Ecotoxicological characterization of surface water, groundwater, and leachate from a sanitary landfill

Abstract

Improper disposal of such waste represents a serious threat to the environment due to its potential to contaminate soil, rivers, and oceans, thereby endangering biota and public health. This study aimed to evaluate the ecotoxicological effects of raw and treated leachate, as well as surface water and groundwater adjacent to a Class II landfill, on germination and early growth parameters of Allium cepa L. and the viability or survival of Aliivibrio fischeri. The tested sample concentrations were 25, 50, 75, and 100%, each diluted in distilled water. In the phytotoxicity assays, A. cepa seeds were exposed to different sample concentrations, with distilled water serving as a negative control. The analyzed parameters included germination percentage, germination speed index, shoot length, root elongation, mitotic index, and chromosomal abnormalities. In the acute toxicity tests, the effect on bacterial luminescence was evaluated. Surface water and groundwater samples did not induce toxicity in A. cepa seeds, whereas raw and treated leachate samples inhibited germination and consequently negatively affected all other evaluated parameters. In A. fischeri assays, leachate samples exhibited toxicity, while surface water and groundwater samples induced a hormesis effect. The results from adjacent water samples demonstrate the landfill’s efficiency in mitigating contamination of water bodies.

Key words
Acute toxicity; cytotoxicity; genotoxicity; Allium cepa L; Aliivibrio fischeri

INTRODUCTION

Consumption in developing countries has increased exponentially in recent decades due to the growth of industrial production and the availability of new technologies, leading to a rise in waste disposal. Consequently, the waste generated by advancements in the productive sector may contain potentially harmful chemical compounds that can enter the environment and disrupt biological cycles due to their toxicity and persistence (Cunha & Pasqualetto 2021). Therefore, the increasing generation of municipal solid waste, its proper disposal, and the management of the environmental liabilities it creates require more effective management strategies.

One of the widely used alternatives is sanitary landfills, which can cause severe environmental impacts in inhabited areas and conservation sites located in their surroundings (Rani et al. 2020). Although modern landfills are designed and engineered to eliminate or minimize these impacts, the generation of leachate, commonly known as landfill leachate, remains a significant challenge. This is because leachate can contain a wide variety of chemical and biological contaminants, either originating from the waste itself or produced during its degradation, facilitating their transport into the soil, surface water, and groundwater (Luo et al. 2020).

Leachate is characterized as a percolate with a complex composition that may contain both organic compounds and inorganic elements, contributing to its toxicity, high organic load, and low biodegradability (Santos et al. 2021). When improperly managed, leachate can become a significant source of pollution. Therefore, its monitoring is essential to prevent negative impacts on terrestrial and aquatic biota, as well as on the health of populations living under its direct or indirect influence (Luo et al. 2020).

In this context, it is noted that leachate monitoring can include both physicochemical and ecotoxicological parameters. Physicochemical parameters are highly important as they allow verification of compliance with legislated environmental standards. Additionally, these parameters help refine water and soil quality monitoring plans in areas influenced by the landfill, enabling the detection of harmful pollutants and contributing to the planning and implementation of control measures (Luo et al. 2020).

On the other hand, ecotoxicological assessments enable the evaluation of deleterious effects on test organisms through their exposure to different simple or complex samples in standardized and validated laboratory tests (Gomes et al. 2012). These tests can be either acute (Tavares et al. 2014) or chronic (Mkandawire et al. 2014), allowing the assessment of different and complementary parameters, thereby predicting and inferring potential environmental damage in impacted areas (Maselli et al. 2015, Rebolledo et al. 2020).

In this context, the objective of this study was to evaluate the toxicity of leachate samples (raw and treated) from a sanitary landfill, as well as surface water and groundwater samples collected from areas adjacent to the landfill. The assessment was conducted using cytotoxicity and phytotoxicity tests with Allium cepa L., acute toxicity tests with Aliivibrio fischeri, and physicochemical parameter analysis.

