# Cytogenotoxicity screening of urban and rural marshes: An integrated in vivo approach coupling fish and plant-based tests adapted for low income countries

Elodie Pepey, Geneviève Conéjéro, Elia Njara Ndrianaivo, Jean-luc Verdeil, Jean-michel Mortillaro, David Boggio, Modestine Raliniaina, Simon Pouil

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## To cite this version:

Elodie Pepey, Geneviève Conéjéro, Elia Njara Ndrianaivo, Jean-luc Verdeil, Jean-michel Mortillaro, et al. Cytogenotoxicity screening of urban and rural marshes: An integrated in vivo approach coupling fish and plant-based tests adapted for low income countries. Environmental Toxicology and Chemistry, 2023, 42 (6), pp.1266-1275. 10.1002/etc.5605. hal-04028206v2

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# Cytogenotoxicity Screening of Urban and Rural Marshes: An Integrated In Vivo Approach Coupling Fish and Plant-Based Tests Adapted for Low-Income Countries

Elodie Pepey,ᵃ,ᵇ,\* Geneviève Conéjéro,ᶜ Elia Njara Ndrianaivo,ᵈ Jean-Luc Verdeil,ᵉ,>f Jean-Michel Mortillaro,ᵃ,ᵇ,>d David Boggio,<fnref n="9" /> Modestine Ralinaina,ᵈ and Simon Pouilʰ

**Abstract:** Effects of anthropogenic activities such as urbanization, population growth, and agriculture on water quality are major concerns particularly in low-income countries where water quality monitoring can be challenging. The purpose of the present study was to evaluate the cytogenotoxic potential of water from urban and rural Malagasy marshes, coupling a fish (Nile tilapia, *Oreochromis niloticus*) and a plant (*Allium cepa*) species as bioindicators. The fish and plants were exposed for 72 h to water sampled in the two locations investigated. Using the comet assay on fish erythrocytes, DNA strand breaks were assessed, while mitotic index and nucleolar alterations were estimated in cells of the plant root apex. Comet assays revealed significant DNA strand breaks to fish erythrocytes in both the marshes investigated while the mitotic index and nucleolar characteristics in the roots of *A. cepa* mainly highlighted potential cytotoxicity in the urban marsh. Our results demonstrate the advantages of coupling *in vivo* biological test systems to screen potential cytogenotoxicity of surface water in low-income countries where comprehensive data sets of aquatic contaminants are often lacking. *Environ Toxicol Chem* 2023;42:1266–1275. © 2023 The Authors. *Environmental Toxicology and Chemistry* published by Wiley Periodicals LLC on behalf of SETAC.

**Keywords:** *Allium cepa*; Comet assay; Madagascar; Mitotic index; Nucleolar alterations; *Oreochromis niloticus*

## INTRODUCTION

Water availability and quality are among the most important concerns for human societies (McLennan, 2021). Water quality has deteriorated as a result of urbanization, population growth, industrialization, and agriculture. Today, poor water quality is widespread and has a major negative effect on many aspects of human society, economy, and the environment (Kirschke et al., 2020). In low-income countries, concerns about the distribution and allocation of water resources and water pollution are

increasing. So far, water monitoring data are lacking in less-developed countries, notably because of capacity challenges to define an enabling environment or prioritizing parameters as well as limiting financial and human means (Capps et al., 2016; Kirschke et al., 2020).

One of the main concerns in water quality deterioration is potential exposure to substances that are both cytotoxic and genotoxic. Cytogenotoxicity can only be evaluated by biologically oriented tests (Hemachandra & Pathiratne, 2017). Among the toxic effects induced by chemicals, one of the main concerns are the mutagenic/genotoxic hazards which could lead to ecological and human health risks (Ohe et al., 2004). Coupling different *in vivo* models, such as fish and plants, and different targeting endpoints with direct exposure to the water samples allows an integrated view of the cytogenotoxic risk for aquatic biomonitoring, as recommended by Orsière et al. (2005).

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

\*Address correspondence to elodie.pepey@cirad.fr
Published online 10 March 2023 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/etc.5605

<footnote>ᵃCIRAD, UMR ISEM, Montpellier, France</footnote>

<footnote>ᵇISEM, Univ Montpellier, CNRS, IRD, CIRAD, Montpellier, France</footnote>

<footnote>ᶜIPSiM, Univ Montpellier, CNRS, Institut Agro Montpellier, INRAE, Montpellier, France</footnote>

<footnote>ᵈDepartment of Zootechnical, Veterinary and Fish Research, FOFIFA, Antananarivo, Madagascar</footnote>

<footnote>ᵉPHIV, CIRAD, UMR AGAP Institut, Montpellier, France</footnote>

<footnote n="5">⁵UMR AGAP Institut, CIRAD, INRAE, Institut Agro, Univ Montpellier, Montpellier, France</footnote>

<footnote n="9">⁹Department of Information Technologies, CIRAD, Montpellier, France</footnote>

<footnote>ʰAgence de Recherche pour la Biodiversité (ARBRE), La Réunion, France</footnote>
Furthermore, several studies already have reported that geno-
toxicity tests based on fish and plants are producing results
showing good correlations with human and other mammalian
test systems (see Dos Reis et al., 2017; Leme & Marin-
Morales, 2009; Vlastos et al., 2016), suggesting that they are
appropriate models for assessing the cytogenotoxicity potential
of contaminated waters to humans.

The potential benefits of using fish as indicators of cyto-
genotoxicity are now widely recognized (de Lapuente
et al., 2015). This is due to their ecological and economic
importance and their similarity to mammals, for which tox-
icological mechanisms are far better described (Martins &
Costa, 2017). The comet assay, consisting in quantifying DNA
breaks by gel electrophoresis (Singh et al., 1988), applied to
fish erythrocytes, is known to be a sensitive way to monitor
aquatic environments affected by pollutants (de Lapuente
et al., 2015; Frenzilli et al., 2009; Martins & Costa, 2017).
Compared with other genotoxicity tests, the advantages of
the comet assay are the detection of slight DNA damages,
the low number of cells required, low cost, precision, ease
of application, reproducibility, and the short period of time
needed to conduct the experiment. In plants, the Allium
cepa assay, which assesses chromosomal damage and
disturbances in the mitotic cycle, is well known among
plant cytogenotoxicity tests and is now used in many
laboratories for a wide range of applications (Leme & Marin-
Morales, 2009). As for the comet assay test, the advantages
of the A. cepa test are that it is fast, easy to use, and
inexpensive and requires minimal sample preparation
(Barbério, 2013; Leme & Marin-Morales, 2009). For these
reasons, all these trials can be conducted in remote
areas from low-income countries because such tests require
limited infrastructures to be performed (Hemachandra &
Pathiratne, 2016, 2017).

