Exo-enzymatic activities and dissolved organic pools in relation with mucilage development in the...

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Science of the Total Environm

Exo-enzymatic activities and dissolved organic pools in relation with

mucilage development in the Northern Adriatic Sea

R. Danovaro a,*, M. Armeni a, G.M. Luna a, C. Corinaldesi a, A. Dell’Anno a,

C.R. Ferrari c, C. Fiordelmondo a, C. Gambi a, M. Gismondi c, E. Manini d,

M. Mecozzi b, F.M. Perrone a, A. Pusceddu a, M. Giani e

a DSM-Dipartimento Scienze del Mare, Universita Politecnica delle Marche, Via Brecce Bianche-60131 Ancona, Italyb ICRAM-Istituto Centrale per la Ricerca Scientifica e Tecnologica Applicata al Mare, via di Casalotti 300, 00166 Rome, Italy

c ARPA-Agenzia Regionale Prevenzione Ambiente Emilia Romagna, V.le Vespucci 2, 47042 Cesenatico FC, Italyd CNR-ISMAR-Istituto Scienze Marine, Sezione Ecosistemi Costieri e Lagunari, Via Pola 4, 70100 Lesina, Italy

e ICRAM-Istituto Centrale per la Ricerca Scientifica e Tecnologica Applicata al Mare, Chioggia, Italy

Available online 12 October 2005

Abstract

We tested the hypothesis that the appearance of mucilage in the Northern Adriatic Sea was related with the accumulation of

dissolved organic compounds released by intensive enzymatic activities and not utilized as direct substrate for microbial growth. To

do this enzymatic activities and dissolved organic and inorganic pools in periods characterized by the presence of mucilage and in

the same seasons but in absence of mucilage were compared. Extracellular enzymatic activities (aminopeptidase, h-glucosidase andalkaline phosphatase), nutrient pool concentrations (total dissolved nitrogen, dissolved organic nitrogen, total dissolved phospho-

rus, dissolved organic phosphorus) and the biochemical composition of particulate and dissolved organic matter (in terms of

proteins and carbohydrates) were determined on a monthly basis over a period of 3 years. Aminopeptidase and alkaline

phosphatase activities displayed higher values in springs preceding the appearance of mucilage than in spring when no mucilage

was observed. h-Glucosidase activity showed significantly higher values in summer periods characterized by the massive

production of mucilage than in summers without mucilage events. The months preceding mucilage events were also characterized

by an increase of the alkaline phosphatase to aminopeptidase activity ratio and by a significant accumulation of dissolved proteins.

These findings, together with the significant increase of the DON/DOP ratio, suggest that mucilage formation is favoured by the

deficiency of organic P. The present study provides compelling evidences that mucilage formation is favoured by the unbalance

between organic matter mobilization by enzymatic activities and the accumulation of labile dissolved organic-N compounds.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Mucilage; Aminopeptidase; h-Glucosidase; Alkaline phosphatase; Dissolved organic matter; Adriatic Sea

1. Introduction

0048-9697/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.scitotenv.2005.09.029

* Corresponding author. Department of Marine Science, Faculty of

Science, Polytechnic University of Marche, Via Brecce Bianche,

60131 Ancona, Italy. Tel.: +39 71 220 4654; fax: +39 71 220 4650.

E-mail address: [email protected] (R. Danovaro).

The appearance of large mucilage aggregates in the

Adriatic Sea has been documented since the 18th

century, but has become increasingly evident in the

last decades (Funari et al., 1999). This phenomenon

has been also increasingly reported in several areas

worldwide, as repeatedly reported from studies carried

ent 353 (2005) 189–203

R. Danovaro et al. / Science of the Total Environment 353 (2005) 189–203190

out in Tyrrhenian Sea (Innamorati, 1995), in the North

Aegean (Gotsis-Skretas, 1995), in the North Sea (Rie-

besell, 1992), in coastal areas of the Pacific Ocean

(Alldredge et al., 2002) and New Zealand (MacKenzie

et al., 2002). In the Adriatic Sea, mucilage aggregates

usually appear in late-spring/summer. Each aggregate

can have a size from few mm up to several meters

extending up to several hundred km2. Mucilage scav-

enging plankton and detrital particles, settling on the

bottom, can determine ipoxic and/or anoxic conditions

particularly in the bottom waters and at the sediment–

mucilage interface. The suffocation of benthic and

epibenthic (including nekton) organisms poses serious

fishery and sanitary problems, with important socio-

economical implications.

