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Mutagenic potential of fine wastes from dimension stone industry
Ecotoxicology and Environmental Safety 125 (2016) 116–120
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Mutagenic potential of fine wastes from dimension stone industry Luara Louzada Aguiar a, Camila Bruschi Tonon a, Erika Takagi Nunes a, Adriane Cristina Araújo Braga a, Mirna Aparecida Neves b, José Augusto de Oliveira David a,n a b
Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, Alegre, Espírito Santo CEP 29500-000, Brasil Departamento de Geologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, Alegre, Espírito Santo CEP 29500-000, Brasil
art ic l e i nf o
a b s t r a c t
Article history: Received 10 July 2015 Received in revised form 26 November 2015 Accepted 27 November 2015
The industrial treatment of dimension stones, such as marbles and granites, includes a stage of plate polishing, in which resins and abrasives are used, producing a fine grained waste with high moisture content. These wastes pass through decantation tanks in order to separate the solid and liquid phases. Until now, there is no knowledge about the mutagenic effects that this effluent can cause to organisms exposed to it. Thus, this study evaluated the mutagenic potential of dimension stone polishing wastes in onion root cells and fish erythrocytes. The onion seeds were germinated in Petri dishes with filter paper moistened in the liquid phase of the effluent. After germination, the onion roots were prepared for analysis of chromosomal aberrations in meristematic cells. The fishes were exposed during 72 h to the solid phase of the effluent diluted in pure groundwater. Blood samples were used for counting of micronucleus and nuclear abnormalities. The onion seeds had similar germination and mitotic index in all treatments. However, it was observed in the seeds exposed to the polishing waste, numbers significantly higher of micronucleus, nuclear buds and other chromosomal aberrations when compared with the negative control. The fishes exposed to the waste showed numbers significantly higher of micronucleus when compared with the negative control. The fishes from all treatments showed significant increase in nuclear abnormalities when compared to the negative control. We concluded that the analysed wastes have mutagenic potential at the studied conditions; this effect can be related to the high content of phenolic compounds identified in the samples. & 2015 Elsevier Inc. All rights reserved.
Keywords: Allium cepa Oreochromis niloticus Mutagenesis Phenolic compounds
1. Introduction Brazil is between the main dimension stone producer countries, exporting blocks and plates of marble and granite for use as surface coverings. From January to October 2014, Brazil exported approximately $1 billion worth of rock plates (Abirochas, 2014) with 75% produced in the state of Espírito Santo (ES). According to Villaschi Filho and Sabadini (2000), the oldest production centre in Brazil is located at Cachoeiro de Itapemirim, a municipality in the southern portion of ES, which is the source of the majority of Brazilian production; however, it has been receiving attention due to the large quantity of waste generated. After sawing of blocks, the produced plates are polished to confer gloss to the final material. This process involves the use of abrasives that are based on magnesium, diamond or phenolic resins. The generated waste is taken to settling tanks where the liquid and solid phases of the wastewater are separated. The liquid phase returns to the processing plant, and the solid phase n
Corresponding author. E-mail address: [email protected] (J.A. de Oliveira David).
http://dx.doi.org/10.1016/j.ecoenv.2015.11.035 0147-6513/& 2015 Elsevier Inc. All rights reserved.
continues to be deposited until the maximum capacity of the tank is reached. When this capacity is reached, the liquid phase is released in water bodies and the solid phase is deposited directly onto soil without proper sealing. Several substances disposed of by industrial processes in water bodies can cause genomic mutation. These substances show binding affinity to the genetic material and can potentially cause DNA damage. They are known as genotoxic or mutagenic agents (Boer And Hoeijmakers, 2000). Among the organisms used to monitor aquatic pollution, the most commonly used are fish, molluscs and plants. Onion, Allium cepa, has stood out among higher plants as a model in tests to detect the mutagenic action of chemical substances or contaminated water (Matsumoto et al., 2006). The efficiency of onion as a test organism is due to the knowledge regarding the duration of its cell cycle, its rapid root growth, high tolerance to various cultivation conditions and large chromosomes in reduced number, characteristics that are essential for genotoxicity studies (Evseeva et al., 2003; Egito et al., 2007). The use of fish as test organisms in toxicological monitoring has also been shown to be efficient because it precisely evaluates agents that potentially cause genetic damage in the aquatic
L.L. Aguiar et al. / Ecotoxicology and Environmental Safety 125 (2016) 116–120
environment. Fish have nucleated erythrocytes, which allows easy visualisation of micronuclei. According to Vijayan et al. (1996), tilapia, Oreochromis niloticus, is a species that is recognised to have high sensitivity for the detection of pollutants, rapidly responding to environmental alterations. The economic relevance of the dimension stone industry and the volume of wastes generated emphasise the necessity of knowing the potential impacts that these materials can cause to the environment. Thus, the present study aimed to analyse the mutagenic potential of the waste generated during processing of dimension stones in onion root meristems and fish erythrocytes.
