DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring

Journal Pre-proof DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring

Valerio Pellegri, Gessica Gorbi, Annamaria Buschini PII:

S0048-9697(19)35775-4

DOI:

https://doi.org/10.1016/j.scitotenv.2019.135780

Reference:

STOTEN 135780

To appear in:

Science of the Total Environment

Received date:

30 August 2019

Revised date:

4 November 2019

Accepted date:

24 November 2019

Please cite this article as: V. Pellegri, G. Gorbi and A. Buschini, DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.135780

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© 2019 Published by Elsevier.

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DNA damage detection by Comet Assay on Daphnia magna: application in freshwater biomonitoring.

Valerio Pellegri1, Gessica Gorbi1, Annamaria Buschini1,2 1

Department of Chemistry, Life Sciences and Environmental Sustainability, Parco

Area delle Scienze 11/a, 43124 Parma, Italy. Centre for Molecular and Translational Oncology-COMT, University of Parma,

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Parco Area delle Scienze 11/a, 43124 Parma, Italy.

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[email protected]

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Annamaria Buschini (Corresponding Author)

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Department of Chemistry, Life Sciences and Environmental Sustainability

Tel. +39 0521 905607

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Fax. +39 0521 905604

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Parco Area delle Scienze 11/a, 43124 Parma, Italy.

Valerio Pellegri [email protected] Department of Chemistry, Life Sciences and Environmental Sustainability Parco Area delle Scienze 11/a, 43124 Parma, Italy. Tel. +39 0521 905618 Fax. +39 0521 905402 1

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Gessica Gorbi [email protected] Department of Chemistry, Life Sciences and Environmental Sustainability Parco Area delle Scienze 11/a, 43124 Parma, Italy. Tel. +39 0521 905618

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Fax. +39 0521 905402

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ABSTRACT

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Monitoring of water genotoxicity still remains underexploited in risk assessment. The

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present study aimed at standardizing and evaluating the sensitivity and applicability

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of the Comet Assay adapted for Daphnia magna in genotoxicological investigations in freshwater environments. Two sampling campaigns (2014-2015) were performed

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in the watercourses of a pilot basin located in the Parma district (Italy). Fourteen

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sampling stations with different Ecological Status and/or EBI values were selected, all with a good Chemical Status according to the EU–Water Framework Directive 2000/60. The Alkaline Comet Assay was performed on 48h-aged daphnids exposed (24h) to 23 water samples. In parallel, the acute toxicity test was carried out. Daphnids exposed to samples, collected upstream the main watercourses in non impacted areas, showed low DNA migration (Tail Intensity percentage – TI% - in the range 2.97-13.21), similar to laboratory controls. An increase in genotoxicity (TI% in the range 20-40) proceeding from the mountain towards the plain area was observed, 2

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in agreement with the land uses and the ES/EBI values of the stations. The highest genotoxic damage was observed after exposure to samples from watercourses of the minor hydrographic network in the plain area and waterbodies receiving wastewater treatment plant outflows. A modified version of the Comet Assay able to identify the presence of genotoxins inducing DNA oxidative damage, after standardization, was applied to daphinids treated with waters from 4 selected monitoring stations. The

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presence of oxidative contaminants was detected downstream a wastewater treatment plant outflow.

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The Comet Assay on D. magna has proven to be sensitive and able to discriminate

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among differently impacted areas and might be applied routinely. The FPG-Comet

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proved to be able to highlight the presence of contaminants causing oxidative stress.

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In our knowledge, this is the first time that Comet assay on Daphnia magna is

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successfully applied for freshwater monitoring.

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KEYWORDS: Genotoxicity; FPG; DNA oxidative damage; EBI; WWTP.

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1. INTRODUCTION Although significant progress has been made to improve freshwater quality, many waterbodies still suffer from the impact of chronic contamination by many toxic pollutants at low concentrations. As a consequence, superficial water bodies can contain mixtures of chemicals that may constitute an ecological risk not predictable on the basis of the concentration of single components (Altenburger et al., 2015;

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Wernersson et al., 2015; Neale et al., 2017). The exposure to mixtures of a large number of chemicals, at concentrations supposed to be safe and environmentally

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acceptable, can cause severe damage to the biota given their additive or even synergic

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effect (Carvalho et al., 2014; Altenburger et al., 2015). Urban/industrial wastewater

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treatment is not able to remove completely a large part of contaminants that can be

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resistant to degradation and might be harmful to the aquatic ecosystems (Hecker and Hollert, 2011; Mijangos et al., 2018). Furthermore, some transformation products or

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metabolites are not monitored and it has been widely proved that countless

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genotoxicants from anthropogenic sources, such as industrial and agricultural activities, find in the aquatic environment the ultimate recipient (Eriksson et al., 2017; Muz et al., 2017). Effect-based assays are able to evaluate the negative impact of complex mixtures, without a prior knowledge of their detailed chemical profile, by analysing the responses of test organisms exposed to extremely complex environmental matrices as a whole (De Zwart et al., 2009; Wernersson et al., 2015; Brack et al., 2018). Effectbased assays can thus be regarded as key complementary tools, in addition to 4

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ecological and chemical investigation, to detect the potential negative impact from toxic contaminants (Altenburger et al., 2019). In particular, genotoxic agents could induce severe effects at very low concentrations, which are more easily studied through effect-based assays. Taking into account the “one health” approach, the presence of genotoxic xenobiotics in the aquatic ecosystems could both negatively influence the fitness of the biota but also pose a severe risk to humans (Destoumieux-

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Garzón et al., 2018).

Several assays employing microorganisms (Ames test and SOS umu/C assay),

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animals (micronucleus test, Comet Assay) and plants (chromosomal aberration and

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micronucleus test) are used, single or in battery, to reveal the genotoxic load of

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waters (Žegura et al., 2009; Boettcher et al., 2010; Radić et al., 2010; Tabrez et al.,

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2011).

