The search for fuels to replace petroleum consumption has caused an increase in the production of biofuels worldwide. The ethanol, which comes from sugarcane, is an energy resource with low polluting potential, but its production generates other environmental problems. On average, 10 to 15 liters of vinasse are generated while preparing each liter of ethanol. Vinasse is the final by-product of the biomass distillation, mainly for the production of ethanol, from different cultures such as sugarcane. Because excessive quantities of vinasse are produced, alternatives have been required for use, for example as fertilizer, in a process known as fertigation. These excessive amounts of vinasse applied in soils have generated adverse effects on soil properties and to the organisms. This study carried out the toxic, cytotoxic and genotoxic potential of sugarcane vinasse obtained from two different harvests (Samples I and II), using the <i>Allium cepa</i> organism test. <i>A. cepa</i> seeds were exposed to raw vinasse (RV) and diluted in different concentrations: control soil + raw vinasse (SV); vinasse diluted in water at 50% + control soil (V 50%); vinasse diluted in water at 25% + control soil (V 25%); vinasse diluted in water at 12.5% + control soil (V 12.5%). The chemical characterization of vinasse samples showed a low pH and high concentration of potassium. The results demonstrate that the two RV samples tested are toxic, since no seeds germination was observed. The cytotoxic potential was observed in the sample II of SV and V (50%). All groups evaluated in samples I and II, induced chromosomal alterations, statistically significant compared with negative control. An increase in frequency of micronuclei in meristematic cells was observed in the SV (Sample I) and all groups evaluated in samples II. Based on the results it is concluded that the genetic material of the test-system was damaged when exposed to sugarcane vinasse, suggesting that one should be very careful in the use of this waste that has been used sometimes indiscriminately in soils.
In recent years worldwide, it has increased the demand for “green” fuel (biofuels) as an alternative to fossil fuels [
The soil is an essential component of ecosystems and is the main substrate used by plants, playing multiple roles, such as regulation of the distribution, drainage, and infiltration of rainfall and irrigation [
A. cepa is one of the best test-systems already developed, due to its high sensitivity to chemical agents and good correlation with mammal test-systems [
In this context, in order to obtain more information about the possible effects of sugarcane vinasse on plants, the present study evaluated the toxic, cytotoxic and genotoxic potential of the raw sugarcane vinasse and diluted added to the soil, of two different harvests (Samples I and II), using the A. cepa test.
Vinasse samples (I and II) were collected at a sugarcane processing facility, located in São Paulo State, Brazil (22˚21'25''S/47˚23'03''W). The samples were maintained in a cold storage chamber (4˚C), at the Department of Biochemistry and Microbiology of the UNESP (São Paulo State University), Rio Claro, São Paulo, to minimize bacterial degradation until beginning of experiments.
The biological material used on the evaluation of sugarcane vinasse toxicity was the seeds of A. cepa (Liliaceae), from the same lot and variety (Baia Periforme). They were stored in the dark at a temperature between 6˚C and 10 ˚C until use.
The soil used for the application of sugarcane vinasse samples, termed control soil (CS), was obtained on the UNESP Rio Claro Campus, São Paulo (22˚24'36''S/47˚33'36''W). For the bioassays, soil samples were homogenized, dried at ambient temperature and sieved with 4-mm mesh sieves and subjected to chemical characterization.
Physicochemical analysis of the control soil (CS), raw sugarcane vinasse (RV), metals analysis of sugarcane vinasse samples and polycyclic hydrocarbons in control soil and the combinations of control soil and sugarcane vinasse samples, it was performed for guidelines of soil quality (mg/Kg) and groundwater quality in São Paulo State, according to the current legislation―CETESB 195/2005-E―to Environmental Sanitation Technology Company (Companhia de Tecnologia de Saneamento Ambiental-CETESB). For the CS samples, 16 priority aromatic polycyclic hydrocarbons established by the Environmental Protection Agency (EPA) were quantified following EPA 8270D method.
The maximum dosage of vinasse used was determined according to the current legislation P4.231 of the CETESB (2005).
Treatments were prepared with two different samples (I and II) of sugarcane vinasse, different controls (CS, NC and PC) and all bioassays were conducted in duplicate.
Groups 1: Negative control (NC)―ultrapure water.
Groups 2: Control soil (CS).
