Aquaculture International (2021) 29:2101–2118

https://doi.org/10.1007/s10499-021-00736-z

Estimation of the potential of Lemna minor for effluent

remediation in integrated multi-trophic
aquaculture using newly developed synthetic
aquaculture wastewater

Simona Paolacci 1,2 & Vlastimil Stejskal

1,2,3
& Marcel A. K. Jansen
1,2

Received: 21 January 2021 / Accepted: 14 June 2021/ Published online: 30 June 2021
# The Author(s) 2021

Abstract
Aquaculture is an important source of animal protein and a key contributor to global food
security. However, aquaculture can exert a negative effect on the aquatic environment due
to the release of effluents containing high nutrient levels. In integrated multi-trophic
aquaculture (IMTA), the waste produced by one species is the input for another, referred
to as extractive species (ES). Potential ES include plants. In the present study, it was
explored whether Lemna minor can be used to remove nitrogen and phosphorus from
aquaculture wastewater. A representative synthetic wastewater was designed based on the
composition of aquaculture effluents found in the literature. Synthetic wastewater was
found to be a suitable medium for growth of Lemna minor, and plants readily took up
NH4+-N, NO3-N and PO43--P. In particular, NH4+-N concentrations rapidly decreased.
The highest removal rates per square meter of water surface, calculated for NH4+-N, NO3-
N and PO43--P, were, respectively, 158, 206 and 32 mg·m-2·day-1, and these rates were
achieved at a plant surface density of 80%. As removal of nutrients is essentially a surface
area–related process, the effect of plant density on nutrient uptake was determined.
Uptake of nutrients per square meter of surface area was highest at the highest plant
density. Yet, when uptake rates were calculated per square meter of water area covered by
Lemna fronds, the highest removal rates were found at the lowest plant density, and this is
likely to be associated with a reduced intraspecific competition. The present work enables
the calculation of potential nutrient uptake by Lemna minor and lays the foundation for a
more scientific approach to the design of duckweed-based aquaculture wastewater
treatment systems.

Keywords Lemnaceae . Aquaculture . Water restoration . Removal rate . Synthetic wastewater

Handling Editor: Gavin Burnell

* Simona Paolacci
[email protected]