The Comet Assay, or Single Cell Gel Electrophoresis assay (SCGE), is a simple,

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quick and inexpensive technique able to detect DNA damage in the form of strand

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breaks generated by the action of genotoxic agents (Singh et al., 1988; Collins et al., 1997; Møller, 2018). Virtually, any eukaryotic cell can be used for the test, without any prior knowledge of the genome structure. It has been a widely and successfully applied test for over 30 years on fungi, plants, invertebrate, fish and mammalians, humans included. It was adopted as a new standard assay by the Organisation for Economic Co-operation and Development (OECD) for mammalian in vivo biomonitoring (OECD, 2014), as well as accepted by the Food and Drug Administration (FDA) and World Health Organization (WHO). This assay has been 5

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fruitfully implemented for model/sentinel species i.e. organisms playing a special role in their natural ecosystems (Gajski et al., 2019). It has been applied on freshwater organisms such as Chironomus riparius (Lee et al., 2008; Im et al., 2019), Gammarus fossarum (Lacaze et al., 2010; Lacaze et al., 2011), bivalves (Frenzilli et al., 2009) such as Corbicula fluminea (Rigonato et al., 2005; Rigonato et al., 2010; de Oliveira et al., 2016), Unio pictorum (Štambuk et al., 2009; Kolarević et al., 2016), Dreissena

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polymorpha (Pavlica et al., 2001; Buschini et al., 2003; Michel et al., 2013), Limnoperna fortune (Villela et al., 2006; do Amaral et al., 2019), as well as the snail

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Biomphalaria glabrata (Grazeffe et al., 2008; Ibrahim et al., 2018), anellidae

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(Mihaljević et al., 2011) and planarians (Guecheva et al., 2001; Prá et al., 2005).

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A Comet Assay protocol adapted to Daphnia magna (Crustacea, Cladocera), a

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worldwide-used model species, was recently developed and standardized (Pellegri et al., 2014), driven by the perspective of employing this test method in further eco-

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genotoxicological studies on freshwater ecosystems. A facilitation for the routinely

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application of this protocol in freshwater monitoring is represented by the fact that daphnid breeding is relatively easy, quite inexpensive and requires little space and time investment. The potential applicability of the Comet Assay with D. magna in detecting DNA damage induced by single compound or mixtures has recently gained attention (Galdiero et al., 2015, Lavorgna et al., 2016; Parrella et al., 2015; Silva et al., 2015), but still there isn’t any application of this bioassay in field biomonitoring. A modification of the standard Comet Assay provides the use of enzymes of the excision repair system for the evaluation of DNA oxidative damage (Collins, 2014). 6

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Formamidopyrimidine [fapy]-DNA glycosylase (FPG) recognizes and cuts oxidized bases, mostly purines (7, 8-dihydro-8-oxoguanine, 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil) (Tchou et al., 1994). When this enzyme nicks DNA at sites of oxidatively damaged nucleotides, it creates strand breaks detectable by the alkaline Comet Assay. In aquatic organisms, DNA oxidative damage has been linked to

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developmental toxicity, in particular in relation with pesticide contamination of waters (Pašková et al., 2011).

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The aims of the present study were i) the set-up of the protocol for FPG-modified

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Comet Assay with D. magna; ii) the evaluation of the applicability of Alkaline and

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FPG Comet Assay for eco-genotoxicological investigations in freshwater

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environments.

In our knowledge, this is the first time that Comet assay on Daphnia magna is

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successfully applied for fresh water monitoring. Introducing new tests to reveal the

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presence of genotoxins using Daphnia magna could improve the use of this well known biosensor in environment monitoring, in line with the EU Water Frame Directive (WFD 2000) that suggests the reduction of the number of bioassays involving medium-sized species.

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2. MATERIAL AND METHODS

2.1 Test organisms Parthenogenetic adult females of the species Daphnia magna Straus were cultured in natural water (from hereafter referred as NW), commercially available and suitable for rearing daphnids, previously aerated for 24 h. The main physical-chemical

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characteristics of NW are reported in Figure S1 (Supplementary material). The cultures were maintained in a climate controlled chamber (temperature =

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20±1°C; photoperiod 16h light: 8h dark provided by 1000 lux cool-white fluorescent

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lamps) at a population density of 3 individuals/100 ml and fed, at renewals, with

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aliquots of stock cultures of the unicellular green alga Raphidocelis subcapitata

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(6105 cells/ml) in exponential growth phase, cultured according to the method by US-EPA (1978), and Saccharomyces cerevisiae (6105 cells/ml) according to the

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method by APAT and IRSA-CNR (APAT IRSA-CNR, 2003). The breeding medium

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was completely renewed twice a week transferring adult females into crystallizing dishes containing fresh NW and food. After a 7-week period, cultures were discarded and started again with newborn organisms.

2.2 Comet Assay on D. magna. 2.2.1 Alkaline Comet Assay: further standardization Alkaline Comet Assay was performed as described in (Pellegri et al., 2014) with minor modifications. 8

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To assess if the soaking time in Buffer P (0.1M phosphate buffer, 0.2% citric acid, 0.1M NaCl, 1mM EDTA, pH=7.8) prior to haemolymph extraction might influence the TI% in control daphnids (i.e. daphnids maintained in NW; 3 replicates), dedicated independent experiments were performed by crushing daphnids after different time of permanence in the buffer, i.e. immediately, 10 min, 20 min and 30 min after transferring from the breeding medium to the buffer, at 20±1 °C temperature. As a

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control of the extraction process, we monitored the presence of small rounded shaped cells characteristic of haemolymph by microscopic analysis. For each treatment, two

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slides were prepared and 50 cells per slide were scored. The median of TI% of 50

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nuclei per slide was calculated.