The objective of the present study was to investigate the
cytogenotoxic potential of water sampled from two marshes
in Madagascar, where the urban population is growing at
twice the rate of that in the rest of the world (+5% per year;
Bastaraud et al., 2018). One of the two marshes is mainly
used to supply water to the capital city of Antananarivo
(>1 300 000 inhabitants), while the second is mainly sur-
rounded by agricultural land. Given the risk for both the
aquatic organisms and the human health through con-
taminants entering the food chain and affecting human
health, we chose to assess in vivo exposure using Nile tilapia,
Oreochromis niloticus, an aquatic species, and, A. cepa, a
terrestrial species, as bioindicators of cytogenotoxicity.
These species are consumed by local populations (Food and
Agriculture Organization, 2022) and are both present in the
two locations.

MATERIALS AND METHODS

Study areas and sample collection

The investigated urban marsh (Masay) is an artificial basin
built in 2004 measuring approximately 1 km² and located in the
northern part of the city of Antananarivo (18°52'5"S, 47°32'23"E);

it acts as a buffer basin for wastewater and runoff from the
surrounding districts (Figure 1). The basin opens into the
Andriantany canal and the Ikopa river, collects rainwater and
untreated domestic wastewater and industrial discharges from
two watersheds, and regulates excess water to prevent
flooding. Masay marsh is surrounded by residential housing,
agri-food industrial establishments, and numerous car garages,
while uncontrolled landfills also exist near the marsh. Locally,
several unpublished studies highlighted the pollution of this
marsh, notably by heavy metals and other contaminants, with
sometimes illegal fishing for self-consumption and sale in local
markets. For the purpose of comparison, water was also col-
lected in a rural marsh located along the Viliahazo River (18°48'
5"S, 47°40'13"E), which is used for agriculture, mostly for rice
and cassava cultivation and fishing (Figure 1). There is also
significant market gardening activity in the area, allowing the
supply of vegetables to the city of Antananarivo, an agricultural
activity requiring much more pesticides compared with other
cropping systems (Razanakoto et al., 2021).

The investigation was carried out in the rainy season,
February 2019. Unfiltered surface water was collected from
the urban and rural marshes at a depth of 50 cm and then,
transported to the laboratory. For technical reasons, the
samples of water were kept oxygenated using air bubbling
and stored for 3 days at room temperature in the dark in
separate 250-L food-grade plastic drums prior to the ex-
periment. Ceretti et al. (2010) found that storage materials
were not associated with the genotoxic properties of the
water even after 10 days of storage at 40 °C. Thus, it is rea-
sonable to assume that our storage conditions had no effect
on water toxicity.

Water analysis

The physical–chemical parameters of the water under each
experimental condition were measured at the beginning of the
experiment. The temperature and the conductivity of the water
were determined using a combined tester (HI 98311; Hanna
Instruments), while pH was measured using a portable digital
pH probe (HI 98128; Hanna Instruments). Dissolved oxygen
was measured using a dissolved oxygen meter (HI 9146; Hanna
Instruments), and the biochemical oxygen demand after 5 days
was analyzed as described by Aminot and Kérouel (2004).

After the storage period, 100 mL subsamples of water
originating from each location were taken from each drum,
filtered using 0.2-µm polyethersulfone filters, and kept in 50-mL
Falcon® tubes with trace element–grade nitric acid (pH <2) for
the analysis of trace metals. All samples were kept at room
temperature in the dark prior to analysis.

As potential cytogenotoxic agents (Tchounwou et al., 2012),
the dissolved and particulate trace metals (i.e., As, Co, Cr, Cu, Fe,
Mn, Ni, Pb, and Zn) were analyzed. In the water samples kept in
Falcon tubes, trace elements were directly analyzed by in-
ductively coupled plasma–mass spectrometry (ICP-MS X series 2;
Thermo Scientific). Trace metals on the filters were digested
using hydrofluoric and perchloric acids following International
<figure><img src="image_5.png" /><figcaption>**FIGURE 1:** Location of Masay and Viliahazo marshes with respect to the capital city of Antananarivo and land uses in the vicinity. The map showing Masay marsh was modified from Dupuy et al. (2017).</figcaption></figure>

Organization for Standardization (ISO) method ISO 14869-1. All quality assurance procedures (e.g., blanks; reference materials SLRS6 and CRM 7004 [Metranal 34]) were applied in each analytical method. Only the elements recovered from reference materials in the 80%-120% range were analyzed.

## Bioassay assembly

Thirty-two adult Nile tilapia (fresh wt 127 ± 48 g) were obtained from a local fish farm. Fish were randomly placed in four 150-L glass aquariums (n = 5/aquarium) filled with dechlorinated tap water and equipped with air-lift filters and hiding places. Fish were acclimated to the laboratory conditions for 10 days under natural light and a natural photoperiod and were fed with a commercial diet (PGR32; Aqua Madagascar Innovation). Feeding was stopped 48 h before the bioassays began. After the acclimation period, the water in two aquariums was replaced by water collected from the urban and rural marshes, while the two other aquariums were used as controls. While dechlorinated tap water was used as a negative control, a further set of fish were exposed to ethyl methanesulfonate (EMS) at 10 mg L⁻¹ and used as positive controls, similarly to a previous genotoxicity investigation in Nile tilapia (Könen & Çavaş, 2008). After the 3-day exposure, five fish from each condition, including controls, were anesthetized with eugenol (0.5 mL L⁻¹), and blood was sampled from the ventral tail vein using a heparinized syringe.

For the A. cepa test, we followed the protocol initially proposed by Fiskesjö (1985). Briefly, the plants were first grown in plastic glasses in tap water. When the roots were at least 1 cm

long, they were transferred to the water collected from the urban and rural marshes, while, as for fish, dechlorinated tap water and EMS at 10 mg L⁻¹ were used as negative and positive controls, respectively. After 72 h of exposure, root tips from all experimental and control conditions (n = 4 bulbs/condition) were processed for microscopy. The test was conducted under laboratory conditions in a shaded area at room temperature.

