The effect of ferric chloride addition in
a shallow eutrophicated peaty lake on
submerged macrophytes
Number of EC: 30
Period: February 2010 – April 2011
Name: Kirsten Mels- Vendrig
Student ID: 5653045
MSc in Biologocal Sciences
Study Programme: Limnology & Oceanography
Examiner: Dr. P. Visser
Supervisors: Dr. L. Bakker & drs. A. Immers
Research institute: The Netherlands Institute of Ecology (NIOO-KNAW)
Date: 08 april 2011
The effect of ferric chloride addition in a shallow eutrophicated peaty lake on
submerged macrophytes
Kirsten Mels-Vendrig
Abstract
In shallow aquatic lakes submerged macrophytes play a key role in the functioning of the ecosystem and
have positive effects on water transparency and aquatic biodiversity. The state and ecological functioning of
aquatic systems like shallow lakes is affected by eutrophication, sulphate pollution and global warming.
Many shallow lakes have responded positively to the reduction of the external nutrient load. However,
delayed recovery often has been observed, caused by internal P-loading, mostly P-release from the
nutrient-rich sediments. The main objective of this study is to investigate if addition of iron chloride (FeCl 3), a
measurement for recover the ecological state by binding the released P, to a freshwater peaty system is
harmful for the growth of macrophytes. In this study, we are investigating the effect of addition of FeCl 3 to
the surface water (1) on growth, biomass and nutrient concentrations in plant tissue of freshwater
macrophytes (2) on nutrient and ion concentrations of sediment pore water and surface water and (3) how
macrophytes change these concentrations in sediment pore water and surface water. To test various
potential methods of FeCl3 concentrations, 90 tanks were placed under laboratory conditions e.g. constant
temperature of 18 °C and constant light intensity. We tested two different plant species: Elodea nuttallii (non
root) and Potamogeton pectinatus (root), and a control (no plants). Ironchloride was added slowly into the
surface water, in some tanks half of end doses of ironchloride was already added to the sediment to
outcompete nutrient processes and to be sure that toxicity is playing a role in the experiment. Addition of
ironchloride, in surface water or sediment was not harmful for the growth of E. nuttallii or P. pectinatus and
the effect of iron addition on the growth of macrophytes is species specific. Significant effects of iron addition
on total biomass of E. nuttallii were not found. Differences in biomass of P. pectinatus were found to be
significantly (p<0,05) between no iron addition and high iron addition. The highest total biomass of P.
pectinatus was measured in the tank with no iron addition. Ratio root/shoot, C, N, P in tissue of shoot and C,
N, P in root of E. nuttallii and P. pectinatus didn’t significantly change with increasing iron concentrations.
Both E. nuttallii and P. pectinatus effected nutrient and ion concentrations of surface water and sediment
pore water. Concentrations of ammonium, nitrate and total nitrogen in surface water and pore water were
low, even in control, which can indicate N-limitation. Also concentrations of PO4 in surface water and pore
water were almost zero at the end of the experiment. Ionconcentrations (Al, Ca, SO4, Fe) were effected
because of iron addition. Relatively small amounts of free iron were present in the surface water. In the pore
water the amounts were in some cases factor 10 higher but still small. The added iron binds with PO4 in the
surface and pore water and precipitates. Significant differences between treatments in surface water and
pore water aluminium and calcium were found, however the differences in concentration are so small (< 1
µmol L -1 ) that they hardly have effect on the growth of E. nuttallii or P. pectinatus.
Fe: PO4 ratio in pore water of 1-3.5 mol mol-1, is a threshold for mobilisation (< 1- 3.5) or immobilisation (> 13.5) of PO4. At the end of our experiment the Fe: PO4 ratio in pore water was > 10 mol mol-1 , which means
immobilisation of PO4.
Introduction
Submerged macrophytes play a key role in the functioning of shallow aquatic ecosystems and have positive
effects on water transparency and aquatic biodiversity. The state and ecological functioning of aquatic
systems like shallow lakes is affected by eutrophication, sulphate pollution and global warming. Phosphorus
availability is one of the most important factors for determining the ecological state of a lake. High loading of
phosphorus leads to high phytoplankton biomass, a turbid state of the lake and often changes in the whole
system, including loss of biodiversity and disappearance of submerged macrophytes (Sondergaard et al.,
2003). Over the last decades, many measures have been taken to restore a clear water state with abundant
macrophytes. These measures can be divided in two approaches: the food web approach and the approach
of directly reducing phosphate availability. Food web manipulation, often done by reducing fish stocks, can
force a lake from a turbid state into a clear state (Jeppesen et al., 1997;Scheffer, 1998; Gulati & van Donk,
2002). Reducing planktivorous fish species leads to increasing top-down control of zooplankton on
phytoplankton. Reducing benthivorous species leads to less stirring up detritus laying on the top of the
sediment. However biomanipulation by reducing fish stock in peaty lakes in the Netherlands has rarely been
successful (Van Liere & Gulati, 1992; Meijer et al., 1999; Gulati & Van Donk, 2002). Short water residence
time and external or internal P-loading counteracted the positive effect.
Many shallow lakes have responded positively to the reduction of the external nutrient load (Moss et al.,
2005; Philips et al., 2005; Jeppesen et al., 2005). However, delayed recovery often has been observed
(Jeppesen, et al., 2005), caused by internal P-loading, mostly P-release from the nutrient-rich sediments.
Methods to reduce P-release from sediments include: (1) dredging the phosphorus-enriched upper sediment
layer and direct remove this dredged sediment (Ryding, 1982; Cook et al., 1986; Van der Does et al., 1992;
Verberk et al., 2007; Hickey & Gibbs, 2009); (2) a method of sediment oxidation focussing on decreasing the
P-release from sediments, using a combined surface sediment treatment by calcium nitrate (Ca(NO3))2,
ferric chloride (FeCl3) and lime (CaCO3) (Ripl, 1976); (3) binding with alum, iron or Phoslock (La-modified
bentonite clay) as “active capping agents” (Robb et al., 2003; Hickey & Gibbs, 2009). Active P capping
agents are designed to replace the reversible P-binding with an irreversible binding to the capping agent and
then settle to the lake bed (Cooke et al., 1993; Lewandowski et al., 2003). Recent mesocosm and field
experiments have shown that adding FeCl3 to the sediment can successfully create P-immobilisation by
binding of PO43- to Fe3+ in the sediments (Boers et al., 1994; Smolders et al., 2001; van der Welle et al.,
2006; van der Welle et al., 2007). However, adding FeCl3 can potentially be harmful for the whole lake
system in sulphate rich systems. Under anaerobic circumstances sulphate can be reduced to sulphite (if
sulphate reducing bacteria are present), which can be highly toxic to plants (Koch & Mendelssohn, 1989;
Koch, Mendelssohn & McKee, 1990; Armstrong, Armstrong & Van der Putten, 1996; Smolders & Roelofs,
1996; Van der Welle et al., 2006) and Fe3+ reduction (to Fe2+ ) can lead to sulphide production and
mobilisation of Fe-bound PO4 (fig 1; Smolders & Roelofs, 1993; Roden & Edmonds, 1997; Wang and
Chapman, 1999; Wetzel, 2001; Lamers et al., 2002a), which can cause serious eutrophication rather than
PO4 immobilisation (Brouwer et al., 1999; Lucassen et al., 2004a; Zak et al., 2006; van der Welle et al.,
2007). Moreover, the addition of FeCl3 to the sediment may be possible in mesocosms, but is a challenge
for a whole lake. Adding FeCl3 to the surface water may be more feasible in case of restoration of a whole
lake. However, the effects of adding FeCl3 to the surface water on various organisms in the ecosystem of a
peaty lake, are not yet known. In this study we investigate the effects of iron addition to the surface water on
macrophytes growing in sediment of peaty lake Terra Nova.
