Ecotoxicology and Environmental Safety 153 (2018) 54–59

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Effects of treated industrial wastewaters and temperatures on growth and T

enzymatic activities of duckweed (Lemna minor L.)
⁎
E. Basiglini, M. Pintore1, C. Forni
Dipartmento di Biologia, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Roma, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The efficacy of the removal of contaminants from wastewater depends on physico-chemical properties of pol-
Lemna minor lutants and the efficiency of treatment plant. Sometimes, low amounts of toxic compounds can be still present in
Biomass the treated sewage. In this work we considered the effects of contaminant residues in treated wastewaters and of
Toxic pollutants temperatures on Lemna minor L. Treated effluent waters were collected, analyzed and used as duckweed growth
Stress response
medium. In order to better understand the effects of micropollutants and seasonal variation, the plants were
Antioxidant enzymes
grown under ambient conditions for seven days in summer and winter. Relative growth rate, pigments and
phenolic compounds concentrations were determined, as well as the activities of catalase (CAT), ascorbate
peroxidase (APX), guaiacol peroxidase (G-POD) and polyphenol oxidase (PPO).
The pollutant concentrations varied in the two seasons, depending on the industrial and municipal activities
and efficiency of treatments. Treated waters contained heavy metals, nitrogenous and phosphorus compounds,
surfactants and hydrocarbons. Compared to the control, duckweed growth of treated plants decreased by 25% in
summer, while in the winter due to the lower temperatures and the presence of pollutants was completely
impeded. The amounts of photosynthetic pigments of treated plants were not significantly affected in the
summer, while they were higher than the control in the winter when the effluent had a high nitrogen amount.
High CAT activity was registered in both seasons. Treated plants had significantly lower APX activity in the
summer (53%) and winter (59%) respect to the controls. The observed inhibition of the peroxidase activities in
the exposed plants, confirms the controversy existing in the literature about the variability of enzymatic response
in stress condition.

1. Introduction determine the possible future impacts of treated wastewaters on the

The discharge of contaminated waters into water bodies can have an
important impact on aquatic ecosystems, being a tremendous hazard to
both natural ecosystems and human health (Calderón-Preciado et al.,
2011; Petrovic et al., 2011); therefore, the wastewaters have to undergo
to treatment before being discharged. These treatments are not always
designed and operated to eliminate completely the pollutants, thus
some residues, consisting of a vast and expanding array of anthro-
pogenic, as well as natural substances, can be still present in the treated
waters. The presence of these residues can endanger the habitats due to
the toxicity of contaminants, that can disrupt many cellular functions
impacting on plant growth (Kohen and Nyska, 2002; Forni, 2014). A
better understanding of the effects of micropollutants on not target
organisms, living in the receiving water bodies, is important to
ecosystems and for the improvement of wastewater treatment pro-
cesses.
Lemnaceae are the world's smallest and fast growing angiosperms
(Ziegler et al., 2015), representing suitable producers of large amount
of biomass. They have a great economic potential and many practical
applications in biotechnological and ecological fields (Cascone et al.,
2004; Ippolito et al., 2007; Forni et al., 2012; Tel-Or and Forni, 2011;
Neagu et al., 2014; Cui and Cheng, 2015). Their morphological and
physiological characteristics are favorable features that allow valuable
bioassays under limited spatial condition in short time, thus re-
presenting model laboratory organisms (reviewed by Forni and
Tommasi, 2016). Lemna species are used as bioindicators for in situ and
ex situ ecotoxicological assays (Scherr et al., 2008; Forni, 2014; Brain
and Solomon, 2007). In these assays growth inhibition, pigments

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; f.w., fresh weight; GI, growth index; G-POD, guaiacol peroxidase; HM, heavy metals; PPO, polyphenol oxidase; RGR, Relative
Growth Rate; ROS, reactive oxygen species
⁎
Corresponding author.
E-mail address: forni@uniroma2.it (C. Forni).
1
Pa.L.Mer, Via Carrara 12/A loc. Tor Tre Ponti, 04013 Latina Scalo (LT), Latina, Italy.