Extended author information available on the last page of the article

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Introduction

Global food security refers to a scenario in which everybody has access to sufficient, safe and
nutritious food (FAO 2001). Food quality is a major consideration in the concept of food
security and, especially, the supply of quality protein is of concern (Tuomisto 2010). Fish in
the broadest sense, i.e. including shellfish, is a key source of animal protein with some 4.5
billion people worldwide acquiring at least 15% of their intake from this source (Naylor et al.
2000). However, the stagnation in capture fisheries has resulted in a situation whereby supply
is not meeting demand (Ahmed et al. 2019). This, in turn, has led to increased emphasis on fish
farming to improve food security (Ahmed and Lorica 2002; Allison 2011; Jennings et al.
2016). The global production of farmed fish increased from 105.46 million tonnes in 2016 to
110.21 million tonnes in 2018 (Tacon 2018). The total global production is valued 243.26
billion of US dollars (FAO 2018). The growth of the aquaculture sector is set to continue, with
the European Union having invested €3.4 billion in the aquaculture sector over the period
2000–2020 (Guillen et al. 2019).
Despite the importance of the aquaculture industry as a contributor to global food security,
the sustainability of current aquaculture practises has been questioned. Negative impacts on
coastal and marine habitats through, amongst others, waste disposal, fish escape and pathogen
invasions have been reported (Valenti et al. 2018). Aquaculture can similarly be a major threat
to the local freshwater environment (Wang et al. 2019). Effluent containing fish excrement and
feed leftovers can impact on receiving waters, causing, amongst others, eutrophication and
oxygen depletion (Correll 1998; Cornel and Whoriskey 1993). Regulatory authorities typically
impose limits on the discharge of nutrient-rich effluents (e.g. Musacchio et al. 2020), and this
necessitates some form of wastewater treatment, representing a substantial cost to the aqua-
culture industry (Turcios and Papenbrock 2014).
In response, integrated multi-trophic aquaculture (IMTA) has been developed. The princi-
ple of IMTA is that the waste produced by one species is the input (e.g. feed source or
fertiliser) for another species, referred to as extractive species (ES). These ES include
heterotrophic filter feeders and detritivores, as well as autotrophic seaweeds and a range of
plant species. In essence, ES perform a form of environmental remediation. The biomass of the
species used for bioremediation can comprise a second source of income for the industry
(Ridler et al. 2007). As a result, IMTA systems can be mostly closed loop, with a reduced
impact on the surrounding environment.
The concept of phytoremediation is well established (Chandra et al. 2017). Aquatic plant
species with remediation potential are of particular interest for integration into aquaculture.
Lemnaceae (common name duckweed) are a family of (mostly) free-floating freshwater plants
that are known for having a high growth rate (duplication biomass in less than 2 days in
optimal conditions (Ziegler et al. 2015) and high protein content (Oron et al. 1985; Mohedano
et al. 2012). Moreover, ammonium, the prevalent form of nitrogen in aquaculture effluent, is
the preferred form of nitrogen for Lemnaceae (Landolt and Kandeler 1987). Lemnaceae
protein has the added advantage of a favourable amino acid profile, which means that biomass
has potential value as a feed source (Zhou and Borisjuk 2019). The combination of remedi-
ation potential and valuable biomass makes Lemnaceae attractive candidates for the role of ES
in IMTA.
In practise, a major challenge for IMTA is that a balance is needed between waste
production by fish and remediation by the ES (Reid et al. 2008). Thus, it is important that
the removal capacity of ES is well established, to underpin the design of IMTA systems. The
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aim of the present study was to develop a synthetic aquaculture wastewater and use it to
investigate the quantitative capacity of Lemna minor to remove nutrients from aquaculture
effluent. The development of the SAW is an important step to enhance reproducibility of
experimental studies. This standardised synthetic wastewater facilitates generation of repro-
ducible data that can be used to develop mathematical models on the balance between waste
production and plant-based removal in IMTA. A standardised synthetic wastewater is a useful
tool to test the effect of different conditions on the performance of duckweed in
phytoremediation systems. For example, it can be used to verify the effect of light and
temperature and to indicate the performance of these plants in different regions and/or in
different seasons. Here, SAW is used to quantify the effect of nutrient availability and plant
density on the removal efficiency.

Material and methods

Plant material and growth conditions

The strain of Lemna minor used for this study was collected in Blarney, Co. Cork, Ireland
(51°56′25.7″N 8°33′49.1″W) and registered in the Rutgers Duckweed Stock Cooperative
(www.ruduckweed.org) as strain number 5500, also referred to as MJ100. Axenic fronds
were cultured on half-strength Hutner’s medium (Hutner 1953), in a growth room at a
temperature of 20°C, a light intensity of 40 μmol m-2 s-1 and a day/night regime of 16-h light
and 8-h darkness.

Synthetic aquaculture wastewater

A synthetic aquaculture wastewater (SAW) was developed on the basis of published data. The
search of peer-reviewed literature centred on studies that analysed the chemical composition of
freshwater aquaculture effluents. The relevant literature was searched using the on-line
scientific database Google Scholar. All searches were completed by January 2020. The
principal search terms used to identify relevant publications were “freshwater, aquaculture
and effluents”. The first 40 search results were examined for papers providing analytical data
on freshwater aquaculture effluents. Additionally, the citation lists of identified publications
were inspected for further relevant papers. The papers were screened for the concentrations of
NO3-N, NH4+-N and PO43--P in aquaculture effluents. Variations in these nutrient concentra-
tions were analysed in order to identify representative concentrations for SAW.

