 30 AM in January 1997), whereas late spring and summer were

characterised by low values (f 6 AM) in late April 1996 and below detection limit (f 0.1

AM) in late April 1997 (Fig. 2c).

a. b.

d.c.

e. f.

Fig. 2. Temporal nutrient concentrations during 1996 (open circles) and 1997 (closed circles) sampling periods.


Ammonium concentrations and timing were slightly different in these 2 years (Fig. 2d).

From late winter of 1996, there was a slow increase in concentration, reaching amaximum of

f 8 AM in early spring (in March). From the maximum, the concentration decreased to the

minimum of 0.2 AM before increasing again to f 6 AM in late May. Sudden decrease was

observed at the end of May. In 1997, there were higher concentrations in mid winter and by

the beginning of spring, in early March, the concentration had decreased to f 1 AM. The


lowest concentration of 0.2 AM was measured in mid-April. In the similar way as in 1996,

from the minimum, the concentration increased to the maximum of 10 AM at the end of

May 1997.

Urea concentrations (Fig. 2e) remained < 1 AM throughout the sampling seasons of

1996. Between February and April 1996, urea concentrations varied from 0.3 to 0.8 AMthen decreased tof 0.3 AM in early May. This concentration was maintained until the end

of June. In 1997, the concentrations varied from 0.9 AM in March to the minimum of

f 0.2 AM in April, before increasing to the highest level of the season, 6 AM in May. This

highest concentration was remarkably exceptional and coincided with a similar increase

observed in the NH4 concentration.

The variations in NO2� concentrations were identical during both years (Fig. 2f). The

maximum concentration was 0.5 AM at the end of March 1996, whereas it was 0.8 AM in

the middle of March 1997. There were persistent fluctuations of concentrations throughout

the sampling periods with a decreasing trend from early spring to early summer. Both

years had elevated concentrations in summer.

3.2. Seasonal changes in phytoplankton biomass

In 1996, chlorophyll a concentrations (Fig. 3) remained less than 2 Ag l� 1 from

February to mid-March and increased to a maximum value of 18 Ag l� 1 between 25 April

and 5 May. In late spring, the chlorophyll a concentrations decreased and remained

constantly low during second half of May and June. In 1997, a threefold denser bloom

developed from early February to late April, with a maximum chlorophyll a concentration

of 52 mg l� 1.

The spring bloom was initiated by diatoms but dominated by the Phaeocystis colonies

in both years (Fig. 4). Diatom appeared in different community assemblages that

composed of different species. Early assemblage composed of Asterionella glacialis,

Fig. 3. Chlorophyll a concentrations following seasonal development, in 1996 (open circles) and in 1997 (closed

circles) season.

Fig. 4. Phaeocystis total biomass (closed squares) and diatoms total biomass (open circles). Comparison of their

temporal variations during the sampling seasons.


A. Kariana, Tharassiosira spp., Tharassionema nitzschoides, Plagiogramma brockmanii

and Skeletonema costatum. Followed by an assemblage of Chaetoceros spp., a final

assemblage to appear composed of Rhizosolenia spp., Guinardia sp. and Ditylum sp.

Each assemblage was clearly separated from another by a lowering of biomass. In 1996,

spring growth started by mid-March with a first moderate diatom development (Fig. 4)

that was followed by a small increase of biomass (63 Ag C l� 1) in early April. The

spring bloom reached maximum values of 174 Ag C l� 1 for diatoms and 521 Ag C l� 1

for Phaeocystis biomass. During the middle of May, both diatoms and Phaeocystis bloom

collapsed and the biomass decreased to the minimum levels that were maintained up to

the middle of June. A short and small increase of Phaeocystis biomass observed at the end

of May corresponded to the development of Phaeocystis nanoflagellates. In July, only

diatoms were observed and their biomass increased towards the middle of the month. In

1997, diatoms started developing by early February and reached their maximum in mid-


February. The total diatom biomass remained always < 100 Ag C l� 1. Furthermore, an

intensive Phaeocystis bloom (1600 Ag C l� 1) occurred in mid-April (Fig. 4).