## Bioassay procedures

**Comet assay using fish.** The alkaline comet assay using Nile tilapia erythrocytes was performed according to Singh et al. (1988) and Osman et al. (2012), with some modifications. Before the assay, frosted microscope slides were precoated with 1% (w/v) of normal-melting agarose and dried at room temperature for at least 2 days. Each fish blood puncture was immediately diluted with phosphate-buffered saline at a ratio of 1:500 (v/v). Then, 40 µL of each diluted blood sample was added to 360 µL of 0.5% (w/v) low-melting agarose, and 150 µL of the mixture was immediately placed on the precoated slide and covered with a coverslip. The slides were incubated at 4 °C for 15 min to allow solidification, then immersed in cold lysing buffer (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid [EDTA], 10 mM trishydroxymethylaminomethane [Tris] base, pH 10, 1% Triton X-100, 10% dimethyl sulfoxide) at 4 °C overnight (Hemachandra & Pathiratne, 2016; Iturburu et al., 2018). Then, the slides were incubated at 4 °C in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH ≥ 13) for 40 min, and electrophoresis was carried out at 0.8 V/cm and 300 mA at 4 °C for 40 min. A neutralization step was then performed consisting of
three successive 10-min incubation periods in 0.4 M Tris buffer (pH 7.5). Slides were rinsed in ultrapure water for 5 min, then immersed in ethanol (≥99.6%) for 10 min to start dehydration before being dried at 45 °C for 1 h. Finally, the slides were stained with 100 µL of SYBR Gold (Thermo Fisher Scientific) diluted 10,000x with Tris/EDTA buffer (pH 8.0), and the comets were viewed under an epifluorescence microscope (Nikon Eclipse NI-E; objective Plan APO 0.75 NA x20). For each fish, we analyzed two slides, each containing 200 cells (i.e., 400 cells/fish and ~2000 cells/condition). The images were analyzed using the plug-in OpenComet with ImageJ software (Gyori et al., 2014), and the extent of DNA strand breaks was expressed as the percentage of DNA in the tail (Kumaravel & Jha, 2006).

**Mitotic index and nucleolar characteristics in the plant.** The 3- to 5-mm-long root tips were cut, and immediately transferred to a fixative solution (ethanol:glacial acetic acid 3:1, v/v), and left overnight (Pathiratne et al., 2015). After fixation, the root tips were transferred to 70% ethanol and stored at 4 °C until analysis. Samples were hydrolyzed in 1 M HCl at 60 °C for 5 min (Creber et al., 1994) and washed three times with distilled water. Samples were processed with a ClearSee clearing protocol to enable visualization of all nuclei in the meristem region (Kurihara et al., 2015). After washing with a phosphate buffer, the root tips were immersed in a ClearSee solution (urea 25% w/v, sodium deoxycholate 15% w/v, xylitol, 10%, w/v) for 5–7 days under gentle shaking. The nuclei were then stained with a 4',6-diamidino-2-phenylindole solution (5 µg mL⁻¹) prepared in ClearSee solution for 60 min. After washing, five root tips were placed on a glass slide and gently squashed in a drop of ClearSee solution by applying very slight pressure to the coverslip. Pictures of the meristem tissues were acquired with a multiphotonic microscope, 880 NLO Zeiss (lena) equipped with an Apochromatic x20 NA 1 objective, at 740 nm of excitation (bandpass emission, 405–495 nm; Plant Cell Imaging Platform, Plateforme d'histo-cytologie et d'imagerie cellulaire végétale, Montpellier Re-ssources Imagerie; ISO9001-certified). A set of confocal images was taken of the specimen in such a way that the area of the image along the x- and y-axes remained the same but the distance from the objective (z-axis) differed in each image (z-stack). Z-stacks were performed on 100 µm with 1 µm between two optical slices (ZEN soft; Zeiss). Because the distance be-tween adjacent images is precisely controlled in the z-stack, it can be used to form a three-dimensional (3D) image. This method allows us to obtain pictures on several intact cell layers. Some pictures were obtained with an x40 LD C-Apo 1.1NA W or an x63 Plan Apo 1.4 NA oil objective to observe the mor-phology of the nuclei.

The mitotic index was used to evaluate the cytotoxic potential in each of the experimental conditions. It was calculated as the percentage of cells undergoing mitotic stages (metaphase, anaphase, and telophase) in at least 1000 cells of meristem tissues observed per root apex and three root apexes analyzed by plant (i.e., ~3000 cells/plant and 10 000–12 000 cells/condition). In complement to the mitotic index,

quantitative analysis of nucleoli has also been proposed as a sensitive biomarker for routine monitoring of cytotoxicity in plants (Arkhipchuk et al., 2000; Lima et al., 2019). In the present study, nucleolar alterations were determined by counting the number of nucleoli per nucleus and expressed as a percentage of approximately 1000 nuclei of root meristem cells per condition (Singh et al., 1988). Cell counting was performed using the Cell Counter plugin in ImageJ-1.53a software.

## Statistical analysis

The differences in tail DNA (in fish), mitotic index, and nucleolar activity (in plant) among the different experimental conditions were evaluated by one-way analysis of variance, followed by Tukey's post hoc test. Data were arcsine-transformed, and average values per individual were used in the statistical analysis. The normality and homogeneity of variances were graphically confirmed and evaluated by the Shapiro-Wilk and Levene's tests. In addition, a nonparametric Spearman's correlation analysis was used with concentrations of trace elements in samples of water collected in the two locations prior to the experiment. All statistical analyses were performed in R, Ver 3.6.3, using the packages "car" and "stats," while the package "ggplot2" was used to create graphics. The level of significance for statistical analyses was always set to α = 0.05. If not specified, the results are expressed as mean ± SD.

## RESULTS

### DNA damage in fish and plants

**Comet assay in fish.** After 72 h of exposure, no mortality was recorded in control and experimental conditions (i.e., water from the urban and rural marshes). Comet assays in blood cells (Figure 2) revealed significant differences in DNA strand breaks between conditions with high variability among individuals (*p* < 0.001). The genotoxicity detected in fish (n = 5) from the two marshes (mean ± SD 43.7 ± 25.8%, median 42.1% for the urban marsh; mean ± SD 39.2 ± 26.6%, median 36.3% for the rural marsh) was lower (*p* < 0.001) than that found in the positive control group (i.e., fish exposed to EMS; mean ± SD 64.1 ± 24.1%, median 71.9%) but higher (*p* < 0.001) than the DNA strand breaks found in the negative group (i.e., fish kept in tap water; mean ± SD 25.3 ± 22.8%, median 17.1%; Figure 2A). The DNA strand breaks in fish exposed to water from the two marshes were then compared with the damage detected in control conditions and confirmed by observations of DNA degradation in erythrocytes by epifluorescence microscopy (Figure 2B).

**Mitotic index and nucleolar characteristics.** Cytotoxicity was assessed by determining both the mitotic index and nucleolar alterations in plants (Figures 3 and 4). Significant differences were found in the mitotic index estimated for the cells of meristem tissues in the root apex of A. cepa between the experimental conditions (*p* < 0.001). Pairwise comparisons revealed that the mitotic index in the plants exposed to water
<figure><img src="image_6.png" /><figcaption>**FIGURE 2:** (A) Percentage of damage to the DNA of erythrocytes of *Oreochromis niloticus* exposed to tap water (negative control), ethyl methanesulfonate (positive control), and water taken from the urban (Masay) and rural (Viliahazo) marshes (n = 5 fish per condition, 400 measurements performed per fish) presented as box plots and violin plots (i.e., probability density of the data at different values). Lowercase letters denote significant differences (p < 0.05) between experimental conditions. (B) Degradation of DNA in *O. niloticus* erythrocytes by epifluorescence microscopy. Scale bar = 20 µm.</figcaption></figure>

sampled from the urban and rural marshes differed significantly (p = 0.035), with values of 1.18 ± 0.75 (n = 4 plants) in plants exposed to water taken from the urban marsh and 3.02 ± 0.64 in plants exposed to water taken from the rural marsh.

