Owing to the complexity of the mixture, it is difficult to determine precisely which compounds are responsible for SS phytotoxicity (Mazzeo et al. 2015). So far, phytotoxicity has been linked primarily to heavy metals (Amin 2011), because they are not easily removed and can be mobilized depending on the physicochemical conditions of the SS and soil in which this residue is applied (Udom et al. 2004). In this study, the concentration of metals in the solid matrix was in accordance with the maximum values established by the Brazilian legislation and previous results (Christofoletti et al. 2013; Martins et al. 2016). These studies show that chemical analyses may not be sufficient to characterize the possible toxic effects of SS because as suggested by Martins et al. (2016), they do not show the synergistic impact of all SS contaminants. The addition of SS increases the extractable form of diethylenetriamine pentaacetic acid, which generates potentially mobile extracts and phytotoxic fractions of heavy metals (Dhanker et al. 2021). Biological tests should complement chemical analyses to better understand the toxic potential of SS (Mazzeo et al. 2015). Moreover, annual and local testing should account for variations in the concentration of heavy metals in each period and location (Gen et al. 2021).
Techniques for heavy metal removal from SS have been tried to ensure the viable and ecologically appropriate use of this substrate in soils. For example, the application of fermentation and bioleaching was tested in anaerobic bioreactors under unregulated pH conditions (acidic or alkaline). In these settings, a combination of ultrasound pretreatment and alkaline fermentation attained the best result, reducing the bioavailability of toxic metals by up to 25% (Yesil et al. 2021). Other tested methods include the application of permeable mesh bags filled with SS and stacked next to corn, banana, and papaya crops. After nine years, more than 94% of heavy metals were found to be retained, the soil had low levels of heavy metals, and those in the harvested fruits were within the designated limits (Lin et al. 2021).
SS can also contain organic compounds with cyto-genotoxic properties, such as organochlorine pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) (Rank and Nielsen 1998). The adverse biological effects of small organic compounds can be attenuated by their rapid volatilization and biodegradation (Smith 2009). However, Mazzeo et al. (2015) monitored the natural attenuation of a domestic SS and reported high concentrations of the semi-volatile organic compound m- and p-cresol in aqueous extracts and in the crude sludge sample for up to two months. The genotoxic activity of SS samples was detected previously by the chromosome aberrations test in Allium cepa. Paraíba et al. (2010) concluded that low-molecular weight PAHs were more likely to accumulate in corn grains than high-molecular weight PAHs. Therefore, SS containing significant concentrations of light PAHs would not be recommended as fertilizers for food crops.
The organic matter of SS in this study was rich in recalcitrant compounds. Although they are highly toxic, some researchers have minimized their contribution to the phytotoxicity of the sludge due to the strong adsorption of these compounds in the solid matrix and, consequently, their lower bioavailability (Smith and Riddell-Black 2007; Smith 2009). However, the effects of these xenobiotics should not be totally neglected, since some of these compounds can be transferred via sludge/soil to plants and eventually, consumers. McLachlan et al. (1994) reported high levels of PCBs and polychlorinated dibenzofuran in milk from cows grazing in soils fertilized with SS. Therefore, we suggest that organic pollutants, when present at high levels in SS, can contribute to phytotoxicity.
According to Oleszczuk (2012), physicochemical properties determining plant development and growth (such as pH and amount of nutrients) should be taken into account during phytotoxicity analysis of SS. These properties may interfere with plant germination and development and erroneously point to other pollutants. Walter et al. (2006) argued that a combination of several factors contributed to the toxicity of SS, such as concentration and availability of heavy metals, high salinity, and excess of ammonium and organic compounds. In addition, the different toxicity responses of the plants can be attributed to the tolerance of each species or the nature of the sludge. For example, the application of SS (69, 138, and 276 kg of N ha− 1) did not influence sunflower (Helianthus annuus L.) germination and had a positive influence on morphological characteristics, growth, and seed yield (Koutroubas et al., 2020). In giant miscanthus (Miscanthus × giganteus Greef and Deuter), SS (160 kg of N ha− 1) had a similar effect on yield as mineral fertilizers (Dubis et al. 2020). In industrial hemp (Cannabis sativa L.), only 25 t ha− 1 of SS had a positive effect on plant performance, and beyond that, it had a toxic effect (Praspaliauskas et al. 2020).
