The rapid urban development combined with agricultural practices and inadequate systems of collection and treatment of effluents has led to a global concern about the pollution of water resources. Considering the interactions between complex chemical mixtures in effluents, conventional analyses of water physicochemical parameters are not suitable for hazard assessment (Silveira et al. 2017). Thus, studies integrating water quality parameters and bioassays are critical in the water quality evaluation.

Plants are excellent biological systems because they are good bioindicators of toxicity, with high sensitivity to detect cytotoxic and mutagenic agents through different endpoints, including effects in root meristematic cells (Matos et al. 2017). The Allium cepa test is a simple and reliable bioassay for evaluating cytotoxicity and mutagenicity of environmental substances (Leme and Marin-Morales 2009; Gupta et al. 2018). In addition, fishes are also often used as biological indicators of water quality, and biomonitors for the presence of genotoxic aquatic pollutants (Lemos et al. 2007). The micronucleus test in fish is widely used to detect DNA-damaging substances in the water (Bolognesi and Hayashi 2011).

In southern Brazil, the Ilha River and the Paranhana River are the main tributaries of the Sinos River – one of the most polluted rivers in Brazil and that supplies water for more than 1.6 million inhabitants. Although several studies have been conducted in the Sinos River basin (Nunes et al. 2011; Bianchi et al. 2015; Souza et al. 2016; Alves et al. 2018; Peteffi et al. 2019), only a few have focused on the water quality of the tributaries (Fontanella et al. 2008; Rodrigues et al. 2016; Dalzochio et al. 2019). The assessment of the water quality of these rivers is important because they are also under anthropogenic impacts, related mainly to sewage and industrial discharges, agricultural runoffs and solid waste disposal and thus, they may contribute negatively to the water quality of the basin. Hence, the objectives of this study were to: (1) assess physicochemical and microbiological parameters of water samples collected at four sites of these two Sinos River tributaries; (2) evaluate micronucleus (MN) and nuclear abnormalities (NA) frequencies in fish exposed under laboratory conditions to the water samples and; (3) analyze mitotic index (MI), micronucleus (MN) and chromosomal aberrations (CA) frequencies in A. cepa seeds exposed to water samples.

Methods and Materials

The Sinos River basin is situated northeast of the State of Rio Grande do Sul, southern Brazil. Sampling sites are located in the Ilha River and Paranhana River. Sites 1 and 2 (S1 and S2) are located in the source and mouth of the Ilha River, respectively (29°33′21.16″S; 50°37′45.55W and 29°40′56.42″S; 50°44′22.86″W). These sites are mainly under agricultural impacts, especially S2. Sites 3 and 4 (S3 and S4, source and mouth, respectively) (29°25’29.39”S; 50°46’17.05”W and 29°39’32”S; 50°48’07”W) are located in the Paranhana River, where discharges of industrial and domestic effluents occur.

Approximately 15 L of water samples were collected at each site for water quality parameters analysis and laboratory experiments with fish and A. cepa in August and November/2015 and February and May/2016. The collection, transport and analysis procedures were in accordance with the recommendations of the Standards Methods for the Examination of Water and Wastewater (APHA 2005). The following physicochemical and microbiological parameters were analyzed: biochemical oxygen demand (BOD5), total phosphorous, ammoniacal nitrogen, aluminum, iron, lead, total chromium, nickel, zinc, total and thermotolerant coliforms. The laboratory where water analyses were performed is accredited by the State Environmental Protection Agency (FEPAM), and also followed the APHA recommendations.

For fish experiments, Astyanax jacuhiensis specimens measuring 3–4 cm and weighing 5 g were obtained from a local fish farm. The animals obtained from this fish farm were apparently healthy (with no damage to the skin and fins) and, in previous studies from our research group, they always showed basal levels of cytogenetic damage (Bianchi et al. 2015, 2019). This fish species was chosen because it is native in southern Brazil (Lima et al. 2003) and has successfully been used as a bioindicator organism in previous studies (Bianchi et al. 2015, 2019; Marins et al. 2020). Before each sampling campaign, fish were fed daily and acclimated in glass aquaria containing 5 L of dechlorinated tap water, under constant aeration and natural photoperiod for seven days at 21 ± 1 °C. Then, fish were exposed to water samples collected at each site (n = 10 per group) for five days. A control group was maintained in dechlorinated tap water during the same period. A semi-static procedure was carried out and 50 % of water volume was renewed with the water from each site (stored at 4°C until renewal) at the third day after the exposure had begun. By the end of the exposure period, fish were killed for blood collection. This study was approved by the Ethics Committee for Animal Experimentation of Universidade Feevale (protocol n. 02.13.022).