Interestingly, while the mitotic indices were similar (p = 0.99) in plants exposed to water taken from the rural marsh and in plants exposed to tap water (i.e., negative controls), the mitotic indices of the plants exposed to water taken from the

<figure><img src="image_7.png" /><figcaption>**FIGURE 3:** (A) Mitotic index of the root meristem cells of *Allium cepa* bulbs (n = 4 per condition, three measurements performed per bulb) exposed to tap water (negative control), ethyl methanesulfonate (positive control), and water from the urban (Masay) and rural (Viliahazo) marshes investigated. Lowercase letters denote significant differences (p < 0.05) between experimental conditions. (B) Three-dimensional image of the meristem region used to calculate the mitotic index from a Z-stack of 109 optical slices and tile scan function (frame size 1570 × 1570 × 110 µm) in the root apex. Inset: Magnified view of a portion of meristem cells showing some mitosis. Scale bar = 100 µm. Values are means ± SD.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**FIGURE 4:** (A) Proportion of cells with three or more nucleoli per nucleus in meristem cells of *Allium cepa* roots (from *n* = 4 bulbs per condition) exposed to tap water (negative control), ethyl methanesulfonate (positive control), and water from urban (Masay) and rural (Viliahazo) marshes investigated. Lowercase letters denote significant differences (*p* < 0.05) between experimental conditions. (B) The nucleus of root meristem cells in each experimental condition. Four bulbs were used per experimental condition. Scale bar = 10 µm. Values are means ± SD.</figcaption></figure>

urban marsh were similar to the indices observed when plants were exposed to EMS (i.e., positive controls; *p* = 0.345; Figure 3). These results show that the mitotic index was only affected by exposure to water taken from the urban marsh.

<table><caption>**TABLE 1:** Summary of water quality parameters and trace elements measured for the tap water (negative control) and water from urban (Masay) and rural (Viliahazo) marshes</caption><thead><tr><th rowspan="2">Parameter</th><th rowspan="2">n</th><th colspan="3">Treatment</th></tr><tr><th>Tap water<br>(negative control)</th><th>Masay</th><th>Viliahazo</th></tr></thead><tbody><tr><td>Water parameters</td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature (°C)</td><td>5</td><td>22.2 ± 0.6<sup>a</sup></td><td>22.0 ± 0.5<sup>a</sup></td><td>22.0 ± 0.4<sup>a</sup></td></tr><tr><td>DO (mg L<sup>-1</sup>)</td><td>4</td><td>4.7 ± 1.0<sup>a</sup></td><td>5.1 ± 0.8<sup>a</sup></td><td>5.4 ± 0.3<sup>a</sup></td></tr><tr><td>pH</td><td>5</td><td>6.1 ± 0.2<sup>a</sup></td><td>6.9 ± 0.2<sup>b</sup></td><td>5.9 ± 0.2<sup>ac</sup></td></tr><tr><td>Conductivity<br>(µS cm<sup>-1</sup>)</td><td>5</td><td>182 ± 50<sup>a</sup></td><td>346 ± 38<sup>b</sup></td><td>89 ± 33<sup>c</sup></td></tr><tr><td>BOD5 (mg L<sup>-1</sup>)</td><td>1</td><td>1.25</td><td>1.80</td><td>1.81</td></tr><tr><td>Trace elements</td><td></td><td></td><td></td><td></td></tr><tr><td>Cr (µg L<sup>-1</sup>)</td><td>1</td><td>0.13</td><td>0.24</td><td>0.22</td></tr><tr><td>Mn (µg L<sup>-1</sup>)</td><td>1</td><td>12.42</td><td>57.22</td><td>16.43</td></tr><tr><td>Fe (µg L<sup>-1</sup>)</td><td>1</td><td>19.14</td><td>44.72</td><td>14.35</td></tr><tr><td>Co (µg L<sup>-1</sup>)</td><td>1</td><td>0.20</td><td>0.38</td><td>0.11</td></tr><tr><td>Ni (µg L<sup>-1</sup>)</td><td>1</td><td>0.46</td><td>0.67</td><td>0.40</td></tr><tr><td>Cu (µg L<sup>-1</sup>)</td><td>1</td><td>1.48</td><td>1.19</td><td>0.98</td></tr><tr><td>Zn (µg L<sup>-1</sup>)</td><td>1</td><td>69.71</td><td>10.58</td><td>38.46</td></tr><tr><td>As (µg L<sup>-1</sup>)</td><td>1</td><td>0.07</td><td>0.21</td><td>0.05</td></tr><tr><td>Pb (µg L<sup>-1</sup>)</td><td>1</td><td>0.76</td><td>0.19</td><td>0.63</td></tr></tbody></table>

Values are means ± SD. No data were recorded after ethyl methanesulfonate addition in tap water in the positive control, to avoid contamination of the instruments. Letters denote significant differences (*p* < 0.05) between experimental conditions.

BOD5 = biochemical oxygen demand after 5 days; DO = dissolved oxygen.

Significant differences were observed between the experimental conditions in the proportion of cells with three or more nucleoli per nucleus (*p* < 0.001). Nucleolar activity in plants exposed to water taken from the two marshes did not differ from the negative control (*p* ≥ 0.07), but the proportion of cells with three or more nucleoli per nucleus was lower in the rural marsh than in the urban marsh (i.e., 1.4 ± 0.7% vs. 12.0 ± 6.0%; *p* = 0.027; Figure 4).

## Correlations between cytogenotoxicity and concentrations of trace elements in the water

The results of water quality measurements and trace element analysis are given in Table 1. Spearman's correlations revealed no significant correlations between cytogenotoxicity results (comet assay in fish, mitotic index and nucleoli activity in plant) and the concentrations of trace elements in water (*p* ≥ 0.13). Among the trace elements we investigated, concentrations of the nonessential elements (i.e., As, Ni, and Pb) in the unfiltered water were always <1 µg L<sup>-1</sup> (Table 1).

## DISCUSSION

To the best of our knowledge, this is the first time cytogenotoxic effects of aquatic environments have been assessed in Madagascar. We combined different approaches for one species of fish (*O. niloticus*) and one species of plant (*A. cepa*) to evaluate the levels of cytogenotoxic pollution in two marshes, one located in an urban area and one in a rural area of the
Malagasy highlands. Such investigation is needed to highlight
the interest of implementing ecotoxicity assessment tools in
the management of surface water in Madagascar.