Lake Terra Nova, a peat lake of 85 ha and part of the Dutch lakes “ Loosdrechtse plassen”, is dominated by
algae and (toxic) cyanobacteria. Biomanipulation of the lake in winter of 2003/2004 (benthic fish taken out,
repeated every year after winter of 2003/2004 for 5 years), resulted in a clear lake with aquatic macrophytes
for several years (Ter Heerdt & Hootsmans, 2007; Van de Haterd & Ter Heerdt, 2007) but has not resulted
in a regime shift, as nutrient and algal concentrations in the lake are still very high. The input of external
phosphorus in Lake Terra Nova is declining, but internal phosphorus loading from the sediment resulted
directly and indirectly in high levels of phosphate in the water column (Table 1, Vendrig in prep). Addition of
FeCl3 to the surface water to reduce internal P-loading is being tried for this lake.
Table 1 Nutrient, Chlorophyll-a and SO4 surface water data and fish stock data of lake Terra Nova.
Nutrient and SO4 data are taken from 5 years before (1999-2003) biomanipulation and 5 years after
(2004-2008) biomanipulation. Nutrient and SO4 data are given as summer average over 5 years and
standard error. pH data is given as year average over 5 years with standard error. Fish stock (all) is
measured in 2003 (before) and 2004 (after) (Ter Heerdt & Hootsmans, 2007).
TP (µg L-1)
PO4 (µg L-1)
TN (mg L-1)
NH4 (mg L-1)
CHLFa (µg L-1)
SO4 (mg L-1)
pH
Fish fresh weight (kg ha-1)
Before
0.07 ± 0.005
0.013 ± 0.002
1.35 ± 0.71
0.03 ± 0.006
60.1 ± 5.42
7.19 ± 0.44
8,17 ± 0,048
244
After
0.08 ± 0.005
0.026 ± 0.004
1.96 ± 0.85
0.07 ± 0.016
60.5 ± 9.45
11.9 ± 0.86
8,22 ± 0,046
48
The main objective of this study is to investigate if addition of iron chloride (FeCl 3) to a freshwater peaty
system is harmful for the growth of macrophytes. In this study, we are investigating the effect of addition of
FeCl3 to the surface water (1) on growth, biomass and nutrient concentrations in plant tissue of freshwater
macrophytes (2) on nutrient and ion concentrations of sediment pore water and surface water and (3) how
macrophytes change these concentrations in sediment pore water and surface water.
We hypothesize that macrophyte growth will be reduced when iron chloride is added. Because of toxicity of
iron we expect that nutrient concentrations in the macrophytes tissue decreases with increasing iron
addition, because of the limitation outside the macrophyte. Accumulation of iron chloride in the sediment will
enhance these responses. When iron chloride is added in high doses we expect to find iron hydroxide on
the root surface and outside the roots and a reduction of macrophyte growth due to iron-toxicity.
We expect that macrophytes may alter the sediment-water nutrient interactions mainly stimulating the
processes of nitrification of NH4 and keeping the sediment aerobic which acts as a barrier for phosphate
input from the sediment to the water layer (Andersen & Olsen, 1994; Christensen & Wigand, 1998). Also,
macrophytes may function as a nutrient pump and transfer nutrients from the sediment to the water column
through the plant.
Macrophyte response to iron chloride the effect of macrophytes on sediment-water interactions may depend
on whether plants are rooted in the sediment or mainly free floating. In this experiment we therefore used
the rooting plant species Potamogeton pectinatus L. and the facultative rooting plant species Elodea nuttallii
(Planch.) St. John and a control without plants. We added three levels of FeCl3 into the tanks. To simulate
situations where the addition of FeCl3 to the water column has resulted in an accumulation of FeCl3 in the
sediment, we added a treatment in which we already added half of the end doses of FeCl3 in the sediment
at the start of the experiment and the rest in the water and compared this with the addition of FeCl 3 only to
the water layer.
Material and methods
Experimental set-up
To test various potential methods of FeCl3 concentrations 90 polyethylene tanks (w x d x h = 0,19 x 0,19 x
0,29 m) were placed under laboratory conditions e.g. constant temperature of 18 °C and constant light
intensity (mean 102.16 µE) with a day-night rhythm. Each tank was filled with upper layer Terra Nova
sediment (N52º 12’ 58.41” E5º 2’ 18.52”) of 2 L (around 5 cm), which was kept for minimum of 2 days in
freezer to eliminate most bottom dwelling animal and plant species. Before the tanks were filled up with
water, first half of end doses of FeCl3 was added to the sediment in the tanks were we wanted to test the
effect of adding FeCl3 to the sediment (form of a quick adding) and half of end doses NaCl was added to the
sediment of the control. All tanks finally where filled up with filtrated (Whatman SF92 Glass fibre prefilter and
ME 24 membrane filter mixed cellulose 0,2 µm) Terra Nova surface water in total 7,3 litre per tank whithout
disturbing the sediment. In every tank ironchloride was added to reach the effect of P-limitation in every
tank. We tested two different plant species: Elodea nutallii (non root) and Potamogeton pectinatus (root),
and a control (no plants). We wanted to test 6 different treatments (see table 2). All macrophytes of one
species were collected on approximately same length and visual conditions. In each tank (five tanks per
treatment) 3 cutted species of E. nuttallii and 3 tubers of P. pectinatus were placed 1 cm in the sediment.
Ceramic Soil moisture samplers ((rhizons) (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands)
were placed in each tank and the sampler was installed in the upper layer (2-3 cm) of the sediment.
Because of evaporation once a week the tanks most be re-filled with approximately 2 litre filtered (Whatman
SF92 Glass fibre prefilter and ME 24 membrane filter mixed cellulose 0,2 µm) Terra Nova water. Table 2
gives an overview of the added concentrations of Fe (from FeCl3*H2O) and Cl (from NaCl) for control in this
experiment (treatments). The final doses in each tank were 51.6 mg Fe (high) and 25.8 mg Fe (low). The
NaCl for control was added because we wanted to exclude the effect of chloride.