Groups 3: Raw vinasse (Samples I and II) (RV).
Groups 4: Raw vinasse (Samples I and II) + control soil (SV).
Groups 5: Vinasse (Samples I and II) diluted in water at 50% + control soil (V 50%).
Groups 6: Vinasse (Samples I and II) diluted in water at 25% + control soil (V 25%).
Groups 7: Vinasse (Samples I and II) diluted in water at 12.5% + control soil (V 12.5%).
Groups 8: Positive control (PC), aneugenic herbicide Trifluralin® (TRIF) (CAS N01582-09-8) at a concentration of 0.019 mg/mL [
To evaluate the toxic, cytotoxic and genotoxic potential of the different groups, A. cepa seeds were used according to a modified version of Grant’s protocol (1982) [
The toxicity was evaluated based on the seed germination index, obtained by the ratio between the germinated seeds number and all the seeds exposed to germination.
Cytotoxicity was assessed based on the quantification of morphological cell alterations indicating cell death and on the mitotic index (MI), characterized by the total number of dividing cells in the cell cycle following the equation: MI = (number of dividing cells/total number of observed cells) × 100.
In the evaluation of the genotoxicity, cells with chromosome alterations were quantified and it was calculated of chromosomal aberration index (CAI) by the formula CAI = (number of cells with CA/total number of observed cells) × 100.
The micronuclei (MN) were counted in meristematic region cells and for the F1 region cells. The observation of MN in the F1 region cells permits examine possible damage fixation.
The mean and standard deviation was calculated from the mitotic, chromosomal aberrations index and MN in meristematic and the F1 region cells. The data do not follow a normal distribution (Shapiro-Wilk) and Kruskal- Wallis test show differences between groups. All groups were compared to the NC by the non-parametric Mann- Whitney test, with the program Statistical Package for the Social Sciences for Windows, version 15.0, (SPSS Inc., Chicago, IL, EUA).
To ensure the correct application of sugarcane vinasse, similar to that applied in the field, the following parameters of fertility and the agronomic potential of the control soil (CS) were measured: pH, organic matter (OM), residual phosphorus (P res), potassium (K), calcium (Ca), magnesium (Mg), exchangeable aluminum (H+Al), sum of bases (SB), cation exchange capacity (CTC), base saturation (V%) and Ca/Mg and Mg/K ratios (
The results of the physicochemical analyses of the control soil and sugarcane vinasse samples are presented in
Metal analyses of the control soil and sugarcane vinasse samples are presented in
None of the 16 priority aromatic polycyclic hydrocarbons (APHs) determined by the Environmental Protection Agency (EPA) was found in the study samples, as shown in
The results from the A. cepa assay, in which the cells were exposed to sugarcane vinasse samples and the negative and positive controls, are shown below in
Samples | pH | g/dm3 | mg/dm3 | mmol/dm3 TFSA | % | Ratios | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Ca/Cl2 | OM | P res | K | Ca | Mg | H + Al | SB | CTC | V | Ca/Mg | Mg/K | |
Crop I | 6.2 | 18 | 3 | 0.8 | 2 | 1 | 88 | 3.9 | 91.9 | 4.2 | 2 | 1.25 |
Crop II | 5.1 | 17 | 3 | 1.7 | 9 | 6 | 30 | 16.6 | 47.1 | 3.5 | - | - |
TFSA: dried soil air; OM: organic matter; P res: residual phosphorus; SB: sum of bases; CTC: cation exchange capacity; V: base saturation.
