1. ORIGINAL PAPER
Growth and nutrient absorption of two submerged
aquatic macrophytes in mesocosms, for reinsertion
in a eutrophicated shallow lake
Adriana Ciurli Æ Paolo Zuccarini Æ Amedeo Alpi
Received: 14 March 2007 / Accepted: 17 March 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Aquatic macrophytes play a central role
in preserving the ecological equilibrium of shallow
lakes and in the restoration of eutrophic lakes that
have switched to phytoplankton-dominated turbid
water. Massaciuccoli Lake, a shallow lake located
along the Tuscan coast in Italy, has shown a constant
and progressive simplification of the submerged plant
community, for anthropogenic reasons, leading, in
recent years, to turbid water. The growth and nutrient
absorption capability of two macrophyte species,
Myriophyllum verticillatum L. and Elodea canadensis
Michaux, in the lake was investigated, with the
prospect of a future lake restoration programme
centred on their replacement. Mesocosm experiments
were conducted to monitor the plant growth and
nutrient (NO2
-
, NO3
-
, NH4
+
, Ntot, PO4
3-
, Ptot) content
in the plant dry matter and water at the beginning and
at the end of the trial. Bacterial activity was analysed
in the water in order to verify the possible nutrient
absorption contribution by organisms other than
plants. Both M. verticillatum and E. canadensis
showed satisfactory growth and nutrient reduction
in the water body. Moreover, their different growth
patterns suggested that optimal replacement can be
performed with their introduction in two steps,
starting with M. verticillatum, which shows the best
capacity to colonise the aquatic environment, due to
its tendency towards lengthening.
Keywords Elodea canadensis Michaux Á
Eutrophication Á Mesocosms Á Myriophyllum
verticillatum L. Á Restoration Á Shallow lakes
Introduction
Aquatic macrophytes play a key role in the preser-
vation of clear water in lakes through a series of
mechanisms connected with both biotic interactions
and the chemical–physical characteristics of water
and sediments. Submerged vegetation provides shel-
ter for zooplankton (Lauridsen et al. 1996; Van Donk
and Van de Bund 2002) and for habitat and the
reproduction of macroinvertebrates, fishes and water-
birds (Rozas and Odum 1988; Kemp et al. 1990). It
competes with microalgae for nutrients, limiting
phytoplankton growth (Smith 1978; Carignan and
Kalff 1980; Blindow et al. 1993) and limits the
activity of benthivorous fish, reducing the resuspen-
sion of sedimented materials (Butcher 1933; Kemp
et al. 1984; Jeppesen 1998). Moreover, it takes part in
the oxygenation of the water column (Rose and
Crumpton 1996), physical stabilisation of the bottom
(Petrini et al. 1996) and active absorption of nutrients
from the bottom and from the water column (Wetzel
1964; Barko and James 1998). For this latter reason,
A. Ciurli Á P. Zuccarini Á A. Alpi (&)
Dipartimento di Biologia delle Piante Agrarie, Sezione
Fisiologia Vegetale, Universita` di Pisa, Via Mariscoglio
34, 56124 Pisa, Italy
e-mail: aalpi@agr.unipi.it
123
Wetlands Ecol Manage
DOI 10.1007/s11273-008-9091-9

2. the authors have proposed the use of harvested
aquatic macrophytes as manure for agriculture (Little
1979).
The replanting of aquatic macrophytes can be a
useful tool for the restoration of lakes that have gone
through the eutrophication processes and switched to
a ‘‘turbid water state.’’ There are several examples of
the success of such a technique in the literature (Brix
and Schierup 1989; Moss 1990; Coops and Doef
1996; Brix 1997), especially when applied in com-
bination with other strategies (Reynolds 1991).
Massaciuccoli Lake, which is shallow (average
depth 2–2.5 m) and covers 700 ha, is located in the
Migliarino San Rossore Massaciuccoli Natural Park,
10 km north of Pisa, along the Tuscan coast in Italy.