Table 2 Added total concentration (mg) per tank and total final concentration added per tank (in total 36
adding days). Mix means added to the sediment before. Low means 25,8 mg added, high means 51,6
mg added. NaCl is added for control. Every treatment has 15 tanks and there are 2 species and a control
we wanted to test, which have 5 replicates.
Added to
Sediment
Surface water
NaCl
Fe low
25.28
NaCl mix
454.99
12.64
Total final concentration
Fe high
0.72
Fe low mix
12.9
0.36
1.44
Fe high mix
25.8
0.72
353.88
631.93
25.8
25.8
51.6
51.6
Every treatment on every species (E. nuttallii, P. pectinatus, control) has five replicates which were
randomly distributed in blocks. One block had eighteen tanks and in total five blocks were placed because
there were five replicates. Every Monday, Wednesday and Friday NaCl en FeCl3*H2O was added in small
doses for 36 times over 2,5 months. pH and conductivity were measured, 3 hours after FeCl3 was added,
with a WTW pH323 meter calibrated against WTW technical buffer solutions pH 4.01 and 7.00 (accuracy
0.01 ± 1 digit). The experiment started on 29th of January 2010 and finished at 22th of April 2010.
Macrophytes that sprouted from the sediment propagule bank during the experiment were counted and
removed and the species was determined.
Sampling
Every two weeks, six times in total (To on 29 January 2010, T1 on 15 February 2010; T6 on 22 April 2010),
surface water and pore water samples were taken from each tank. Surface water samples were taken in 50
ml centrifuge tubes, than samples were split up. For analyzing ions 50 ml sample was filtrated over a 0,45
um membrane filter and filtrate was stored in polyethylene bottles with 1 ml nitric acid (HNO3) 2 mol/l. For
analyzing chloride 20 ml was filtrated over pre-weighted 45 um membrane filters and filtrate was stored in
polyethylene bottles. For analyzing nutrients 10 ml sample was filtered over glass microfibre GF/C
(Whatman) filters and filtrate was stored in 15 ml centrifuge tubes. For analyzing precipitated Fe 10 ml of
sample was filtered over glass microfibre GF/C (Whatman) filters. The wet glass microfibre filters were
collected and placed for 3 days in the stove by 60 °C. Filters were again weighted when dry, then placed in
50 ml centrifuge tubes. All filtrates and membrane filters were stored in the dark at – 18 °C until analyses
could be done. Samples taken for measuring pH and alkalinity were put in 50 ml centrifuge tubes and
measured immediately after sampling by titration of 25 ml of water with 0,01 M HCl down to pH 4,2 with
Radiometer analytical TIM840 titration manager.
Sediment pore water was collected anaerobically using 50 ml vacuum syringes connected to ceramic soil
moisture samplers (rhizons) (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands). Because
sampling with the vacuum syringes is a slow process samples were taken in two days and overnight. Taken
samples were stored in polyethylene bottles (ions and chloride) and centrifuge tubes (15 ml for nutrients and
50 ml for filters) in the dark at -18 C until analyses could be done. Table 3 gives the amount of sample
needed for the divers analysis. Almost every sample time (sample time 2 & 3 were not included) the amount
of plant mass in each tank was estimated (in %) and written down. Also the amount of zooplankton was
estimated in 4 classes (1-5 species, 5-20 species, 20 – 40 species or > 40 specimen) and we tried to
estimate the amount of filamentous algae (in %).
Table 3 Amount of sample needed for each analysis and total amount of sample needed. Both pore water
and surface water needed the same volume of sample for analyzing.
Analysis
pH, Alkalinity
Ions (ICP)
Cl (Spectrofotometric)
Nutrients (auto analyzer)
Total ml
Pore water/surface water
25 ml
50 ml
20 ml
10 ml
110 ml
Chemical analysis
NO2, NO3, NH4 and PO4 were determined colour metrically using Quaatro auto-analyzer (Seal analytical)
according to Quaatro method 541502714100 (NO2/NO3), 541502714000 (NH4) and Q-031-04 Rev.1
(Murphy & Riley, 1962) (PO4). Dissolved Fe, dissolved Al, Ca and SO4 were measured with a Inductively
Coupled Plasma –spectrometer (Varian, Liberty II, Varian) with Atomic Emission Spectroscopy (ICP-AES)
according to the Dutch NEN-EN-ISO 17294. Chloride was measured spectrofotometrically (Aquakem 250,
Thermo Scientific), using mercury(II)thiocyanate to form mercury (II)chloride, then using iron(II)nitrate to
form a iron(III)thiocyanate- complex, which was measured by extinction at 480 nm.
Before measuring precipitated Fe, the residu on the glass microfibre filter was solved in 8 ml concentrated
nitric acid and 42 ml demi-water and heated, then analyzed on ICP-AES as described above.
Ratio C:N in plant tissue was measured by combustion technique on EA elemental analyzer euro EA3000.
Before measuring, plant tissue of P. pectinatus and E. nuttallii was weighed in pressed tin capsules (total
amount between 0,8 and 1,1 mg) and incinerated by 500 °C. Before P in plant tissue was measured on
autoanalyzer described above, plant material was weighed in pressed silver capsules (total amount between
0,8 and 1,15 mg) and incinerated by 500 °C. The incinerated plant material was digested in H2O2 (2,5 %)
(Murphy & Riley, 1962).
Plant measurements
For measuring growth of plants we used plants biomass. When E. nuttallii shoots and P. pectinatus young
sprout were placed in the tanks at time 0, we also measured the fresh and dry weight of 10 extra shoots of
E. nuttallii and 10 extra young sprout of P. pectinatus. With mean fresh: dry weight ratio of the 10 shoots for
E. nuttallii or young sprout for P. pectinatus we could calculate the dry weight of all shoots or young sprout
with were placed in the tanks. At the end of the experiment we took all plants from the tanks and also
weighted the fresh and dry weight of the plants. We used fresh:dry weight ratio for determining growth of
both macrophytes.
Statistical analysis
Statistical analysis were carried out with SPSS version PASW Statistics 18 and Statistica for Windows
version 9 (Kruskal-Wallis test and subsequent post hoc tests). All data were tested for normal distribution
with a Kolmogorov-Smirnov test and homogeneity of variance with a Levene’s test. Data were log(x+1)
transformed to obtain homogeneity of variances and a normal distribution. We tested the effect of iron
addition on macrophyte growth with a two-way analysis of variance (ANOVA) and subsequent Tukey’s posthoc test, with level of iron addition and place of addition as fixed factors. When data were not normally
distributed or there was no homogeneity of variances even after transformation we used a Kruskal-Wallis
test and subsequent post hoc tests with level of iron addition and place of addition as fixed factors. Two
variables that were measured (PO4 and Fe) were taken of time T5 instead of time T6 because on T6 most
data was measured below detection limit and therefore almost always zero. For calculating ratio’s these
values can not be used. Correlations between variables were analysed using Pearson’s correlation
coefficients (r).