Parameters | Sample I | Sample II | ||||
---|---|---|---|---|---|---|
CS (mg/Kg) | RV (mg/Kg) (V) | CS (mg/Kg) | RV (mg/Kg) (V) | Method | ||
Ammonia (mg/L) | USEPA 440/5-85-001 | |||||
Calcium (mg/L) | 29.3 | 719 | 42.8 | 671 | SM21 3120 B | |
COD (mg/L) | 5046 | 7941 | SM21 5210 B | |||
BOD (mg/L) | 13380 | 25225 | SM21 5220 D | |||
Hardness (mg CaCO3/L) | 2493 | 276 | SM21 2340 B | |||
Total phosphate (mg/L) | 317 | 1.3 | NE | SM21 4500-P C | ||
Potassium (mg/L) | 437 | 2056 | <0.008 | 3401 | SM21 3120 B | |
Non-filtrable residue (mg/L) | 2765 | 1800 | SM21 3120 D | |||
Sodium (mg/L) | 50.2 | 114 | SM21 3120 B | |||
Sulfate (mg/L) | 710 | <0.5 | 2993 | SM21 4500- | ||
Organic Carbon | 12.6 | 32.3 | SSSA Cap40 | |||
Electric Conductivity (µs/cm) | 115 | 13530 | 97.9 | 15110 | SM21 3120B | |
Total Sulfur | 151 | 1219 | 123 | 1681 | SM21 3120B | |
Total Phosphorus | 182 | 317 | 207 | SM21 3120B | ||
Total Magnesium | 237 | 264 | SM21 3120B | |||
Nitrate (mg/Kg) | 4.4 | 1.3 | 8.14 | 1.49 | SM21 4500- | |
Nitrite (mg/Kg) | 0.06 | 0.008 | 0.043 | 0.03 | SM21 4500- | |
Amoniacal Nitrogen (mg/Kg) | 31.8 | 49.6 | SM21 4500 NH3 E | |||
Nitrogen Kjeldal (mg/Kg) | 476 | 276 | 922 | 171 | SM21 4500-Norg B | |
Nitrate (mg/Kg) | 4.4 | 1.3 | 8.14 | 1.49 | SM21 4500- | |
Nitrite (mg/Kg) | 0.06 | 0.008 | 0.043 | 0.03 | SM21 4500- | |
pH | 6.2 | 3.9 | 5.1 | 4.37 | EPA 4095 C | |
Total Potassium | 406 | 2056 | 3401 | SM21 3120B | ||
Total Sodium | 50.2 | 114 | SM21 2540B | |||
Total Solids | 0.86 | 0.93 | SM21 2540B | |||
Total Volatile Solids | IV | 0.08 | SM21 2540B | |||
Solid content | 0.86 | 0.93 | SM21 2540B | |||
Moisture (g/g) | 0.14 | 0.06 | SM21 2540B |
CS: control soil; RV: raw vinasse; IV: inconsistent value; NE: data not evaluated; QL: quantification limit; SM: standard methods of the water and wastewater; RV: guidelines of soil quality (mg/Kg) and groundwater quality in São Paulo State, according to CETESB (195/2005-E); EPA: Environmental Protection Agency, US.
Parameters | Sample I | Sample II | |||||
---|---|---|---|---|---|---|---|
CS (mg/Kg) | RV (mg/Kg) (V) | CS (mg/Kg) | RV (mg/Kg) (V) | Method | VR (mg/Kg) | ||
Arsenic | 16.8 | SM21 3120B | 3.5 | ||||
Barium | 5.91 | 0.41 | SM21 3120B | 75 |
Cadmium | <0.16 | SM21 3120B | <0.5 | |||
---|---|---|---|---|---|---|
Lead | 49.3 | 42.7 | SM21 3120B | 17 | ||
Copper | 37.2 | 0.35 | 76.5 | 0.76 | SM21 3120B | 35 |
Chromium | 31.2 | 0.04 | 108 | 3.56 | SM21 3120B | 40 |
Total Sulfur | 151 | 1219 | 123 | 1681 | SM21 3120B | |
Mercury | 0.0019 | 0.065 | EPA 470A | 0.05 | ||
Molybdenum | 3.64 | 0.008 | 9.6 | SM21 3120B | <4 | |
Nickel | 13 | 0.03 | 24.2 | SM21 3120B | 13 | |
Selenium | 52.1 | SM21 3120B | 0.25 | |||
Total Sodium | 50.2 | 114 | SM21 2540B | |||
Zinc | 23.2 | 1.66 | 96 | SM21 3120B | 60 |
CS: control soil; RV: raw vinasse; SM: standard methods of the water and wastewater; EPA: Environmental Protection Agency, US; QL: quantification limit; RV: guidelines of soil quality (mg/Kg) and groundwater quality in São Paulo State, according to CETESB (195/2005-E)