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2.2.2 FPG-modified Comet Assay: standardization

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A modification of the Comet Assay has been developed to assess the oxidative damage to DNA in Daphnia magna.

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Starting from the protocol described in Pellegri et al. (2014) and evidences obtained

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from published papers (Barata et al., 2005; Gómez-Oliván et al., 2014) the following steps were investigated:

- washing buffer: after lysis and before the enzymatic treatment, slides were washed 3 times (5 min each) at 4°C in a washing buffer. Two different buffers were tested: Trevigen Buffer FlareTM and 4-(2-hydroxyethyl)-1-piperazinee ethane sulfonic acid (HEPES), both at pH 7.4 and 8.0. - cleaving enzyme: Endonuclease III (ENDOIII) Trevigen® and Formamidopyrimidine DNA glycosylase (FPG), kindly provided by Prof. Andrew 9

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Collins, were considered the candidate enzymes to evaluate oxidative damage. Enzyme stock solutions (1U/100μl) were stored at -80°C in 1.5 ml microtubes and defrosted on the day of the assay. - Reaction Buffer: ENDOIII was diluted in HEPES (pH = 7.4) or NAR (10 mM TrisHCl, 100 mM NaCl, 1 mM EDTA); FPG was diluted in HEPES (pH = 8.0) prepared according to Gómez-Oliván et al. (2014): 40 mM HEPES, 0.1 M KCl, 0.5 mM

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EDTA, 0.2 mg/ml Bovine Serum Albumin (BSA). 25 μl of enzyme solution was inoculated on the 22x22 mm agarose spots containing the lysed nucleoids.

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- Control Buffer: untreated spots were inoculated with 25 μl of Phosphate Buffered

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Saline (PBS) or the same reaction buffer in which the enzyme was diluted.

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- Incubation time: slides were covered with a squared cover slip to spread the enzyme

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homogeneously over the LMA (Low Melting Agarose) layer and placed in a moist box at 37°C. The following incubation times were tested: 30 min (ENDOIII), 20 min

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(FPG) or 45 min (Gómez-Oliván et al., 2014) (FPG).

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- Unwinding, electrophoretic time and pH: 7 and 10 minutes of unwinding were tested, together with 10 and 15 minutes of electrophoretic run at pH>13 or 12.1. - Reference oxidative agent and concentrations: H2O2 was tested at 1 and 2 µM; Menadione (2-methyl-1,4-naphtoquinone; vitamin K3) at 200 and 400 µg/l, due to its 48h LC50 equal to 491.3 µg/l; Cu2+ was tested at 10 and 20 µg/l, due to its 48h LC50 equal to 37.9 µg/l (Barata et al., 2005) - Image magnification: due to the additional DNA fragmentation caused by the enzymatic activity, 200x and 400x magnification were evaluated when scoring with 10

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Comet Assay IV software (Perceptive Instruments Ltd, UK) under a Leica DMLS fluorescence microscope (excitation filter BP 515-560 nm, barrier filter LP 580 nm), after staining agarose spots with 20 μl of ethidium bromide (10 μg/ml). Three replicate per condition were performed in each experiment. For each replicate, two slides were prepared and 50 cells per slide were scored. The median of TI% of 50 nuclei per slide was calculated.

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2.3 Freshwater monitoring 2.3.1 Study area

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The study area, located in the Parma province (Northern Italy) covers two hydro-

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ecoregions, namely HER_6 (Pianura Padana) and HER_10 (Appennino

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Settentrionale), and is part of the largest River Basin in Italy (ITB, Po River Basin).

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On one hand, the Parma province is hosting several natural areas that are particularly important for their ecosystem quality and services. On the other hand there are 330

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WWTPs (Fava, 2007), with 2 main plants insisting on the area: East-WWTP

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(130,000 population equivalent) and West-WWTP (168,000 population equivalent) whose presence was taken into account in this study. The hydrographic network is composed of three major watercourses: Parma Stream (a Po River tributary; 815 km2 area, 94 km length), which is the main watercourse, and two tributaries, Baganza (225 km2 area, 50 km length) and Cinghio (36 km2 area, 22 km length) stream. Alongside, watercourses belonging to the minor hydrographic system were sampled. All waterways, except one canal, are gathering into the main stream (Parma Stream) before its inflow into the Po River. 11

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2.3.2 Water and macroinvertebrate samplings Fourteen stations (Fig. 1, Table 1) were selected in order to cover a wide typology of water bodies subjected to different pressures as identified in the area. PS0, BS0 and CS0 stations, being located in a mountain area, not so far from the head of the rivers, are presumably not contaminated; in particular, PS0 and BS0 stations were located in river sections designated for specific use (i.e. fisheries). Four stations (NC0, NC1,

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NC2 and AC) were located on minor watercourses receiving the outflow from the two main WWTPs, and were therefore considered as presumably polluted.

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Sampling of all surface waters was carried out during 2014 and 2015, in spring or

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autumn (Table 2). The most critical stations were sampled more than once during the

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two-year monitoring period. Each sample was collected at least 2 weeks after major

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precipitation in clean 25 l high-density polyethylene (PEHD) tanks, leaving no headspace, and immediately transported to the laboratory, in refrigerated containers,

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to record pH and conductivity values and to perform acute toxicity assays. In

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addition, a dedicated and simultaneous water sampling campaign, carried out on a single day, was performed on the watercourse receiving the East-WWTP discharge (NC stations). Samples were pre-filtered on GF/A glass microfiber filters (Whatman), then filtered on 0.45 μm mixed cellulose ester sterile filter to minimize the presence of particulate matter (thus reducing the total bacteria load and undesired/uncontrolled food sources) and stored in darkness at 4°C in clean PEHD containers.