Results
Visible effects
Typical symptoms that indicate iron toxicity, like brown necrotic spots on the leaves (Laan et. al., 1991) were
not observed on all E. nuttallii and P. pectinatus macrophytes. Both, E. nuttallii and P. pectinatus have
grown in shoot as well as in root size in every treatment. Filamentous algae came up after 2 weeks, no
blooms of phytoplankton or cyanobacteria were seen in the whole experiment (figure 1). After 2 weeks also
few macrophytes came up from propagules in the sediment. These were namely Chara virgata, Chara
globularis, Nitella mucronata, P. pectinatus and some seedling of Nuphar lutea or Nymphaea alba.
Zooplankton was also growing and throughout the experiment, different generations of Daphnia and
Copepods were developing.
(a)
(b)
(c)
(d)
Figure 1. Photos of different treatments at T=5. Situation of P. pectinatus with no addition of iron
(control)in one of the five tanks (a), Situation of E. nuttallii with high addition of iron and mixed
sediment in one of the five tanks (b), Situation of P. pectinatus with high iron addition in one of the five
tanks, on photo the precipitation of ironphosphate is visible (c) and the situation of E. nuttallii with high
filamentous algae amount (d).
Changes in biomass
Significant effects of iron addition on total biomass (figure 2a) and ratio shoot:root of E. nuttallii were not
found. Differences in tissue C, N or P and root C, N or P in E. nuttallii were also not significant between
treatments. Differences in biomass of P. pectinatus were found to be significantly (p<0,05) between no iron
addition and high iron addition. The highest total biomass of P. pectinatus was measured in the tank with no
iron addition (figure 2b). Also differences in tissue C, N or P and root C, N or P in P. pectinatus were not
significant different between treatments. Root/shoot ratio of both E. nuttallii and P. pectinatus does not
significantly change with increasing iron concentrations (figure 2d).
Addition responses of physical parameters are presented in appendix III.
2,5
1,4
biomass increase (g)
biomass increase (g)
1,6
1,2
1,0
0,8
0,6
0,4
0,2
0,0
25
(a)
b
1,5
1,0
0,5
50
0
root/shoot ratio (g g -1 d.w.)
2,0
1,5
1,0
0,5
0,0
0
0 mix
25
25
mix
50
50
mix
0
E. nutallii
0 mix
25
25
mix
50
50
mix
50
iron addition
2,5
2,0
1,5
1,0
0,5
0,0
0
P . pectinatus
treatment
25
(b)
iron addition
2,5
biomass increase (g)
ab
2,0
0,0
0
(c)
a
0 mix
25
25
mix
50
50
mix
0
E. nutallii
(d)
0 mix
25
25
mix
50
P . pectinatus
treatment
Figure 2 Biomass increase of E. nuttallii (a), P. pectinatus (b) biomass increase in all treatments (c) and
root/shoot ratio (d). Biomass and root/shoot ratio is given in dry weight in g or g g-1 (d.w.). Dry weight
is measured as dry weight end – dry weight begin. SEM is given in the figure. Significant differences
between treatments, as tested by Tukey (P<0,05) are indicated by different letters.
Changes in nutrient concentrations
pH response during iron addition
Terra Nova water has a pH of around 8.2 (see table 1) and can reach pH of 9.5 in summer. During addition
of iron the response of pH in time, is increasing at T1 and T2 and after T3/T4 decreasing. pH reaches never
<6.5 in every treatment. This is important because binding of phosphate to iron depends on pH and is
stronger when pH is above 6.5. Therefore, neutralize the pH in this experiment wasn’t necessary.
In surface water, only the pH in treatment with E. nuttallii and high iron addition reaches a pH < 7 at T5 and
T6. In pore water pH is always lower then the pH in surface water. Only in treatment with macrophytes pH
can reach <7 (figure 3), after T=4.
50
mix
pH
pH
(a)
(b)
Figure 3. Boxplots of pH during the whole experiment per treatment in surface water and pore water. (a)
without mixed sediment, (b) mixed sediment. C=control, E=Elodea, P=Potamogeton, 25=low iron
addition, 50=high iron addition.
Response of nutrients and ions during iron addition in surface water
The results of chemical measurements in surface water in tanks with E. nuttallii showed that Fe (s), SO4
(aq) and Al (aq) were found to be significantly (p<0.05) correlated with Fe additions. For Fe (aq), Ca (aq)
and Cl (aq) the effect of addition was significant (p<0.05) but dependent on the place were the iron was
added. For all other parameters measured, no differences between the treatments were found.
The results of chemical measurements in surface water in tanks with P. pectinatus showed that Al (aq), Ca
and ratio Fe:PO4 (T=5) were found to be significant (p<0.05) correlated with Fe additions. For Fe (aq), Fe
(s) and Cl the effect of addition was significant (p<0.05) but dependent on the place were the iron was
added. For all other parameters measured, no differences between the treatments were found. The addition
responses of biochemistry in surface water are presented in appendix I.
surface water PO4 (µmol Lˉ¹)
E--
0,16
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
E-M
E 25 E 25 M
E 50 E 50 M
T0
T1
T3
T5
T6
time
surface water TON (µmol Lˉ¹)
(a)
70
60
60
P 25 -
50
P 25 M
40
P 50 M
----M
P -M
- 25 - 25 M
50
P 50 -
- 50 - 50 M
40
30
30
20
20
10
10
0
0
T0
(b)
70
P --
T1
T2
T3
treatment
T5
T6
T0
(c)
T1
T2
T3
T5
T6
treatment
Figure 4 (a,b,c). Effects of various treatments on the surface water biogeochemistry in tanks with E.
nuttallii, P. pectinatus and control. Phosphate concentrations over time in all tanks with E. nuttallii as
example (a), total N concentrations over time in tanks with P. pectinatus as example (b), total N
concentrations over time in control tanks (c). The following treatments were applied in (a,b,c): E = E.
nuttallii, P = P. pectinatus, - = control, - - control, – M control with mixed sediment, 25 – 25 mg iron
addition, 25 M 25 mg iron addition and mixed sediment, 50 – 50 mg iron addition, 50 M 50 mg iron
addition and mixed sediment. In all figures SEM is given.
In time PO4 concentrations in all tanks are decreasing and after T=5 (70 days) concentrations of PO4 are
almost zero (figure 4a). In time the concentrations of TON also decreased in all tanks and from T=3 (44
days) in tanks with E. nuttallii and P. pectinatus concentrations are almost zero (figure 4b), in control tanks
concentrations of TON are low but never reach zero (figure 4c).
Addition of iron in tanks with E. nuttallii resulted in higher dissolved iron concentrations in tanks with iron
addition and mixed sediment than in tanks without addition (figure 5a), except in tanks with high iron addition
and no mixing. Addition of iron in tanks with E. nuttallii resulted in higher precipitated iron concentrations in
tanks with iron addition than in tanks without addition (figure 5c). Iron concentrations are always higher in
tanks with addition over time. Addition of iron in tanks with P. pectinatus resulted in significant higher
dissolved Fe concentrations in tanks with low iron addition and mixed sediment than in tanks with addition of
high iron (figure 5 b). In tanks with P. pectinatus and with high iron addition, without mixed sediment, the
concentration of precipitated iron is significant higher than the concentration of precipitated iron in tanks with
no addition of iron (control) (figure 6d).