https://doi.org/10.1016/j.ecoenv.2018.01.053
Received 15 May 2017; Received in revised form 22 January 2018; Accepted 28 January 2018
0147-6513/ © 2018 Published by Elsevier Inc.
E. Basiglini et al.
content are the most common endpoints considered.
In the literature the oxidative stress, induced by stressors like pol-
lutants and high temperatures, is considered an important subject both
for terrestrial and aquatic toxicology and for determining plant toler-
ance (Apel and Hirt, 2004; Valavanidis et al., 2006; Radić et al., 2011).
Plant exposure to environmental stressful conditions can produce the
unbalance between reactive oxygen species (ROS) production and
scavenging, that causes oxidative stress. In plants, cell molecular and
physiological responses are elicited to counteract the oxidative stress
induced by ROS overproduction, these include antioxidant defenses
(enzymatic and non enzymatic).
Since the presence of even low load of pollutants in treated waters
may represent an environmental risk, in this work the complex effluent
samples from industrial wastewater treatments were analyzed, and
these waters were used as duckweed growth medium. Thus, to de-
termine the extent to which L. minor can withstand the presence of
multipollutants in natural conditions, the responses of duckweed grown
in treated wastewaters were analyzed with reference to changes of
pollutant load and seasonal temperatures. We have considered the
following parameters: (1) pollutant load of wastewaters; (2) plant
growth rates; (3) contents of chlorophylls, carotenoids and phenolic
compounds; and (4) the activities of antioxidant enzymes, such as as-
corbate peroxidase (APX) and guaiacol peroxidase (G-POD), catalase
(CAT) and polyphenol oxidase (PPO).
2. Materials and methods
2.1. Plant growth conditions
Lemna minor plants were collected in a pond in the Latium Region
(Italy), duckweed was maintained as stock cultures and prior the ex-
periments, plants were acclimated in Hoagland medium, pH 6.5 (Forni
et al., 2001).
Depending on possible variation of the effluents over time, due to
the industrial activities and performance of wastewater treatment
plants, we decided to sample the effluent, from Industrial District of
Latium Region, in two different period, e.g. summer and winter.
To keep the conditions as close as possible to the natural ones, the
experiments were performed using wastewater samples “as is” (Wang,
1990). The experiments were undertaken indoor under environmental
conditions in summer (July) and in winter (December). During the
summer the registered ambient temperatures ranged between 28 °C
(min.) and 38 °C (max.). In the winter the minimum of temperature was
18 °C and maximum was 20 °C.
For the experiments, 4 g/L duckweed plants, corresponding to 80%
surface coverage, were rinsed and inoculated in plastic bowls (20 cm in
diameter), containing 900 mL of 1/10 strength Hoagland medium, pH
6.5 (controls) (Forni et al., 2001) or 900 mL treated wastewaters
(treated). Evapotranspiration rate was also considered. Each treatment
was performed in triplicate and a randomised block design was applied
to the experiments.
Plants were grown over a period of a week, when the consequent
potential growth inhibition and other effects can be estimated. At the
end of the experiments the plants were sampled, rinsed, gently blotted
with paper and frozen at −80 °C until the analyses reported below.
All chemicals were purchased from Sigma, unless otherwise stated.
2.2. Growth and pigments concentration of the duckweed
After 7 days of treatment, plants were collected, surface-dried by
gentle blotting between layers of paper towels and fresh weight was
detected. Growth was determined as Relative Growth Rate (RGR, g d
−1
) as follows: RGR = log N final fresh weight (g.) – log N initial fresh
weight (g.)/ time (d).
Concentrations of photosynthetic pigments were determined in