A constant and progressive simplification of the
macroalgal and submerged macrophyte community
has been shown over the last 50 years, leading, in
recent years, to a phytoplankton-dominated turbid
water condition. Several causes of this phenomenon
have been identified, all of anthropogenic origin, such
as the excessive turbidity of the water, due to the
suspension of minute particles brought about by soil
erosion (Pensabene et al. 1997) and by the phyto-
plankton-dominated state (Cuppen et al. 1997; Mason
1997); the accumulation of civil and agricultural
pollutants in the lake (Ceccarelli et al. 1997;
Vaithiyanathan and Richardson 1997); the loss of
cohesion of the bottom (Schutten et al. 2005); the
introduction of the crayfish Procambarus clarkii
Girard, which contributed to the disappearance of
aquatic vegetation through grazing (Barbaresi and
Gherardi 2000). The combination of the above-
mentioned phenomena shifted the lake to a turbid
water phytoplankton-dominated state, characterised
by an abnormal increase in the phytoplankton pop-
ulation (including cyanobacteria), impoverishment of
the submerged macrophytic community, reduction in
diversity and the abundance of aquatic invertebrates.
The fish community experienced a reduction in
species diversity, simplification of age classes and
biomass of individual species. The macrophytic
community, in particular, virtually disappeared from
the lake, and the species Elodea canadensis Michaux
and Myriophyllum verticillatum L. were the last to
disappear.
The ultimate goal is the reintroduction of macro-
phytes in the lake, so the intermediate objective is to
understand how such plants could grow in lake water.
The working hypothesis was that the macrophytes
could be grown in mesocosms filled with lake water.
This paper tests this hypothesis and also evaluates
the biomass produced by the two plants, measures the
effects of the growing plants on the water level of
phytoplankton and evaluates the nutrient absorption
capacity of the two species.
The results obtained could be important for the
reintroduction of submerged vegetation in the lake if
the causes of its degradation can be removed.
Materials and methods
Plant material
Elodea canadensis Michaux and Myriophyllum ver-
ticillatum L. were chosen as the model species, being
the most indicated in view of a subsequent forced
reinsertion in the lake since they had been the last to
disappear. They are also characterised by good
rusticity and the ability to colonise (Barrat-Segretain
and Amoros 1996; Strand and Weisner 2001), to the
point that E. canadensis is considered to be a weed in
several European countries (Abernethy et al. 1996).
Samples of both plants were collected in small
streams around Massaciuccoli Lake: E. canadensis
in a drainage channel in Stiava, Viareggio, and
M. verticillatum in a small stream flowing into the
lake itself, in the ‘‘La Piaggetta’’ area.
The apical parts of the plants were cut and
transported in plastic containers filled with water
from the streams, and they were washed with tap
water before being put into the propagation aquaria in
the laboratory.
Preparation of the mesocosms (aquaria)
Two sets of aquaria were prepared, one for plant
propagation and preliminary density trial and one for
the real experiment.
Two separate propagation aquaria were used for
each species, with dimensions of 40 9 100 9 50 cm
and a 200-l capacity, filled with a layer of expanded
clay enriched with oligoelements (AcqualineÒ
) and
an upper layer of fine gravel, which aided the
oxygenation of the bottom. Tap water was used and
the filtration system consisted of a pump with a
submerged filter having a 400-l flow per hour. The
Wetlands Ecol Manage
123

3. lighting plant for each aquarium was composed of
two fluorescent phytostimulating Dennerle lamps,
Trocal 3085, 30 W with reflecting parabola, con-
nected to a timer set to 10 h of light per day. The
temperature was kept at 18–20°C with a 150-W
immersion heater (Visi-Therm Mod VTN 50, Aquar-
ium SystemÓ
). The plant scions did not show
transplant shock and were periodically trimmed to
keep them at a constant length of about 30 cm.
Smaller aquaria (dimensions 25 9 30 9 33 cm
with a 30-l capacity) were used for the preliminary
density test and for the experiments. They were
covered with wire netting in order to avoid the
intrusion of insects, and filled with water collected
from the centre of the lake. Plant scions (10 cm in
length) were fixed with raffia to a wire net placed at
the bottom of each aquarium, in order to force the
plants to absorb nutrients directly from the water
column. The lighting plant for each aquarium was
composed of one fluorescent lamp, like the ones used
for the propagation aquaria, connected to a timer
set to 10 h of light per day. The temperature was kept
at 18–20°C with a 150-W immersion heater
(Visi-Therm Mod VTN 50, Aquarium SystemÓ
).