Experimental design

The nitrogen and phosphorus removal ability of L. minor in SAW was determined at four
different plant densities: 20% (0.9 g·l-1), 60% (1.8 g·l-1), 40% (2.7 g·l-1) and 80% (3.6 g·l-1) of
water surface area). The 100% density was not tested as, from a management point of view, it
would be complicated to maintain this density at all times. In fact, growth would lead to a
multi-layered mat of duckweed and cause degradation of the lower layers of duckweed and the
release of nutrients in these fronds. In this paper, the plant density refers to the percentage of
the surface area of the plastic culture container that was covered by fronds. The surface area of
the culture containers in which the experiments were carried out was 36 cm2 (6 × 6 cm), and
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the four densities were obtained by adding fronds such that they covered, respectively, 7.2,
14.5, 21.6 and 28.8 cm2 of the available surface. These areas were covered using approxi-
mately 90, 180, 270 and 360 mg of fresh biomass, respectively. These weights were obtained
using approximately 70, 140, 210 and 280 fronds of L. minor. Each culture container was
filled with 100 ml SAW.
The experiment started with four culture containers for each plant density. Every 24 h, one
culture container per density was used to perform water analysis. The concentrations of NO3-
N, NH4+-N and PO43--P were quantified in order to determine the amount of nutrients
removed. In the other culture containers, the plant density was kept constant by removing
every day as many fronds as had grown during the previous 24 h. The experiment lasted 4 days
and was independently replicated twelve times. During the experiment, the plants were kept
under the same growth conditions described in “Plant material and growth conditions” with the
exception of the light intensity. The intensity during the experiment was increased to 300 μmol
m-2 s-1.

Water quality analysis

Nitrate, ammonia and phosphate concentrations in the medium were determined using a
HACH DR2800 machine. NO3 -N was determined by the cadmium reduction method (HACH
method 8171) using NitraVer®5 Nitrate Reagent Powder Pillows (absorbance measured at
500 nm, detection range between 0.3 and 30.0 mg ·l-1). The NH4+-N concentration was
determined by the Nessler method (HACH Method 8038) using Nessler reagent, mineral
stabilizer and polyvinyl alcohol dispersing agent (absorbance measured at 420 nm, detection
range of 0.02–2.50 mg ·l-1). PO43 -P was determined by the ascorbic acid method (HACH
method 8048) using PhosVer® 3 Phosphate Reagent Powder Pillows (absorbance measured at
880 nm, detection range of 0.02 to 2.50 mg ·l-1).

Calculations of N and P uptake rates

The removal rate of NO-3-N, NH4+-N and PO43--P was calculated for plants kept at four
different plant densities. Removal rates of N and P were calculated per square meter of water
surface (covered by different plant densities) per day, as well as per square meter of water area
covered by fronds of Lemna minor (at different densities) per day. The following formula was
used to calculate the uptake rates:
RWS ¼ ðCi−Cf Þ*V=Aw*t
where RWS is the removal rate per water surface, Ci is the initial concentration of the nutrient, Cf is
the final concentration of the nutrient, V is the volume in litre, t is the time in days and Aw is the
surface area of water available to the plants in square meter (area of the culture container).

RDS ¼ ðCi−Cf Þ*V=Ad*t

where RDS is the removal rate per square meter of water area covered by Lemna fronds, Ci is
the initial concentration of the nutrient, Cf is the final concentration of the nutrient, V is the
volume in litre, t is the time in days and Ad is the water area covered by Lemna fronds in
square meter.
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The relationship between removal rates and concentrations of NO-3-N and NH4+-N in the
SAW was analysed in more detail for the two highest plant densities (60% and 80%), for
which removal rates per surface area were highest and significantly different from the removal
rates at 20% and 40%. For PO43--P, all the four densities were included in the analysis.

Statistical analysis

All statistical analysis was performed using IBM SPSS statistics 26. A two-way repeated
measures ANOVA was conducted to determine the effects of plant density and time on the
removal of NO3 -N, NH4+-N and PO43 -P. For the simple two-way interactions and simple
main effects, a Bonferroni adjustment was applied. Statistical significance was accepted with
95% confidence level.
The removal rates of NO3 -N, NH4+-N and PO43 -P at different plant densities were
compared using a one-way ANOVA. The normality of the rates was assessed by the
Shapiro-Wilk test and the homogeneity of variances by Levene’s test. Post hoc analysis was
performed with a Tukey test.
Linear regression was used to explore the effects of different concentrations of NO3 -N,
NH4+-N and PO43 -P on removal rates. The linearity of the relation between the two variables
was assessed by visual inspection of the scatter plot, and homoscedasticity and normality of
the residuals were verified. When the variables failed to show a clear pattern on the scatter plot,
a Kendall tau-b test was carried out to confirm the lack of correlation. The proportion of the
variation in the removal rate explained by the concentration was determined, and dependent
variable values were predicted from new independent variable values.