1,4
b
not mixed
mixed
bc
1,2
abc
1,0
0,8
0,6
0,4
abc
ab
0,2
1,6
surface water dissolved
Fe (µmol Lˉ¹)
surface water dissolved
Fe (µmol Lˉ¹)
1,6
a
1,4
not mixed
mixed
1,2
a
1,0
0,8
0,6
0,4
ab
ab
0
(a)
25
50
0
(b)
not mixed
mixed
b
b
b
a
b
a
0
25
iron addition
50
surface water precipitated
Fe (µmol Lˉ¹)
surface water precipitated
Fe (µmol Lˉ¹)
b
25
50
iron addition
iron addition
(c)
b
0,2
0,0
0,0
0,26
0,24
0,22
0,20
0,18
0,16
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
ab
(d)
0,26
0,24
0,22
0,20
0,18
0,16
0,14
0,12
0,10
0,08
0,06
0,04
0,02
0,00
ab
not mixed
mixed
b
ab
a
ab
a
0
25
50
iron addition
Figure 5 (a,b,c,d). Effects of various treatments on the surface water biogeochemistry in tanks with E.
nuttallii and P. pectinatus. Dissolved iron concentrations in tanks with E. nuttallii at T=6 (a), dissolved
iron concentrations in tanks with P. pectinatus at T=6 (b), precipitated iron concentrations in tanks with
E. nuttallii at T=6 (c) and precipitated iron concentrations in tanks with P. pectinatus at T=6 (d). In
(a,b,c,d) iron addition is given belong the X-axis (o = control, 25 = 25 mg iron addition, 50 = 50 mg iron
addition). In all figures SEM is given. Significant differences between treatments, as tested by KruskalWallis (P<0,05) are indicated by different letters in (a,c). Significant differences between treatments, as
tested by Tukey (c) or Kruskal-Wallis (d) (P<0,05) are indicated by different letters in (b,d).
Addition of iron in tanks with E. nuttallii resulted in significant higher SO4 concentrations in the tanks with
high iron addition and lower SO4 concentrations in tanks without iron addition (figure 6a). In time the
concentrations of SO4 in tanks with E. nuttallii, P. pectinatus and in control tanks increased in all treatments
(figure 6b). Addition of iron in tanks with E. nuttallii and in tanks with P. pectinatus resulted in significant
lower dissolved Al concentrations in tanks with high iron addition (figure 6c,d respectively). In time
aluminium is increasing and from T=4 decreasing in all tanks and all treatments.
Addition of iron in tanks with E. nuttallii resulted in higher Ca in tanks with high iron addition and mixed
sediment than in the treatment with no addition of iron (figure 6c). Concentration of Ca in tanks with P.
pectinatus, with high iron addition, are significant higher than Ca concentrations in tanks with low or no iron
addition (control) (figure 7d).
0,8
0,7
not mixed
mixed
a
ab
ab
0,9
b
surface water SO4 (mmol Lˉ¹)
surface water SO4 (mmol Lˉ¹)
0,9
b
a
0,6
0,5
0,4
0,3
0,2
0,1
0
25
(a)
surface water Ca (mmol Lˉ¹)
0,2
0,1
T1
T2
T3
surface water Al (µmol Lˉ¹)
a
a
b
b
T4
T5
T6
time
a
1,6
not mixed
mixed
a
1,4
a
ac
1,2
1,0
bc
0,8
b
0,6
0,4
0,2
0,0
25
50
0
b
ab
1,6
ab
50
iron addition
2,0
not mixed
mixed
a
25
(d)
a
surface water Ca (mmol Lˉ¹)
surface water Al (µmol Lˉ¹)
a
a
ab
0,8
0,6
0,4
0,2
1,8
1,6
a
a
1,4
a
a
b
not mixed
mixed
b
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0,0
0
(e)
0,3
(b)
iron addition
1,2
1,0
0,4
1,8
(c)
1,4
0,5
T0
not mixed
mixed
0
1,8
0,6
50
iron addition
2,0
0,7
--M
25 25 M
50 50 M
0,0
0,0
2,4
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0,8
25
iron addition
0
50
(f)
25
50
iron addition
Figure 6 (a,b,c,d,e,f). Effects of various treatments in surface water biogeochemistry in tanks with E.
nuttallii and P. pectinatus. Sulphate concentrations in tanks with E. nuttallii at T=6 (a), sulphate
concentrations in all tanks over time (b), dissolved aluminium concentrations in tanks with E. nuttallii at
T=6 (c), dissolved aluminium concentrations in tanks with P. pectinatus at T=6 (d) calcium
concentrations in tanks with E. nuttallii at T=6 (e) and calcium concentrations in tanks with P.
pectinatus at T=6 (f). In (a,c,d,e,f) iron addition is given belong the X-axis (o = control, 25 = 25 mg iron
addition, 50 = 50 mg iron addition). The following treatments were applied in (b): E - - Elodea control, E
– M Elodea control with mixed sediment, E 25 – Elodea with 25 mg iron addition, E 25 M Elodea with 25
mg iron addition and mixed sediment, E 50 – Elodea with 50 mg iron addition, E 50 M Elodea with 50 mg
iron addition and mixed sediment. SEM is given in all figures. Significant differences between treatments,
as tested by Kruskal-Wallis (a,c,d,f) or Tukey (e) (P<0,05) are indicated by different letters (a,c,d,e,f).
Response of nutrients and ions during iron addition in pore water
The results of chemical measurements in pore water in tanks with E. nuttallii showed that Al (aq), Ca (aq)
and Cl (aq) were found to be significantly (p<0,05) correlated with Fe additions. The results of chemical
measurements in pore water in tanks with P. pectinatus showed that Al and Ca were found to be
significantly (p<0,05) correlated with Fe additions. For Cl (aq) the effect of addition was significant (p<0.05)
but dependent on the place were the iron was added. The addition responses of biochemistry in pore water
are presented in appendix II.
TON and PO4 are decreasing in time to almost zero at the end of the experiment in all tanks and
treatments. The pore water dissolved iron concentrations are between 6 – 10 times higher than dissolved
iron concentrations in surface water. Like in surface water also in pore water the SO4 concentrations are
increasing in time of all treatments with identical concentrations (at the end 70 mg/l).
Significant lower Al concentrations in pore water are present in all tanks (E. nuttallii, P. pectinatus and
control) with addition of iron (figure 7a). Like in surface water, concentrations of aluminium are increasing in
time and from T=4 decreasing in all tanks (figure 7b). Concentrations of aluminium in pore water are always
lower in all treatments than the concentrations in surface water.