The POC and PON concentrations are given in Table 1. For the period between 29

March and 31 May 1996, the C/N ratios were close to the Redfield ratio (f 6.6). This

suggested that there was no pronounced influence of non-phytoplanktonic particulate

matter in the organic matter composition. In 1997, we observed a significant departure

from the Redfield value between 3 April and 25 April. This could be due to the influence

of detrital materials but also to the build-up of Phaeocystis colonies exceeding that of

1996. Indeed, the polysaccharide mucous, formed by Phaeocystis, is a source of excess

carbon to the biomass values (Van Boekel, 1992; Thingstand and Billen, 1994; Lancelot

and Rousseau, 1994).

3.3. Nitrogen uptake rates

Incubation experiments were carried out between 29 March and 31 May in 1996 and

between 13 March and 7 May in 1997 (Fig. 5a–c). Nutrient concentrations measured in

the incubation bottles after 24 h showed that the regeneration of ammonium and nitrate

was sufficiently low (see estimations in the annex) such that the uptake rates could

reasonably be approached by the classical equations of Dugdale and Goering (1967) and

Dugdale and Wilkerson (1986).

In 1996, specific NO3� uptake rates (mNO3

) varied between 0.001 and 0.005 h� 1 with no

particular trend, except some small fluctuations (Fig. 5a). In 1997, mNO3decreased from

0.032 h� 1 at the end of March to a minimum of < 0.001 h� 1 at the beginning of May.

Similar variation patterns were observed for the absolute NO3� uptake rates (qNO3

), with

the maximum uptake rates of 0.031 AM h� 1 in early May 1996 and of 0.197 AM h� 1 in

mid-March in 1997, respectively.

The specific uptake NH4 rates (mNH4) varied in similar seasonal trends during both years

(Fig. 5b). A maximum mNH4of 0.0046 h� 1 was observed on 22 May 1996 and of 0.0056

h� 1 on 25 April 1997. The corresponding qNH4were 0.0154 and 0.070 AM h� 1 in 1996

and 1997, respectively. In both years, the minimum uptake of NH4 was in April and a peak

value was developing in late spring (April–May). The lag in the trend of 1996 uptake as

compared to 1997 was a result of late development of bloom of both diatom and

Phaeocystis in 1996.

Table 1

Particulate organic nitrogen and carbon during days of incubation experiment for 2 years

Date PON

(AM)

POC

(AM)

C/N

ratio

Date PON

(AM)

POC

(AM)

C/N

ratio

29 March 1996 3.54 28.18 7.97 7 March 1997 6.44 48.09 7.47

4 April 1996 5.32 46.12 8.68 13 March 1997 6.15 49.32 8.02

15 April 1996 2.04 14.60 7.14 21 March 1997 7.50 53.54 7.14

26 April 1996 8.56 51.74 6.05 3 April 1997 12.20 123.23 10.10

6 May 1996 7.58 53.43 7.05 18 April 1997 13.47 132.62 9.84

22 May 1996 3.40 19.47 5.73 25 April 1997 12.44 138.79 11.16

31 May 1996 3.31 20.07 6.06 7 May 1997 4.49 35.97 8.01

Fig. 5. Specific and absolute uptake rates determined by 15N incubation experiment for the 1996 (open circles)

and 1997 (closed circles) spring season.


The urea uptake rates had no specific seasonal patterns (Fig. 5c). However, a slight

trend for a maximum uptake was observed during late April. When the uptake trends were

compared to the biomass of planktonic bacteria in 1996 (Fig. 6), there was a good

correspondence but not with the trend of available urea concentrations (Fig. 2e). The

maximum absolute urea uptake rate was 0.0097 AM h� 1 in April 1996 and 0.0191 AMh� 1 in late April 1997.