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The extended biotic index (EBI) of each section was obtained from ARPA (PR) reports on surface water quality (Regione Emilia Romagna, 2015); when not available the EBI was calculated by sampling the macrobenthic fauna in situ on the day of the water sampling. Macroinvertebrates were qualitatively sampled using a Surber net (mesh size 500 µm) in four sampling points on the watercourse station following a linear transect that took into account riffle and pool areas to cover as

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much as possible different microhabitats and different current velocity. When the water column was too deep to reach the bottom sediment by hand, a kick-sample was

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performed. Right after sampling, macrobenthic invertebrates were pooled into 100 ml

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glass bottles and fixed with 96% ethanol until further taxonomic identification that

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was carried out under a stereomicroscope in the laboratory. EBI index was calculated

2.3.2 Acute Toxicity

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according to the APAT IRSA-CNR guideline (APAT IRSA-CNR, 2003).

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Acute toxicity tests on environmental samples were performed in accordance to the

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standard protocol by APAT IRSA-CNR (APAT IRSA-CNR, 2003). Ten daphnids aged ≤ 24h, born to adults bred in standard condition as described in par 2.1. (the first 3 broods were discarded), were placed in 100 ml of unfiltered water samples on the very same day of sampling. Control replicates were performed in NW. No food was added. The tests were carried out at the same conditions as for the cultures. The number of immobilized or dead specimens was recorded after 24, 48 and 72h exposure. The appropriate sensitivity of animals has been regularly checked using potassium dichromate as reference chemical (Figure S2, Supplementary material). 13

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2.3.3 Alkaline and FPG-Comet Assay. The Alkaline Comet Assay was performed on filtered samples, within 48h after sampling, according to the method by Pellegri et al.(Pellegri et al., 2014), taking into account the procedure improvements reported in the present study. Laboratory control replicates were performed in NW. Three replicates, each consisting of 10 juveniles (48 h age) of D. magna, were used. Only alive daphnids at the end of the

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exposure time (24 h) were collected for haemolymph extraction. As positive genotoxic agent H2O2 (10 µM) was used (Fig S3, Supplementary material).In

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addition, to assess if sample storing time might alter the overall genotoxicity,

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dedicated experiments were executed on filtered and unfiltered samples both on the

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very same day of sampling and after 2 months of storage at 4°C.

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To evaluate the oxidative damage, FPG-Comet Assay was applied to selected environmental samples. In this case, as representative parameter of DNA oxidative

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stress, ΔTI% was calculated, for each sample, as the difference between the DNA

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migration (TI%) registered on enzyme (FPG)-treated slides and the TI% registered on slides without enzymatic treatment..

2.4 Statistical analysis The software IBM SPSS Statistics® v.21 was used for statistical analysis. To compare the results (TI%) obtained from assays on daphnids exposed to environmental samples from the various stations of the studied basin, data from the two sampling campaign were pooled. Levene’s test was firstly applied to evaluate 14

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variance homogeneity. When homoscedasticity was verified, comparison was performed by ANOVA (Log TI%) and Tukey’s test as post hoc. The Student’s t test was applied to evaluate the significance of the differences between TI% values from

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+FPG and –FPG assays and between preserved and non-preserved samples.

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3. RESULTS 3.1 Alkaline Comet Assay: further standardization Independent experiments were performed to assess if permanence of daphnids in the buffer prior to haemolymph extraction might influence the TI% in control replicates. Daphnids were crushed immediately, 10 min, 20 min and 30 min after their transfer from the breeding medium to the buffer. TI% values in control replicates

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progressively decreased with the increase of the soaking time in buffer (Supplementary material: Figure S4), suggesting that a period of at least 20 min of

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soaking prior to haemolymph extraction ought to be introduced when performing the

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Comet Assay on D. magna. No significant difference was detected between the

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independent experiments.

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3.2 Standardization of the oxidative Comet Assay The first set of experiments, performed to identify a suitable reference oxidative

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agent for the oxidative Comet Assay, highlighted that H2O2 (hydrogen peroxide),

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using ENDOIII as candidate enzyme, was not adequate for this purpose (Tab. 2). The mean TI% values were coupled with too high standard deviations. For this reason, Cu2+ and Menadione were tested in substitution. Menadione was a far better reference oxidative agent than Cu2+ (data not shown), inducing a higher TI% compared to the replicates spotted without ENDOIII either in NAR or HEPES, witnessing the effective cleaving action performed by the enzyme at electrophoresis pH=12.1 (Supplementary material: Fig. S5). For this reason,

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Menadione was chosen as the reference oxidative agent for the subsequent experiments. In addition, FPG as cleaving enzyme and HEPES as reaction and control buffer were used to improve the methodology. FPG activity was amplified after 45 min of incubation compared to 20 min and the best combination of unwinding and electrophoresis timings were 10 min + 15 min, yielding good results especially when

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the electrophoresis buffer was at pH=12.1. Controls with a high level of integrity, facilitate the discrimination between basal DNA damage and the main target of the

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assay that is to highlight the enzyme activity related amount of oxidised pyrimidines,

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particularly 8-OH-dGs.