MATERIALS AND METHODS

Study area characterization

The regional sanitary landfill is located in the southern region of the state of Minas Gerais, Brazil (21°30’50”S 45°55’21”W). This landfill receives municipal solid waste from 11 municipalities, with a total population of 258,857 inhabitants (IBGE 2021). The facility processes 4,500 tons of waste per month, generating 1,200,000 liters of leachate, which undergoes treatment in an anaerobic lagoon (raw leachate) before flowing into a facultative lagoon (treated leachate). In the facultative treatment lagoon, the leachate is collected and transported to a wastewater treatment plant (WWTP) managed by a third-party company, which is responsible for its final disposal.

Sample collection

The sampling points are distributed within the sanitary landfill site, with samples collected from seven locations, characterized as follows: Upstream Surface Water (USW), Downstream Surface Water (DSW), Upstream Groundwater Point 1 (UGP1), Downstream Groundwater Points 1 and 2 (DGP1 and DGP2), Raw leachate (RL), and Treated leachate (TL). The coordinates of the sampling points are presented in Figure 1.

Figure 1
Location of the sanitary landfill, highlighting the sampling points. USW - 21°31′00″S, 45°55′29″W; DSW - 21°31′01″S, 45°55′12″W; UGP1 - 21°30′44″S, 45°55′20″W; DGP1 - 21°30′44″S, 45°55′20″W; DGP2 - 21°30′58″S, 45°55′19″W; RL - 21°30′55″S, 45°55′29″W; TL - 21°30′57″S, 45°55′25″W.

Physicochemical analysis of the samples

During sampling, the samples were evaluated for pH, conductivity, and temperature. In the laboratory, they were subjected to analyses for the quantification of chlorides, settleable solids, dissolved oxygen, organic compound parameters and organic loads, nutrients, trace elements, and pollution indicators. The analyses were conducted following the Standard Methods for the Examination of Water and Wastewater (Rice et al. 2012), Method No. 1060, and the ABNT NBR 9898 standard (ABNT 1987) for the preservation and sampling techniques of liquid effluents in receiving bodies. For surface water and groundwater analyses, the obtained values were compared with CONAMA Resolution No. 357/2005 (Brasil 2005) and CONAMA Resolution No. 396/2008 (Brasil 2008), respectively, which are the prevailing regulations in the region where the enterprise is located.

Phytotoxicity test

The phytotoxicity test was performed according to the protocol established by Cunha Neto et al. (2023), with adaptations. A total of 30 A. cepa cv. “Baia Periforme” seeds were placed in a Petri dish (Ø 6.5 cm), with three replicates. For each experimental unit, 3 mL of each sample, solubilized in distilled water at concentrations of 0, 25, 50, 75, and 100%, were added.

The evaluated germination parameters included the first germination count (2nd day), final germination percentage (10th day), and the germination speed index (GSI), calculated according to Maguire (1962) at 12-hour intervals. For the assessment of root length and shoot length, ten seedlings from each replicate were analyzed at the end of the experiment using the Fiji-ImageJ software, and their respective values were obtained for each treatment.

The experiments were conducted in a B.O.D. (Biochemical Oxygen Demand) germination chamber at a temperature of 25°C and a photoperiod of 12 hours.

Cytotoxicity test

Cytogenotoxic evaluations were performed using root meristems obtained from the germination of A. cepa seeds, exposed to the same experimental conditions described previously. The root tips were fixed in Carnoy solution (3:1) and stored at -18°C. Cytological preparations were conducted using the squash method, as described by Cunha Neto et al. (2023). Based on cell counting, the EC50% for the mitotic index was determined.

Acute toxicity test

The acute toxicity tests with A. fischeri were conducted in accordance with the technical standard NBR 15411-3, Aquatic Ecotoxicology – Determination of the Inhibitory Effect of Water Samples on the Light Emission of Vibrio fischeri (ABNT 2021), following the protocol established by the MICROTOX® Omni Software, version 4.1.