Fig. 6. Seasonal trend of bacteria biomass in the 1996 sampling period.


3.4. Bacteria biomass

The development of bacteria biomass (Fig. 6) was similar to diatoms from early

February, and increased to the maximum together with the diatom and Phaeocystis spring

bloom. The highest biomass of bacteria was observed between 26 April and 31 May 1996.

Although the highest range of biomass (50–54 Ag C l� 1) was far small to influence the

total microbial biomass (diatoms + Phaeocystis + bacteria), the simultaneous bloom

appearance with the phytoplankton should be indicating dependence or preferences for

the same environmental factors. The maximum biomass of bacteria was maintained even

after the Phaeocystis bloom. Possibly, that appearance was also supported by other organic

compounds. Examples hereof are the photosynthetic products, excreted from the Phaeo-

cystis cells mainly in the form of small molecules, which can directly be utilised by

planktonic bacteria (Lancelot and Mathot, 1987). These favour bacterial growth at a period

of Phaeocystis bloom and even later, when there is mucous release resulting from colony

disintegration.

4. Discussion

The observed elevated nutrient concentrations at station 330 are not startling, since the

Belgian coastal region is largely affected by anthropogenic nutrient inputs. Especially in

winter when biological uptake is at the lowest, the nutrient levels are significantly

enhanced. The typical winter values measured in the North Atlantic waters entering the

North Sea are 12 AM of NO3�, 0.8 AM of PO4

3� and 6 AM of Si (North Sea Task Force,

1993). In comparison with this, the maximum winter concentrations of f 40 AM for

NO3�, f 1.5 AM for PO4

3� and f 30 AM for Si measured at Station 330 signify a

doubled PO43� concentration and threefold to fivefold increases in NO3

� and Si con-

centrations. Riverine supply is presumably the first reason for enhanced nutrient concen-


trations. The phytoplankton development in these coastal waters has been related to the

nutrient enrichment (Billen et al., 1991; Riegman et al., 1992; Lancelot, 1995). However,

year to year variations in the phytoplankton biomass development were observed to occur

even when similar increase in the nutrients was experienced during 1996–1997 sampling

years.

4.1. Phytoplankton and nutrient dynamics

Figs. 2 and 4 show that variations of spring nutrient concentrations were under the

classical control of biological uptake. This is particularly clear for NO3�, NO2

� and Si

whose highest concentrations are recorded before the growth season. The sharp decrease

of Si, NO3�, NO2

� and NH4 observed in early March 1996 was due to the development of

the first diatom community. The development of Phaeocystis and diatoms started when Si

and NH4 were at low levels (4 and 0.3 AM, respectively) and when NO3� concentration

was 28 AM. When Phaeocystis bloom was declining, NH4 and PO43� concentrations

increased strongly, suggesting a significant mineralization. In 1997, there was a poor

development of diatoms, but in contrast, higher biomass and earlier appearance of

Phaeocystis were experienced. Phaeocystis developed the highest biomass when nutrient

concentrations were at the lowest levels (NO3� 1.2 AM, NO4 0.2 AM and urea 1.2 AM) and,

generally, the ambient concentrations were lower than those of 1996.

The difference in uptake patterns is well illustrated by both the specific and absolute

NO3� uptake rates (Fig. 5a), the corresponding ambient NO3

� concentrations available for

phytoplankton uptake as well as by the stock to uptake rate ratios. As a matter of fact,

dividing the ambient stock by the corresponding uptake rate gives the time required for

complete exhaustion of the stock, assuming that phytoplankton continued removing NO3�

at a constant rate. The period would amount to some 80 days (approximately 12 weeks) in

1996 and to 5 days in 1997. Indeed, the observed high NO3� abundance in 1996 requires

the theoretical prolonged period for exhaustion of the NO3� pool. Similarly, low NO3

�

concentrations and exhaustion in 1997 are in agreement with rapid removal. These

observations connote regulation of the autotrophic processes by something else other

than NO3� concentration (and PO4

3�, which remained above 0.1 AM in 1996 and above 0.5

AM in 1997 throughout spring).