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Due to the enzymatic activity and the relatively small dimension of D. magna’s

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nucleoids, comet tails are longer and nucleoids fluorescence is weaker compared to the classic Comet Assay on mammalian cells. Therefore, 200x and 400x

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magnifications were compared during scoring to evaluate feasibility and

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representativeness of this stage. Since no substantial differences were observed in TI% values (data not shown) when scoring the same spot at the two conditions, a 200x magnification, that allows a faster scoring, is advisable. The FPG-Comet Assay protocol adapted for D. magna is summarized in the Figure S6 (Supplementary material). 3.3 Freshwater quality monitoring 3.3.1 Chemical and ecological status

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The fourteen monitored stations are subjected to various levels of anthropogenic pressure and have different Ecological Status (from good to bad, sensu Water Framework Directive 2000/60/EC) and/or EBI values (from 10-9 to 2) (Table 2). Downstream, in the plain area, all the watercourses of the studied basin are suffering from diffused (pesticides, atmospheric deposition, pollutant leaching from fertilised lands) and point (WWTPs, discharges of industrial and livestock farming activities)

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sources of pollution. According to the report by the Regional Agency for Environmental Protection (Regione Emilia Romagna, 2015), which refers to the

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period 2010-2013, heavy metals do not seem to pose major threats for freshwater

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quality, whereas organic (herbicides, insecticides, brominated diphenyl ethers) and

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inorganic compounds (nitrates, ammonium, total phosphorus) are considered as

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criticalities (Table 1). Nevertheless, where the chemical status (CS) was monitored it resulted “good” according to the EU – Water Framework Directive 2000/60.

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The level of water pH was slightly/moderately alkaline, falling in the range suitable

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for Daphnia magna (pH 6-9; OECD, 1984). The electrical conductivity increased from the mountain sites (PS0, BS0, CS0) downstream to the plain stations (Table 2). 3.3.2 Acute Toxicity Acute toxicity was found in two samples from stations located close to the WWTPs outflows (NC1 and AC sites); in most of the samples no or negligible (less than 20%) acute toxicity was observed (Supplementary material: Fig. S7). In the toxic samples, daphnid immobilization increased with the exposure time and, in the sample from NC1 station, reached almost 90% within 48 h; this percentage is, however, 18

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considered acceptable from the Italian legislative point of view since it was below 50% after 24 h (Testo Unico Ambientale 152/06, 2006). 3.3.3 Alkaline Comet Assay The levels of genotoxicity of freshwater samples collected in the pilot basin are reported, as TI%, in Fig. 2. Daphnids exposed to mountain samples (PS0, BS0 and CS0) generally showed low

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TI% values, not statistically different from laboratory controls (i.e organisms maintained in NW). This response was observed in all samples collected over the

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two-year campaign in mountain stations. Low TI% values were also observed in

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daphnids exposed to the samples from PS1 and BS1 stations, located in hill areas

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moderately impacted by anthropic activities. Samples from stations CS1 and CS2,

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located on Cinghio Stream, caused instead a significantly higher DNA damage respect to control and the up-stream station CS0.

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The highest genotoxic effect was observed after daphnid exposure to samples from

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watercourses of the minor hydrographic network (GC and MC stations) in the plain area and from stations of courses receiving wastewater treatment plant outflows (NC1, NC2 and AC). By comparing the effect of the samples from NC stations, simultaneously-collected during the dedicated campaign in 2015, a significant increase in TI% values appears in daphnids exposed to the water from stations downstream the implant discharge, both close to (NC1) and 11 km far (NC2) from it, with respect to the upstream station (NC0), highlighting the detrimental impact of the wastewater treatment plant effluent on this waterbody and its inability of resilience 19

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(auto-depuration) (Fig. 2). Repeated samples from these stations, as well as station PS2 (located at basin closure just before Po river), always showed a high genotoxicity over the two-year monitoring, suggesting a consistent contamination by genotoxic substances at relevant levels. With regards to the effect of the storage time span (at 4°C) on sample overall genotoxicity, filtered samples showed no significant alterations in mean TI% values

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after 2 months of storage (Fig. 3). On the contrary, a significant reduction in genotoxicity was observed when filtration was not performed before storing.

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3.3.4 FPG-Comet Assay

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Selected samples collected in the pilot basin were subjected to FPG-Comet to

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measure oxidative stress (pH = 12.1). (Fig. 4). No oxidative damage was detected in

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daphnids exposed to the sample from the mountain station PS0, confirming the data returned from the Alkaline Comet Assay. Interestingly, sample from station NC2

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showed a marked difference in TI% with or without enzyme treatment (Δ

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TI%=11.62) due to the cleaving action of FPG. This witnesses the presence of genotoxic agents that significantly and specifically act on DNA causing oxidative stress.

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4 DISCUSSION

4.1 Set up of the FPG-Comet Assay Comet assay could be improved in its ability to detect DNA alterations introducing extra steps of enzyme digestion to identify specific DNA lesions, such as base alkylation and in particularly base oxidation. Generally, endonuclease III (ENDOIII)

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is used to detect oxidized pyrimidines and formamidopyrimidine and DNA glycosylase (FPG) to detect oxidized purine, mainly 8-oxoguanine (Collins, 2014).

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We have successfully adapted the FPG-Comet Assay to D. magna. This extension of

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the Alkaline Comet Assay is promising since oxidative stress seems to be one of the

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major contributors to DNA damage in a variety of studies dealing with polluted

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environments (Kolarević et al., 2016). It is important to remind that the generation of reactive oxygen species is one of the principal mode of action of pollutants present in

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the list of priority chemicals of the WFD 2000 (Mitchelmore and Chipman, 1998;

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Napierska et al., 2018) .