It has been hypothesised that mucilage aggregates are

an exacerbated and evolving stage of the bmarine snowQ(Herndl and Peduzzi, 1988; Herndl, 1992; Fogg, 1995),

appearing in environmental conditions able to favour the

progressive coalescence of marine aggregates. Although

the phenomenon of mucilage appearance has been al-

ready well described, little is known yet about mechan-

isms triggering its massive formation. Although there is

a converging opinion that mucilage production is the

result of synergistic effects of biological and physico-

chemical factors, several other hypotheses have been

Fig. 1. Sampling area and station locati

proposed. Among them the most accredited hypotheses

are: (i) altered inorganic nutrient supply and N/P ratio,

(ii) excess of ungrazed primary production, (iii) high

viral infection and consequent release of dissolved or-

ganic matter, (iv) accumulation of DOC and malfunc-

tioning of the microbial loop (Azam, 1997; Degobbis et

al., 1999). In the last years, the malfunctioning of the

microbial loop has been investigated in more detail

(Herndl et al., 1992; Azam, 1997). In fact, bacterio-

plankton is known to produce exo- and ecto-enzymes,

which represent a key step for the transformation of

complex organic macromolecules into low molecular

weight compounds (dissolved organic matter) available

for their assimilation and growth (Azam and Long,

2001). A crucial step for mucilage formation appears

to be the aggregation from dissolved organic com-

pounds into larger flocs. This process becomes expo-

nential when DOM concentrations reach threshold

levels and the aggregation and coalescence is facilitated

by the stability of the water column (Fogg, 1995).

Enzymatic activities and the breakdown of organic

particles are therefore essential steps in the release of

dissolved organic matter, but enzymatic activity alone

cannot explain the formation of mucilage. In fact, the

accumulation of DOM is possible only when there is an

imbalance between DOM production (by enzymatic

ons in the Northern Adriatic Sea.

R. Danovaro et al. / Science of the Total Environment 353 (2005) 189–203 191

cleavage of macromolecules) and bacterial uptake.

Such uncoupling, whose reasons are still under debate,

enhances the accumulation of DOM and TEP leading to

the coalescence of organic matter into large aggregates

(Decho and Herndl, 1995).

We investigated for 3 years, on a monthly basis, a

transect of stations crossing coast to coast the Northern

Adriatic Sea, in a sector frequently interested by muci-

lage formation. We analyzed the interactions between

dissolved and particulate organic matter pools (i.e.,

proteins and carbohydrates), in relation with extra-

cellular enzymatic activities, such as aminopeptidase

(which hydrolyses peptides and proteins), h-glucosi-dase (a key enzyme in carbohydrate degradation) and

alkaline phosphatase (which hydrolyses organic phos-

phoesters). These variables were investigated also in

relation with dissolved organic and inorganic N and P

Total Dissolved

0

5

10

15

20

25

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

µM-N

C4 surface C

0

5

10

15

20

25

µM-N

C7 surface C

a

b

0

5

10

15

20

25

µM-N

C12 surface C

c

Fig. 2. Temporal patterns of total dissolved nitrogen concentrations (TDN) at

C4 (a), station C7 (b) and station C12 (c).

nutrient pools in order to better understand the role of

the mechanisms influencing dynamics of organic pools

and their role in the formation of mucilage aggregates

in the Adriatic Sea.

2. Materials and methods

2.1. Study area and sampling activities

Sampling was carried out on a monthly basis from

June 1999 to June 2002 in the Adriatic Sea, along a

transect between Senigallia (Italian coast) and Susak

(Croatian coast; Fig. 1). The transect consisted of three

stations: C4 (bottom depth 59 m); C7 (depth 70 m) and

C12 (depth 65 m). At each station, water samples were

collected using Niskin bottles (capacity 12 l) on a

rosette sampler, at the surface, at 10 m depth and at 1

Nitrogen (TDN)D

ec 0

0

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

4 10 m C4 bottom

7 10 m C7 bottom

12 10 m C12 bottom

the three sampling depths (surface, intermediate and bottom) at station


m above the bottom. Samples were processed immedi-

ately after collection, as described below.