plant samples (200 mg f.w.) homogenized in methanol. Extracts were
55
Ecotoxicology and Environmental Safety 153 (2018) 54–59
incubated in the dark and shaken (100 rpm, New Brunswick, Orbital
Shaker, USA) for 1 h. Supernatants were collected from centrifuged
samples and their absorbances at 665 nm (Chl a), 652.4 nm (Chl b) and
470 nm (carotenoids) were determined by spectrophotometer (Cary 50
Bio UV–Visible Spectrophotometer). Pigments concentration was de-
termined according to Lichtenthaler (1987). Results are expressed as
milligrams of chlorophylls or carotenoids per gram of plant fresh
weight. The data are expressed as mean values ± ES.
2.3. Extraction and quantification of phenolic compounds
200 mg of plants were homogenized in liquid nitrogen (LN) and
suspended in 4 mL 0.1 N HCl (Legrand, 1977). Samples were incubated
and shaken (100 rpm, New Brunswick, Orbital Shaker, USA) at 5 °C for
1 h, and then centrifuged. Supernatants were collected and pellets re-
suspended in 4 mL di 0.1 N HCl and then centrifuged again. Phenols
amount was determined in the two pooled supernatants by Folin Cio-
calteu method (Booker and Miller, 1998), using chlorogenic acid (CA)
as standard, for which a calibration curves was carried out with dif-
ferent concentration solutions of this compound (R2 = 0.999, y =
0.0031x + 0.011, calibration curve of July; R2 = 0.998, y = 0.0021x –
0.014, calibration curve of December). Results are expressed as milli-
grams of CA equivalents per g of plant fresh weight (Forni et al., 2012).
The data are expressed as mean values ± ES.
2.4. Determination of enzymatic activities and total soluble proteins
All enzymatic activities were determined in both treated and un-
treated plants at the end of the experiments. Protein concentration was
determined by Bradford protein assay (Bradford, 1976), using bovine
serum albumin as standard.
2.4.1. Ascorbate peroxidase (APX, E.C. 1.11.1.11)
The crude enzyme was extracted basing on the method of Nakano
and Asada (1981). 250 mg f. w. were homogenized in 2 mL extraction
buffer (Corsi et al., 2015). The reaction buffer (1.5 mL total volume)
was composed by 250 µl of the extract, 1 mM EDTA, 0.5 mM ascorbic
acid and 1 mM H2O2. The decrease in absorbance was followed at
290 nm for 300 s by a spectrophotometer (model Cary 50 Bio UV–Vi-
sible Spectrophotometer, Varian, Netherlands). One enzyme unit (EU)
is the amount of enzyme that oxidized 1 μmol of ascorbate per minute
at test condition (Corsi et al., 2015). The results were expressed as EU
per microgramme of proteins.
2.4.2. Catalase (CAT, EC 1.11.1.6)
The enzyme was extracted and its activity detected according to the
method of Aebi (1984), modified as follows. 250 mg f.w. were homo-
genized on ice and suspended in 3 mL extraction buffer, containing
0.1 M phosphate buffer pH 7.3, 3 mM EDTA and 1% (v/v) Triton X-100.
The extracts were centrifuged and 75 µl, of the supernatant was added
to reaction buffer containing 0.1 M phosphate buffer, pH 7.3, 3 mM
EDTA, 10 mM H2O2.
The activity was determined spectrophotometrically at 240 nm
(model Cary 50 Bio UV–Visible Spectrophotometer, Varian,
Netherlands). The activity is expressed as µmol/mL of H2O2 consumed
/minute, extinction coefficient ɛ = 0.036 mM−1 cm−1.
2.4.3. Guaiacol peroxidase (G-POD, EC 1.11.1.7) and Polyphenol oxidase
(PPO, EC 1.14.18.1)
250 mg (f.w.) of samples were homogenized in LN and suspended in
2 mL extraction buffer containing 50 mM phosphate buffer pH 6.5,
0.4 mg MgCl2 6H2O, 100 mM EDTA, 20 mg polyvinil pirrolidone
(PVPP) and 6% (v/v) Triton X-100 (Ayaz et al., 2008). The extracts
were kept at 5 °C for 1 h. and shaken at 100 rpm. Then the extracts were
centrifuged and the supernatants were collected.
To determine G-POD activity, three millilitres of reaction mixture
E. Basiglini et al. Ecotoxicology and Environmental Safety 153 (2018) 54–59