4.2. Phaeocystis and diatoms dominance

In 1996, it was observed that the development of the diatoms bloom appeared at the

same time as Phaeocystis and that the bloom collapsed in spite of non-limiting Si

concentrations (>5 AM). Apart from several other governing parameters, strong photo-

inhibition, which is likely to happen during late spring, may have induced the collapse of

diatoms in 1996 when Si were sufficiently available. Diatoms are known to be affected by

high light intensities (Lancelot and Mathot, 1987).

In 1997, however, the diatoms bloom occurred earlier before the Phaeocystis develop-

ment and their biomass decrease mirrored the lowering of Si concentrations. This early

appearance of diatoms, where they were followed by Phaeocystis before the appearance of

their second peak, has been explained to be a characteristic of the station (Rousseau et al.,


1996). Considering lower ambient Si and NO3� concentrations in spring 1997 as compared

to 1996, a poor diatom development was reasonably understandable, but it was difficult to

understand the development of Phaeocystis biomass, which was three times higher than

that of 1996, while NO3� concentrations were much less.

In 1997, there was intensive NO3� uptake which was followed by NO3

� depletion,

during late spring (Figs. 2c and 5a). Contrary to that, in 1996, high NO3� availability

coincided with poor NO3� uptake and there was no NO3

� depletion. The pertinent question

for understanding the ecosystem’s functioning is why did phytoplankton fail to consume

NO3�? Several factors are known to impair NO3

� uptake and subsequently lowering

primary production (Cullen, 1991; Dugdale and Wilkerson, 1991, 1992). It is believed that

uptake by phytoplankton is lowered when NH4 availability exceeds a threshold value.

Earlier studies in different environments such as the Subarctic Pacific (Wheeler and

Kokkinakis, 1990), the North Atlantic (Elskens et al., 1997) and the Southern Ocean

(Goeyens et al., 1995) have already explained this role of NO4 in regulating phytoplankton

production.

In this study, the difference between incomplete and complete exhaustion of NO3�

corresponds to high and poor abundances of NH4. Highest uptake rates were measured in

March and April 1997, a period that was characterised by the absence of enhanced NH4

and urea. Any inhibition of oxidised nitrogen (Nox, mainly NO3�) uptake by enhanced

reduced nitrogen (Nred) is, therefore, unlikely during that particular period. The Nox/Nred

ratios (Fig. 7) demonstrate a change of trend from higher values (>10) during early spring,

to lower values at the end of the sampling season, with few values being < 1. Additionally,

a graph of specific NO3� uptake rates versus Nred illustrates the inhibiting effect of the Nred

(Fig. 8). The lowest values (V 0.001 h� 1) indicate very poor capacity of the phytoplankton

to utilise NO3� as their nitrogen source. These cannot be explained as a consequence of

exhaustion, since these lowest uptake rates (observed in 1996) occurred NO3� at

concentrations exceeding 5 AM. On the contrary, the hyperbolic trend of mNO3versus

Nred in Fig. 8 suggests a considerable inhibition at higher Nred availability (>2 AM). The

Fig. 7. Logarithmic change of seasonal variation of nutrient ratios between oxidized nitrogen concentrations and

reduced nitrogen concentrations for 1996 (closed circles) and 1997 (open circles).

Fig. 8. Scatter plot showing the relationship between specific nitrate uptake rate versus the concentration of total

reduced nitrogen.


highest Nred concentrations generally occurred during late spring (May 1996 and April–

May 1997), with some smaller increases in late winter and early spring (March 1996 and

March 1997).