Addition of iron in tanks with E. nuttallii resulted in significant higher Ca concentrations in tanks with high
iron addition than in tanks without addition or with low addition. In tanks with P. pectinatus significant higher
Ca concentrations are present in tanks with high iron addition than in tanks without addition.
pore water Al (µmol Lˉ¹)
0,9
0,8
2,2
2
not mixed
mixed
a
a
0,7
0,6
0,5
0,4
b
b
0,3
b
b
0,2
pore water Al (µmol Lˉ¹)
1
0,1
1,2
1
0,8
0,6
0,4
0,2
0
0
0
(a)
1,8
1,6
1,4
E- E- M
E 25 E 25 M
E 50 E 50 M
25
iron addition
50
T0
(b)
T1
T2
T3
T4
T5
T6
time
Figure 7 (a,b). Effects of various treatments in pore water biogeochemistry in tanks with E. nuttallii, P.
pectinatus or control. Dissolved aluminium concentrations at T=6 in all tanks (a) and dissolved
aluminium concentrations over time in all tanks (b). In (a) iron addition is given belong the X-axis (o =
control, 25 = 25 mg iron addition, 50 = 50 mg iron addition). The following treatments were applied in
(b): - - control, – M control with mixed sediment, 25 – 25 mg iron addition, 25 M 25 mg iron addition
and mixed sediment, 50 – 50 mg iron addition, 50 M 50 mg iron addition and mixed sediment. SEM is
given in all figures. Significant differences between treatments, as tested by Tukey (P<0,05) are
indicated by different letters (a).
Pore water ratio Fe:PO4 in tanks with iron treatments and with E. nuttallii or P. pectinatus is high at T=6. At
T=5 in all tanks the ratio was < 1 (figure 8).
E. nuttallii
P. pectinatus
level 1
1,2
1
0,8
0,6
0,4
0,2
0
control
140
pore water Fe:PO4 (mol mol-1)
pore water Fe:PO4 (mol mol-1)
control
E. nuttallii
P. pectinatus
120
level 1
level 3.5
100
80
60
40
20
0
0
0 mix
25
25 mix
treatment
50
50 mix
0
0 mix
25
25 mix
50
50 mix
treatment
Figure 8. Fe:PO4 ratio (mol mol-1) in pore water in control tanks and tanks with E. nuttallii and P.
pectinatus at T=5 (a) and T=6 (b). Iron addition is given belong the X-axis. The following treatments
were applied: o = control, 0 mix = control and mixed sediment, 25 = 25 mg iron addition, 25 mix = 25
mg iron addition and mixed sediment, 50 = 50 mg iron addition and 50 mix = 50 mg iron addition and
mixed sediment). SEM is given in all figures.
Discussion
Effects of iron addition on growth, biomass and nutrient concentrations in plant tissue of freshwater
macrophytes were different between E. nuttallii and P. pectinatus.
There was no effect on biomass or nutrient concentrations in plant tissue of E. nuttallii between treatments.
In Xing et al. (article in press, 2010) results showed that growth of E. nuttallii was concentration dependent.
Plants were put in containers and 0.1, 1, 10, 100, 500 and 1000 mg L-1 ironchloride was added at once.
Plants were exposed for 36 hours. Growth of E. nuttallii was promoted by low iron concentration (1-10 mg
L-1 [Fe3+]), but growth inhibition was observed when iron concentration was beyond 10 mg L -1. In our
experiment iron was added slowly in small concentrations, spread over 12 weeks, therefore E. nuttallii did
grow in the first weeks, but at the last weeks of the experiment the growth of E. nuttallii seems to stop
(visual). The biomass increase therefore is less (figure 2c) than the biomass increase of P. pectinatus. This
can indicate that when E. nuttalli is exposed longer than 36 hours to iron concentrations below 10 mg L-1
this concentration can inhibit growth of Elodea instead of growth. Van der Welle et al., (2007) found out that
growth of E. nuttallii was stimulated by iron or iron plus sulphate addition. They put enclosures into a peat
ditch and put 50 g Fe m -2 once a year. In their experiments Elodea did not grow with addition of only
sulphate due to sulphide toxicity or sulphide-induced iron defiency. Elodea formed only roots when iron was
added. E. nuttallii in our experiment also formed roots, like in the case of Van der Welle et al. (2007), these
roots however, are never formed in the sediment, but were formed on the stem of E. nuttallii in the surface.
We can therefore conclude that E. nuttallii forms roots by addition of iron, even if addition is in small
amounts.
There are no effects in nutrient concentrations in tissue of E. nuttallii between treatments. E. nuttallii tissue
C:N:P was between C: 33 – 36 %, N: 0.6 – 0.8 % and P: 0.8 – 0.9% (all % of d.w) in our study when
finishing the experiment. Gerloff and Krombholz (1966) found in their experiments a critical P in tissue of
0.13 % ( of d.w.) and a critical N in tissue of 1.3 % (d.w.). They found in Elodea spec. tissue of Wisconsin
lakes between 3.02 and 1.78 % N, and between 0.33 and 0.12 % P (% in d.w.). In our experiment much
lower N en more P was found in the tissue. The lower, in tissue found, N can indicate N- limitation in our
experiment. The higher, in tissue found, P can indicate high P availability in sediment and maybe storage of
P in macrophytes.
In the case of P. pectinatus biomass was affected by addition of iron. P. pectinatus did grow in all tanks but
biomass in tanks with high iron addition was lower than biomass in tanks without iron addition. Differences in
total biomass between no addition and addition of iron however, were small. P. pectinatus formed long roots
in the sediment in all treatments.
There are no effects in nutrient concentrations in tissue of P. pectinatus between treatments. P. pectinatus
C:N:P was between C: 37 – 39 %, N: 0.7 – 0.9 % and P: 1 – 2 (all % of d.w.) in our study when finishing the
experiment. Van Wijck et al. (1992) found C, N and P content in P. pectinatus tissue between 33.27 SD 4.36
and 35.4 SD 1.74, between 2.07 SD 0.32 and 1.47 SD 0.25 and between 0.25 SD 0.01 and 0.21 SD 0.02
respectively. C, N and P content in P. pectinatus root was 35.4 SD 1.74, 1.47 SD 0,25 and 0.13 SD 0.01
respectively (all % of d.w.). Also in P. pectinatus the found N in tissue was much lower and the found P was
higher than in Van Wijck et al. (1992). We can conclude that there is possible N- limitation for both plants in
our experiment.
Batty and Younger (2003) found a significant effect of external Fe concentration on root phosphate
concentrations in Phragmites australis. Phosphate concentrations of roots were lower when exposed to less
than 1 mg L-1 Fe. Phosphate concentration of roots was consistently higher than those of shoots when
exposed to 2 mg L-1 or higher. Batty and Younger (2003) also found that there was no significant difference
in phosphate content of shoots between treatments with addition of >1 mg L-1 and 20 mg L-1. In our case no
significant effects are present in N and P in shoot or root between different treatments. Apparently addition
of iron doesn’t effect nutrient concentrations in root or shoot of submerged macrophytes.