were prepared in sodium phosphate buffer (0.2 M, pH 6.5) by adding
50 µl enzyme extract, 5.3 mM guaiacol and 30 mM H2O2. The enzyme
kinetic was followed at 470 nm for 200 s by a spectrophotometer
(model Cary 50 Bio UV–Visible Spectrophotometer, Varian,
Netherlands), extinction coefficient of oxidized guaiacol ɛ =
26.6 mM−1 cm−1.
To determine the activity of PPO 50 µl of the extract were added to
the reaction buffer containing 50 mM phoshate buffer pH 6.5 and
50 mM pyrocathecol. Enhancement of the absorbance was detected
spectrophotometrically at 420 nm for 200 s. The activity (EU) is ex-
pressed as enzymatic unit (EU) corresponding to 1 µM product /minute,
extinction coefficient ɛ = 1150 M−1 cm−1.
The data of the enzymatic activities are the mean of
determinations ± ES.
2.5. Determination of pollutants in the wastewaters
Wastewaters from industries of Latium Region were used as growth
medium of the macrophytes. The waters are a mixture of urban and
industrial (textile, food processing, chemicals etc.) sewage waters. They
were collected after treatments to remove pollutants from a treatment
plant of Latium Region (Italy), where the wastewater treatment tech-
nologies and design consist of a combination of chemical, physical and
biological (activated sludges) methods.
Samples of treated wastewaters were collected in two different
periods (June-July and October-November). The compositions of was-
tewaters were determined according to the official guidelines and
methods as reported in Table 1. The analyses were carried out ac-
cording to the recommended ISO methods. The data on the reliability of
the various methods used for wastewater measurement are presented in
Table 2. In particular, the accuracy of the various parameters reported
is expressed as a percentage of Relative Standard Deviation (RSD) of 6
replicates conducted in a laboratory on a real sample of wastewater.
Reproducibility and accuracy were extracted from a proficiency test
carried out on a sample of wastewater analyzed by over 250 Italian
laboratories to which the laboratory participates periodically. In par-
ticular, the reproducibility of the various parameters reported is ex-
pressed as the percentage ratio between the RSD and the associated
value that corresponds to the average of all data considered valid ac-
cording to Huber tests. The accuracy was calculated as the percentage
ratio between the data acquired by the laboratory and the relative value
assigned by the proficiency test.
2.5.1. Heavy metals and phosphorus determination
Determination of HM and total phosphorus content of the waste-
waters was performed by Inductively Coupled Plasma and mass spec-
trometry as detector Perkin Elmer (model ELAN DRC-e, Waltham,
Massachusetts, U.S.A) previously acidification of 100 mL sample with
0.1 mL of HNO3 concentrated and filtration.
The standard solutions used for instrument calibration were
purchased from Perkin Elmer.
2.5.2. Total nitrogen determination
The total nitrogen determination was carried out after digestion of
the sample under alkaline conditions in oxidant solution in order to
convert all the organic and inorganic nitrogen compounds to nitrate.
Subsequently the sample was filtered and it was measured the absor-
bance at 220 nm with spectrophotometer UV–VIS VARIAN Mod. CARY
50 SCAN (Palo Alto, California, USA). The value read has been inter-
polated with a calibration curve obtained by the aid of standard solu-
tion of known concentration of total Nitrogen.
2.5.3. Total Hydrocarbon determination
The determination was conducted by liquid-liquid extraction with
n-esane.
250 mL of sample was spilled in sepatory funnel and extracted twice
with 100 mL of esane. The organic phase was filtered on a column
containing silica gel. The silica gel retains from extract oily substances
but letting the fraction of hydrocarbons. The latter was collected on a
ball pre- weight and pre-weighed. The content of Total hydrocarbons
was obtained from the flask weight difference before and after the
collection of the eluate.
2.5.4. Total surfactants determination
The total surfactants are represented by the sum of the anionic and
nonionic surfactants. The following describes the procedures for the
acquisition of these parameters.
– Anionic Surfactant
The specific surfactants represent the fraction of the anionic sur-
factants which generates a blue colored complex with methylene blue.
The complex can be quantitatively extracted into chloroform defined
precisely Methylene Blue Active Substances (MBAS).
100 mL of sample was spilled in separatory funnel and added with
phosphate buffer 0.07 M at pH 10.0 and 5 mL di methylene blue solu-
tion (0.35 g/L) and 15 mL of chloroform. The solution was shaken for
1 min and the organic phase was spilled second time and added with
110 mL of distilled water and methylene bleu solution dissolved in
acidic solution (6.5 mL of 96% H2SO4 solution in 1 L of distilled water).
The organic phase was once more shaken for 1 min until when the
organic phase was separated from not organic ones. The organic phase
was spilled in a flask using a funnel contain flock of cotton embedded of
chloroform. The filtration was repeated 3 times and filtered was spilled
in a flask, added with chloroform up to 50 mL of total volume.
Subsequently the sample was filtered and it was measured the ab-
sorbance at 650 nm with spectrophotometer UV–VIS VARIAN Mod.
CARY 50 SCAN (Palo Alto, California, USA). Surfactant concentration
was detected through a calibration curve obtained by standard solution
of known concentration of Sodium Dodecyl Sulphate (SDS).