2.2. Analyses of dissolved inorganic nitrogen and

phosphorus

Water samples for nutrient determinations were fil-

tered utilizing HA 0.45 Am Millipore membrane filters

and then stored frozen until laboratory processing,

which was usually carried out within 3 days after sam-

pling. Total dissolved nitrogen (TDN), total dissolved

phosphorus (TDP), total nitrogen (TN) and total phos-

phorus (TP) were measured according to Koroleff

(1983) after persulfate digestion. Analyses of inorganic

nutrient concentrations (NO2, NO3, NH4, PO4) were

carried out spectrophotometrically using a Bran Luebbe

autoanalyzer (Traacs 800 and AAIII). Dissolved organic

Dissolved Organi

0

5

10

15

20

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

µM-N

C4 surface C

0

5

10

15

20

µM-N

C7 surface C

0

5

10

15

20

µM-N

C12 surface C

a

b

c

Fig. 3. Temporal patterns of dissolved organic nitrogen concentrations (DO

station C4 (a), station C7 (b) and station C12 (c).

nitrogen (DON) and dissolved organic phosphorus

(DOP) were calculated as the difference between TDN

and inorganic nitrogen (NO2+NO3+NH4) and between

TDP and inorganic phosphorus (PO4), respectively.

2.3. Analyses of particulate and dissolved organic

matter

For the analysis of total particulate proteins (PPRT),

water samples (from 0.3 to 1 l) were filtered onto

Whatman GF/F membranes (nominal pore size 0.45

Am), which had been pre-combusted in a muffle furnace

(480 8C, 2 h). Protein determination was carried out

according to Hartree (1972), using a UNICAM UV–

VIS spectrophotometer. For total dissolved proteins

(DPRT), aliquots of samples were put into polyethylene

bottles and stored at �20 8C until analyses, which were

c Nitrogen (DON)D

ec 0

0

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

4 10 m C4 bottom

7 10 m C7 bottom

12 10 m C12 bottom

N) at the three sampling depths (surface, intermediate and bottom) at


carried out by adapting the procedure described for

sediment samples by Lawrence et al. (1995). For the

analysis of total particulate carbohydrates (PCHO),

water samples (1 l) were filtered onto Whatman GF/F

membranes, which had been pre-treated in a muffle

furnace (480 8C), and stored at �20 8C until analyses.

Filters were then processed according to a colorimetric

method (Mecozzi et al., 2004), using a double beam

DMS 200 Varian UV–VIS spectrophotometer. For total

dissolved carbohydrates (DCHO), aliquots of samples

(25 ml) were put into plastic tubes and stored at �20 8Cuntil analyses, which were carried out as described by

Mecozzi et al. (1999). Standard used for protein and

carbohydrates quantification were solutions of BSA

(Bovine Albumin Serum) and D(+)glucose, respective-

ly. All biochemical analyses were carried out on three

replicates.

Total Dissolved P

0.0

0.4

0.8

1.2

1.6

2.0

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

µM-P

0.0

0.4

0.8

1.2

1.6

2.0

µM-P

0.0

0.4

0.8

1.2

1.6

2.0

µM-P

C4 surface C

C7 surface C

C12 surface C

c

b

a

Fig. 4. Temporal patterns of total dissolved phosphorus concentrations (TD


2.4. Analyses of exo-enzymatic activities

Extracellular enzymatic activities (aminopeptidase,

h-glucosidase and alkaline phosphatase) were deter-

mined by cleavage of analogue fluorogenic substrates

(l-Leucine-4-methylcoumarinyl-7-amide, Leu-MCA;

4-MUF-h-d-glucopyranoside, Glu-MUF; and 4-MUF-

P-phosphate, MUF-P, respectively; all substrates from

SIGMA), as described in Corinaldesi et al. (2003).

After evaluation of saturating concentrations, measure-

ments were carried out in triplicate by adding 100 Al ofGlu-MUF and Leu-MCA (final concentration 200 AM)

and 50 Al of MUF-P (final concentration 50 AM) in a

final volume of 5 ml of seawater. Substrate incubations

were performed in the dark at in situ temperature for 1

h. Samples were analyzed fluorometrically (at 380 nm

excitation, 440 nm emission for Leu-MCA and 365 nm

hosphorus (TDP)

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

4 10 m C4 bottom

7 10 m C7 bottom

12 10 m C12 bottom

P) at the three sampling depths (surface, intermediate and bottom) at


excitation, 455 nm emission for Glu-MUF and MUF-P)

using a Shimadzu RS-1501 fluorometer. Fluorescence

of samples was measured immediately after addition of

the substrate and after incubation. The increase of

fluorescence units during incubation was converted

into activity by comparison with standard curves of 7-

amino-4-methylcoumarin for Leu-MCA and of 4-

methylumbelliferone for both Glu-MUF and MUF-P.