Parameters | Sample | Method | Concentration allowed in the soil (mg/Kg) | ||
---|---|---|---|---|---|
CS | SV | CB | CO | ||
Acenaphthene (µg/Kg) | EPA 8270 D | - | - | ||
Acenaphthylene (µg/Kg) | EPA 8270 D | - | - | ||
Anthracene (µg/Kg) | EPA 8270 D | - | - | ||
Benzo (a) anthracene (µg/Kg) | EPA 8270 D | 0.025 | 0.025 | ||
Benzo (a) pyrene (µg/Kg) | EPA 8270 D | 0.052 | 0.052 | ||
Benzo (a) fluoranthene (mg/Kg) | EPA 8270 D | 0.38 | - | ||
Benzo (a) perylene (mg/Kg) | EPA 8270 D | 0.57 | |||
Benzo (a) fluoanthene (µg/Kg) | EPA 8270 D | 0.38 | 0.38 | ||
Chrysene (mg/Kg) | EPA 8270 D | 8.1 | - | ||
Dibenzo (a,h) anthracene (mg/Kg) | EPA 8270 D | 0.08 | - | ||
Phenanthrene (µg/Kg) | EPA 8270 D | 3.3 | 3.3 | ||
Fluoranthene (µg/Kg) | EPA 8270 D | - | - | ||
Fluorenone (µg/kg) | EPA 8270 D | - | - | ||
Indeno (1,2,3-cd) pyrene (µg/Kg) | EPA 8270 D | 0.031 | 0.031 | ||
Naphthalene (µg/Kg) | EPA 8270 D | 0.12 | 0.12 | ||
Pyrene (µg/Kg) | EPA 8270 D | - | - |
CS: control soil; SV: control soil + vinasse CB: guidelines (prevention) for soils in São Paulo State according to CETESB 9195/2005-E); CO: maximum concentration allowed in the soil, according to CONAMA (375/2006); EPA: Environmental Protection Agency, US; QL: quantification limit.
Parameters analysed/5000 cells | ||||
---|---|---|---|---|
Groups | MI | CAI | MN (M) | MN (F1) |
NC | 29.1 ± 3.7 | 0.2 ± 0.2 | 0.2 ± 0.4 | 0.4 ± 0.5 |
CS | 29.9 ± 1.7 | 0.4 ± 0.2 | 0.4 ± 0.5 | 0 ± 0 |
SV | 29.4 ± 2.6 | 3.8 ± 0.6* | 1.2 ± 0.4* | 0.4 ± 0.5 |
V (50%) | 41.7 ± 13.4 | 1.4 ± 0.6* | 0.4 ± 0.5 | 2.2 ± 1.5 |
V (25%) | 34.5 ± 3.3 | 1.6 ± 0.7* | 0.6 ± 0.9 | 0.8 ± 0.8 |
V (12.5%) | 42.4 ± 10.5 | 1.3 ± 0.4* | 0.6 ± 0.5 | 1 ± 1 |
TRIF | 19.4 ± 1.4* | 10.2 ± 1.6* | 10.4 ± 1.3* | 2.6 ± 1.1* |
NC: negative control; TRIF: trifluralin-positive control; CS: control soil; SV: control soil + vinasse (Sample I); V: vinasse (Sample I); MI: mitotic index; CAI: chromosomal aberration index; MN (M): micronuclei in meristematic cells; MN (F1): micronuclei in cells of the region F1. *p < 0.05. Values statistically significant, compared to negative control with the Mann Whitney test.
Parameters analysed/5000 cells | ||||
---|---|---|---|---|
Groups | MI | CAI | MN (M) | MN (F1) |
NC | 50.4 ± 7.7 | 0.3 ± 0.2 | 0.4 ± 0.5 | 0.2 ± 0.4 |
CS | 57.2 ± 13.5 | 0.5 ± 0.5 | 0.8 ± 0.8 | 0 ± 0 |
SV | 64.0 ± 7.9* | 1.3 ± 0.6* | 2.8 ± 1.5* | 1.0 ± 0.7 |
V (50%) | 63.2 ± 9.8* | 2.6 ± 1.1* | 3.0 ± 2.8* | 1.0 ± 1.0 |
V (25%) | 55.2 ± 7.9 | 2.2 ± 0.7* | 2.6 ± 0.5* | 0.2 ± 0.4 |
V (12.5%) | 61.7 ± 3.7 | 3.7 ± 1.4* | 1.8 ± 0.8* | 2.2 ± 3.3 |
TRIF | 37.8 ± 9.6 | 9.1 ± 3.3* | 19.8 ± 13.1* | 3.6 ± 1.8* |
NC: negative control; TRIF: trifluralin-positive control; CS: control soil; SV: control soil + vinasse (Sample II); V: vinasse (Sample II); MI: mitotic index; CAI: chromosomal aberration index; MN (M): micronuclei in meristematic cells; MN (F1): micronuclei in cells of the region F1. *p < 0.05. Values statistically significant, compared to negative control with the Mann Whitney test.