Verification of predicted values

Real aquaculture wastewater was collected from a fish farm in Ireland, farming rainbow trout
(Oncorhynchus mykiss) and European perch (Perca fluviatilis). The wastewater contained
3.75, 1.9 and 3.6 mg·l-1 of, respectively, NH4+-N, NO3 -N and PO43 -P. Duckweed at densities
between 60 and 80% were placed in a culture container containing 100 ml of real aquaculture
wastewater. The nutrient content was measured after 24 h, and the uptake rates were calculated
in order to compare the rate of uptake from real aquaculture medium with that predicted from
the experiments using synthetic aquaculture medium. The experiment was replicated 12 times.

Results

Development of a synthetic aquaculture wastewater

The literature was mined for data on the composition of freshwater aquaculture effluents and
specifically concentrations of NO3 -N, NH4+-N and PO43--P. A total of 16 articles contained
relevant data for the purpose of the present study. These articles contained data on the effluents
from farms culturing rainbow trout (Oncorhynchus mykiss), salmon smolt (Salmo salar),
brown trout (Salmo trutta), arctic char (Salvelinus alpinus), European eel (Anguilla Anguilla),
pike perch (Sander lucioperca), sturgeon (Acipenser sp.), Nile tilapia (Oreochromis niloticus),
European perch (Perca fluviatilis), European sea bass (Dicentrarchus labrax), catfish
(Ictalurus sp.) and bluegill (Lepomis macrochirus) (supplementary table I). There was
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substantial variation in effluent concentrations of NO3 -N, NH4+-N and PO43--P (Fig. 1a–c).
The mean values for NO3 -N, NH4+-N and PO43--P , in the papers reviewed, were, respec-
tively, 25.7, 5.7 and 2.7 mg ·l-1. The highest concentration of NH4+-N was found in a study by
Gendel and Lahav (2013). The authors farmed 105 tilapia in 500 l of medium and the
concentration of NH4+-N in the water reached 20 mg·l-1. The highest concentration of NO3 -
N (200 mg·l-1) was found in a study by Dalsgaard et al. (2013) in which rainbow trout was
farmed at a density of 50–80 kg·m-3. The highest concentration of PO43--P in effluent was
found by Ng and Chan (2018). The authors observed a concentration of 19 mg·l-1 PO43--P in a
fish farm in China; however, they did not specify which species was farmed and at which
density.
Based on the information obtained from the literature (Fig. 1, Suppl. Table I), a synthetic
aquaculture waste effluent was designed that contains 25, 8 and 2 mg ·l-1 of NO3-N, NH4+-N
and PO43--P, respectively. These values are the rounded up mean values calculated for the
papers reviewed. SAW was supplemented with common macro- and micronutrients, to
resemble eutrophic wastewater (Table 1). Other elements necessary for the growth of
Lemnaceae were added in the amount and proportions suggested by Hutner (1953). The pH
of the synthetic wastewater was 5.5.

Lemna minor–mediated removal of nutrients from SAW

The growth of L. minor on SAW during the 4 days of experiment was substantial. Every day, a
number of fronds had to be removed in order to maintain the chosen densities. Approximately
7, 14, 20 and 25 fronds of L. minor were removed daily from the containers with plants at the
densities of, respectively, 20%, 40%, 60% and 80%. The NH4+-N, NO3 -N and PO43 -P
concentrations in the SAW were measured every day. NH4+-N depletion was substantial
(Fig. 2a). In most of the experiments with a plant density of 60% and 80%, the concentration
of NH4+-N in the medium fell to just above zero after 4 days. The decrease in the concentration
of NH4+-N ranged between 6.17 ± 0.33 (at a plant density of 20%) and 7.67 ± 0.13 (at a plant

Fig. 1 Concentrations in freshwater aquaculture effluents of NH4+-N (a), NO3 -N (b) and PO43--P (c) found in the
literature. The data were collected from 16 articles (Supplementary Table I). All the concentrations were converted
in milligrams per litre. The x indicates the mean, the horizontal line within the box indicates the median, and the
lower and upper extremes of the box indicate respectively first and third quartiles. Grey circles are outliers
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Table 1 Composition of synthetic aquaculture wastewater