Experimental scheme
A preliminary test was conducted in order to evaluate the
optimal plant density for growing in the small aquaria:
the growth of 30, 60 and 150 plants per aquarium,
corresponding to 250, 500 and 1,250 plants m-2
,
respectively, was compared and each thesis was
repeated six times.
The main experiment was set up according to the
density test results. Four treatments were compared:
control (water from the lake), lake water with
E. canadensis, lake water with M. verticillatum and
filtered control (lake water filtered with 0.2-l filters).
Each thesis was repeated six times.
Measurements
Height, leaf area and dry weight of all of the plants
were measured at the beginning (T0) and at the end of
the experiment (T4, four weeks later). The leaf area
was measured using a leaf area meter—DT Area
Meter MK2, Delta T-Devices, Burwell, UK; the dry
weight at T0 was estimated for each species as the
average value of 50 scions, 10 cm in length each,
collected from the propagation aquaria and kept in a
forced draft oven at 70°C for a week.
The water content of algal pigments (chlorophyll-a
and pheophytin) was measured at T0 (only in the
unfiltered water) and at T4 (in the filtered water also),
through filtration, extraction in acetone and spectro-
photometry (Lorenzen and Jeffrey 1980).
The ion content in water was analysed at the
A.R.P.A.T. (Regional Agency for Environmental
Protection in Tuscany), in Piombino (Livorno), with
an automatic chemical analyser lMAC-1000 for
NO3
-
, NO2
-
, NH4
+
and PO4
3-
, and a programmable
digester (VELP Scientific) for the total nitrogen and
total phosphorus. The ion content was analysed only
at T0 in the unfiltered water and at T4 in the filtered
water.
Chemical analyses were performed on plant dry
matter at the beginning (50 scion samples, collected
from the propagation aquaria) and at the end
(plants from the experimental aquaria) of the trial.
The total-P (nitro-perchloric digestion and spectro-
photometry: Johnson and Ulrich 1959), total-N
(Kjeldahl method: Stuart 1936) and nitrates (salicylic
acid method: Cataldo et al. 1975) were analysed.
Analysis of bacterial activity
Analysis of the activity of nitrifying bacteria (nitri-
tifying and nitratifying) was performed at the
Division of Agricultural Microbiology at the Faculty
of Agriculture, University of Pisa, Italy. Samples of
water from each of the four kinds of aquaria (control,
M. verticillatum, E. canadensis and filtered control)
were put into 1-ml test tubes containing a specific
mineral substrate for chemoautotrophic microorgan-
isms (Schmidt and Belser 1982) at 11 levels of
dilution (from 1 to 10-10
), and each dilution was
repeated three times. The test tubes were incubated at
28°C for 3 weeks, after which, the presence of
bacteria was investigated by colorimetric way. The
bacterial number was determined through the most
probable number (MPN), using McCrady’s tables
(McCrady 1915; Peeler et al. 1992).
Statistical analysis
The data were analysed using the SAS package: a
paired t-test was performed to check the significance
of the variations in plant growth, water-biochemistry
Wetlands Ecol Manage
123

4. parameters (algal pigments and ion content) and ion
content in plant tissues over time; one-way analysis
of variance (ANOVA) was used to compare the
reductions in algal pigments and ion content in water
carried out by M. verticillatum and E. canadensis
(SAS Institute 1990).
Results
Density test
The preliminary density test indicated that 30 plants/
aquarium was the optimal density for both
M. verticillatum and E. canadensis, allowing the
highest growth in terms of dry weight, height and leaf
area (Fig. 1). All plants showed a reduction in growth
at higher densities, but at the highest density
(150 plants aquarium-1
), M. verticillatum was higher
than at 60 plants/aquarium, despite the lower levels of
leaf area and dry weight. This clearly shows that, at
excessive densities, plants tend to develop in height
without consistent biomass production. For this reason,
the subsequent nutrient-uptake experiment was set up
with the minimum plant density. The leaf area was less
sensitive to the different densities, but the highest
values were still recorded at 30 plants aquarium-1
.