Table 1
Composition of the treated wastewaters. The data are the average of the determinations (n = 6) ± S.D.

Parameters Analytical methods Summer concentration Winter concentration Unit

pH APAT CNR IRSA 2060 Man 29 2003 8.22 ± 0.02 8.00 ± 0.02 UpH
Chemical Oxygen Demand (COD) APAT CNR IRSA 5130 Man 29 2003 68 ± 4 74 ± 4 mg/L
Biochemical Oxygen Demand (BOD5)* APAT CNR IRSA 5120 Man 29 2003 31 ± 1 26 ± 1 mg/L
Total Phosphorus (P) UNI EN ISO 17294-1:2007 + UNI EN ISO 17294-2:2016 2.9 ± 0.3 4.1 ± 0.4 mg/L
Total Nitrogen * APAT IRSA 4060 A2 Man 29 2003 12.2 ± 0.5 103 ± 4 mg/L
Nichel UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 4.5 ± 0.1 15.0 ± 0.4 μg/L
Lead UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 0.10 ± 0.01 1.10 ± 0.01 μg/L
Copper UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 8.4 ± 0.2 25.0 ± 0.5 μg/L
Zinc UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 17.4 ± 0.4 41.0 ± 0.9 μg/L
Total Surfactants APAT CNR IRSA 5170 Man 29 2003 + APAT CNR IRSA 5180 Man 29 0.60 ± 0.04 0.60 ± 0.04 mg/L
2003
Total Hydrocarbons EPA 1664 Rev B 2010 <1 <1 mg/L

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E. Basiglini et al. Ecotoxicology and Environmental Safety 153 (2018) 54–59

Table 2
Reproducibility and accuracy of the analytical methods used to determine the wastewater composition. The data are based on proficiency test used by 250 Italian laboratories.

Parameters Analytical methods Precision Reproducibility Accurancy

(CV %) (CV %) (%)

pH APAT CNR IRSA 2060 Man 29 2003 0.27 2.97 97.65

Chemical Oxygen Demand (COD) APAT CNR IRSA 5130 Man 29 2003 5.57 8.00 90.57
Biochemical Oxygen Demand (BOD5)* APAT CNR IRSA 5120 Man 29 2003 4.57 13.54 85.58
Total Nitrogen * APAT IRSA 4060 A2 Man 29 2003 9.89 8.01 102.55
Total Phosphorus (P) UNI EN ISO 17294-1:2007 + UNI EN ISO 17294-2:2016 4.04 7.87 104.99
Nichel UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 2.71 10.00 107.53
Lead UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 1.13 14.98 107.49
Copper UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 2.01 10.00 103.70
Zinc UNI EN ISO 17294-1: 2007 + UNI EN ISO 17294-2: 2016 2.15 8.00 100.62
Total Surfactants Anionic Surfactants APAT CNR IRSA 5170 Man 29 2003 5.89 9.21 103.54
Non Ionic Surfactants APAT CNR IRSA 5180 Man 29 2003 13.41 20.01 112.14
Total Hydrocarbons EPA 1664 Rev B 2010 13 20.43 97.85