For the MN test, blood smears were prepared and fixed in absolute alcohol for 10 min and air-dried overnight. Slides were then stained with Giemsa 5% for 10 min and rinsed in tap water. Slides were coded and analyzed under light microscopy according to criteria described by Al-Sabti and Metcalfe (1995). A total of 2000 erythrocytes per fish were counted at 1000× magnification under an oil-immersion objective. Other nuclear anomalies, e.g. invaginations, buds and binucleated cells were assessed as described by Carrasco et al. (1990), and were grouped as nuclear abnormalities (NA) (Seriani et al. 2015; Vieira et al. 2016).

For A. cepa bioassay, seeds were previously germinated in Petri dishes containing filter paper with distilled water at 25°C. Roots measuring approximately 1 cm were exposure to water samples for 24 h. A control group was maintained in distilled water. After exposure, roots were fixed in Carnoy solution overnight and stored in alcohol 70% at 4°C. For slides preparation, roots were hydrolyzed with HCl 1 N at 60°C for 10 min, rinsed with distilled water and stained with acetic orcein 1% for 30 min. A total of 10 slides containing two roots were prepared for each group. The microscopic analysis included cytotoxicity evaluation, where the number of cells under division was recorded (analysis of 1000 cells) – corresponding to the mitotic index (MI), and the genotoxicity evaluation, where the frequency of micronucleated cells (analysis of 1000 interphase cells) and CA (analysis of 100 anaphase-telophase cells) were estimated. CA included chromosome losses, bridges and chromosome laggards (Silveira et al. 2017; Liman et al. 2020).

Statistical analysis was performed using the Kruskal-Wallis test, followed by the Dunn’s multiple comparison test, when appropriated. All analyses were performed using the Statistical Package for the Social Sciences – SPSS 22 considering a significance level of p ≤ 0.05.

Results and Discussion

Water physicochemical and microbiological analysis revealed mean values of aluminum and iron above the limits established by the Brazilian legislation at all sampling sites. Mean values of thermotolerant coliforms also exceeded the limits at all sites, except for S1, and nickel was found above the limits only at S2 (Table 1). Ammoniacal nitrogen, copper, total chromium and lead were not detected in the samples. Previous studies conducted on the basin also detected concentrations of iron, aluminum and thermotolerant coliforms above the limits established by the Brazilian legislation (Dalla Vecchia et al. 2015; Souza et al. 2016; Bianchi et al. 2019). These studies reported higher levels of such parameters at sites located in the middle and lower section of the basin, where anthropogenic activities are more pronounced. The high levels of metals, such as aluminum, iron and nickel, could be related to the lack of basic sanitation, discharges of industrial effluents and disposal of solid waste near the rivers, as well as to the soil composition of the region (Jorgensen et al. 2012; Oliveira and Henkes 2013). In general, higher values of thermotolerant coliforms were detected at the mouth of rivers (S2 and S4), suggesting contamination by sewage discharge and/or fecal material of swine and cattle farms (Blume et al. 2010). These results highlight the lack of basic sanitation in the tributaries, which can negatively affect the basin.