Lyophilized bacteria were obtained from SDI and stored at -20°C. Each bacterial ampoule contained a culture of 10⁸ cells. The 50% effective concentrations (EC50) were determined by measuring bacterial luminescence after 30 minutes of exposure to six sample dilutions (81.9%, 40.95%, 20.48%, 10.24%, 5.12%, and 2.56%).

To assess toxic effects, the software compares the light emitted by the sample at different dilutions. The lower the emitted light, the higher the sample toxicity. Thus, the relative toxicity of the sample is expressed as a percentage of inhibition compared to the control (Stolte et al. 2012).

Experimental design and statistical analysis

The experimental design followed a completely randomized design (CRD) with a two-factor factorial arrangement.

For the phytotoxicity test, the factorial design was 7 × 5, corresponding to the sampling points (Upstream Surface Water, Downstream Surface Water, Upstream Groundwater Point 1, Downstream Groundwater Points 1 and 2, Raw leachate, and Treated leachate) and the sample dilutions (0%, 25%, 50%, 75%, and 100%).

For the acute toxicity test, the factorial design was 7 × 6, corresponding to the same sampling points as in the phytotoxicity test and the sample dilutions (81.9%, 40.95%, 20.48%, 10.24%, 5.12%, and 2.56%).

The data were subjected to analysis of variance (ANOVA) and regression analysis for model fitting at a 5% significance level using the Sisvar software (Ferreira 2019). The EC50% values were determined using the regression equations obtained, calculating the concentration at which a 50% reduction occurred in the evaluated parameters, including the luminescence of A. fischeri, phytotechnical parameters, and the mitotic index of A. cepa.

Ethics statement

Ethics committee approval was not required because the study used environmental samples, Allium cepa seeds, and Aliivibrio fischeri and did not involve human participants or vertebrate animals.

RESULTS AND DISCUSSION

Physicochemical analyses

The quality of the leachate generated depends on a variety of factors, such as waste composition, landfill age, temperature exposure, operational practices, moisture content, oxygen availability, regional climate, among others. All these factors directly affect the physicochemical characteristics of the leachate (Sharma & Dubey 2005).

Initially, it was observed that no suspended solids, oils, or greases were found in the raw leachate, unlike the treated leachate, in which these parameters were quantified at 330 mg L⁻¹ and 15,000 mg L⁻¹, respectively. The temperature of the samples also changed after treatment, with the treated leachate showing a reduction to 24.3°C compared to 26.2°C in the raw leachate.

Regarding pH, both samples exhibited values characteristic of basicity. The data for Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) indicate a significant reduction in values from the raw to the treated leachate: from 2291 mg L⁻¹ O₂ to 869 mg L⁻¹ O₂ for BOD and from 5728 mg L⁻¹ O₂ to 2172 mg L⁻¹ O₂ for COD. Additionally, ammonia nitrogen levels decreased from 975 mg L⁻¹ in the raw leachate to 138 mg L⁻¹ in the treated leachate.

Regarding trace elements, when detected, they were found in lower concentrations, which do not significantly impact the composition of the samples (Table I).

Table I
Physicochemical characterization of raw and treated leachate.