Table 3 to purple. The resulting solution was titrated with 0.0005 N NaPDC, the
Relative Growth Rate (RGR) of the plants at the end of the experiments. The data are the equivalence point was acquired potentiometrically by pH-meter Hanna
average of the determinations (n = 3) ± S.E.
Instruments mod. HI2550 (Italy).
Season Sample RGR
2.6. Statistical analysis
Summer Control 0.091 ± 0.005
Treated 0.068 ± 0.005
Winter Control 0.00 ± 0.00
The accuracy and efficacy of the methods, utilized to determine
Treated −0.032 ± 0.0014 wastewater composition, are described and reported in paragraph 2.5
and Table 2.
The results of the experiments are the mean of at least three re-
– Non -Ionic Surfactant plications with standard errors.
The normality was checked by Shapiro-Wilk test. For the data not
The specific surfactants represent the fraction of non-ionic ethoxy- showing normal distributions the variance analysis was performed by
lated surfactants that form with Dragendorff reagent (KBiI4 + BaCl2 in the Kruskal-Wallis non-parametric one way analysis of variance (PAST
glacial acetic acid), a precipitate where the combination of Bi-surfac- version 2.17c; Hammer et al., 2001; http://folk.uio.no/ohammer/past).
tant ratio is about 1: 1. The precipitate was dissolved and the bismuth All analyses were considered significant at p ≤ .05.
was potentiometric titrated with Pyrrolidindithiocarbamate sodium
(NaPDC) that the complex in the ratio 3: 1 (3 NaPDC: 1BI). 3. Results and discussion

2.5.4.1. Extract purification and analysis. To 1 L of sample are added
100 g of NaCl and 5 g of NaHCO3, the solution was transferred into a
sublator system with 100 mL of ethyl acetate. The sublation was carried
under nitrogen flow for 15 min after which the organic phase was
recovered.
This operation was repeated 2 more times with 100 mL of ethyl
acetate collecting the organic extract in a 250 mL flask. Then the extract
was dried with a rotavapor, and the residue was dissolved in 60 mL of
methanol and purified from possible anionic and cationic surfactants,
respectively, on a cation exchange column and subsequently the
column of anion exchange.
The purified extract in methanol was dried on a rotavapor and so-
lubilized in 5 mL of methanol and 40 mL of distilled water acidified
with 0.5 mL of 1% HCl.
To this solution was added 30 mL of Dragendorff reagent. The
precipitate obtained was filtered on a 0.8 µm filter. The filter was
transferred into beakers of 250 mL and the precipitate was solubilized
with a warm aqueous solution (80 °C) of ammonium tartrate to a final
volume of 200 mL. To this solution was added a few drops of bromine
cresol purple and NH3 at 16% until the indicator changes from yellow
Treatments of urban and industrial wastewaters are usually applied
with the aim to remove pollutants before discharging the effluents in
water bodies (Boari et al., 1997). The efficiency of the treatment can
vary according to both load of pollutants and efficacy of treatments, but
sometimes residues of contaminants are still present in the treated
waters (Batt et al., 2006; Luo et al., 2014); thus their presence may
represent a serious threat for the aquatic organisms.
The analyses of the treated wastewaters, sampled in the summer and
in the winter, showed diverse arrays of contaminants that may elicit
different toxicity mechanisms. The wastewaters contained different
amounts of HM, nitrogenous and phosphorus compounds, surfactants
and hydrocarbons (Tables 1, 2). Their concentrations varied in the two
seasons, depending on the industrial and municipal activities and effi-
ciency of treatments (Tables 1, 2). The levels of pH of water samples
were alkaline. COD as well as total phosphorus, nitrogen and HM (lead,
copper, nichel, zinc) were higher in the wastewater sampled in winter
(Table 1). The HM detected in the treated waters belong to both redox-
active (i.e. Cu) and non redox active (i.e. Ni, Zn) metals. The first can
directly generate oxidative injury, while the non-redox active metals
can indirectly cause oxidative stress (Valko et al., 2005; Bielen et al.,

Table 4
Amounts of photosynthetic pigments and phenolics at the end of the exposure.