Table 1 Mean, minimum and maximum values (in parenthesis) of physicochemical and microbiological parameters of four water samples collected at four sites located in the Ilha River (S1 and S2) and Paranhana River (S3 and S4)
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Plant and fish bioassays are tools that can demonstrate potential toxic effects reflecting interactions among mixtures of contaminants present in water resources. Considering the genotoxic effects in fish, frequencies of MN were low and varied from 0 to 0.27‰. Statistically significant differences were observed only in Nov/15, when higher MN frequencies were found in S4 in comparison to control group (p = 0.03). Frequencies of NA varied from 0.83 to 3.77‰ and there were no statistical differences among groups (Table 2). Microscopic findings are demonstrated in Fig. 1. Low frequencies of MN and NA were also found in other studies which aimed to assess the genotoxic potential of the Sinos River using A. jacuhiensis. Bianchi et al. (2015) found mean frequencies of MN below 0.25‰ and of NA below 1 ‰ in fish exposed for 96 h to water samples collected at different sites of the basin. Frequencies of MN ranging from 0 to 0.4 ‰ and NA ranging from 0.83 to 5,44‰ were also found in A. jacuhiensis exposed for 72 h to water samples collected at five sites of the basin (Bianchi et al. 2019). A. jacuhiensis is a native species, widely distributed in aquatic environments in Brazil and is tolerant to oscillations of water physicochemical parameters (Bemvenuti and Moresco, 2005), thus it has been successfully used in ecotoxicological studies (Bianchi et al. 2015; Marins et al. 2020), as well as in the present study. Nonetheless, the increased frequency of MN at S4 could the related to contamination by urban effluents as a consequence of the presence of industries and higher population density in the area in comparison to S3.

Table 2 Micronucleus (MN) and nuclear abnormalities (NA) in Astyanax jacuhiensis exposed for five days to water samples collected at four sites located in the Ilha River (S1 and S2) and Paranhana River (S3 and S4) and control group (tap water). Data are expressed as mean ± standard deviation
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Fig. 1
figure 1

Representative images of Astyanax jacuhiensis erythrocytes. A micronucleated cell (arrow); B notched nucleus (asterisk); C nuclear bud (arrow head). Bar = 10 μm

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In the A. cepa bioassay, MI varied from 5.98 to 10.86. It was possible to observe a significant decrease of MI in the mouth of the rivers (S2 and S4) in comparison to the sources (S1 and S3) and control group in all sampling periods (p < 0.001), except for Aug/15. However, a significant decrease of MI was found in the mouth of both rivers in comparison to control group in Aug/15 (p < 0.001). Dourado et al. (2017) and Nunes et al. (2007) have also evidenced inhibition of MI in roots exposed to water samples from contaminated areas. In this context, metals could induce disturbances in cell cycle or dysfunction of chromatin by interaction of metal and DNA (Glinska et al. 2007). Morever, similar findings were observed by Rodrigues et al. (2016) who observed cytotoxic effects in A. cepa roots exposed to water samples collected in the Ilha River.

Frequencies of MN varied from 0.4 to 1.3, whereas frequencies of CA varied from 0 to 0.80. However, there were no significant differences among groups (Table 3). Similarly, Nunes et al. (2007) also observed only cytotoxic effects in A. cepa roots exposed to water samples from the Sinos River. Thus, although metals and other substances could have induced cytotoxic effects in the present study, they did not cause mutagenic and genotoxic damage in A. cepa roots cells exposed to water samples. Microscopic findings are demonstrated in Fig. 2.

Table 3 Mitotic index (MI), micronucleus (MN) and chromosomal aberrations (CA) in Allium cepa seed exposed for 24 h to water samples collected at four sites located in the Ilha River (S1 and S2) and Paranhana River (S3 and S4) and control group (tap water). Data are expressed as mean ± standard deviation
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Fig. 2
figure 2

Representative images of Allim cepa meristematic cells. A Cell in interphase with micronucleus (arrow); B cell in anaphase with chromosomal bridge (asterisk); C cell in telophase with chromosomal loss (arrow head). Bar = 10 μm

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In this study, the analysis of water quality parameters evidenced the poor water quality of the tributaries of the Sinos River, especially considering the levels of iron, aluminum and thermotolerant coliforms. In addition, a spatial variation in the cytogenotoxic potential of pollutants was observed using the fish and plant bioassays. However, it is important to highlight that only two sampling sites were selected for each tributary and that, in order to better assess the status of the water quality, more sampling sites should be further evaluated. The most relevant results were the toxicity on mitotic activity of A. cepa root meristem induced by water samples of the four sampling sites. The cytotoxicity observed in the mouth of both rivers raises environmental concerns. Thus, the use of different approaches to assess the water quality is important for evaluating the environmental risks posed by contaminants.