Root:shoot ratio of both E. nuttallii and P. pectinatus did not change in our experiment with increasing iron
addition. These findings correspond with Snowden & Wheeler (1993), who found out that for most
monocotyledons shoot:root dry weight ratio was little affected by iron concentration.
Macrophytes do effect nutrient and ion concentrations of surface water and sediment pore water. In our
case nutrients (TON, PO4) and some ion concentrations (Al, Ca, SO4, Fe) are changing in time or been
different between treatments.
Concentrations of NO3, total N and NH4 in all tanks were low almos zero), even in control. Geurts et al. (20
found in his enclosure experiments, in a peaty lake when added 50 or 100 g Fe m -2, an increase of NH4 up
to 250 µmol L-1, but no changes between different additions. Aeration enhances nitrification, and thus nitrate
availability. This process is important for macrophyes because macrophytes use nitrate as their nitrogen
sources (Schuurkes, Kok & Den Hartog, 1986). All tanks in our experiment had aerobe circumstances,
however these aerobe circumstances never leads to higher N availability. The cause of this phenomenon
can be that the process to N-limitation already took place in the sediment from Terra Nova, before the
experiment started. Results in Vendrig (in prep) showed that N-limitation is one of the processes that plays a
role in Lake Terra Nova.
Relatively small amounts of free iron were present in the surface water in our experiment. In the pore water
the amounts were in some cases factor 10 higher but still small. It seems that the added iron binds with PO4
in the surface and pore water and precipitates (see figure 1c). When PO4 reaches zero in the surface and
pore water and iron is still been added, iron can’t bind PO4 anymore. There are however indications (figure 5
a-d) that iron is accumulating in the tanks as precipitated iron and dissolved iron. There were no indications
that iron is also accumulating in the plants (E. nuttallii as well as P. pectinatus showed no brown colour of
the leaves or necrotic spots on leaves).
Sulphide toxicity might play a role when root vitality is decreasing, in sediments with very low concentrations
of free iron, like in our experiment. Sulphide toxicity is known to affect the roots of aquatic macrophytes
(Allam & Hollis, 1972; Yoshida & Tadano, 1978; Koch & Mendelssohn, 1989). In our case the amount of free
iron is less than 50 µmol L-1. In our case sulphate levels in surface and pore water are increasing from 20
mg L-1 at the beginning of the experiment till 70 mg L-1 at the end of the experiment. This could be an
indication of the findings of Smolders & Roelofs (1995,1996): In sediments with low dissolved iron levels (<
20 µmol L-1), sulphide levels are generally high, in sediments with iron levels higher than 50 µmol L-1
sulphide levels are very low. There are however no indications that root vitality is decreasing. When Fe 3+
reduces to Fe2+, Fe2+ binds to sulphur and forms pyrite. This is the case when calcium concentrations are
high. In Terra Nova sulphate levels are low in surface water, but in some area in Terra Nova, sulphur is
present as gas from sediment. Negative effects of Fe2+ and S2- on biomass production of submerged
macrophytes have not been reported. Submerged macrophytes have several adaptations to aquatic
environments: gas exchange over the entire plant surface (Sculthorpe, 1967), nutrients up-take by the
above-ground parts of the plant, as well as by the roots (Gunnison and Barko, 1989; Van Wijk, 1989), and
oxygen transport from above-ground parts to the roots by the intercellular air-space system. This allows the
plant to influence the redox potential of the rhizosphere (Sand-Jensen et al., 1982; Kemp and Murray,
1986).
Significant differences between treatments in surface water and pore water aluminium and calcium were
measured, however the differences in concentration are so small (< 1 µmol L -1 ) that they hardly have effect
on the growth of E. nuttallii or P. pectinatus.
In our experiments we can conclude that all mobilized P is bound to the added iron.
The results of Van der Welle et al. (2007) showed that phosphate mobilisation takes place at Fe:P ratios in
the pore water below 1. A study by Smolders et al. (2001) showed that phosphate mobilisation from the
sediment to the water layer occurred when Fe:P ratios in the pore water were below 1. In that study it was
also shown that when Fe:P ratios exceed 10, hardly any phosphate is mobilised from the sediment as was
also the case in the Fe treatment of Van der Welle et al., (2007). Nutrient mobilization measurements
(Geurts et al., 2010) showed that PO4 mobilization from the sediment to the water layer becomes much
higher below Fe: PO4 ratios of 1 mol mol-1. This is consistent with other studies, which found that the risk of
PO4 mobilization increases below pore water Fe: PO4 ratios of 1-3.5 mol mol-1 (Lofgren & Bostrom, 1989;
Smolders et al., 2001; Zak et al., 2004; Geurts et al., 2008). Where there is insufficient Fe in the pore water
to bind PO4 (Fe:PO4 ratio < 1 mol mol -1), PO4 mobilization from the sediment to the water layer will be
mainly determined by diffusion of pore water PO4 (Reddy et al., 1996). This is comparable with mobilization
of mineral N from the sediment to the water layer, which is largely determined by diffusion (Geurts et al.,
2010).
The main objective of this study was to investigate if addition of iron chloride (FeCl 3) to a freshwater peaty
system is harmful for the growth of macrophytes. In our study, addition of ironchloride, in surface water or
sediment (25 mg or 50 mg in a tanks of 7,3 litre filtered water) was not harmful for the growth of E. nuttallii or
P. pectinatus and the effect of iron addition on the growth of macrophytes is species specific (figure 1). Van
Wijck et al. (1992) tested the influence of FeCl3 additions (1.2 mg g -1 (sediment d.w.)) to the sediment on P.
pectinatus, in tanks which were placed outside, and observed growth in all treatments during the entire
experiment. All plants showed root elongation and increasing shoot numbers. Neither mortality nor dead
tissue occurred in any of the plants.
In our experiment organic reducing sediment (from Terra Nova) was used. Species with high oxidizing
abilities have high iron-excluding capacities (Bartlett, 1961; Yoshida & Tadano, 1978) and often show iron
hydroxide precipitations on the root surface (Bartlett, 1961; Armstrong & Boatman, 1967; Green &
Etherington, 1977; Chen, Dixon & Turner, 1980; Taylor et al., 1984; Laan et al., 1989; Laan, Smolders &
Blom, 1991; St-Cyr et al., 1993), which was not the case with E. nuttallii or P. pectinatus in our experiment.
The amount of radial oxygen loss from the roots of a species however seems to depend on the reductivity of
the sediment on which the species occurs (Smits et al., 1990). Species from organic reducing sediments
have lower oxidizing abilities than species from oxidizing sediments (Smits et al., 1990; Smolders & Roelofs,
1996) because species from oxidizing sediments leak oxygen along the entire length of their roots and
maintaining the oxidized status of the sediment (Smolders & Roelofs, 1996). Both E. nuttallii and P.
pectinatus in our case were dealing with lower oxidizing abilities, because of the organic reducing sediment,
both species might be more prevented against radical oxygen losses. Prevention of radial oxygen losses will
also hinder the uptake of nutrients from the sediments, but availability of nutrients in the surface water of
organic reduced sediments is mostly very high (De Lyon & Roelofs, 1986).