Season Samples Chlorophylls (mg /g f. w.) Ratio a /b Carotenoids (mg/g.f.w.) Phenolics (mg/g.f.w.)

Summer Control 1.062 ± 0.016 a 2.79 0.21 ± 0.004 a 1.73 ± 0.09 a

Treated 1.044 ± 0.037 a 2.71 0.21 ± 0.01 a 1.43 ± 0.13 a
Winter Control 0.832 ± 0.015 a 2.58 0.15 ± 0.00 a 1.50 ± 0.04 a
Treated 0.960 ± 0.02 b 2.73 0.18 ± 0.001 b 1.96 ± 0.02 b

The data are the average of the determinations (n = 3) ± S.E. Different letters within the same season indicate significant difference at p ≤ .05.

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E. Basiglini et al. Ecotoxicology and Environmental Safety 153 (2018) 54–59

Table 5
Antioxidant enzymatic activities in the control and treated plants detected in the summer and the winter.

Season Samples CAT APX G-POD PPO

(EU /μg proteins) (EU /μg proteins) (EU /μg proteins) (EU /μg proteins)

Summer Control 65.27 ± 3.84 a 0.63 ± 0.06 a 7.83 ± 0.72 a 0.38 ± 0.00 a
Treated 68.63 ± 6.67 a 0.33 ± 0.03 b 3.27 ± 0.09 a 0.59 ± 0.01 a
Winter Control 38.44 ± 0.75 a 0.49 ± 0.07 a 11.57 ± 0.06 a 0.61 ± 0.01 a
Treated 43.97 ± 2.95 a 0.29 ± 0.04 b 7.52 ± 0.51 b 0.32 ± 0.04 a

The data are the average of the determinations (n = 3) ± S.E. Different letters within the same season indicate significant difference at p ≤ .05.