Enzymatic activities were expressed as nmol of sub-

strate hydrolysed l�1 h�1.

2.5. Statistical analyses

A Spearman-Rank correlation was performed to test

relationships between all the investigated variables.

Differences in the investigated variables were investi-

Dissolved Organic

0.0

0.4

0.8

1.2

1.6

2.0

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

µM-P

0.0

0.4

0.8

1.2

1.6

2.0

µM-P

0.0

0.4

0.8

1.2

1.6

2.0

µM-P

C4 surface

C7surface C

C12 surface C

a

b

c

Fig. 5. Temporal patterns of dissolved organic phosphorus concentrations (D


gated by means of two-way analysis of variance

(ANOVA) after testing the homogeneity of variance.

The data set was then subdivided into different periods

according to appearance of mucilage (late spring/sum-

mer) and to the period preceding mucilage formation

(spring) during all sampling period (i.e., covering years

in which mucilage occurred and years in which muci-

lage did not occur). The data set was then processed for

a Multi-Dimensional Scaling analysis (PRIMER, Ver-

sion 5.0, Plymouth) in order to identify the variable/s

better associated to mucilage occurrence.

3. Results

Mucilage formation is a long-term process in which

organic particle production and their coalescence typ-

Phosphorus (DOP)D

ec 0

0

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

C4 10 m C4 bottom

7 10 m C7 bottom

12 10 m C12 bottom

OP) at the three sampling depths (surface, intermediate and bottom) at


ically start in spring. Therefore, from the data set

composed of 35 oceanographic cruises, we selected

the late-spring/summer (June, July and August) and

the spring (March, April and May) seasons as period

better describing the starting process of mucilage for-

mation. For data elaboration and statistical analyses

we used seven sampling periods: summer 1999 (no

mucilage observed), spring 2000 (before mucilage

appearance), summer 2000 (during a massive muci-

lage event), spring 2001 and summer 2001 (no mu-

cilage observed), spring 2002 (before the second

mucilage event) and summer 2002 (during the second

mucilage event). Average values were calculated for

each season mediating values observed along the 3

stations and integrated throughout the entire water

column.

Particulate p

0

100

200

300

400

500

600

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

µg L

-1

0

100

200

300

400

500

600

µg L

-1

0

100

200

300

400

500

600

µg L

-1

C4surface C4

C7 surface C

C12 surface C

a

b

c

Fig. 6. Temporal patterns of particulate protein concentrations (PPRT) at the

(a), station C7 (b) and station C12 (c). Standard deviations (FS.D.) are rep

3.1. Dissolved nitrogen and phosphorus

Patterns of total dissolved nitrogen (TDN), dis-

solved organic nitrogen (DON), total dissolved phos-

phorus (TDP) and dissolved organic phosphorus

(DOP) during the 3-year study are shown in Figs. 2–

5. TDN ranged from 0.22 (station C4) to 22.63 AM(station C7; Fig. 2). DON ranged from 0.17 to 15.16

AM (station C4; Fig. 3). TDP ranged from 0.03 (station

C4) to 1.88 AM (station C12; Fig. 4). DOP ranged

from 0.01 (station C4) to 1.81 AM (station C12; Fig.

5). TDN and DON concentrations showed significant

differences both among stations and among sampling

months (ANOVA, pb0.05). However, no differences

were observed comparing the seven periods investigat-

ed, except that in spring and summer 2002, when

roteins (PPRT)D

ec 0

0

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

10 m C4 bottom

7 10 m C7 bottom

12 10 m C12 bottom

three sampling depths (surface, intermediate and bottom) at station C4

orted.


lower values of TDN were observed (ANOVA,

p b0.05). No significant differences in inorganic nitro-

gen concentrations (ANOVA, ns) were observed in

periods characterized by the presence of mucilage.

TDP did not display a significant temporal and spatial

variability throughout the 3 years of investigation, with

the exception of a strong and significant increase ob-

served in spring 2001 at all stations and all sampling

depths (year in which no mucilage was observed;