2. Materials and methods The experiments were performed with wastes collected at a dimension stone processing company located in the municipality of Cachoeiro do Itapemirim, in the southern portion of the state of Espírito Santo (ES), Brazil. The water that supplies the processing plant comes from a lake located near the property. After polishing marble and granite plates, the water that circulates through the polisher is directed to the settling tanks to decrease the quantity of suspended sediment, after which the water is reused in the process. The settling tanks are emptied when their storage capacity is exhausted, and the settled solid waste is removed for disposal. Filling of the settling tanks is then reinitiated. 2.1. Study of the liquid phase of the waste Two sampling points were selected for the liquid phase of the polishing waste, one at the inlet of the settling tank (TI) and another at the outlet (TF). One sampling point was in the supply lake of the company (LA). Water temperature (t), pH, electrical conductivity (EC), turbidity (T) and dissolved oxygen (DO) were measured in situ using portable equipment. Sample collection was performed according to NBR 9898 (ABNT, 1987), and the samples were sent to the Hydrogeology Laboratory of the Centre for Agrarian Sciences of the Federal University of Espírito Santo (Laboratório de Hidrogeologia do Centro de Ciências Agrárias da Universidade Federal do Espírito Santo) for analyses of alkalinity (ALK), chloride (CHLO) and total phenols (PHEN). Alkalinity was determined by titration with a base according to NBR 13736 (ABNT, 1996), and the analyses of chloride and total phenols were performed by colorimetry using a scanning UV–vis spectrophotometer. 2.2. Experiment with Allium cepa Seeds of Allium cepa from the same lot were germinated on Petri dishes lined with filter paper moistened in a solution according to the following treatments: (1) Control–negative control group containing distilled water; (2) Lake-water collected from a lake that supply the company; (3) Waste-liquid phase of the polishing waste collected at the inlet of the settling tank; and (4) Resin-dilution of the polishing resins with distilled water (2.5 mL L 1). In each plate were placed 60 seeds. The plates were kept under a 12 h light/dark cycle and controlled temperature (24 °C) in incubator. After reaching approximately 2.0 cm, the total number of germinated seeds was counted and the germination index (GI) was calculated, which represents the percentage of germinated seeds in each treatment, represented by the formula:
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The roots were fixed in alcohol:acetic acid (3:1) for 24 h. Next, they underwent hydrolysis in 1 N HCl for 5 min at room temperature. The meristematic region (the first three millimetres of the root) from each of five roots was cut off, stained with a drop of acetic orcein and covered with a coverslip. The material was crushed, providing better spreading of the cells, and was analysed with a light microscope at 400 magnification. To assess the cytotoxic potential a total of 2000 cells were counted per slide, differentiating cells at interphase and at different stages of mitosis to calculate the mitotic index (MI), which corresponds to the ratio between the number of dividing cells (DC) and the total number of cells observed (CO). During counts the micronucleated cells, nuclear budding and chromosomal aberrations were quantified, in order to determine mutagenicity. The data obtained with the onion root meristems were subjected to statistical analysis using Bioestat 5.0 software and the non-parametric Mann–Whitney test for comparisons between pairs of treatments. 2.3. Study of the solid settled waste The solid phase of the waste was collected according to the procedures of NBR 9898 (ABNT, 1987), the concentrations of lead, chromium, cadmium, silver, aluminium, iron, sodium, phenol, fluoride and chloride were measured by leaching test according to NBR 10005 (ABNT, 2004a), and a solubilisation test was performed according to NBR 10006 (ABNT, 2004b). The results were used for classification of the wastes according to NBR 10004 (ABNT, 2004c). 2.4. Experiment with Oreochromis niloticus The experiment with O. niloticus was conducted with animals approximately 10.0 cm in length obtained from the fish farming department of the Federal Institute of Espírito Santo-Alegre Campus (Instituto Federal do Espírito Santo-Campus de Alegre). Fishes were exposed for 72 hours in 50 L tanks, in four treatments with 10 individuals each: (1) Control-containing pure groundwater; (2) Lake-containing 50 L of pure groundwater mixed with 2.0 Kg of sediment collected in the lake that supplies the marble factory; (3) Waste-containing 50 L of pure groundwater mixed with 2.0 Kg of polishing waste collected from the inlet of the settling tank; and (4) Resin-containing 50 L of pure groundwater and 10 mL of the resin used in the polishing process. The tanks were kept under constant aeration. After 72 h of exposure, a 0.1 mL blood sample from each fish was used for the preparation of two slides with a blood smear per individual. The slides were stained with panoptic dye and were analysed under a light microscope with 1000 magnification. A total of 6000 erythrocytes were counted per fish (3000 per slide). Micronuclei that were smaller than the nucleus, clearly separated, not refringent and that showed the same staining as the nucleus were quantified. Cells bearing other nuclear alterations were also analysed according to Carrasco et al. (1990). The data obtained with the fish erythrocytes were subjected to statistical analysis using Bioestat 5.0 software and the non-parametric Mann–Whitney test for comparisons between pairs of treatments.