Assays	Raw leachate	Treated leachate	Unit	Method
Suspended Solids	-	330	mg/L	SM 2540 D
Settleable Solids	0.3	0.7	mL/L	SM 2540 F
Sample Temperature	26.2	24.3	°C	SM 2550 B
pH	8.84	7.99	-	SM 4500 H+B
Oils and Greases	-	15000	mg/L	SM 5520 B
Conductivity	39900	15880	μS/cm	SM 2510 B
Biochemical Oxygen Demand (BOD)	2291	869	mg/L O₂	SM 5210 B
Chemical Oxygen Demand (COD)	5728	2172	mg/L O₂	SM 5220 D
Escherichia coli	<1	<1	CFU/100 mL	SM 9213 D
Surfactants	1.34	<0.10	mg/L	SM 5540 C
Total Aluminum	-	0.6	mg/L	SM-3120 B
Total Arsenic	-	0.059	mg/L	SM-3120B
Total Barium	-	0.17	mg/L	SM-3120B
Total Boron	-	1.69	mg/L	SM-3120B
Total Cadmium	0.0031	0.0016	mg/L	SM-3120B
Total Lead	0.065	<0.005	mg/L	SM-3120 B
Total Cyanide	-	<0.02	mg/L	SM 4500-CN
Chlorides	5.66	3.56	mg/L	SM 4500-Cl B
Total Cobalt	-	0.019	mg/L	SM-3120B
Soluble Copper	0.065	0.018	mg/L	SM-3120 B
Total Copper	-	0.013	mg/L	SM-3120 B
Hexavalent Chromium	-	<0.01	mg/L	SM 3500 Cr
Total Chromium	0.57	0.026	mg/L	SM-3120 B
Total Tin	-	<0.5	mg/L	SM-3120B
Soluble Iron	-	5.7	mg/L	SM-3120 B
Total Phosphorus	18.78	11.96	mg/L	SM 3120B
Total Lithium	-	<0.1	mg/L	SM-3120 B
Total Mercury	-	<0.0002	mg/L	SM-3112 B
Total Nickel	0.18	0.094	mg/L	SM-3120B
Nitrate	<1.0	<1.0	mg/L NO₃⁻	SM 4500 NO₃⁻ E
Ammonia Nitrogen	975	138	mg/L N-NH₃	SM 4500NH3 F
Total Silver	-	<0.01	mg/L	SM-3120B
Total Selenium	-	<0.01	mg/L	SM-3120 B
Total Vanadium	-	0.11	mg/L	SM-3120 B
Total Zinc	0.56	0.1	mg/L	SM-3120 B

The absence of suspended solids in raw leachate, in contrast to the 330 mg L⁻¹ found in treated leachate, suggests a higher biodegradation of organic compounds due to the action of aerobic microorganisms (Ali et al. 2021). This increased degradation is evidenced by the reduction in the concentrations of nitrogenous compounds and total phosphorus in the treated leachate compared to the raw leachate.

The aerobic process, used for the removal of organic matter and nutrients from water, can lead to a higher concentration of suspended particles, resulting in the formation of flocs that can aggregate and settle. This phenomenon also explains the increase in settleable solids in the treated leachate (Zeng et al. 2024).

The reduction in the temperature of the treated leachate (24.3°C) compared to the raw leachate (26.2°C) may indicate thermal stabilization, which is associated with the biological degradation process occurring in the facultative lagoon (Tałałaj et al. 2019).

Reductions in Biochemical Oxygen Demand (BOD - 2291 to 869 mg L⁻¹ O₂), Chemical Oxygen Demand (COD - 5728 to 2172 mg L⁻¹ O₂) and ammonia nitrogen (975 to 138 mg L⁻¹) were accompanied by a decrease in phytotoxic effects in Allium cepa, as reflected by higher EC50 values for germination (20.05 to 65.98%), shoot length (16.50 to 53.14%), and root elongation (19.09 to 39.19%). This indicates that the reduction in organic load was sufficient to attenuate toxicity in the plant-based assay.

In contrast, the bacterial assay showed increased sensitivity to the treated leachate, with Aliivibrio fischeri presenting a lower EC50 (14.43%) than the raw leachate (31.86%), indicating that reductions in BOD, COD and ammonia nitrogen did not correspond to a decrease in bacterial toxicity. This divergence between bioassays suggests that toxicity was not governed solely by bulk physicochemical parameters. This behavior may be associated with the persistence of recalcitrant organic compounds not fully captured by BOD and COD, as well as the formation of intermediate by-products during biological treatment.