The pH range indicated for phytotoxicity studies is between 5.0 and 7.0 (Baumgarten and Spiegel 2004). Dhyèvre et al. (2014) documented that primary roots of Vicia faba were necrotized and secondary roots did not exhibit any growth in soil with pH 10.1. Liming helps stabilize SS while also reducing the content of pathogens and augmenting pH (Costa and Costa 2011). However, the stabilized alkaline SS can adversely affect plant growth due to nutritional deficiency (Samaras et al. 2008) from a smaller humic fraction (Ramos et al. 2015). The highest concentrations of sludge exhibited an elevated pH, which can explain the significant inhibition of germination and initial development of L. sativa and P. alata. This effect was observed with respect to increased electrical conductivity. The soluble salts present in SS can negatively affect the growth of plants, reducing the osmotic potential and compromising the development of the species (Baumgarten and Spiegel 2004).
The results obtained in the present study corroborate those observed by Martins et al. (2016) in A. cepa grown in commercial substrate mixed with SS, whereby 28% and 56% reduction in germination was observed at SS concentration of 320 t ha− 1 and 520 t ha− 1, respectively. Oleszczuk et al. (2012) observed that Sinapis alba (white mustard) seeds presented germination inhibition of 60% in sandy soil treated with SS (90 t ha− 1), whereas Kou et al. (2020) reported that the percentage and germination index of Brassica campestris L. ssp. Chinensis Makino (Chinese white cabbage) seeds decreased with an increased concentration of sludge, together with increased inhibition of root elongation. In the present study, the deleterious effect of SS addition was more pronounced in P. alata. Our data are similar to those observed by Prado and Natale (2005), who concluded that SS, at doses above 10 t ha− 1, promoted death of passion fruit due to the presence of metals and excess sodium and potassium ions. In this study, sodium was also present at a very high concentration. Dias et al. (2012) reported that salinity strongly affected passion fruit growth and should be carefully monitored.
SS may also cause disturbances in the plant’s cell cycle (Martins et al. 2016). Alterations in cell division can indicate the level of cytotoxicity of environmental pollutants, which compromise metabolic processes, growth, and development (Leme and Marin-Morales 2009; Singh et al. 2009). Faschineto et al. (2007) observed that reduction in the mitotic index coincided with decreased root growth. An increased frequency of interphase cells at SS concentration of up to 120 t ha− 1 suggests that repair mechanisms lock the cells at this stage to prevent mitosis in cells with possible genetic abnormalities (Palmieri et al. 2014). When repair processes fail, cell death pathways can be activated. Here, the genotoxic effect of elevated SS concentrations was manifested by the presence of condensed nuclei in L. sativa root cells (Fig. 5), which led to cell death.
Other commonly observed cell cycle alterations were induction of c-metaphase (84%), followed by the presence of micronuclei (14%), bridges (11%), sticky chromosomes (6%), and chromosome loss (3%). Tests detecting mitotic and chromosomal abnormalities help identify the mechanism of action employed by toxic compounds. These may be clastogenic, with abnormalities, such as chromosome breakage and bridges or aneugenic, leading to anomalies, such as chromosomal losses, c-metaphase, and sticky chromosomes (Leme and Marin-Morales 2009; Rossato et al. 2010). It is possible to infer that the studied SS concentrations employed both aneugenic and clastogenic mechanisms of action (Fig. 5), although the former were prevalent. These results are similar to those found by Amin et al. (2009), who studied V. faba meristem cells and found changes in the mitotic spindle after consecutive SS applications to the soil. Moreover, Amin (2011) demonstrated the occurrence of aneugenic effects in meiocytes of Zea mays after exposure to SS.
A micronucleus can be formed by chromosomal fragments or by the loss of entire chromosomes. As no breakage was observed in this study, it appears that micronuclei were formed by the loss of entire chromosomes, further confirming the aneugenic effect of SS.
The increase in cyto-genotoxicity observed in root meristem cells of L. sativa as a function of increased pH and addition of SS corroborates the results of Dhyèvre et al. (2014). According to the authors, under extreme conditions of pH (3.5, 4.3, and 9.0) root cells of V. faba exposed to soil contaminated with 5 µg kg− 1 maleic hydrazine showed a higher frequency of micronuclei and a lower mitotic index. At the highest tested concentrations of maleic hydrazine, the cell proliferation rate decreased dramatically, indicating double stress, i.e., extreme pH and xenobiotic concentration.