4.2 Application in monitoring freshwater quality To our knowledge, the present study constitutes the first application of the Comet Assay with D. magna in monitoring campaigns on a complex network of stations. Various authors have recognized the high sensitivity of the Comet Assay in identifying DNA damage, especially as exposure biomarker, with respect to other genotoxicity assays (Kim and Hyun, 2006; Frenzilli et al., 2009; Vasquez, 2010;) 21

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Kračun-Kolarević et al. (2016) reported that Comet Assay, performed in fish, had a higher sensitivity in comparison with micronucleus test, showing the highest potential in discrimination among river stations under low and high extent of anthropogenic pressure. Significant correlations between concentrations of hazardous priority compounds and levels of DNA damage in mussel haemocytes and fish erythrocytes from specimens collected in the Danube river were reported by several

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authors (Kolarević et al., 2016 and Deutschmann et al., 2016). Although in situ exposure of transplanted organisms or analysis of native specimens can be regarded

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as representative of the ecosystem complexity and take into account all co-occurring

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environmental variables, organism responses might be influenced by natural factors,

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such as temperature (Buschini et al., 2003; Pellacani et al., 2006; Kolarević et al.,

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2013), oxygen level (Pellacani et al., 2006), water flow (Guidi et al., 2010), metabolic status and physiological adaptation (Regoli, 1998). In addition, native species can not

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be evenly distributed, in space and time, in the studied basin or can display avoidance

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behaviour or migration. In both cases, for the reasons reported above, the identification of a reference station may encounter difficulties due to the possible lacking of pristine sites/populations in the studied basin. In our study, daphnids were exposed in vivo to water samples under laboratory constant/controlled conditions. The in vivo exposure, avoiding sample manipulations, assures ecologically relevance to the test and the use of highly standardized procedure/condition, at the same time, allows excluding natural confounding factors.

22

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Two mountain stations located in the main watercourse and its principal tributary (PS0 and BS0) and one located ahead of a minor tributary (CS0) were selected as possible reference stations, according to physical-chemical and biological data provided by the Emilia Romagna Regional authority (Regione Emilia Romagna, 2015). Daphnids exposed to water samples from these stations showed a DNA damage similar to laboratory controls over the two-year campaign, proving that these

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stations can be regarded as reliable reference sites. The identification of reference stations would be undeniably preferable in the application of the Comet Assay in

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freshwater monitoring, although the “best available site” concept (Kolarević et al.,

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2016) could be adopted. However, in our study, the similarity between the response

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(TI% values) of organisms exposed to the superficial water from not-impacted areas

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and laboratory control organisms suggests that the latter could be used as surrogate reference when reference stations can not be found/achieved. A significant reduction

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in genotoxicity was observed in preserved unfiltered samples, suggesting that, to

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minimize alterations in genotoxicity, it is advisable to filter water just after sampling. Comparison between daphnid responses to filtered and unfiltered preserved samples could thus give information about the persistence of the global genotoxic load. At a basin scale, acute toxicity was found in water sampled close to WWTPs outflows and a general trend of increase in genotoxicity proceeding from the mountain towards the plain area was observed, in agreement with the ES (sensu WFD) and EBI values of the various stations, land uses and anthropogenic pressures. In daphnids exposed to the samples from stations having a good ES and/or a high 23

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EBI value, constantly absent/negligible genotoxic effect was observed, while significant DNA damage was evidenced in daphnids exposed to samples from stations impacted by industrial and agricultural activities and characterized by poor/bad ES/EBI despite a good CS. The only exception was the sample from station NC0: despite a bad ES and very low EBI values, as the other stations of Naviglio Canal, water samples from this station did not cause significant DNA damage and/or

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acute toxicity. The ES, however, does not only depend on water quality: this means that, by the use of these simple assays, water quality (toxic and/or genotoxic

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contamination) can be excluded as the cause of a non WFD-compliant ES of NC0

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hydromorphological alterations).

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station and requalification intervention can be driven on other possible factors (e.g.

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Despite all the studied stations showed a good CS (sensu WFD) (as reported in Regione Emilia Romagna, 2015), contamination by a number of toxic substances

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(herbicides, insecticides, fungicides and heavy metals), even if at concentrations

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below their single acceptance criterion, was frequently detected in waters from many stations located in the plain area (stations CS2, AC, GC, NC2 and PS2). It is remarkable to highlight that the network of minor watercourses falls outside the area of surveillance according to the WFD. Nevertheless, the station located at the basin closure (station PS2) could suffer the impact of contaminant load from the confluence of Naviglio and Galasso canals. Surface waters are at the same time used for and impacted by agricultural practices and, to a lesser extent, are a source of drinking water. Serious concern arises when witnessing that waters flowing in watercourses 24

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receiving WWTP outflows can actually be directly reused for irrigation as long as specific physical/chemical/microbiological criteria are fulfilled in the effluent, according to Italian legislation. As a whole, the results demonstrate the sensitivity of the Comet Assay with D. magna and its capability to discriminate among different environmental situations, suggesting that this assay can represent a useful tool in aquatic biomonitoring. The

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daphnid response occurs within a brief time period (24 h), so that the test is quick, relatively cheap and can be applied routinely. In addition, it is based on a world-wide

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used model organism regarded as one of the most sensitive species to aquatic

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contaminants.

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These characteristics can make the Comet Assay with Daphnia magna a suitable

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candidate for a bioassay battery to be applied in water quality evaluation. As suggested by Brack et al. (2017), there is a need “to investigate and define a

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minimum and a higher tiered bioassay battery for the evaluation of water samples

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within the WFD”, since effect-based tools are able to reveal the risk due to nondetected compounds and to unknown mixtures.