The alkaline in vivo comet assay can be used to detect DNA breaks in aquatic species following exposure to genotoxicics (Frenzilli et al., 2009) and is recommended for use by regulatory agencies (Speit et al., 2015). The Nile tilapia is known to respond rapidly to environmental alterations (Vijayan et al., 1997), making it a good candidate for genotoxic studies, while the erythrocyte-based comet assay has already been successfully used for cytogenotoxicity assessments (de Campos Ventura et al., 2008; Christofoletti et al., 2009; Hemachandra & Pathiratne, 2016; Matsumoto et al., 2006). Cytogenotoxic effects can be detected very soon after exposure to contaminated waters. For instance, using the comet assay, cytogenotoxic effects have been detected in common carp (*Cyprinus carpio*) after 24 h of exposure to industrial plant effluents (SanJuan-Reyes et al., 2015), while Matsumoto et al. (2006) found genotoxicity and mutagenicity in Nile tilapia exposed for 72 h to water contaminated with tannery effluents.

In the present study, we found that a greater cytogenotox-
icity was induced by the contaminants in the water taken from
the urban and rural marshes we investigated when compared
with the negative control condition. According to de Souza
Trigueiro et al. (2021), the median value of the percentage of
DNA damage in the tail suggested that most of the damage
was minimal or low for fish coming from the negative control
condition, while fish exposed to the water from the two
marshes exhibited mostly high damage. Such genotoxic effects
in fish can lead to several cellular and physiological alterations,
such as mutations causing changes in the life span and func-
tions of circulating erythrocytes as well as cell death (Çavaş &
Könen, 2007; da Silva et al., 2019; de Souza Trigueiro
et al., 2021). The slight changes observed in the genotoxicity
results between the two marshes combined with the absence
of correlations between comet assay results and concentrations
of trace elements suggest that (1) the genotoxic agents in-
volved are not trace elements; (2) the genotoxic agents are
present in both the urban and rural marshes, although probably
not at the same concentrations in the marshes; and/or (3) the
genotoxic effects observed were not caused by the same
contaminants in the two locations.

The A. cepa test is one of the most efficient, feasible, and
low-cost methods for the assessment of the cytogenotoxic ef-
fects of mixtures of contaminants in aquatic environments
(Leme & Marin-Morales, 2009). In the A. cepa test, a decrease
in the mitotic index in the root tip cells points to cytotoxicity,
while induction of chromosomal abnormalities, micronuclei,
and nuclear buds in the root cells indicate cytogenotoxicity
and can also be used to check for mutagenicity (Leme &
Marin-Morales, 2009). In the present study, we combined
measurement of the mitotic index and observations of nucle-
olar abnormalities with a focus on the number of nucleoli per
nucleus.

Nucleolar activity is sometimes considered to be a more
sensitive parameter than the parameters mentioned above in

cytotoxicity studies (Lima et al., 2019). In A. cepa, some trace
elements, including Al, Cd, Cu, and Pb, have been reported to
disrupt the synthesis of ribosomal RNAs but also to inhibit
nucleolus reconstruction at the end of the telophase (Jiang
et al., 2014; Qin et al., 2010; Wang et al., 2016). At the root
apex, the nuclear cells mainly have two nucleoli, which some-
times merge at the end of the telophase (Jiang et al., 2014).
Thus, observations of three or more nucleoli per nucleus in root
meristem cells point to cytotoxicity (Lima et al., 2019). The 3D
images obtained thanks to multiphoton microscopy allowed us
to observe the nuclei in all their thickness and to count the
exact number of nucleoli, which are not always in the same
plane in each nucleus, unlike what is visible in the two-
dimensional images commonly used for such measurements.
Overall, the mitotic index and nucleolar activity measured in A.
cepa indicated potential cytotoxicity of urban marsh water that
primarily inhibited cell division, whereas no cytotoxicity of rural
marsh water was found.

Cytogenotoxicity tests performed in fish and plants pointed
to increasing toxicity effects ranked as follows: tap water
(negative control) ≤ rural marsh ≤ urban marsh ≤ EMS (positive
control), with several discrepancies between the comet assay
performed in fish and the other cytotoxicity markers in plants.
There are several possible explanations for these differences.
Physical barriers in plants (i.e., cell walls and plasma membrane
of the root) can prevent certain chemicals from entering,
whereas in fish chemicals present in the water can enter the
body directly (Matsumoto et al., 2006). Furthermore, de-
pending on the contaminants involved and their respective
concentrations, cytogenotoxic effects may occur in fish but not
in plants. Radić et al. (2013) found that although genotoxicity
responses to the exposure of fertilizer factory effluents were
mostly in agreement in common carp and duckweed (Lemna
minor), several discrepancies were observed. The authors
linked these to differences in the sensitivity of model organisms
or in experimental conditions.

The trace elements measured in the samples of water col-
lected from the two marshes and from drinking water were
within the Malagasy drinking water standards (Ministère de
l’Eau, de l’Assainissement et de l’Hygiène, 2004) and do not
exceed the World Health Organization guidelines for drinking
water quality (World Health Organization, 2017). Nevertheless,
both in vivo test systems (fish erythrocyte and A. cepa root-
based tests) confirmed the cytogenotoxicity of the marshes'
waters. The absence of correlations with the trace elements in
water suggests that the cytogenotoxic effects are not due to
only one of the elements measured. However, because our
chemical characterization of these water samples was not
comprehensive, it is difficult to conclude on the actual chemical
contamination status because a complex mixture of chemicals
at trace concentrations can have cytogenotoxic impacts. In
addition to trace elements (Barbosa et al., 2010), a wide range
of contaminants can have cytogenotoxic effects that are likely
to be present in Madagascar and potentially in our study
locations.

Anthropogenic activities produce other contaminants of
concern in Madagascar, including persistent organic pollutants
that are likely to have cytogenotoxic effects (Baqar et al., 2017). Through a screening of environmental pollutants in wastewater, Rasoazanany (2008) found that, by comparison to the rural environment, the urban environment of Antananarivo is disrupted by polluting elements including Cr and Pb. The pollutants occurring in an urban area and a rural area may differ. The agricultural practices used in cultivating vegetable crops in the vicinity of the rural marsh (Figure 1) included slash and burn, which may leach toxic pyrrolizidine alkaloids into the soil (Letsyo et al., 2021), and the use of pesticides, which could also explain the cytogenotoxic effects observed in this water body. A more comprehensive data set of contamination levels of Malagasy water bodies is needed to fully explain our observations.

It has also been shown that the toxicity of surface water can vary with the season (Wu et al., 2004). Meteorological and hydrological factors may greatly affect water quality. In the present study, the experiment was performed during the rainy season. We can thus hypothesize that rains may dilute pollutants dissolved in the water. Further investigations carried out during the dry season would be relevant to assess such changes. Indeed, several studies performed in tropical areas highlighted that induction of DNA damage in fish erythrocytes and gill tissues is higher in summer than in winter (Nagpure et al., 2015; Scalon et al., 2013). Our findings suggest that, although specific priorities need to be considered depending on countries, cytogenotoxicity assessments in tropical areas should consider potential seasonal or time-specific changes, especially in the context of global climate change.