Additionally, residual ionic species and complex mixtures of contaminants may exert combined or synergistic effects, particularly in more sensitive organisms, whose response is influenced by changes in chemical composition rather than total organic load alone (Narciso et al. 2023).

Furthermore, the reduction in ammonia nitrogen levels may indicate the effectiveness of the treatment system in mitigating nitrogenous contaminants, which are associated with significant ecotoxicological impacts (Ramli et al. 2021). However, the maximum allowable limits for BOD and COD in treated wastewater are 40 mg/L and 120 mg/L, respectively, according to a study evaluating different methodologies for the treatment and removal of toxic pollutants from wastewater (Saravanan et al. 2021).

The analysis of chlorophyll-a in surface water samples collected both upstream (USW) and downstream (DSW) of the landfill showed concentrations of 57.5 mg m⁻³ and 58.7 mg m⁻³, respectively, whereas no detectable values were found in groundwater samples for this parameter. This difference between water samples was also observed for E. coli, with surface water presenting higher values—240 CFU/100 mL and 680 CFU/100 mL—compared to groundwater, which showed lower concentrations of 6 CFU/100 mL, 3 CFU/100 mL, and less than 1.0 CFU/100 mL.

The electrical conductivity results obtained from the analysis of surface water and groundwater samples reflect variations in the ionic load of these waters. Specifically, the DSW and USW samples exhibited conductivities of 18.36 μS/cm and 19.25 μS/cm, respectively. In contrast, groundwater samples, collected from different depths and proximities to the landfill (UGP1, DGP1, and DGP2), showed higher conductivity values, reaching 61.71 μS/cm, 26.55 μS/cm, and 63.3 μS/cm, respectively (Table II).

Table II
Physicochemical characterization of surface water and groundwater. DSW – Downstream Surface Water; USW – Upstream Surface Water; UGP1 – Upstream Groundwater Point 1; DGP1 and DGP2 – Downstream Groundwater Points 1 and 2.

Assays	DSW	USW	UGP1	DGP1	DGP2	Unit	Method
Surfactants	<0.1	0.5	-	-	-	mg/mL	SM 5540 C
Chlorophyll-a	57.5*	58.7*	-	-	-	mg/m³	SM 10200 H
Biochemical Oxygen Demand (BOD)	3	<2.0	-	-	-	mg/L O₂	SM 5210 B
Cyanobacteria Density	<1.0	<1.0	-	-	-	Cells/mL	SMEWW 10200 A-F
Chemical Oxygen Demand (COD)	8	<5.0	-	-	-	mg/L O₂	SM 5220 D
Escherichia coli	240	680	6**	3**	<1.0**	CFU/100 mL	SM 9213 D
Water Level	0	0	12.5	2.3	11.32	m	(-)
Oils and Greases	<10*	<10*	-	-	-	mg/L	SM 5520 B
Dissolved Oxygen	7.04	7.01	-	-	-	mg/L O₂	SM 4500-O G
pH	6.38	6.29	6.12	6.21	6.18	-	SM 4500 H+B
Total Cadmium	<0.0005	<0.0005	<0.0005	<0.0005	<0.0005	mg/L	SM-3120B
Total Lead	<0.005	<0.005	<0.005	<0.005	<0.005	mg/L	SM-3120 B
Chlorides	-	-	1	3.3	1	mg/L	SM 4500-Cl B
Soluble Copper	<0.005	<0.005	<0.005	<0.005	<0.005	mg/L	SM-3120 B
Conductivity	18.36	19.25	61.71	26.55	63.3	μS/cm	SM 2510 B
Total Chromium	<0.01	<0.01	<0.01	<0.01	<0.01	mg/L	SM-3120 B
Total Phosphorus	0.015	0.011	-	-	-	mg/L	SM 3120B
Total Nickel	<0.01	<0.01	-	-	-	mg/L	SM-3120B
Nitrate	1	<1.0	<1.0	<1.0	<1.0	mg/L NO₃⁻	SM 4500 NO₃⁻ E
Nitrite	-	-	<0.03	<0.03	<0.03	mg/L NO₂⁻	SM 4500-NO₂ B
Ammonia Nitrogen	<0.05	<0.05	<0.05	<0.05	<0.05	mg/L N-NH₃	SM 4500NH3 F
Total Zinc	0.34*	0.3*	0.52	0.49	0.37	mg/L	SM-3120 B