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5. CONCLUSIONS

The Comet Assay with D. magna has proven to be a promising tool in freshwater biomonitoring. The results of the two-year monitoring campaign in the pilot basin show high level of genotoxic pollution in the majority of the watercourses in the plain area, where anthropic activities (agriculture, industry and livestock farming) and

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WWTP outflows are severely and intensely impacting aquatic ecosystems, downgrading the Ecological Status. The results of this first application of the Comet

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Assay with D. magna in a spatial and time scale monitoring are promising because of

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a number of positive aspects:

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impacted sections;

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i) the test has proven to be sensitive and able to discriminate among differently

ii) it is quick and might be applied routinely;

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iii) D. magna is one of the most used species in aquatic toxicology, even for

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regulatory measures such as in evaluating the acceptability of effluent discharges in surface waters, and the present study suggests a possible extension of its application in the biomonitoring with a little effort required by the operators; iv) water samples can be possibly preserved at 4°C after filtration even on the long-term without appreciable changes in overall genotoxicity; v) specific oxidative damage can be detected using the FPG-Comet Assay we set up as described in this paper. 26

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Our results show that waters receiving WWTP outflows are consistently highly genotoxic and, in some cases, cause acute toxicity in D. magna. The effective safety and harmlessness of the practice of treated wastewater reuse for irrigation of products for human consumption needs to be further investigated, as it is an urgent task. In light of our evidences, the inclusion of genotoxic bioassays in the battery of tests required by legislation to evaluate the suitability of this typology of surface water is

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highly recommendable, since neither acute toxicity tests nor threshold concentrations (MAC, AA) or microbiological parameters are able and suitable to estimate the risk

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posed by genotoxic agents.

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AKNOWLEDGMENTS

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We thank the Parma section of ARPAE (Emilia Romagna) for useful advice and

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technical assistance. We thank Dr Mirca Lazzaretti for graphical assistance.

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Funding: This work has been carried out in the frame of the activities of the ‘COMPHUB’ initiative, funded by the ‘Department of Excellence’ Project of the Italian Ministry for Education, University and Research (MIUR).

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planarians. Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 585, 79–85. doi:10.1016/j.mrgentox.2005.04.002 Radić, S., Stipaničev, D., Vujčić, V., Rajčić, M.M., Širac, S., Pevalek-Kozlina, B., 2010. The evaluation of surface and wastewater genotoxicity using the Allium cepa test. Sci. Total Environ. 408, 1228–1233. doi:10.1016/j.scitotenv.2009.11.055 Regione Emilia Romagna -Assessorato Difesa del Suolo e della Costa, P.C. e P.A. e della M., 2015. Valutazione dello stato delle acque sotterranee 2010 - 2013. 37

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Loureiro, S., 2015. Ecotoxicity and genotoxicity of a binary combination of triclosan and carbendazim to Daphnia magna. Ecotoxicol. Environ. Saf. 115,

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Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. doi:10.1016/0014-4827(88)90265-0 Štambuk, A., Pavlica, M., Vignjević, G., Bolarić, B., Klobučar, G.I.V., 2009. Assessment of genotoxicity in polluted freshwaters using caged painter’s mussel, unio pictorum. Ecotoxicology 18, 430–439. doi:10.1007/s10646-009-0297-2 Tabrez, S., Shakil, S., Urooj, M., Damanhouri, G.A., Abuzenadah, A.M., Ahmad, M., 2011. Genotoxicity testing and biomarker studies on surface waters: An 38

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overview of the techniques and their efficacies. J. Environ. Sci. Heal. - Part C Environ. Carcinog. Ecotoxicol. Rev. doi:10.1080/10590501.2011.601849 Tchou, J., Bodepudi, V., Shibutani, S., Antoshechkin, I., Miller, J., Grollman, A.P., Johnson, F., 1994. Substrate Specificity of Fpg Protein: Recognition and cleavage of oxidatively damaged DNA. J. Biol. Chem. 269, 15318–15324. doi:10.1021/bi1014453

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and repair in haemolymph cells of golden mussel (Limnoperna fortunei) exposed to environmental contaminants. Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 605, 78–86. doi:10.1016/j.mrgentox.2006.02.006 Wernersson, A.S., Carere, M., Maggi, C., Tusil, P., Soldan, P., James, A., Sanchez, W., Dulio, V., Broeg, K., Reifferscheid, G., Buchinger, S., Maas, H., Van Der Grinten, E., O’Toole, S., Ausili, A., Manfra, L., Marziali, L., Polesello, S., Lacchetti, I., Mancini, L., Lilja, K., Linderoth, M., Lundeberg, T., Fjällborg, B., Porsbring, T., Larsson, D.G.J., Bengtsson-Palme, J., Förlin, L., Kienle, C., Kunz, 39

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P., Vermeirssen, E., Werner, I., Robinson, C.D., Lyons, B., Katsiadaki, I., Whalley, C., den Haan, K., Messiaen, M., Clayton, H., Lettieri, T., Carvalho, R.N., Gawlik, B.M., Hollert, H., Di Paolo, C., Brack, W., Kammann, U., Kase, R., 2015. The European technical report on aquatic effect-based monitoring tools under the water framework directive. Environ. Sci. Eur. 27, 1–11. doi:10.1186/s12302-015-0039-4

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1460. doi:10.1016/j.chemosphere.2009.02.041

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Figure legends

Fig. 1. Pilot basin. White circles=sampling stations. Diamond = Parma town. WWTP = WasteWater Treatment Plant. 1 column

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Fig. 2. Alkaline Comet Assay in D. magna. Genotoxicity of waters sampled in the various stations of the studied watercourses. Error bars represent the standard

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deviation. **: significantly different from the laboratory control (ANOVA and

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Tukey’s test; p<0.01). Different letters label significantly different (p<0.05) stations

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within each watercourse; letters were omitted when no difference was detected.

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Positive control (H2O2 10µM) TI% = 20.37 ± 7.59 (mean±sd of independent

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2 columns

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experiments performed in 2014 and 2015).

Fig. 3. Comet Assay on D. magna. DNA damage (TI%) after daphnids exposure (24h) to filtered/unfiltered freshwater samples maintained for a different time span at 4°C. Error bars represent the standard deviation. Positive control (H2O2 10µM) TI% = 24.14 ± 6.68. Statistical analyses between fresh and stored samples were performed; * p>0.05 Student’s t test. 1 columns

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Fig. 4. FPG Comet Assay on D. magna. DNA damage (TI%) after daphnids exposure (24h) to freshwater samples without (-FPG) or with (+ FPG) digestion with FPG glycosylase. Unwinding and electrophoresis pH = 12.1. Error bars represent the standard deviation. In brackets relative Δ TI% (TI% +FPG – TI% -FPG). Positive control: Menadione (Men) 200µg/L. Statistical analyses between FPGtreated and untreated samples were performed; * p>0.05 Student’s t test.