## CONCLUSION

All of the bioassays we performed revealed cytogenotoxic potential, particularly in the marsh that received urban, industrial, and artisanal waste from the capital city Antananarivo. We demonstrated the interest in using fish and plant tests combined to assess cytogenotoxicity. Indeed, while the comet assay performed on fish erythrocytes showed genotoxicity for both marshes, the tests on A. cepa showed differential cytotoxicity between the waters from the two sources. The present study provides scientific evidence for the urgent need to include ecotoxicity assessment tools in the management of surface water used for human activities in Madagascar. In addition, the present study shows that in vivo test systems combining fish and plants are efficient in low-income countries where comprehensive water quality monitoring programs can be complex to achieve because of limited resources.

**Acknowledgments**—We are most grateful to M.-C. Raivomanana and F. Andrianarimandimby (Centre National de Recherche Appliquée au Développement Rural, Madagascar), M. Lorcin (Institut national de recherche et de sécurité), M. Canonne (Centre de coopération internationale en recherche agronomique pour le développement [CIRAD]), and M. Laraud (CIRAD) for their technical assistance and to H. de Verdal (CIRAD) for his advice on the experimental procedures

implemented for animal welfare. We are also grateful to the nongovernmental organization APDRA Pisciculture Paysanne for technical and logistic assistance before or during fieldwork in Madagascar. We acknowledge the imaging facility Montpellier Ressources Imagerie, member of the national infrastructure France-Biolmaging, supported by the French National Research Agency (ANR-10-INBS-04). We are grateful to the CIRAD Water, Soil and Plant Analysis Laboratory (US Analyses, CIRAD, Montpellier, France) for the analyses of trace elements. This is publication ISEM 2023-048.

**Author Contributions Statement**—Elodie Pepey: Conceptualization; Methodology; Formal analysis; Investigation; Writing—original draft; Supervision; Writing—review & editing; Funding acquisition. Geneviève Conéjéro: Conceptualization; Methodology; Formal analysis; Investigation; Writing—original draft; Visualization; Writing—review & editing; Funding acquisition. Elia Njara Ndrianavo: Investigation; Resources. Jean-Luc Verdeil: Methodology; Formal analysis; Writing—review & editing. Jean-Michel Mortillaro: Resources; Writing—review & editing. David Boggio: Software; Formal analysis. Modestine Raliniaina: Resources. Simon Pouil: Conceptualization; Formal analysis; Writing—original draft; Visualization; Writing—review & editing.

**Data Availability Statement**—The data and methodology supporting the results of the present study are available within the article. Regarding the cytogenotoxicity data, the nature of our study did not generate large data sets requiring the use of data repositories. However, the corresponding author (E. Pepey; elodie.pepey@cirad.fr) may be contacted for further inquiries regarding the data.

## REFERENCES

Aminot, A., & Kérouel, R. (2004). *Hydrologie des écosystèmes marins: Paramètres et analyses*. Editions IFREMER.

Arkhipchuk, V. V., Malinovskaya, M. V., & Garanko, N. N. (2000). Cytogenetic study of organic and inorganic toxic substances on Allium cepa, Lactuca sativa, and Hydra attenuata cells. *Environmental Toxicology*, 15(4), 338–344. https://doi.org/10.1002/1522-7278(2000)15:4<338::AID-TOX10>3.0.CO;2-R

Baqar, M., Arslan, M., Sadef, Y., Mahmood, A., Qadir, A., & Ahmad, S. R. (2017). Persistent organic pollutants in Pakistan: Potential threat to ecological integrities in terms of genotoxicity and oxidative stress. *Human and Ecological Risk Assessment*, 23(6), 1249–1271. https://doi.org/10.1080/10807039.2017.1292843

Barbérío, A. (2013). Bioassays with plants in the monitoring of water quality. In W. Elshorbagy & R. Chowdhury (Eds.), *Water treatment* (pp. 317–334). InTech.

Barbosa, J. S., Cabral, T. M., Ferreira, D. N., Agnez-Lima, L. F., & De Medeiros, S. B. (2010). Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals. *Ecotoxicology and Environmental Safety*, 73(3), 320–325. https://doi.org/10.1016/j.ecoenv.2009.10.008

Bastaraud, A., Rakotondramanga, J. M., Mahazosaotra, J., Ravaonindrina, N., & Jambou, R. (2018). Environmental factors and the microbial quality of urban drinking water in a low-income country: The case of Madagascar. *Water, 10*(10), Article 1450. https://doi.org/10.3390/w10101450

Capps, K. A., Bentsen, C. N., & Ramírez, A. (2016). Poverty, urbanization, and environmental degradation: Urban streams in the developing world. *Freshwater Science*, 35(1), 429–435. https://doi.org/10.1086/684945
Çavaş, T., & Könen, S. (2007). Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (*Carassius auratus*) exposed to a glyphosate formulation using the micronucleus test and the comet assay. *Mutagenesis*, **22**(4), 263-268.

Ceretti, E., Zani, C., Zerbini, I., Guzzella, L., Scaglia, M., Berna, V., Donato, F., Monarca, S., & Feretti, D. (2010). Comparative assessment of genotoxicity of mineral water packed in polyethylene terephthalate (PET) and glass bottles. *Water Research*, **44**(5), 1462-1470. https://doi.org/10.1016/j.watres.2009.10.030

Christofoletti, C. A., David, J. A. O., & Fontanetti, C. S. (2009). Application of the comet assay in erythrocytes of *Oreochromis niloticus* (Pisces): A methodological comparison. *Genetics and Molecular Biology*, **32**, 155-158. https://doi.org/10.1590/S1415-4757200900500020

Creber, H. M. C., Davies, M. S., Francis, D., & Walker, H. D. (1994). Variation in DNA C value in natural populations of *Dactylis glomerata* L. *New Phytologist*, **128**(3), 555-561. https://doi.org/10.1111/j.1469-8137.1994.tb03001.x

da Silva, G. S., de Matos, L. V., da Silva Freitas, J. O., de Campos, D. F., & de Almeida E Val, V. M. F. (2019). Gene expression, genotoxicity, and physiological responses in an Amazonian fish, *Colossoma macropomum* (CUVIER 1818), exposed to Roundup® and subsequent acute hypoxia. *Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology*, **222**, 49-58.