Plant growth
The two species showed different growth patterns
(Fig. 2): M. verticillatum developed more vertically,
reaching the water surface quite rapidly, while
E. canadensis tended to form dense carpets on the
bottom, producing new buds in several directions.
Moreover, the total dry biomass production of
M. verticillatum was pronouncedly higher than that
of E. canadensis: the former more than tripled
(P = 0.001) from the beginning to the end of the
trial, while the latter almost doubled (P = 0.005).
The leaf area of E. canadensis showed the strongest
increase.
Chlorophyll and pheophytin
The chlorophyll values dropped significantly in all of
the aquaria from the beginning to the end of the trial
(Fig. 3). The reduction in the control aquarium (lake
water) was about -14% (P = 0.041), while more
consistent reductions were measured in the presence
of E. canadensis and M. verticillatum (-22.4 and
-39.1%, respectively, corresponding to the P-values
of 0.034 and 0.012). The maximum drop in chloro-
phyll content was obviously in the aquaria filled with
filtered water, where the reduction reached -75%
(P = 0.001), bringing the chlorophyll concentration
below the minimum alert level. The pheophytin
0.000
0.025
0.050
0.075
0.100
0.125
E. canadensis M. verticillatum
DryWeight(g)
0
5
10
15
20
25
Height(cm)
T0 T4 30 pt T4 60 pt T4 150 pt
0
10
20
30
40
Plant Density
LeafArea(cm
2
)
a
b
c
Fig. 1 (a–c) Effects on the growth of Myriophyllum verticill-
atum and Elodea canadensis (a: dry weight; b: height and
c: leaf area) grown in aquaria with artificial sediment (Aqualite
and gravel) of different implant densities; in the abscissa, the
number of plants placed (30, 60 and 150) per aquarium is
reported. T0: beginning of the experiment; T4: end of the
experiment (4 weeks later). The bars represent ±SE of the
means of six replicate aquaria
Wetlands Ecol Manage
123

5. trends closely followed those of chlorophyll, with
five times smaller concentrations on average.
Ion content in plant dry matter
The nitrogen content in plant dry matter increased in
both species during the experiment, in terms of both
the total and nitric nitrogen (Fig. 4). The increase was
proportionally more pronounced in E. canadensis
plants, and regarded total-N (P = 0.023) more than
NO3
-
–N (P = 0.030). The total phosphorus content
underwent a slight but not significant reduction in
both E. canadensis (P = 0.061) and M. verticillatum
(P = 0.054) at the end of the trial. This reduction
(-19.1 and -23.7%, respectively) was, in any case,
proportionally less pronounced than the increases in
the dry weight of the two plants, which—as men-
tioned previously—doubled and tripled, respectively.
0.00
0.05
0.10
0.15
DryWeight(g)
0
10
20
Height(cm)
E T0 E T4 M T0 M T4
0
5
10
15
20
25
30
35
40
LeafArea(cm
2
)
Plant and Time
a
b
c
Fig. 2 (a–c) Growth (a: dry weight; b: height and c: leaf area)
of M. verticillatum and E. canadensis in the experimental
aquaria. E: E. canadensis; M: M. verticillatum; T0: beginning
of the experiment; T4: end of the experiment (4 weeks later).
The bars represent ±SE of the means of six replicate aquaria
T0 Contr.T4 Elo.T4 Myr.T4 0.2µT4
0
5
10
15 Chlorophyll-a
Pheophytine
Treatments
Pigment
Concentration(µg/l)
Fig. 3 Chlorophyll-a and pheophytin content in water from
the aquaria corresponding to the different treatments at the
beginning and at the end of the experimental period. Contr.:
control (unfiltered water from the lake with no macrophytes);
Elo.: E. canadensis; Myr.: M. verticillatum; 0.2 l: filtered
control (water from the lake passed through 0.2-l cellulose
filters); T0: beginning of the experiment; T4: end of the
experiment (4 weeks later). The bars represent ±SE of the
means of six replicate aquaria
E T0 E T4 M T0 M T4
0.00
0.05
0.10
NO3
-
-N N-tot P-tot
0.5
1.5
2.5
Plant and Time
IonContentin
PlantTissues(%d.m.)