ANOVA, p b0.05). The same trend was evident for

DOP, but not for orthophosphate, whose concentrations

did not show a significant seasonal variability neither

differences among periods of presence and periods of

absence of mucilage (ANOVA, ns). TDN/TDP ratios

were 2–3 times higher in spring 2000 and 2002 (66 and

Dissolved p

0

500

1000

1500

2000

2500

3000

Jun

e 9

9

July

99

Aug

99

Se

pt 9

9

Oct

99

No

v 99

Jan

00

Fe

br

00

Ma

rch

00

Ap

ril 0

0

Ma

y 00

Jun

e 0

0

July

1 0

0

July

2 0

0

Aug

00

Oct

00

No

v 00

µg L

-1

0

500

1000

1500

2000

2500

3000

µg L

-1

0

500

1000

1500

2000

2500

3000

µg L

-1

C4 surface

Jun

e 9

9

July

99

Au

g 9

9

Se

pt 9

9

Oct

99

Nov

99

Jan

00

Fe

br

00

Ma

rch

00

Ap

ril 0

0

Ma

y 00

Jun

e 0

0

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

Nov

00

C7 surface

Jun

e 9

9

July

99

Aug

99

Se

pt 9

9

Oct

99

Nov

99

Jan

00

Fe

br

00

Ma

rch

00

Ap

ril 0

0

Ma

y 00

Jun

e 0

0

July

1 0

0

July

2 0

0

Aug

00

Oct

00

Nov

00

C12 surface

a

b

c

Fig. 7. Temporal patterns of dissolved protein concentrations (DPRT) at the

(a), station C7 (b) and station C12 (c). Standard deviations (FS.D.) are rep

42, respectively) than in spring 2001 (22). The same

trend was observed for DON/DOP ratios, which values

during springs of years characterized by the presence of

mucilage (103 and 61 during spring 2000 and 2002,

respectively) were much higher than in spring 2001

(DON/DOP was 13), when no mucilage was observed.

Such differences were, however, not evident in any

summer period of the 3 investigated years.

3.2. Particulate and dissolved proteins and

carbohydrates

Patterns of particulate and dissolved proteins and

carbohydrates during the three sampling years are il-

lustrated in Figs. 6–9. PPRT ranged from 19.6F0.1

roteins (DPRT)

De

c 00

Jan

01

Fe

br

01

Ma

r 01

Ap

ril 0

1

Ma

y 01

Jun

e 0

1

July

01

Aug

01

Se

pt 0

1

Oct

01

No

v 01

Jan

02

Ma

r 02

Ap

ril 0

2

Ma

y 02

Jun

e 1

02

Jun

e 2

02

C4 10 m C4 bottom

Dec

00

Jan

01

Fe

br

01

Ma

r 01

Ap

ril 0

1

Ma

y 01

Jun

e 0

1

July

01

Au

g 0

1

Se

pt 0

1

Oct

01

Nov

01

Jan

02

Ma

r 02

Ap

ril 0

2

Ma

y 02

Jun

e 1

02

Jun

e 2

02

C7 10 m C7 bottom

Dec

00

Jan

01

Fe

br

01

Ma

r 01

Ap

ril 0

1

Ma

y 01

Jun

e 0

1

July

01

Aug

01

Se

pt 0

1

Oct

01

Nov

01

Jan

02

Ma

r 02

Ap

ril 0

2

Ma

y 02

Jun

e 1

02

Jun

e 2

02

C12 10 m C12 bottom

three sampling depths (surface, intermediate and bottom) at station C4

orted.


(station C12) to 503.6F35.4 Ag l�1 (station C4; Fig.

6). DPRT ranged from 64.8F0.0 to 2854.9F360.8 Agl�1 (both values reported at station C4; Fig. 7). PCHO

ranged from 1.8 at station C4 to 922.4 Ag l�1 (both

values reported at station C12; Fig. 8). DCHO ranged

from 59.4 at station C4 to 6246.1 Ag l�1 (both values

reported at station C7; Fig. 9). Concentrations of PPRT

and DPRT displayed a wide variability among sampling

depths, stations and periods (ANOVA, p b0.05). No

significant differences in PPRT concentrations were

observed comparing all spring and late-spring–summer

periods of the 3 years investigated (ANOVA, ns). On

the contrary, DPRT concentrations typically doubled in

all springs preceding mucilage events when compared

with values observed in springs without mucilage (i.e.,

on average 697.7F45.6 Ag l�1 in 2000 and 2002 vs.

Particulate Carb

0

200

400

600

800

1000

C4 surface

0

200

400

600

800

1000

0

200

400

600

800

1000

a

b

c

µg L

-1µg

L-1

µg L

-1

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

C7 surface

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

C12 surface

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Fig. 8. Temporal patterns of particulate carbohydrate concentrations (PCHO


390.6F22.8 Ag l�1 in 2001). It is worth noting that

such differences were not evident comparing summer

periods. PCHO and DCHO displayed a wide temporal

and spatial variability, and no differences were ob-

served comparing average spring concentrations of

the three sampling years (ANOVA, ns). Conversely in

summers characterized by mucilage events (i.e., 2000

and 2002) PCHO concentrations were significantly

higher (ANOVA, p b0.05) than in summers without

mucilage (i.e., 1999 and 2001). Such differences were

not significant for DCHO.