de Campos Ventura, B., de Angelis, D. D. F., & Marin-Morales, M. A. (2008). Mutagenic and genotoxic effects of the atrazine herbicide in *Oreochromis niloticus* (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. *Pesticide Biochemistry and Physiology*, **90**(1), 42-51. https://doi.org/10.1016/j.pestbp.2007.07.009

de Lapuente, J., Lourenço, J., Mendo, S. A., Borràs, M., Martins, M. G., Costa, P. M., & Pacheco, M. (2015). The comet assay and its applications in the field of ecotoxicology: A mature tool that continues to expand its perspectives. *Frontiers in Genetics*, **6**, Article 180. https://doi.org/10.3389/fgene.2015.00180

de Souza Trigueiro, N. S., Gonçalves, B. B., Dias, F. C., de Oliveira Lima, E. C., Rocha, T. L., & Sabóia-Morais, S. M. T. (2021). Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide induces DNA damage and mutagenic effects in the guppy (*Poecilia reticulata*). *Environmental Toxicology and Pharmacology*, **81**, Article 103521. https://doi.org/10.1016/j.etap.2020.103521

Dos Reis, G. B., Andrade-Vieira, L. F., de Campos Moraes, I., César, P. H. S., Marcussi, S., & Davide, L. C. (2017). Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents. *Ecotoxicology and Environmental Safety*, **142**, 110-116. https://doi.org/10.1016/j.ecoenv.2017.04.004

Dupuy, S., Defrise, L., Gaetano, R., & Burnod, P. (2017). *Antananarivo—* Cartographie de l'occupation du sol 2017. CIRAD Dataverse (Version 1). https://doi.org/10.18167/DVN1/NHE34C

Fiskesjö, G. (1985). The *Allium* test as a standard in environmental monitoring. *Hereditas*, **102**(1), 99-112. https://doi.org/10.1111/j.1601-5223.1985.tb00471.x

Food and Agriculture Organization. (2022). *FishStatJ: Universal software for fishery statistical time series*.

Frenzilli, G. I. A. D. A., Nigro, M. A. R. C. O., & Lyons, B. P. (2009). The comet assay for the evaluation of genotoxic impact in aquatic environments. *Mutation Research/Reviews in Mutation Research*, **681**(1), 80-92. https://doi.org/10.1016/j.mrrev.2008.03.001

Gyori, B. M., Venkatachalam, G., Thiagarajan, P. S., Hsu, D., & Clement, M. V. (2014). OpenComet: An automated tool for comet assay image analysis. *Redox Biology*, **2**, 457-465. https://doi.org/10.1016/j.redox.2013.12.020

Hemachandra, C. K., & Pathiratne, A. (2016). Combination of physicochemical analysis, *Allium cepa* test system and *Oreochromis niloticus* erythrocyte based comet assay/nuclear abnormalities tests for cyto-genotoxicity assessments of treated effluents discharged from textile industries. *Ecotoxicology and Environmental Safety*, **131**, 54-64. https://doi.org/10.1016/j.ecoenv.2016.05.010

Hemachandra, C. K., & Pathiratne, A. (2017). Cytogenotoxicity screening of source water, wastewater and treated water of drinking water treatment plants using two in vivo test systems: *Allium cepa* root based and Nile tilapia erythrocyte based tests. *Water Research*, **108**, 320-329. https://doi.org/10.1016/j.watres.2016.11.009

ImageJ [Computer software]. National Institutes of Health.

Iturburu, F. G., Simoniello, M. F., Medici, S., Panzeri, A. M., & Menone, M. L. (2018). Imidacloprid causes DNA damage in fish: Clastogenesis as a

mechanism of genotoxicity. *Bulletin of Environmental Contamination and Toxicology*, **100**(6), 760-764. https://doi.org/10.1007/s00128-018-2338-0

Jiang, Z., Zhang, H., Qin, R., Zou, J., Wang, J., Shi, Q., Jiang, W., & Liu, D. (2014). Effects of lead on the morphology and structure of the nucleolus in the root tip meristematic cells of *Allium cepa* L. *International Journal of Molecular Sciences*, **15**(8), 13406-13423. https://doi.org/10.3390/ijms150813406

Kirschke, S., Avellán, T., Bärlund, I., Bogardi, J. J., Carvalho, L., Chapman, D., Dickens, C. W. S., Irvine, K., Lee, S., Mehner, T., & Warner, S. (2020). Capacity challenges in water quality monitoring: Understanding the role of human development. *Environmental Monitoring and Assessment*, **192**(5), 1-16. https://doi.org/10.1007/s10661-020-8224-3

Könen, S., & Çavaş, T. (2008). Genotoxicity testing of the herbicide trifluralin and its commercial formulation Treflan using the piscine micronucleus test. *Environmental and Molecular Mutagenesis*, **49**(6), 434-438. https://doi.org/10.1002/em.20401

Kumaravel, T. S., & Jha, A. N. (2006). Reliable comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. *Mutation Research/Genetic Toxicology and Environmental Mutagenesis*, **605**(1-2), 7-16. https://doi.org/10.1016/j.mrgentox.2006.03.002

Kurihara, D., Mizuta, Y., Sato, Y., & Higashiyama, T. (2015). ClearSee: A rapid optical clearing reagent for whole-plant fluorescence imaging. *Development*, **142**(23), 4168-4179. https://doi.org/10.1242/dev.127613

Leme, D. M., & Marin-Morales, M. A. (2009). *Allium cepa* test in environmental monitoring: A review on its application. *Mutation Research/Reviews in Mutation Research*, **682**(1), 71-81. https://doi.org/10.1016/j.mrrev.2009.06.002

Letsyo, E., Adams, Z. S., Dzikunoo, J., & Asante-Donyinah, D. (2021). Uptake and accumulation of pyrrolizidine alkaloids in the tissues of maize (Zea mays L.) plants from the soil of a 4-year-old *Chromolaena odorata* dominated fallow farmland. *Chemosphere*, **270**, Article 128669. https://doi.org/10.1016/j.chemosphere.2020.128669

Lima, M. G. F., Rocha, L. C., Silveira, G. L., Alvarenga, I. F. S., & Andrade-Vieira, L. F. (2019). Nucleolar alterations are reliable parameters to determine the cytogenotoxicity of environmental pollutants. *Ecotoxicology and Environmental Safety*, **174**, 630-636. https://doi.org/10.1016/j.ecoenv.2019.03.033

Martins, M., & Costa, P. M. (2017). The comet assay in aquatic (eco)genotoxicology using non-conventional model organisms: Relevance, constraints and prospects. In M. L. Larramendy (Ed.), *Ecotoxicology and genotoxicology: Non-traditional aquatic models* (pp. 1-32). Royal Society of Chemistry.

Matsumoto, S. T., Mantovani, M. S., Malaguttii, M. I. A., Dias, A. L., Fonseca, I. C., & Marin-Morales, M. A. (2006). Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish *Oreochromis niloticus* and chromosome aberrations in onion root-tips. *Genetics and Molecular Biology*, **29**, 148-158. https://doi.org/10.1590/S1415-47572006000100028

McLennan, M. (2021). The global risks report 2021 (16th ed.). World Economic Forum.