• Values exceeding the maximum allowed limits in *Table I of CONAMA Resolution No. 357/2005, Class II - Freshwater; **Annex 1 of CONAMA Resolution No. 396/2008 for human consumption.

Although electrical conductivity is not a legally regulated parameter under CONAMA Resolution No. 396/2008, this variable can indicate an increase in aquatic pollution, as it is directly related to the concentration of total dissolved solids and turbidity. Turbidity reflects the amount of light retained by suspended particles (Slaets et al. 2014), which can, in turn, dissolve and consequently increase the electrical conductivity of the water body, influencing the total dissolved solids values (Paula-Filho et al. 2020).

This trend is observed in this study, where the conductivity values of both raw and treated leachate, as well as suspended solids, are higher, particularly when compared to surface water and groundwater. Conductivity values may also be influenced by the geological characteristics and surrounding environment of the study site. The results obtained in this study are consistent with those reported in other aquatic environments in the region (Silva et al. 2018, Melo et al. 2022). According to Von Sperling (2007), freshwater bodies typically exhibit conductivity levels ranging from 10 to 100 µS/cm.

The high concentrations of chlorophyll-a found in surface water can be explained by the greater availability of light and nutrients at the surface, which favors the growth of phytoplankton, for which chlorophyll-a is a direct indicator (Manzano-Sarabia & Salinas-Zavala 2008). Conversely, in groundwater, no detectable values were recorded for this parameter, which is expected, as the absence of light prevents photosynthesis and, consequently, the production of chlorophyll-a. These results align with the inherent characteristics of surface water and groundwater environments, where abiotic conditions and the presence of photosynthetic organisms differ substantially (Fernandes et al. 2023).

The analysis of E. coli highlights the presence of microbiological contaminants in surface water. This can be attributed to surface water runoff, which transports organic matter and fecal waste into surface water bodies, particularly during periods of intense rainfall (Bhatnagar & Thakral 2023).

On the other hand, groundwater, filtered through various soil layers, generally exhibits lower microbial contamination due to the natural filtration process, which removes or significantly reduces bacterial loads. However, this reduction is not sufficient to ensure suitability for human consumption, according to CONAMA Resolution No. 396/2008 (Da’ana et al. 2021).

The electrical conductivity results show a significant variation between surface water and groundwater. Surface-water samples had a lower concentration of dissolved ions. In contrast, groundwater exhibited significantly higher conductivity, which can be attributed to the leaching of minerals from the soil and surrounding rocks, increasing the ionic load of these waters (Zhang et al. 2022).

Ecotoxicological tests

In the A. cepa test, the first germination count, conducted on the second day of the experiment, already revealed differences depending on the type of sample to which the seeds were exposed. Regarding the concentrations of different water samples, a significant difference was observed at 100% sample concentration, where groundwater induced a higher germination rate, totaling 60%, compared to surface water, which resulted in 40% seed germination.

For leachate samples, a reduction in seed germination was already evident at the 25% concentration, with only 17% of seeds germinating in contact with raw leachate, whereas 43% germination was observed in seeds exposed to treated leachate at the same concentration. At 100% leachate concentration, no seed germination was observed (Figure 2a).

Figure 2
a) First germination count; b) final germination percentage; and c) germination speed index of Allium cepa seeds exposed to different concentrations of samples from Upstream Surface Water (USW), Downstream Surface Water (DSW), Upstream Groundwater Point 1 (UGP1), Downstream Groundwater Points 1 and 2 (DGP1 and DGP2), Raw leachate (RL), and Treated leachate (TL).