3.3. Exo-enzymatic activities

Temporal patterns of aminopeptidase, h-glucosidaseand alkaline phosphatase activities are shown in Figs.

ohydrates (PCHO)

C4 10 m C4 bottom

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

C7 10 m C7 bottom

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

C12 10 m C12 bottom

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

) at the three sampling depths (surface, intermediate and bottom) at

Dissolved Carbohydrates (DCHO)

0

1000

2000

3000

4000

5000

6000

7000

8000

0

1000

2000

3000

4000

5000

6000

7000

8000

0

1000

2000

3000

4000

5000

6000

7000

8000

a

b

c

µg L

-1µg

L-1

µg L

-1

C4 surface C4 10 m C4 bottom

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Fig. 9. Temporal patterns of dissolved carbohydrate concentrations (DCHO) at the three sampling depths (surface, intermediate and bottom) at



10–12. Aminopeptidase activity ranged from 2.3F0.0

(station C7) to 228.7F10.4 nmol l�1 h�1 (station C4;

Fig. 10). h-Glucosidase activity ranged from 0.1F0.0

to 39.6F3.1 nmol l�1 h�1, both at station C4 (Fig. 11).

Alkaline phosphatase activity ranged from 1.7F0.0 to

531F50.6 nmol l�1 h�1 (both values observed at sta-

tion C4; Fig. 12). Overall, all enzymatic activities dis-

played wide spatial and depth-related variability, with

highest values generally observed at surface and lowest

at intermediate or bottom depths. The coastal station C4

generally displayed the highest enzymatic activities.

Aminopeptidase activity was significantly higher in

springs preceding mucilage events (65.7F8.2 nmol

l�1 h�1, average of springs 2000 and 2002; ANOVA

p b0.05) than in spring 2001 (43.4F5.2 nmol l�1 h�1)

when mucilage did not appear. These differences were

not observed for summer periods (on average 21.9F6.5

nmol l�1 h�1; ANOVA, ns). h-Glucosidase activity did

not show differences among spring periods (on average

6.53F0.0 nmol l�1 h�1; ANOVA, ns), but increased

significantly in summer characterized by the presence

mucilage (i.e., 2000 and 2002; ANOVA, p b0.05). Al-

kaline phosphatase activity was significantly higher in

springs preceding mucilage appearance (81.22F8.2

nmol l�1 h�1, average of springs 2000 and 2002;

ANOVA, p b0.05) than in spring when no mucilage

was observed, whereas did not show significant differ-

ences among summer periods (on average 45.8F6.5

nmol l�1 h�1; ANOVA, ns). Aminopeptidase was sig-

nificantly correlated with the concentration of particu-

Aminopeptidase

0

50

100

150

200

250

300n

mo

l L-1

h-1

nm

ol L

-1h

-1n

mo

l L-1

h-1

0

50

100

150

200

250

300

0

50

100

150

200

250

300

a

b

c

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02



Fig. 10. Temporal patterns of aminopeptidase activity at the three sampling depths (surface, intermediate and bottom) at station C4 (a), station C7

(b) and station C12 (c). Standard deviations (FS.D.) are reported.


late proteins and particulate carbohydrates (n =268;

p b0.01); h-glucosidase was significantly correlated

with particulate and dissolved carbohydrates (n =268;

p b0.01), whereas alkaline phosphatase was signifi-

cantly correlated with particulate proteins and carbo-

hydrates (n =268; p b0.01). No relationships were

observed between the enzymatic activities and the

nutrient pools. Summarizing, aminopeptidase and al-

kaline phosphatase activities were significantly higher

in all spring periods preceding mucilage events (2000–

2002), whereas h-glucosidase was significantly higher

in the corresponding summer periods.

4. Discussion

The bmicrobial hypothesisQ suggests that mucilage

formation in the Adriatic Sea is also the result of

interactions occurring at microscale between bacteria

and organic matter, which can have consequences on

biogeochemical cycling at macroscale (Azam et al.,

1999). In particular this hypothesis focuses on the

uncoupling between a fast microbial degradation of

particulate organic matter (with consequent large re-

lease of polysaccharides and peptides) and the limited

utilization of the substrates released by bacteria. The

consequent accumulation of dissolved compounds and

long-lived polysaccharides would represent a factor

favouring coalescence and aggregation processes lead-

ing to the formation of mucilage.