The pronounced decreases in NO3� uptake rates, observed in 1997, were not compen-

sated by similar increases in Nred uptake. When the decrease of qNO3

� was f 0.2 AM h� 1,

the increase of qNH4

+ and urea were only f 0.05 and f 0.02 AM h� 1, respectively.

The variability in nitrogen uptake rates is shown by the f-ratios, which vary strongly

from 1 year to the other (Table 2, p. 35). Bacteria are well known to manifest great

preferences for urea. Moreover, NH4 can be utilised by bacteria as well and its uptake have

occasionally been observed to represent 20–75% of the total uptake (Laws et al., 1985;

Harrison et al., 1992; Kirchman et al., 1989, 1994). In the North Atlantic Ocean, 4–14%

of the total NO3� uptake was attributed to bacterial consumption (Kirchman et al., 1994).

Therefore, bacteria inherently present in every sample could have obscured the phyto-

plankton uptake patterns. The agreement between planktonic bacteria peak abundances

and high urea uptake rates suggests that bacteria contributed especially to the uptake of

Table 2

f-ratio for 1996 and 1997 samples calculated by considering contribution of the three nitrogenous nutrients, NO3�,

NH4 and urea

Date of sampling f-ratio f-ratioa Date of sampling f-ratio f-ratio*

29 March 1996 0.3 0.3 13 March 1997 0.9 0.9

4 April 1996 0.2 0.2 21 March 1997 0.8 0.9

15 April 1996 0.8 0.9 3 April 1997 0.8 0.9

26 April 1996 0.3 0.5 18 April 1997 0.7 0.8

6 May 1996 0.6 0.7 25 April 1997 0.3 0.4

23 May 1996 0.2 0.2 7 May 1997 0.0004 0.001

31 May 1996 0.2 0.3

a f-ratio = f-ratio when urea is excluded.


urea. However, there is very little difference between f-ratios calculated with and without

the uptake of urea (Table 2), which signified that urea uptake rates by phytoplankton and

bacteria were low compared to the total N uptake. Therefore, bacteria uptake did,

probably, not affect significantly the uptake rate results.

At this study area, there is no clear distinction between new and regenerated production

(Dugdale and Goering, 1967), since autochthonous and allochthonous sources supply both

Nox and Nred (see the nitrate and ammonium load of the Schelde River mentioned in

Section 1). Therefore, C-ratios are only used to illustrate the contribution of NO3� in the

total nitrogen uptake. In 1996, f-ratios remained almost constant < 0.5 (predominance of

Nred uptake) with the exception of a short period from mid-April to early May. On the

contrary, in 1997 all ratios were >0.5 (predominance of NO3� uptake), except those at the

very end of the season. When f-ratios were plotted versus the Nred concentration, they

displayed a pronounced decreasing trend with increasing Nred values (Fig. 9). This is

largely due to the effect of ambient Nred concentrations to NO3� uptake rate (herein).

Different types of phytoplankton living in well-mixed coastal waters show distinct

requirements for nutrient assimilation and growth (Hutchinson, 1961), especially in

conditions of nutrient deficiency. We assumed that the observed nitrogen uptake regime

is a representative of the biological activities of the dominant phytoplankton community,

which we defined as that accounting for more than 50% of the phytoplankton biomass. In

this investigation, only two communities were considered; diatoms and Phaeocystis.

Obviously, other communities could also be important. However, however it is generally

understood that diatoms and Phaeocystis are the major components of the phytoplankton

assemblage in the Belgian coastal waters during spring.