2013).
Lemna spp. have been reported to be a good tool both for analyzing
the toxicity of contaminants and for phytoremediation (Forni and
Tommasi, 2016). However, most of the studies have been performed in
the presence of single ion or molecule using artificial media (Forni
et al., 2012; Cvjetko et al., 2010; Hou et al., 2007; Drost et al., 2007;
Parra et al., 2012), and just few considered the industrial effluents
(Sasmaz and Obek, 2009; Radić et al., 2010).
In this work we wanted to determine in L. minor the effects of low
amounts of multipollutants, that are still present in the wastewaters
after the treatment, and of seasonal variation. For this purpose, duck-
weed was grown over a period of 7 days in treated industrial waste-
waters and in Hoagland medium (control) in summer and winter.
Respect to control medium, nitrogen content of effluents was lower
(56%) in the summer, but higher (273%) in winter samples. While
phosphorus content of effluents was always lower (76% summer; 66%
winter) than that of control medium, although it was in the wide range
of phosphorus concentration detected in waters where Lemnaceae are
living (Landolt, 1986).
Even though total nitrogen amount of effluent sampled in the
summer was higher than the nitrogen level reported by Landolt (1986),
the growth of treated plants compared to the control ones decreased by
25% in summer (Table 3). In winter the growth of the treated plants
was completely impeded (Table 3) because of higher load of pollutants
and lower temperatures. The latter also affected the growth of control
plants, in agreement to different authors, who reported seasonal var-
iation in growth rates of L. gibba and L. minor (Wang, 1987; Scherr
et al., 2008).
In the summer if compared to the controls, the amounts and ratio of
pigments of treated samples were not significantly different (Table 4). It
may hypothesized that the low amounts of pollutants and P did not
influence the chlorophylls content in this season. The data confirm that
decline of chlorophylls can be detected when the concentration of
contaminants is beyond certain level (Hou et al., 2007; Parra et al.,
2012; Hou et al., 2007; Forni et al., 2008). Vice versa, in the winter
chlorophylls were significantly higher in the treated plants: e.g. 13%
more chlorophylls and 17% more carotenoids than the controls
(Table 4). This may be probably due to the higher amount of nitrogen in
treated waters. It is worthwhile to observe that under our experimental
conditions no chlorosis was observed in the exposed plants.
Plant exposure to pollutants can cause a stress and an over-
production of ROS; consequently the balance between ROS production
and scavenging is altered (Mittler, 2002); such unbalance affects the
physiology of the plants eliciting a response (Emamverdian et al.,
2015). Plant response to counteract this stress involves the synthesis of
antioxidant molecules or the enhancing of the activity of antioxidant
enzymes (Emamverdian et al., 2015).
The acknowledgement of the importance of free radical damage in
the mechanisms of toxicity by contaminants provided a tool for the
application of biomarkers of oxidative stress in environmental studies
(Valavanidis et al., 2006). Therefore, the determination of the pro-
duction of antioxidant molecules, as phenolic compounds, and the de-
tection of changes in the activities of enzymes, like superoxide dis-
mutase (SOD), CAT, APX and G-POD, have been reported in several
studies (Forni et al., 2012; Forni and Tommasi, 2016; Ippolito et al.,
2007; Razinger et al., 2008).
Phenolics have a role as antioxidant molecules in plants, acting
likely in concert with other antioxidant molecules and scavenger en-
zymes in providing protection against oxidative stress. We detected an
increase (24%) in the amount of phenolics in the treated plants respect
to the control in the winter (Table 4), when the pollutants load of
wastewater was higher. While in the summer, the amount of phenolics
of treated samples was lower, even though not significantly.
The role of different antioxidant enzymes in counteracting the toxic
effect of ROS in stressful conditions has been evidenced by several
authors (Bernard et al., 2015; De Pinto et al., 2015). In our experiments,
seasonal variation affected significantly only G-POD activity of control
plants. Except for CAT in both seasons and PPO in the summer, treated
plants had lower enzymatic activities than the untreated ones (Table 5).
Enhancement of CAT activity in Lemna, grown in the presence of zinc,
has been reported by Uruc Parlak and Demirezen Yilmaz (2012), sug-
gesting a role of this enzyme in oxidative stress tolerance. PPO enzyme
is usually activated in the presence of pathogens, wound and pollutant
(Forni et al., 2012; Lavid et al., 2001). The combination of high tem-
perature and pollutants enhanced PPO activity of the treated plants
(36%) in the summer.
Even though the contemporary presence of multipollutants can in-
crease their toxicity (Cvjetko et al., 2010), and antioxidant enzymes
have been exploited as biomarkers (Radić et al., 2010), differently from
the reports by other authors (Uruc Parlak and Demirezen Yilmaz,
2012), the activities of peroxidases, APX and G-POD, were significantly
lower in the treated plants respect to the controls (Table 5). The ob-
tained results confirm that the upregulation of APX and G-POD depends
on the species, their level of tolerance, plant growth stage and con-
centration of contaminants (Gill and Tuteja, 2010; Forni et al., 2012).
4. Conclusions
Treated wastewaters can still contain low amounts of a mixture of
pollutants, with an over time fluctuating concentrations due to the
changing of contaminant load and efficacy of the treatment. The variety
of results suggests that besides nutrient/pollutant determining factor,
the effect may vary with season. In our work, the contemporary pre-
sence of multicontaminants and lower temperatures affect mostly the
growth, confirming the importance of environmental conditions on the
performance of duckweed. The data on the changes in the enzymatic
activities confirm the controversy existing in the literature about the
variability of enzymatic response in stress condition (Van Doorn and
Ketsa, 2014), thus the relevance of the use of biomarkers needs to be
carefully considered.
This study underlines the importance of the effluent composition
(nutrient status and pollutants) in determining the balance between
toxicity and growth inhibition, and shows that the margin between
growth and toxicity inducing concentrations can be enlarged taking in
consideration seasonal variability.

58
E. Basiglini et al.
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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