Concentration (mg · l-1)

(NH4)2SO4 40
KNO3 73
Ca(NO3)·4H2O 186.15
KH2PO4 8.7
CaSO4 328.6
MgSO4·4H2O 738
K2SO4 471.5
ZnSO4·7H2O 1
MnSO4 H2O 0.1
CuSO4 5H2O 0.03
Na2MoO4 2H2O 0.1
Ferric citrate 1
EDTA Na 0.029
H3BO3 1

density of 80%) mg ·l-1. The biggest decrease in the concentration of NH4+-N was observed
during the first 24 h. Overall, less NH4+-N was removed from SAW at lower plant densities. A
two-way repeated measures ANOVA revealed a significant interaction between time and
density (p < 0.01). The simple main effects of the plant density and time on NH4+-N
concentration were significant, with both p < 0.01. Plants at a density of 80% removed more
NH4+-N than plants at density 40% (p < 0.01) and 20% (p < 0.01). Plants at a density of 60%
removed more NH4+-N than plants at density 40% (p < 0.001) and 20% (p < 0.01). Plants at a
density of 40% removed more NH4+-N than plants at density 20% (p < 0.001).
The concentration of NO3 -N in the medium decreased slowly over the 4 days of monitoring
(Fig. 2b). The concentration of NO3 -N in the SAW reached values as low as 13.5 ± 1.6 mg·l-1
at the highest plant density and after 4 days. A two-way repeated measures ANOVA
highlighted that there was significant interaction between time and density on NO3 -N con-
centration (p = 0.047). The simple main effects of time and density were also significant (p =
0.005 and 0.02, respectively). Plants at a density of 80% removed more NO3 -N than plants at
density 20% (p < 0.011) and 40% (p = 0.035).
The daily decrease in PO43 -P concentration was constant during the 4 days of experiment,
(Fig. 2c). The concentration of PO43 -P in the SAW reached values as low as 0.89 ± 0.31
mg·l-1 after 4 days and at the density of 60%. There was no significant two-way interaction
between time and plant density on the PO43 -P concentration. Only time had a significant
effect, with p < 0.001.

Daily nutrient removal per square meter of water surface and per square meter
of water area covered by Lemna fronds

The absolute nutrient removal rate during the first 24 h of the experiment was calculated based
on the volume of SAW used in the experiment and expressed per square meter of water surface
(Fig. 3a) or per square meter of water area covered by Lemna fronds (Fig. 3b). The rates
calculated were plotted as a function of the plant density. When the removal rates of both
forms of N were calculated per square meter of water surface, they increased with increasing
plant density. The highest removal rates of NH4+-N were 157.61 ± 10.57 mg·m-2·day-1,
achieved at a plant density of 80%. The lowest rate of NH4+-N removal was 99.12 ± 6.41
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Fig. 2 Decrease over time of NH4+-N (a), NO3-N (b) and PO43--P (c) in synthetic aquaculture wastewater with
L. minor at four different densities (20%, 40%, 60% and 80%). Slightly higher nutrient concentrations on day 0
relate to carry-over from standard medium to experimental medium. N = 12, bars are standard errors
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Fig. 3 Nutrient removal rate per square meter of water surface (a) and per square meter of L. minor covering the
water surface (b), at different plant densities (20%, 40%, 60%, 80%) and measured during the first 24 h of
experiments. Different letters indicate significant differences. N = 12, bars are standard errors
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R Fig. 4. Simple scatter plot of daily NO3 -N (a), NH4+-N (b) and PO43--P (c) removal rates expressed per square
meter of water surface area as a function of nutrient concentration in the medium. For NH4+-N, the fit lines and
the regression equations are also indicated. Dots represent individual measurements. NH4+-N concentrations of 8
mg·l1, NO3 -N concentrations of 25 mg·l1 and PO43--P concentrations of 2.5 mg ·l1 represent the initial
concentrations used in uptake experiments. Concentrations higher to SAW concentrations are due to carry-
over from standard medium to experimental medium