Fig. 4 Ion content in submerged macrophytes, expressed as
the percentage of plant dry matter (% d.m.), at the beginning
and at the end of the experiment. E: E. canadensis; M:
M. verticillatum; T0: beginning of the experiment; T4: end of
the experiment (4 weeks later). The bars represent ±SE of the
means of six replicate aquaria
Wetlands Ecol Manage
123

6. Ion content in the water column
The presence of both E. canadensis and M. verticillatum
caused a significant drop in the ion content in the water
columnforalloftheanalysedions(Fig. 5).Inparticular,
the total-P content dropped below the minimum
eutrophication level (0.035 mg l-1
). M. verticillatum
generally showed the most remarkable effects, despite
the fact that the nitrogen content in its tissues increased
less than in E. canadensis and that the total-P content in
dry mass tends to decrease from the beginning to the
end of the trial in both of the species. If we consider
nitrogen, M. verticillatum reduced the nitric form
more significantly (P = 0.013), while in the presence
of E. canadensis, the nitrous form underwent the most
severe drop (P = 0.005). A significant reduction in the
total-P was shown by both plants, but the effect due to
E. canadensis was more evident (P = 0.009) than that
due to M. verticillatum (P = 0.025). All of the analysed
ions, with the exception of NO2
-
–N and total P,
underwent a slight but significant drop in the control
aquaria too, while no significant reduction in ion content
was observed in the aquaria filled with filtered water.
ANOVA tests, performed to compare the efficiency
of the two plants (grouping variable) in lowering
the nutrients in the water column, indicated that
M. verticillatum was the most effective, and this was
particularly evident with regards to NO3
-
(P = 0.003)
and N-tot (P = 0.009).
Analysis of bacterial activity
The analysis of bacterial activity gave negative or
very modest results in all of the aquaria correspond-
ing to the different treatments, as shown in Table 1.
The data indicate that M. verticillatum and
E. canadensis have a strong inhibition effect on
nitritifying and nitratifying bacteria.
Discussion
The experiments gave interesting results in terms of
plant growth and nutrient reduction in the water
column, emphasising how the reintroduction of the
two macrophytes can be a valid biological tool from
the perspective of an integrated approach to the
restoration of an eutrophicated shallow lake. In
particular, the different growth patterns of the two
T0 Contr.T4 Elo.T4 Myr.T4 0.2µT4
0.000
0.025
0.050
0.075
NO2
-
-N
NO3
-
-N
NH4
+
-N
N-tot
PO4
3-
-P
P-tot
0.25
0.75
1.25
1.75
2.25
2.75
Treatments
WaterIonContent(mg/l)
Fig. 5 Ion content in water from the aquaria corresponding to
the different treatments, at the beginning and at the end of the
experimental period. Contr.: control (unfiltered water from the
lake with no macrophytes); Elo.: E. canadensis; Myr.:
M. verticillatum; 0.2 l: filtered control (water from the lake
passed through 0.2-l cellulose filters); T0: beginning of the
experiment; T4: end of the experiment (4 weeks later). The
bars represent ±SE of the means of six replicate aquaria
Table 1 Activity of nitrifying bacteria in water from the
aquaria corresponding to the different treatments at the
beginning and at the end of the experimental period. Control:
unfiltered water from the lake with no macrophytes; 0.2 l:
filtered control (water from the lake passed through 0.2-l
cellulose filters); T0: beginning of the experiment; T4: end of
the experiment (4 weeks later)
Bacterial activity
T0 T4
Nitritifying Nitratifying Nitritifying Nitratifying
Control 0.9 0.45 0.9 7.5
M. verticillatum 0.0 0.11 0.0 0.0
E. canadensis 0.0 0.9 0.0 1.5
0.2 l 0.0 0.0 0.9 0.0
Wetlands Ecol Manage
123

7. plants suggest that they can be reinserted during two
different time periods in nature for a better efficiency.