The final germination count, conducted on the tenth day, showed no difference in the number of germinated seeds exposed to different concentrations of groundwater and surface water samples, with 90% germination observed in both cases. However, regarding the germination behavior of seeds exposed to leachate, a concentration-dependent response was observed for raw leachate, with increasing toxicity as concentration increased, ultimately preventing germination at concentrations of 50% and higher. A similar trend was observed for treated leachate, although it exhibited lower toxicity. Notably, at a 25% concentration, treated leachate still allowed 77% of the seeds to germinate (Figure 2b).

Similar to what was observed in the first germination count, the germination speed index differed between groundwater and surface water only at the 100% concentration. When comparing water samples with leachate samples, it was observed that both raw and treated leachate reduced this index and exhibited a concentration-dependent response (Figure 2c).

It is also noted that the shoot and root length of A. cepa seedlings exhibited similar development. When exposed to different concentrations of surface water and groundwater samples, their lengths were comparable to the negative control, with no statistically significant difference. Due to the absence of seed germination at concentrations of 50% or higher of raw leachate, shoot and root growth did not occur in these treatments. In contrast, for treated leachate, where germination was observed, a reduction in the length of these structures was noted due to the toxicity of the samples (Figure 3). A study evaluating the toxic effects of leachates from municipal solid waste on the growth of two tree species found root growth inhibition when directly exposed to leachate solutions at concentrations above 70% (Palm et al. 2022).

Figure 3
Allium cepa L. seedlings exposed to dilutions of 0, 25, 50, 75, and 100% of each sample: a) shoot length; b) root length; c) groundwater points (UGP1, DGP1, and DGP2); d) surface water (USW and DSW); e) treated leachate (TL); and f) raw leachate (RL). Scale bar = 10 mm.

Considering the above and based on the equations obtained for the phytotechnical parameters and mitotic index, the EC50% values were calculated for the raw and treated leachate samples. This test is used to assess the toxicity of the sample by determining the concentration required to produce a specific effect in 50% of the tested organisms/parameters within a given period.

For all phytotechnical parameters, the percentages were similar, ranging from 16.50% to 20.05% for raw leachate and from 39.19% to 65.98% for treated leachate, both classified as toxic. Regarding the mitotic index, raw leachate inhibited seed germination, making it impossible to establish the EC50% for this parameter. In contrast, treated leachate, despite its toxicity, allowed germination and resulted in an EC50% of 96.73% (Table III). At lower concentrations, germination was stimulated, a phenomenon primarily attributed to the reduced presence of toxic compounds and the availability of organic and mineral nutrients, which initially promote seedling growth (Sharma & Dubey 2005).

Table III
EC50% values for phytotechnical variables. RL - raw leachate; TL - treated leachate.

Parameter	Equation (RL)	RL	Equation (TL)	TL
First germination count	y = 0.0034x² - 0.5001x + 16.746	17.40	y = -0.1666x + 17.596	51.17
Germination	y = 0.0048x² - 0.7331x + 26.175	20.05	y = -0.0008x² - 0.1616x + 27.546	65.98
Germination Speed Index	y = 0.0012x² - 0.1797x + 6.0889	17.79	y = 0.0005x² - 0.1193x + 7.3611	39.73
Shoot Length	y = 0.0006x² - 0.0936x + 3.0717	16.50	y = -6E-05x² - 0.0274x + 3.3157	53.14
Root Elongation	y = 0.0004x² - 0.0578x + 1.8477	19.09	y = 0.0001x² - 0.0279x + 1.83	39.19
Mitotic Index	*	*	y = -0.08x + 15.41	96.73

• *Values not obtained for this parameter.