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1 column

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Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof Table 1. Codes and characteristics of stations located in the pilot basin. Organic and inorganic criticalities: substances that represent a warning for the Italian Environmental Agency. Numbers in brackets represent the mean concentrations recorded in freshwaters during the 2013-2014 period by the Regional Environmental Agency of Emilia Romagna. LRA = Land Reclamation Authority.

CODE

PS0

WATER COURSE

Parma Stream

CHARACTERISTICS

ORGANIC

INORGANIC

CRITICALITIES

CRITICALITIES

Reference station located in a

PS1

Parma Stream

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mountainous area (785 msl) Moderately impacted area by food industries (165 msl) Located at the basin closure (24 msl) CS0

Cinghio Stream

Cinghio Stream

Station

located

ahead

the

Impacted area by agricultural

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and livestock farming activities (110 msl) Cinghio Stream

Impacted area by agricultural,

Herbicides, insecticides

na

CS2

livestock farming and industrial

P-tot (0.27 mg/l)

Reference station located in a

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Baganza Stream

N- NO3 (11.3 mg/l) N-NH4 (0.76 mg/l)

activities (88 msl) BS0

N-NO3 (3.76 mg/l)

herbicides and insecticides

watercourse (271 msl) CS1

Brominated diphenyl ethers,

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Parma Stream

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PS2

mountainous area (804 msl) Baganza Stream

Moderately Impacted area by

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BS1

agricultural

and

livestock

farming activities (304 msl)

NC0

Naviglio Canal

Waterbody

of

the

minor

network in the plain area (40 msl) subjected to urban and industrial impacts; this station was

located

1 km before

WWTP1 outflow NC1

Naviglio Canal

Waterbody

of

the

minor

network in the plain (38 msl) subjected

to

urban

Herbicides, insecticides

N-NO3 (5.89 mg/l) N-NH4 (0.92 mg/l)

and

P-tot (0.66 mg/l)

industrial impacts; this station

Nickel (11 μg/l),

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located

600

m

Zinc (35 μg/l)

after

WWTP1 outflow NC2

Naviglio Canal

Waterbody

of

the

minor

network in the plain (30 msl) subjected

to

urban

and

industrial impacts;11 km after WWTP1 outflow AC

Abbeveratoia

Waterbody

of

Canal

network in the plain (46 msl) subjected

to

the

minor

urban

and

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industrial impacts; 170 m after WWTP 2 outflow Minor waterbody flowing in animpacted msl)by

plain

area

agricultural

(26 and

livestock farming activities Minor waterbody (plain area, 30 msl) highly impacted by

farming

and

livestock

activities;

Herbicides, insecticides

N-NO3 (9.16 mg/l) N-NH4 (0.38 mg/l)

it

accepts

na

occasionally

N-NH4 (0.27 mg/l)

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agricultural

(Chloroform)

Abbeveratoia Canal outflow, according to LRA.

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Galasso Canal

N-NO3 (3 mg/l)

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GC

Trichloromethan

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Milanino Canal

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MC

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Journal Pre-proof Table 2. Codes, sampling date, Ecological Status (ES), Chemical Status (CS), values of Extended Biotic Index (EBI), pH and conductivity of stations located in the pilot basin. n.r.= not reported by the Regional Environmental Agency ARPA-ER. n.d.= not detected. Code

Watercourse

Sampling

ES

CS

EBI

pH

Conductivity (µS cm-1)

Parma Stream

Mar 2014

Good

8.35

256

Apr 2015

8.47

242

Oct 2015

8.39

240

8.81

302

8.18

965

8.18

811

n.d.

7.97

503

6

7.96

1355

5

8.00

1128

8.19

1245

8.42

288

8.44

264

8.31

288

PS1

Parma Stream

Apr 2015

Moderate

PS2

Parma Stream

Mar 2014

Moderate

Oct 2015 May 2014

n.r.

CS1

Cinghio Stream

May 2014

n.r.

CS2

Cinghio Stream

May 2014

BS0

Baganza Stream

Sept 2014

Poor

na

Apr 2015

n.r.

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May 2015

Good

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Cinghio Stream

Good

n.r.

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CS0

Good

Good

10-9

7

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PS0

Good

Good

6

10

Moderate

Good

8

8.40

317

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Oct 2015 BS1

Baganza Stream

Apr 2015

NC0

Naviglio Canal

Bad

Good

2

7.81

967

NC1

Naviglio Canal

Sept 2014

n.r.

n.r.

2

8.03

972

7.81

1049

8.37

931

8.48

956

8.04

1280

7.90

1300

8.25

790

8.33

944

8.10

738

8.07

801

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May2015

May2015 NC2

Naviglio Canal

Sept 2014

Bad

Good

2

May2015 AC

Abbeveratoia Canal

June 2014

n.r.

n.r.

4

June 2015 MC

Milanino Canal

Sept 2014

Bad

Good

4

June 2015 GC

Galasso Canal

Sept 2014 June 2015

Poor

Good

6

46

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Graphical abstract

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HIGHLIGHTS  Water toxicity and genotoxicity was assessed in a Po River tributary  Comet Assay with D. magna was applied for the first time in freshwater monitoring  The FPG-Comet Assay was standardized to detect DNA oxidative stress in D. magna  The Comet Assay on D. magna was able to discriminate differently impacted stations  The test is quick (24 h exposure) and routinely applicable

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Figure 1

Figure 2

Figure 3

Figure 4