mg·m-2·day-1, achieved at a plant density of 20%. Plants at the density of 80% removed
significantly more NH4+-N than plants at a density of 20% (p < 0.01) and 40% (p < 0.01). The
highest removal rate of NO3 -N was 205.99 ± 30.71 mg·m-2·day-1, achieved at a plant density
of 80%, while the lowest was 103.61 ± 20.27 mg·m-2·day-1, achieved at the density of 20%.
Plants at the density of 80% removed significantly more NO3 -N than plants at the density of
20% (p = 0.045).
In contrast to what was observed for rate calculated per square meter of water surface, when
the removal rate was calculated per square meter of plants covering the water surface, it
decreased with the increasing plant density. The highest removal rates of NH4+-N were 495.6 ±
32.05 mg·m-2·day-1, achieved at a plant density of 20%. The lowest rate of NH4+-N removal
was 197.01 ± 13.21 mg·m-2·day-1, achieved at a plant density of 80%. Plants at the density of
20% removed significantly more NH4+-N than plants with a density of 80% (p < 0.01), 60% (p
< 0.01) and 40% (p < 0.01); the removal of NH4+-N at 40% was also significantly higher than
the removal at 80% (p = 0.025). The highest removal rate of NO3 -N was 518.05 ± 101.39
mg·m-2·day-1, achieved at a plant density of 20%, while the lowest was 257.49 ± 38.38
mg·m-2·day-1, achieved at the density of 80%. Plants at the density of 20% removed signifi-
cantly more NO3 -N than plants at the density of 80% (p = 0.045).
The removal rate of PO43 -P per square meter of water ranged between 20.87 ± 7.72 and
32.43 ± 9.1 mg·m-2·day-1, with no significant differences between densities. When calculated
per square meter of plant covering the water surface, the highest removal rate, 139.46 ± 24.15
mg·m-2·day-1, was observed at the plant density of 20%. At this density, the removal rate was
significantly higher than at 40% (p < 0.01), 60% (p < 0.01) and 80% (p < 0.01).
The detailed results of the statistical analyses are reported in tables contained in supple-
mentary material 1.

Removal rates at different nutrient concentrations in the SAW

The daily nutrient removal rate for all sampling time-points and recalculated per square
meter of water surface was plotted as a function of the measured concentrations of
NH4+-N, NO3 -N and PO43 -P in SAW. For NH4+-N and NO3 -N, data are presented
for plants kept at 60 and 80%, at which densities the uptake rates are most substantial.
For PO43 -P, the removal rates at all the densities tested were included as no
significant differences between densities were highlighted in the previous analysis. A
visual inspection of the scatter plots highlighted a linear relationship between daily
NH4+-N removal rate and NH4+-N concentration (Fig. 4a). This relationship was further
analysed using regression analysis. The analysis shows that the concentration of NH4+-
N in the medium significantly predicts the removal rate of the compound in the water
(p < 0.01). The medium concentration accounted for 86% (R2 = 86%) of the variability
in removal rate. A Kendall tau-b correlation was run to determine the relationship
between concentration of NO3 -N or PO43--P and daily removal rate. This test was
selected as a visual inspection of the scatter plots failed to show a linear relationship
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between variables. There was a weak association between both nutrients and daily
removal rate, which was not statistically significant (τb = 0.127, p = .681, and τb =
0.111, p = .702 respectively). Because of the lack of correlation between variables, the
regression analysis was not carried out for NO3 -N and PO43--P data (Fig. 4b, c).
The observed regression line was used to predict the uptake rate of NH4+-N from real, farm-
collected, aquaculture wastewater (containing 3.75 mg·l-1 of NH4+-N). The predicted uptake
rate was 58.8 mg·m-2·day-1. The observed uptake rate of NH4+-N by duckweed grown in the
real aquaculture wastewater was 53.6 ± 0.4 mg·m-2·day-1 (Fig. 5).