The germination in the treated groups (V 50%, V 25% and V 12.5%) and control groups (CS and NC) was over 90%. In the RV seeds did not germinate and in SV, the germination index was below 5%.
The MI was analyzed, which represented the number of dividing cells. In the Sample I, no significant differences and in the Sample II, the groups SV and V 50% showed significant differences were observed when comparing the treatments with the negatives controls (p < 0.05).
The genotoxic potential was evaluated by the CAI for all treatments (SV, V 50%, V 25% and V 12.5%) and was statistically significant when comparing with the negatives controls (p < 0.05). The CAs and nuclear abnormalities observed in the present study were visualized at all stages of the cell cycle. Several types of aberrations were considered [
It was also quantified the presence of MN in meristematic and F1 region cells (
Several alternatives have been developed for the use of vinasse from sugarcane in Brazil, in order to the large volumes that are produced daily. Therefore, every day becomes more necessary the assessments of possible damage to exposed ecosystems. Therefore, this study intended to contribute to a better understanding of the toxicity that this residue derived from the ethanol industry can have on soil.
Thus, the initial chemical analysis of the soil is of great importance to identify and quantify the different chemical elements present in the soil samples used in this study. Also is very important to determine the chemical and physico-chemical characteristics of the vinasse sample study which vary depending on the harvest. This analysis allowed to reproducing the correct amount of application of vinasse in soils on the recommendation of the legislation (CETESB P4.231/2005).
The addition of sugarcane vinasse on the soil can bring harmful effects in the terrestrial ecosystems, for example in the seed germination and alterations in the genetic material of exposed organisms [
This study did not reveal a significant reduction in the MI at the evaluated vinasse samples, being this a parameter that allows for the estimation of the frequency of cellular division, used for identify the presence of cytotoxic pollutants in the environment [
Chromosome loss and polyploidy are events that can derive from problems in cytoplasmic microtubules [
Polyploid cells have a large chromosomal imbalance due to the diversion of chromosomal number. In this case, the chromosomes tend to be condensed [
A substance capable of inducing the formation of micronucleus may be considered a clastogenic or aneugenic compound. The claustogenic action of a substance is demonstrated by the presence of micronuclei from chromosome breaks during the process of cell division. The aneugenic action, on the other hand, is characterized by the inactivation of the mitotic fuse, which results in loss of entire chromosomes that become absent in the main nucleus of the cell [
Genotoxicity studies are very important and have been several reports of damage to the genetic material from different organisms exposed to vinasse for example in Drosophila melanogaster [
Souza et al. (2009) [
In the other hand, Christofoletti et al. (2013) [
Based on the results obtained, this evaluation indicates the importance of studies to assess the toxic, cytotoxic and genotoxic potential of different residues disposed in the environment. These residues may induce alterations that cause irreversible damage to organisms and ecosystems.
After the quantification of the seeds germination and chromosomal aberrations in the test system here applied, it was concluded that the sugarcane vinasse in natura and in different dilutions showed a toxic and genotoxic potential for the A. cepa species. Maybe the low pH, electric conductivity, and chemical elements present in sugarcane vinasse may cause changes in the chemical and physical-chemical properties of soils. Results of this study reinforce the need for more research to evaluate the biological effects of sugarcane vinasse discharged into the environment in different ecosystems compartments, as well as different levels of biological organization.
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), processes 2009/ 53047-9, 2009/50578-3 and 2012/50197-2), for financial support and Thays Casimiro Fernandes for suggestions.
Janaína Pedro-Escher,Cintya A. Christofoletti,Yadira Ansoar-Rodríguez,Carmem S. Fontanetti, (2016) Sugarcane Vinasse, a Residue of Ethanol Industry: Toxic, Cytotoxic and Genotoxic Potential Using the Allium cepa Test. Journal of Environmental Protection,07,602-612. doi: 10.4236/jep.2016.75054