According with this process the first step is repre-

sented by enzyme-mediated microbial degradation of

the particulate organic pools. The measured enzymatic

activities displayed high values typically reported for

highly productive coastal and estuarine areas (Murrell,

ß-glucosidase

0

10

20

30

40

50

0

10

20

30

40

50

0

10

20

30

40

50

a

b

c

nm

ol L

-1h

-1n

mo

l L-1

h-1

nm

ol L

-1h

-1

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Fig. 11. Temporal patterns of h-glucosidase activity at the three sampling depths (surface, intermediate and bottom) at station C4 (a), station C7 (b)

and station C12 (c). Standard deviations (FS.D.) are reported.


2003; Ammerman and Glover, 2000). Alkaline phos-

phatase values observed in springs preceding a mucilage

development were from one to two orders of magnitude

higher than those reported for the Adriatic Sea by Zac-

cone and Caruso (2002) in periods characterized by the

absence of mucilage (February 1998; mean values

13.6F5.0 and 2.4F0.5 nmol l�1 h�1 in the Northern

and Southern Adriatic Sea, respectively). The ob-

served increase in the alkaline phosphatase activity

before the appearance of mucilage suggests a faster

organic P remineralization. In spring periods preceding

mucilage events (2000 and 2002), an increase in the

alkaline phosphatase/aminopeptidase (APA/AMA) ac-

tivity ratio was also observed. Such increase was more

evident in spring 2000 (on average 4.1) than in spring

2002 (on average 1.8). The APA/AMA activity ratio

has been recently proposed as a proxy of P deficiency

in microbial assemblages as it has been observed that

the APA/AMA activity ratio increases in P-deficient

treatments, and decreases after inorganic P addition

(Sala et al., 2001). The high values of the APA/AMA

activity ratio observed in our study suggest the presence

of phosphorus limitation. This conclusion was also

supported by the analysis of the N/P ratio in DOM,

which increased significantly (ANOVA, p b0.01) in

both springs preceding mucilage appearance (but not

in corresponding summers). In addition, P limitation

could have further stressed phytoplankton determining

an increased release of exudates (Myklestad, 1999;

Azam et al., 1999). The significant relationships ob-

served between enzymatic activities (i.e., aminopepti-

dase and h-glucosidase) and particulate proteins and

carbohydrates concentrations (respectively) suggest that

enzymatic activities are induced by the availability of

organic substrate in particulate form. Therefore, our

results indicate that, in the Adriatic Sea, the production

Alkaline phosphatase

0

100

200

300

400

500

600

0

100

200

300

400

500

600

0

100

200

300

400

500

600

a

b

c

nm

ol L

-1h

-1n

mo

l L-1

h-1

nm

ol L

-1h

-1

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02


Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

Oct

01

No

v 01

Jan

02

Mar

02

Ap

ril 0

2

May

02

Jun

e 1

02

Jun

e 2

02

Jun

e 99

July

99

Au

g 9

9

Sep

t 99

Oct

99

No

v 99

Jan

00

Feb

r 00

Mar

ch 0

0

Ap

ril 0

0

May

00

Jun

e 00

July

1 0

0

July

2 0

0

Au

g 0

0

Oct

00

No

v 00

Dec

00

Jan

01

Feb

r 01

Mar

01

Ap

ril 0

1

May

01

Jun

e 01

July

01

Au

g 0

1

Sep

t 01

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v 01

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e 2

02



Fig. 12. Temporal patterns of alkaline phosphatase activity at the three sampling depths (surface, intermediate and bottom) at station C4 (a), station

C7 (b) and station C12 (c). Standard deviations (FS.D.) are reported.


of labile particulate organic pools is the first step of the

mucilage formation process.

The second step of the bmicrobial hypothesisQ is

represented by the accumulation of dissolved organic

compounds. This was confirmed by our study as we

found a significant accumulation of dissolved organic

matter, evident in terms of dissolved proteins, in both

springs preceding mucilage events (2000 and 2002).

Such accumulation was also coupled to significant

increases of the DON/DOP ratios. Therefore, springs

preceding mucilage events tend to display a strong

accumulation of organic N. Such shift in DON/DOP

ratio has been repeatedly reported in the Adriatic Sea

but the limited spatial and time scale did not allow to

identify cause–effect relationships with mucilage occur-

rence (Lipizer et al., 1999).