In case of Terra Nova water managers keep in mind that the lake can be N- limited. This factor is not yet
known in peaty lakes like Terra Nova. In many lakes water managers want to increase the transparency and
solve problems with Cyanobacteria. Addition of iron, best slowly added, can be helpful and enhance growth
of macrophyes, but in an N-limited lake, macrophytes can have problems with their growth because of the
lack of nutrients. Also precipitation of iron-phosphate can be a problem for the growth of macrophytes
because they cover the macrophytes and therefore take out light. In Terra Nova also the concentration of
SO4 is important, because sulphate toxicity can play a role in this lake.
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,
Appendix I
P-values of iron addition effects (iron), effect of place were iron is added (place) and interaction effects (iron*place) for
all chemical variables measured in surface water, as tested by ANOVA with p < 0,05.
Analysis
iron
Elodea
PO4 (µmol L-1)* **
NH4 (mmol L-1)*
0.911
0.018
SO4 (µmol L-1)
Fe (aq) (µmol L-1)*
Fe (aq) (µmol L-1)* **
0.000
Fe (s) (µmol L-1)l*
TON (µmol L-1)*
0.967
0.000
Al (aq) (µmol L-1)*
Ca (mmol L-1)
0.000
Cl (mmol L-1)
Fe:PO4 (µmol/l)* **
Potamogeton
0.015
PO4 (µmol L-1)* **
NH4 (mmol L-1)*
0.751
SO4 (mmol L-1)
0.121
Fe (aq) (µmol L-1)*
Fe (aq) (µmol L-1)* **
Fe (s) (µmol L-1)*
TON (µmol L-1)*
0.273
0.000
Al (aq) (µmol L-1)*
-1
0.002
Ca (mmol L )
0.000
Cl (mmol L-1)*
0.007
Fe:PO4 (µmol L-1)* **
Control
PO4 (µmol L-1)* **
0.516
NH4 (mmol L-1)*
0.955
0.003
SO4 (mmol L-1)
-1
Fe (aq) (µmol L )*
Fe (aq) (µmol L-1)* **
Fe (s) (µmol L-1)*
TON (µmol L-1)*
0.094
Al (aq) (µmol L-1)*
Ca (mmol L-1)
0.000
Cl (mmol L-1)*
Fe:PO4 (µmol L-1)* **
* log(+1) transformed
** PO4 and Fe (aq) T5 were used
iron*place
place
0.859
0.237
0.271
0.694
Tukey (F2,24)
Kruskal-Wallis
0.0179
4.77
0.0003
0.7295
0.813
0.616
0.104
0.081
0.791
0.586
10.866
0.000
0.000
111.428;26.871;17.316
16.706
0.0013
0.0032
0.233
0.886
0.383
0.366
0.876
0.972
4.989
0.0189
0.0130
0.0045
0.808
0.551
0.508
0.000
0.213
0.127
0.022
0.440
0.034
0.020
0.049
0.270
0.068
0.672
0.967
0.167
33.662
8.585
19.174
6.054
3.430
7.750
0.0105
0.0188
0.1913
0.662
0.782
0.0004
0.0612
0.000
0.195
65.389;13.144
0.0325
Appendix II
P-values of iron addition effects (iron), effect of place were iron is added (place) and interaction effects (iron*place) for
all chemical variables measured in pore water, as tested by ANOVA with p < 0,05.
Analysis
iron
Elodea
PO4 (µmol L-1)* **
NH4 (mmol L-1)
0.247
SO4 (mmol L-1)
Fe (aq) (µmol L-1)
0.385
Fe (aq) (µmol L-1)**
0.773
TON (µmol L-1)*
0.150
0.000
Al (aq) (µmol L-1)*
-1
0.000
Ca (mmol L )
0.000
Cl (mmol L-1)
Fe:PO4 (µmol L-1)**
0.639
Potamogeton
PO4 (µmol L-1)* **
NH4 (mmol L-1)
0.315
SO4 (mmol L-1)
0.571
Fe (aq) (µmol L-1)*
Fe (aq) (µmol L-1)* **
0.971
TON (µmol L-1)*
-1
0.000
Al (aq) (µmol L )*
0.014
Ca (mmol L-1)*
0.000
Cl (mmol L-1)*
-1
Fe:PO4 (µmol L )**
0.602
Control
PO4 (µmol L-1)* **
0.639
NH4 (mmol L-1)
0.661
0.008
SO4 (mmol L-1)
Fe (aq) (µmol L-1)
0.227
Fe (aq) µmol/l* **
0.335
-1
TON (µmol L )*
0.240
0.000
Al (aq) (µmol L-1)*
Ca (mmol L-1)
-1
0.000
Cl (mmol L )
Fe:PO4 (µmol L-1)**
0.421
* log(+1) transformed
** PO4 and Fe (aq) T5 were used
Iron*place
place
0.947
0.630
0.616
0.584
0.514
0.867
0.635
0.000
0.249
0.720
0.966
0.950
0.857
0.380
0.007
0.688
0.444
0.479
0.230
0.538
0.802
0.895
0.203
0.123
0.000
0.927
0.756
0.444
0.009
0.897
0.953
0.536
0.823
0.613
0.687
0.682
0.349
0.518
0.796
0.068
0.556
0.707
0.485
0.725
0.004
0.533
0.196
0.207
Tukey (F2,24)
Kruskal-Wallis
0.7295
0.1504
11.906
18.324
103.801;19.109;8.591
0.8758
0.7668
0.5098
13.465
5.161
51.391;18.388;8.108
6.008
12.664
0.1068
36.139;7.195
Appendix III
P-values of iron addition effects (iron), effect of place were iron is added (place) and interaction effects (iron*place) for
all physical variables measured, as tested by ANOVA with p < 0,05.
Analysis
Elodea
Biomassa
root:shoot
C:N tissue
C:N root
N:P tissue
N:P root
P tissue
P root
N tissue
N root
C tissue
C root
Potamogeton
Biomassa
root:shoot
C:N tissue
C:N root
N:P tissue
N:P root
P tissue
P root
N tissue
N root
C tissue
C root
iron
Iron*place
place
0.190
0.481
0.332
0.085
0.911
0.147
0.401
0.844
0.243
0.071
0.191
0.314
0.076
0.166
0.296
0.533
0.440
0.964
0.588
0.994
0.206
0.458
0.499
0.611
0.781
0.641
0.913
0.712
0.976
0.559
0.803
0.602
0.945
0.742
0.825
0.778
0.016
0.324
0.469
0.913
0.188
0.659
0.058
0.343
0.451
0.803
0.999
0.912
0.453
0.370
0.128
0.822
0.347
0.798
0.597
0.351
0.184
0.867
0.836
0.747
0.936
0.349
0.538
0.090
0.713
0.590
0.358
0.350
0.712
0.097
0.348
0.562
Tukey (F2,24)
4.897
Kruskal-Wallis