Discussion

A key challenge in the design of IMTA systems is the balance between fed and extractive
species (Wei et al. 2017; Fang et al. 2019; Nardelli et al. 2019). Where ES are algae or plants,
it is essential to consider their intrinsic growth and nutrient uptake characteristics. These
characteristics vary in response to changes in the composition of the wastewater (Kwon
et al. 2013), as well as in response to environmental parameters such as temperature and light
(Iasimone et al. 2018). Growth and nutrient uptake are also species specific (Khatun et al.
2016; Yongpisanphop et al. 2017; Queiroz et al. 2020). Here, uptake of NO3 -N, NH4+-N and
PO43 -P by L. minor was analysed under controlled conditions using a synthetic aquaculture
wastewater and various plant densities, in order to obtain quantitative information on the
remediation potential of this species.

Development of a synthetic aquaculture wastewater for controlled experiments

The development of synthetic wastewater to enable reproducible testing of the efficiency of

different water remediation systems is increasingly prevalent (e.g. Visvanathan et al. 2008;

Fig. 5. Nutrient removal rate per square meter of real aquaculture wastewater surface at different plant densities
(20%, 40%, 60%, 80%), measured during the first 24 h of experiments. The wastewater contained 3.75, 1.9 and
3.6 mg·l-1 of, respectively, NH4+-N, NO3 -N and PO43--P
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Saleem et al. 2011; Ansari et al. 2017; Barnharst et al. 2018; Walsh et al. 2020). The use of
such a standardised medium facilitates studies on the impacts of other parameters, such as
plant species, plant density, climatic factors, microbial populations and IMTA system design
parameters. Previously, a SAW was used by Ng and Chan (2018) to test the phytoremediation
ability of Spirodela polyrhiza, Salvinia molesta and Lemna sp. The authors based the NH4+-N
and PO43 -P concentrations (20 and 19 mg·l-1 respectively) in their synthetic wastewater on the
concentrations measured at a local fish farm. In the current study, a SAW was developed on
the basis of realistic concentrations of NH4+-N, NO3 -N and PO43 -P in freshwater aquaculture
effluents. Selected papers refer to a variety of fish farms, farming different species at different
densities. Thus, the developed SAW is a generic medium that can be used as a proxy
representing effluent from a broad range of freshwater fish farms.

Removal of nutrients from SAW

In this study, the removal rate of nutrients was determined by measuring the concentration of
the nutrients in the SAW every day. The experiment was carried out in aseptic conditions.
Therefore, it is reasonable to assume that the nutrients removed from the SAW were taken up
by the plants as the microbial activity was negligible. Given the small scale of the experiments,
it was not possible to determine the amount of nutrients in the plant biomass.
The developed SAW is a suitable growth medium for L. minor, with both plant growth and
nutrient removal being substantial. A comparison of the NH4+-N uptake rate from real farm
aquaculture wastewater (58.8 mg·m-2·day-1 at a concentration of 3.75 mg·l-1) and the rates
obtained using SAW (53.6 ± 0.4 mg·m-2·day-1 at 2.4 mg·l-1) shows good agreement. Thus, the
developed SAW proved to be a reliable representative of real aquaculture wastewater.
Rapid removal of NH4+-N and PO43 -P from SAW was observed during the first 24 h of
the experiment. Comparatively, the decrease of NO3¯-N in the SAW was lower. The relatively
slow removal of NO3¯-N is most likely related to the presence of NH4+-N in the SAW. When
both forms of nitrogen are present in a medium, Lemnaceae prefer to take up NH4+-N (Feller
and Erismann 1971), probably as the direct conversion of ammonia into protein is a more
energy-efficient process (El-Shafai et al. 2007). The measured removal rates of the two
combined forms of nitrogen from SAW are slightly lower than those reported in the literature.
For example, in the literature, values range from 500 to 2100 mg·m-2·day-1 (Körner and
Vermaat 1998; Cheng et al. 2002; Mohedano et al. 2012). The phosphorus removal rates
measured in this study are also in the lower portion of the published range (i.e. from 20 to 590
mg·m-2·day-1) (Körner and Vermaat 1998; Cheng et al. 2002; Mohedano et al. 2012). The
absence of bacterial and algal activity under the used experimental conditions is probably
responsible for the relatively low removal of nutrients.
Following the first 24 h, removal rates of all nutrients decreased, and this was associated
with lower nutrient concentrations in the used stationary system. To analyse the relationship
between removal rates and nutrient concentration in the SAW in more detail, removal rates
(mg·m-2·day-1of water surface) of NH4+-N, NO3 -N and PO43 -P were plotted against the
concentration of the compounds in the SAW. For NO3 -N and PO43 -P, the relationships
between removal rate and the concentration were not well defined. However, the removal rate
of NH4+-N linearly decreased with decreasing concentration in the medium. These data cannot
be directly interpreted as meaning that NH4+-N is limiting for growth, as growth is likely to be
more closely related to internal redistribution of nutrients, rather than uptake (Kufel et al.
2012). Nevertheless, the ability to predict removal rates at different nutrient concentrations is
2114 Aquaculture International (2021) 29:2101–2118