When P becomes limiting, bacteria tend to increase

exo-enzymatic production (Hoppe, 1983), enabling a

faster degradation of organic particles with the subse-

quent release of direct substrate, but can also utilize

inorganic P forms to balance their nutritional require-

ment (Danovaro, 1998). The consequent P-turnover

increase is expected to cope with P limitation. How-

ever, this was apparently not the case of our study as

the years of mucilage formation displayed an unbal-

ance between substrate released and microbial uptake,

which could have been responsible of the positive

feedbacks, for which enzyme production was further

stimulated.

It is worth noting that in springs preceding muci-

lage events only the accumulation of labile organic N

as dissolved proteins was evident, whereas dissolved


carbohydrates (which, in the Adriatic Sea, account for

15–20% of DOC; Pettine et al., 1999; Pettine et al.,

2001) did not change. The accumulation of dissolved

proteins would lead to the increase of DOC that,

however, has never been demonstrated before muci-

lage formation. Such discrepancy might be explained

because the protein contribution to DOC (1%, Pettine

et al., 1999) is much lower than that of dissolved

carbohydrates.

Therefore our results clearly indicate that the two

steps favouring the possible formation of mucilage

were actually observed in springs preceding mucilage

occurrence in 2000 and 2002. This was further con-

firmed by a multivariate analysis (MDS ordination;

Fig. 13) performed on a data set composed by

DON/DOP and N/P ratios, AMA and APA activities,

particulate and dissolved proteins and carbohydrate

pools, which clearly pointed out the presence of a

cluster of stations characterized by the presence of

mucilage. The plot, indeed, clearly segregated summer

periods (on the right half), from spring periods (left

half) and highlighted the group of station preceding

mucilage events (lower left).

Being P regeneration mediated by alkaline phos-

phatases we utilized APA activities for discriminating

different sampling periods and we superimposed the

values of APA activities to the MDS output. This

allowed also discriminating between springs of 2000

and 2002, providing quantitative differences related

201.93

253.8

150.06

75.7579.2

182.09

220.21

143.97 53.2

73.35

68.

88.03

MASSIVE MUCILAGE EVENT

2002

2000

MUCILAGE EVENT2002

SPRING

NO MUCILA

NO MUCILAGE

201.93

253.8

150.06

75.7579.2

182.09

220.21

143.97 53.2

73.35

68.

88.03

SPRING

201.93

253.8

150.06

75.7579.2

72.3

182.09

220.21

143.97 53.2

73.35

68.

88.03

SPRING

Fig. 13. Multi-dimensional scaling representation based on a subset of releva

concentrations, aminopeptidase (AMA) and alkaline phosphatase (APA) act

with the different extension and relevance of the

mucilage phenomenon in the 2 years. Moreover, our

results indicate that mucilage formation (visible only

in summer) starts when DON/DOP ratios in the spring

period trespass the threshold level of 50 and APA

activities are above the threshold level of 100 nmol

l�1 h�1, thus enabling to identifying possible tools for

monitoring and predicting the future occurrence of

mucilage.

The present intensive 3-year study provides compel-

ling evidence of the actual consequences of the unbal-

ance between organic matter degradation and uptake,

and highlights the importance of organic P dynamics in

mucilage formation in the northern Adriatic Sea. It is

reasonable also to hypothesise that a more limited

sampling effort would have hampered the demonstra-

tion of such mechanisms, suggesting that the successful

understanding of complex phenomena (such as the

those related with mucilage and/or dystrophic events

in the Adriatic Sea) requires long-term and high-reso-

lution investigations.

Acknowledgments

The study was performed in the frame of the MAT

Project (Mucilages in the Adriatic and Tyrrhenian seas)

coordinated by Istituto Centrale per la Ricerca Scienti-

fica e Tecnologica Applicata al Mare (ICRAM) and

financed by the Italian Ministry of Environment.

27.16

31.96

22.36

34.5641.3927.73

3

33.11

57

49.11

Stress: 0.08

SUMMER

GE

27.16

31.96

22.36

34.5641.3927.73

3

33.11

57

49.11

Stress: 0.08

27.16

31.96

22.36

34.5641.3927.73

3

33.11

57

49.11

Stress: 0.08

nt parameters: N/P ratio, DON/DOP ratio, PPRT concentrations, DPRT

ivities. Values reported refer to APA activities. Stress value 0.08.


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