The tendency of M. verticillatum to reach remarkable
heights—and, consequently, adequate amounts of
light for photosynthesis—in a relatively short time
indicates that it is the ideal plant to be reinserted first,
when the water is still very turbid. The prolonged
growth of M. verticillatum can present problems
connected with mechanical damage due to water
movements (Coops and Doef 1996); therefore, the
subsequent insertion of E. canadensis can be bene-
ficial, as it forms dense carpets on the bottom and is
more resistant to physical disturbances and compe-
tition (Abernethy et al. 1996).
The reduction of chlorophyll-a and pheophytin in
the water indicates competition between plants and
microalgae. In fact, the slight but significant drop of
algal pigments in the control aquaria from the
beginning to the end of the trial suggests that the
nutrient content of the lake water is not sufficient to
support the growth and spreading of phytoplankton,
and that a central role in this mechanism is played in
nature by the long-term nutrient release from the
sediments (Pitt et al. 1997; Søndergaard et al. 2002).
The significant reduction in chlorophyll content
to below 10 lg l-1
(mesotrophy threshold, Ministe-
rial Decree 391/2003) in the aquaria where
M. verticillatum and E. canadensis were present is
proof of this nutritional competition (Schriver et al.
1995; Petr 2000), and the contribution of an allelo-
pathic effect is not to be excluded, as shown by some
authors (Jasser 1995; Ko¨rner and Nicklisch 2002;
Lu¨rling et al. 2006). The most pronounced drop of
algal pigments, recorded in the aquaria filled with
filtered water, is due to the ability of 0.2-l filters to
stop phytoplankton (Nayar and Chou 2003).
The greater increase in total and nitric nitrogen in
E. canadensis is probably due to its inherent low
growth rate and biomass production compared to
M. verticillatum, whereas the latter has the highest
nutrient absorption capacity. Nitric-N content in both
M. verticillatum and E. canadensis becomes signif-
icantly lower at the end of the trial, in contrast with a
generally higher total nitrogen level. This suggests
that higher biomass production leads to the efficient
conversion of nitric nitrogen into its organic form.
The reduction in phosphate content in both
M. verticillatum and E. canadensis is probably due
to the fact that their growth rates were proportionally
higher than the P-absorption rates, leading to the
apparently contradictory result of a drop in the P
concentration in plant tissues. The assimilation of
part of the ions absorbed by the macrophytes by
periphyton and epiphyton communities should not be
excluded either (Howard-Williams and Allanson
1981).
With regards to the nutrient absorption capacity of
the two aquatic plants, M. verticillatum generally
showed more remarkable results than E. canadensis
in reducing the nutrient content in water, particularly
for nitrogen. If we consider that, in our experiment,
no sediment was used as a substrate, this is in
accordance with Best and Mantai (1978), who
observed that the absorption of N by M. verticillatum
occurred at both the root and shoot level, while P was
absorbed principally from the sediment. On the other
hand, the higher ability of E. canadensis to reduce the
P content in water is confirmed by the literature
(Eugelink 1998; Thiebaut 2005). The fact that the
absolute P-absorption was higher in E. canadensis
tissues rather than in M. verticillatum provides
evidence of the tendency of the former to absorb
proportionally more phosphorus and of the latter to
absorb more nitrogen from the water column. The
slight but significant nutrient reduction in control
water, and the simultaneous lack of reduction in 0.2-l
filtered water, suggests the presence of microorgan-
isms that could have contributed to nutrient
absorption, both in the presence and in the absence
of aquatic macrophytes, even though the microbio-
logical analyses show that this effect is not
attributable to nitrifying bacteria. Further experi-
ments are essential in order to explain the nature of
this microbial interference in nutrient absorption,
taking into account the difficulties related to working
in mesocosms because of their limited buffering
power against chemical, physical and biological
perturbations.
Acknowledgement This work was funded by Parco Naturale
di Migliarino San Rossore Massaciuccoli, Pisa, Italy.
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