For surface water and groundwater samples, the mitotic index (14.91%) did not differ from the control (15.09%), indicating that the water samples did not exhibit toxicity. This finding aligns with the phytotechnical parameters (Figure 3).

The EC50% values were also calculated for the luminescence of the bacterium A. fischeri. Similar to the other evaluations, the leachate samples were classified as toxic (31.86% for raw leachate and 14.43% for treated leachate). However, for the other samples, it was not possible to determine the EC50% percentage, and their effect was classified as hormesis (Table IV).

Table IV
EC50% values obtained for leachate samples from acute toxicity tests with Aliivibrio fischeri. RL – Raw leachate; TL – Treated leachate; DSW – Downstream Surface Water; USW – Upstream Surface Water; UGP1 – Upstream Groundwater Point 1; DGP1 and DGP2 – Downstream Groundwater Points 1 and 2; TU – toxicity unit.

Samples	Exposure Time	EC50%	TU	Classification
RL	30	31.86	3.378	Toxic
TL	30	14.43	7.083	Toxic
DSW	30	-	-	Hormesis
USW	30	-	-	Hormesis
UGP1	30	-	-	Hormesis
DGP1	30	-	-	Hormesis
DGP2	30	-	-	Hormesis

Considering that the objective of this study was not to evaluate the reasons for the occurrence of toxicity, no additional investigations were conducted to justify the difference in toxicity between raw and treated leachate. It is known that toxic metabolites may emerge during treatment processes, potentially affecting one organism while not impacting another (Cleuvers 2003, Yang et al. 2008, Godoy et al. 2015).

Moreover, regardless of the degree of toxicity itself, both raw and treated leachate were observed to be toxic to A. fischeri, highlighting the need for attention and additional measures in their management.

It can be observed that some of the tests in this phase indicated a phenomenon known as hormesis, which refers to a positive deviation exhibited by the test organism in the presence of the contaminant. It is important to emphasize that the detection of hormesis does not indicate that the contaminant is beneficial to the organism; on the contrary, it may be associated with a compound that exhibits toxicity in chronic toxicity tests or in acute toxicity tests at higher concentrations (Calabrese 2008). Furthermore, according to Calabrese (2008), hormesis is considered an evolutionary mechanism, as it represents a compensatory or adaptive response of organisms to overcome an induced imbalance and prevent species extinction.

CONCLUSIONS

This study assessed the toxicity of raw and treated leachate, as well as surface water and groundwater adjacent to a landfill in the southern region of Minas Gerais, using phytotoxicity tests with Allium cepa L. and acute toxicity tests with Aliivibrio fischeri. The results indicated that leachate, in both its raw and treated forms, had significant adverse effects on the germination and seedling growth of A. cepa, highlighting the toxicity of the effluents generated. In particular, raw leachate exhibited a pronounced inhibition of germination, while treated leachate, although less toxic, still negatively affected the evaluated parameters. On the other hand, surface water and groundwater samples did not show significant toxicity, and even exhibited hormesis effects, suggesting that the presence of nutrients and less toxic compounds may have a positive effect at lower concentrations.

The results obtained highlight the importance of continuous water quality monitoring in areas adjacent to landfills, as well as the need for improvements in leachate treatment processes to mitigate environmental impacts. Thus, this study contributes to the understanding of the ecotoxicological effects of leachate and the waters surrounding landfills, emphasizing the urgency of effective management practices to ensure the protection of water resources and public health.

Acknowledgements

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Funding Code 001], CAPES/BRASIL PDPG No. 1026/2022, CAPES/BRASIL PDPG-POSDOC No. 2930/2022, CAPES/BRASIL MEC/SESu/FNDE/PET. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) [BPD00571-22].

• Data availability
  The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Edited by

• Handling editor
  Luis Pacheco

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Publication Dates

• Publication in this collection
  14 Sept 2026
• Date of issue
  2026

History

• Received
  27 Sept 2025
• Accepted
  18 May 2026