The general characteristics of the N uptake regime, inhibition of NO3� uptake by

increased Nred availability (Fig. 8) and uptake do not apply in a similar way for these

Fig. 9. Phaeocystis sp. dominated samples (open circles) and diatoms dominated samples (closed circles). The

influence of reduced nitrogen (Nred) to the f-ratios. Circle size represents percentage of biomass of a community to

the total phytoplankton biomass.


two communities. Both groups displayed an intensive NO3� uptake capacity during

periods of abundant NO3� and deficient Nred, with diatoms being probably favoured in

comparison with the Phaeocystis. Really, in Phaeocystis-dominated assemblages, the

maximum mNO3� was 0.0104 h� 1, whereas that of diatom-dominated was 0.0320 h� 1. On

the other hand, when the mNH4

+ of Phaeocystis and diatom-dominated assemblages were

compared with the increase of concentrations (Fig. 10a and b), both communities

displayed ability to increase the uptake rate, but Phaeocystis showed higher uptake

ability than diatoms. The maximum specific uptake rate (mmax) of Phaeocystis was not

reached even when the concentration of NH4 was about 6 AM, whereas diatoms showed

lower mmax value and the saturation point was reached when NH4 concentration was

about 1 AM. Therefore, it can be understood that diatoms are characterised by lower Ks

Fig. 10. Phaeocystis sp. and diatom dominated communities. Trends of specific NH4 uptake rates at different

concentrations of NH4. (a) When >50% of biomass are Phaeocystis (or < 50% are diatoms); (b) when >50% of

biomass are diatoms (or < 50% are Phaeocystis).


and mmax values compared to Phaeocystis. Consequently, Phaeocystis showed ability to

increase their NH4 uptake capacity when more NH4 became available while diatoms

failed to do so as they were already at saturating ammonium concentration.

However, as explained earlier, this increase of NH4 uptake rates does not compensate

for the inhibitory effect on the NO3� uptake rate, with a total N uptake rates remaining

lower in Nred rich environments. The limited number of data appeals for extreme care with

the interpretation of the values, but, equally important, low uptake capacity at low

concentration (< 0.7 AM) and high concentration (>2.5 AM) of NH4 support a different

nitrogen uptake regime of diatoms when compared to Phaeocystis.

These different responses of the two communities to the variable Nred concen-

trations can be used to explain the observed phytoplankton biomass patterns. In 1996,

Si limitation to diatom growth did not really occur, but in spite of this, the diatom

community was out-competed by Phaeocystis. The diatom bloom collapsed at the end

of spring (Fig. 4). A similar observation was reported earlier by Escavarage et al.

(1995). These authors carried out mesocosm experiments and demonstrated that in

spite of high ambient Si concentrations in their samples, the diatom bloom collapsed

and was subsequently replaced by a more intensive Phaeocystis bloom. Investigating

natural environments, Peperzak et al. (1998) also found that the termination of a

diatom bloom in the Dutch coastal zone and its substitution by a denser Phaeocystis

bloom could not be explained by silicate limitation. Several possible explanations

were forwarded, including competition for light, selective grazing of diatoms, vitamin

deficiencies and as previously mentioned, the limitation of diatom growth by high

light intensity. We add another regulatory parameter to this far from exhaustive list of

controls of phytoplanktonic processes, the increasing concentration of Nred. Indeed, in

1996, we observed that the proportion of Nred increased drastically from early April

till mid-May and during the bloom of both communities the Nox/Nred ratio changed

from 47 to f 0.2. The combination of silicate exhaustion and a higher Ks and mmax

for Nred made Phaeocystis the successful competitor in Belgian coastal waters. Their

massive colonies formed during their peak of bloom are not grazed efficiently and,

therefore, Phaeocystis are hindering efficiency of energy transfer to the higher trophic

levels.

5. Conclusion

The bloom biomass of both diatoms and Phaeocystis are particularly supported by

NO3�, which is the major dissolved form of nitrogen. When the concentrations of NH4

are low, diatoms shows higher NO3� uptake ability than Phaeocystis. In this case, it is

generally observed that the uptake rates of NO3� by Phaeocystis and diatoms decrease

when the concentrations of Nred increase and, therefore, lessening the importance of in

primary production. Phaeocystis can increase their NH4 uptake rates as the NH4

concentration increases, but not enough to compensate the reduced rate on NO3�,

whereas diatoms increasing uptake ability were limited to low NH4 concentrations.