of fundamental importance when designing duckweed-based phytoremediation system as it

can inform attempts to achieve a balance between fed and extractive species.

Effect of plant density on the removal rates

As removal of nitrogen and phosphorus is essentially surface area–related processes, the plant
density in the system needs to be considered. In the present study, the removal rate was
calculated both per square meter duckweed covering the water surface and per square meter of
water surface. The two different approaches reflect two different aspects of duckweed-based
wastewater systems. The removal rate per square meter of water is an important tool to predict
the efficiency of the system from the water-restoration point of view. On the other hand, the
analysis of the removal rate calculated on the basis of plant surface reveals physiological
characteristics that can potentially reflect on the nutritional values of the plants.
The duckweed density relates directly to system management and harvest protocols. More
plants will result in more nutrient uptake capacity. Indeed, the data show that the removal rate
per square meter of water surface was higher at higher plant densities. However, the removal
rate per square meter of duckweed was highest at the lowest densities. It has been argued that
the increase in plant density leads to intraspecific competition, which is associated with a
slowdown in nutrient removal and/or growth (Clatworthy and Harper 1962; Rejmánek et al.
1989). The higher removal rate at low plant density could be associated with different
biochemical compositions of the biomass produced, and this aspect should be investigated
further. It has been shown that the most efficient plant density for nutrient removal is around
80% (Verma and Suthar 2015). Furthermore, it has been shown that at high-density plants not
only maximise nutrient removal, but also block sunlight penetration in the water column,
impeding growth of phytoplankton and other algae (Roijackers et al. 2004).
Nutrient removal by Lemnaceae needs to be analysed in more detail in terms of impacts on
growth and nutritional value. Duckweed is considered a potential crop plant for animal feed as
well as human nutrition (De Beukelaar et al. 2019), particularly in the context of the circular
economy. The optimization of the nutritional value of these plants is therefore of major interest
(Appenroth et al. 2018).

Conclusions

The present work lays the foundation for a more scientific approach to the design of
duckweed-based aquaculture wastewater treatment systems. The results presented here high-
light the importance to consider the load of nutrients in the wastewater and its effect on the
plant’s ability to remove them, in order to design an efficient system.

Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1007/s10499-021-00736-z.

Author contribution Conceptualization and methodology: Simona Paolacci and Marcel Jansen; formal anal-
ysis and investigation: Simona Paolacci and Vlastimil Stejskal; writing and original draft preparation: Simona
Paolacci; writing, review and editing: Marcel Jansen, Vlastimil Stejskal and Simona Paolacci; funding acquisi-
tion: Marcel Jansen; supervision: Marcel Jansen.
Aquaculture International (2021) 29:2101–2118 2115

Funding Open Access funding provided by the IReL Consortium. The present study was funded by Bord
Iascaigh Mhara, Irish agency for fishery and seafood (grant 17/KGS/005).

Data availability The datasets used and/or analysed during the current study are available from the corre-
sponding author on reasonable request.

Declarations

Ethics approval Not applicable.

Consent to participate Not applicable.

Consent to publish Not applicable.

Competing interests The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and
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in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or
exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy
of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Affiliations

Simona Paolacci 1,2 & Vlastimil Stejskal 1,2,3 & Marcel A. K. Jansen 1,2
1
School of Biological, Earth and Environmental Sciences, University College Cork, Cork, Ireland
2
Environmental Research Institute, Cork, Ireland
3
Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and
Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, University of South
Bohemia in Ceske Budejovice, Na Sádkách 1780, 370 05 České Budějovice, Czech Republic