Ministère de l'Eau, de L'Assainissement et de l'Hygiène. (2004). Décret n°2004-635 du 15 juin 2004 portant modification du décret n°2003-941 du 09 Septembre 2003 relatif à la surveillance de l'eau, au contrôle des eaux destinées à la consommation humaine et aux priorités d'accès à la ressource en eau.

Nagpure, N. S., Srivastava, R., Kumar, R., Dabas, A., Kushwaha, B., & Kumar, P. (2015). Assessment of pollution of river Ganges by tannery effluents using genotoxicity biomarkers in murrel fish, *Channa punctatus* (Bloch). *Indian Journal of Experimental Biology*, **53**(7), 476-483.

Ohe, T., Watanabe, T., & Wakabayashi, K. (2004). Mutagens in surface waters: A review. *Mutation Research/Reviews in Mutation Research*, **567**(2-3), 109-149. https://doi.org/10.1016/j.mrrev.2004.08.003

Orsière, T., Sari-Minodier, I., Decome, L., Botta, C., Iarmarcovai, G., & Botta, A. (2005). De la génotoxicologie à la biosurveillance. *Journées Nationales de Santé au Travail dans le BTP, Annales*, **28**, 25-28.

Osman, A. G., Abuel-Fadl, K. Y., & Kloas, W. (2012). In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in *Oreochromis niloticus* *niloticus* (Linnaeus, 1758) and *Clarias gariepinus* (Burchell, 1822). *Mutation Research/Genetic Toxicology and Environmental Mutagenesis*, **747**(1), 14-21. https://doi.org/10.1016/j.mrgentox.2012.02.013

Pathiratne, A., Hemachandra, C. K., & De Silva, N. (2015). Efficacy of *Allium cepa* test system for screening cytotoxicity and genotoxicity of industrial
effluents originated from different industrial activities. *Environmental Monitoring and Assessment*, **187**(12), 1–12. https://doi.org/10.1007/s10661-015-4954-z

Qin, R., Jiao, Y., Zhang, S., Jiang, W., & Liu, D. (2010). Effects of aluminium on nucleoli in root tip cells and selected physiological and biochemical characters in *Allium cepa* var. *agrogarum* L. *BMC Plant Biology*, *10*(1), 1–11. https://doi.org/10.1186/1471-2229-10-225

R: A language and environment for statistical computing (Version 3.6.3) [Computer software]. (2022). R Foundation for Statistical Computing.

Radić, S., Gregorović, G., Stipaničev, D., Cvjetko, P., Šrut, M., Vujčić, V., Oreščanin, V., & Klobučar, G. I. V. (2013). Assessment of surface water in the vicinity of fertilizer factory using fish and plants. *Ecotoxicology and Environmental Safety*, *96*, 32–40. https://doi.org/10.1016/j.ecoenv.2013.06.023

Rasoazanany, E. O. (2008). *Highlighting of pollutants elements of atmospheric air and industrial wastewater in Antananarivo and analysis of a forest plants of the east coast of Madagascar* [Doctoral thesis, University of Antananarivo]. INIS Repository. https://inis.iaea.org/search/searchsinglerecord.aspx?recordsFor=SingleRecord&RN=42087671

Razanakoto, O. R., Raharimalala, S., Sarobidy, E. J. R. F., Rakotondravelo, J.-C., Autfray, P., & Razafimahatratra, H. M. (2021). Why smallholder farms' practices are already agroecological despite conventional agriculture applied on market-gardening. *Outlook on Agriculture*, *50*(1), 80–89. https://doi.org/10.1177/0030727020972120

SanJuan-Reyes, N., Gómez-Oliván, L. M., Galar-Martínez, M., García-Medina, S., Islas-Flores, H., González-González, E. D., Cardoso-Vera, J. D., & Jiménez-Vargas, J. M. (2015). NSAID-manufacturing plant effluent induces geno- and cytotoxicity in common carp (*Cyprinus carpio*). *Science of the Total Environment*, *530*, 1–10. https://doi.org/10.1016/j.scitotenv.2015.05.088

Scalon, M., Rechenmacher, C., Siebel, A. M., Kayser, M. L., Rodrigues, M. T., Maluf, S. W., Rodrigues, M. A. S., & Basso da Silva, L. (2013). Genotoxic potential and physicochemical parameters of Sinos River, southern Brazil. *Scientific World Journal*, 2013, Article 2097737. https://doi.org/10.1155/2013/2097737

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. *Experimental Cell Research*, **175**(1), 184–191. https://doi.org/10.1016/0014-4827(88)90265-0

Speit, G., Kojima, H., Burlinson, B., Collins, A. R., Kasper, P., Plappert-Helbig, U., Uno, Y., Vasquez, M., Beevers, C., De Boeck, M., Escobar, P. A., Kitamoto, S., Pant, K., Pfuhler, S., Tanaka, J., & Levy, D. D. (2015). Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT). *Mutation Research/Genetic Toxicology and Environmental Mutagenesis*, **783**, 6–12. https://doi.org/10.1016/j.mrgentox.2014.09.006

Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K., & Sutton, D. J. (2012). Heavy metal toxicity and the environment. *Molecular, Clinical and Environmental Toxicology*, *101*, 133–164. https://doi.org/10.1007/978-3-7643-8340-4_6

Vijayan, M. M., Pereira, C., Grau, E. G., & Iwama, G. K. (1997). Metabolic responses associated with confinement stress in tilapia: The role of cortisol. *Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology*, **116**(1), 89–95. https://doi.org/10.1016/S0742-8413(96)00124-7

Vlastos, D., Antonopoulou, M., & Konstantinou, I. K. (2016). Evaluation of toxicity and genotoxicity of 2-chlorophenol on bacteria, fish and human cells. *Science of the Total Environment*, **551–552**, 649–655. https://doi.org/10.1016/j.scitotenv.2016.02.043

Wang, Q. L., Liu, D. H., & Yue, J. Y. (2016). The uptake of cadmium by *Allium cepa* var. *agrogarum* L. and its effects on chromosome and nucleolar behavior in root tip cells. *Phyton—International Journal of Experimental Botany*, **85**, 155–161. https://doi.org/10.32604/phyton.2016.85.155

World Health Organization. (2017). Guidelines for drinking-water quality (4th ed., incorporating the 1st addendum).

Wu, J. Y., Shen, L., Gao, G., Lin, G. F., Ke, X., & Shen, J. H. (2004). A season-dependent variation of genotoxicity of surface water samples from Taihu Lake, Yangzte delta. *Environmental Monitoring and Assessment*, **98**(1), 225–234. https://doi.org/10.1023/B:EMAS.0000038188.16088.f3

ZEN soft [Computer software]. Zeiss.