Diatoms failure to take-up the increasing NH4 concentrations was a result of their lower

Ks and mmax, which is reached at lower NH4 concentrations. This also disfavoured


diatoms in the use of nitrogen at elevated NH4 concentrations. It is therefore, important

to note that the increase of Nred concentration, especially NH4, destabilises the natural

nitrogen uptake behaviour of diatoms and Phaeocystis. These changes give some

advantages to the Phaeocystis development. They can also be added to the list of

important factors controlling succession and domination between these two commun-

ities.

Acknowledgements

The present study framework is within the objectives of the EC research programme

COMWEB (CT960052) and the national Belgian research programme ‘‘Sustainable

Management of the North Sea’’. Our thanks to all crews of Belgica ship for their

contribution in the sampling. We also acknowledge the contribution of Jean-Yves Parent

for analysis of the samples. [RW]

Appendix A. Annex

A.1. Estimation of ammonium regeneration rates

This estimates the influence of ammonium regeneration during 24-h incubations on our

ammonium uptake rate results. It is true that, depending on the season, 24-h incubations

can have influence on the NH4 uptake rates due to high regeneration rates and hence

isotope dilution of the ammonium pool. Some experimental works have shown that effects

are particularly important when nutrient concentrations are low (Harrison, 1978; Glibert,

1982).

The measured concentrations of ammonium, before incubation and at the end of the

incubation, helped us to establish the possible error on uptake rates at the end of each

incubation, introduced by the regenerated ammonium. In all the experiments, the final

concentrations were lower than the initial concentration except in one experiment which

was found with 9% excess NH4 (see the table below).

Ammonium concentrations before (Ci) and after (Cf) incubation for 24 h in the 1996

and 1997 incubation experiments

Date Ci Cf Date Ci Cf

29 March 1996 4.25 3.01 13 March 1997 0.81 0.34

4 April 1996 2.91 1.96 21 March 1997 1.21 0.47

15 April 1996 0.56 0.24 3 April 1997 0.31 0.34

26 April 1996 0.82 0.33 18 April 1997 0.65 0.55

6 May 1996 1.41 0.68 25 April 1997 0.49 0.38

23 May 1996 5.97 5.31 7 May 1997 6.51 5.83

31 May 1996 2.75 1.02


Taking the results on 3 April 1997 as an extreme case of NH4 regeneration, we can

make a simple calculation to estimate the introduced error, by considering the following

equation for balanced processes:

Cf ¼ Ci þ RDt � UDt

where Ci = initial concentration of ammonium, Cf = final concentration of ammonium,

R = regeneration rate of NH4, U = absolute uptake rate and Dt = incubation time.

From this, we can calculate R for different results and correct values of 15-N abundance

due to dilution.

In our worst case scenario, initial concentration was 0.31 AM and after the incubation it

was 0.34 AM. The corresponding absolute uptake rate was 0.008 AM h� 1.

Then from the above equation:

R ¼ Cf � Ci þ UDt

Dt

R ¼ 0:03ðAMÞ þ 0:19ðAMÞ24 h

R ¼ 0:009 AM h�1:

Assuming R was constant throughout the experiment and the regenerated ammonium

had the natural abundance for nitrogen, then the average 15N abundance after 24 h was

26.3% instead of 35.65%, which was the initial abundance.

Hence, the uptake rate, corrected for regeneration, changed from 0.008 to 0.0108 AMh� 1. This rate is 25% higher than the one uncorrected for regeneration.

All calculated uptake rates can be corrected, but they will all be lower, reasonably much

lower than 25%. In any case, regeneration does not have a major influence on the reported

difference of diatoms and Phaeocystis ammonium uptake rate behaviours. The difference in

highest ammonium uptake rate between diatoms and Phaeocystis is at a factor of about 2–3.

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