3. Results and discussion
GI=TN x 100/PN
3.1. Effects of the liquid phase of the waste on germination of A. cepa
where TN is the total number of germinated seeds and PN the total number of seeds in each plate.
Results from the analyses of the liquid phase of the waste from the processing plant and the supply water are shown in Table 1. In
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Table 1 Analysis of the wastewater liquid phase from the inlet of the settling tank (TI), the outlet of the settling tank (TF) and the supply lake water (LA). Sample
t (°C)
pH
T (FTU)
EC (mS)
TDS (mg L 1)
CLO (mg L 1)
ALK (mg L 1CaCO3)
DO (%)
PHEN (mg L 1)
TI TF LA
30 30 32
10.0 10.1 8.6
707.0 78.0 4.9
4.44 4.49 1.07
2352.4 2254.4 473.6
1544.9 1739.6 415.3
702.80 700.93 55.07
84.0 74.2 70.3
1.976 0.811 0.265
(t¼ temperature, T ¼ turbidity, EC ¼electrical conductivity, TDS¼ total dissolved solids, CLO ¼chlorides, ALK ¼alkalinity, DO ¼dissolved oxygen, PHEN¼ total phenol).
order to evaluate water and wastewater quality, the data were compared with thresholds fixed by the National Council of Environment (Conselho Nacional de Meio Ambiente–CONAMA). Such limits are in the Resolution no. 375/2005 (Brasil, 2005), which addresses the classification of water bodies and gives environmental guidelines for their classification and Resolution no. 430/ 2011 (Brasil, 2011), which addresses the conditions and standards for wastewater disposal. The acceptable pH range for this standards is between 5.0 and 9.0 (Brasil, 2011). The water collected in the supply lake is within this range, but the samples of the wastewater showed pH values higher than permitted limits. The pH of the processing dimension stone wastes is typically elevated due to the use of lime in the process of sawing the rock. CONAMA resolution no. 357/2005 for Class 2 rivers, that is, waters that can be used to supply human consumption (among other purposes) after conventional treatment, recommends turbidity values below 100 formalin turbidity units (FTU). The water at the tank inlet has values around seven times higher than that expected for natural water. At the tank outlet, after sedimentation of the majority of the particulate matter, the turbidity falls to acceptable but still high levels, contrasting with the values measured in the lake water. Thus, it is necessary to recommend that before water from the tank inlet is released to bodies of water, it must remain in the settling tank for sufficient time to reach levels below 100 FTU. Although there is no limit defined for electrical conductivity, it can be considered an indirect measure of water quality. Generally, values above 100 mS cm 1 indicate a polluted environment with a high concentration of dissolved salts (CETESB, 2010). Thus, all of the analysed mixtures indicate a saline environment, which is corroborated by the concentrations of total dissolved solids (TDS) and chlorides. The TDS values in the samples from the settling tank are above the maximum value permitted (MPV) by CONAMA resolution no. 357/2005 (MPV¼500 mg L 1), and the values are close to this limit in the supply lake. The TDS concentration reflects the quantity of dissolved salts in the water, and the high values observed are corroborated by the high concentrations of chlorides measured, including in the lake water. The inputs used in the process of finishing the plates are magnesium-based and have chloride in their composition. Although the MPV of chlorides is 250 mg L 1, the chlorides recorded in the lake water reached almost the double of this value, which may be explained by the use of magnesia compounds in the finishing process of the plates, these compounds present chloride in its composition (Roxo et al.,2006). CONAMA resolution no. 357/2005 does not impose limits on the alkalinity of water, but the values commonly found in surface waters are between 30 and 500 mg L 1 CaCO3. Higher values can be found in domestic and industrial wastewater. In the studied area, the high alkalinity of the liquid phase of the wastewater, similar to its pH, is due to the lime used in the process of sawing the rocky blocks, and it can alter the alkalinity of any body of water that receives the wastewater. In contrast, the dissolved oxygen concentrations were found to be adequate at all points studied (minimum value permitted by
CONAMA, 2011 is 70%), including in the waste settling tank, where the constant movement of the liquid phase causes aeration of the environment, favouring oxygenation. Total phenols concentration was very high in all samples when compared to the established by CONAMA resolutions no. 430/2011 (MPV of 0.5 mg L 1 for wastewater) and no. 357/2005 (MPV of 0.003 mg L 1 for Class 2 bodies of water). At the inlet of the settling tank, this value reached 1.976 mg L 1, and it dropped to 0.811 mg L 1 at the tank outlet, probably due to movement of the wastewater and exposure to the oxidising conditions of the fluid. The total phenol content present in the liquid phase of the wastewater originates from the phenolic resins used to seal small fissures and orifices commonly present in the rocks. However, there must be a small contribution from the water that supplies the facility, as a phenol concentration of 0.265 mg L 1 was also found in the lake samples. The presence of phenols in the lake water may be from natural processes considering that there is no entry of industrial or domestic wastes into the lake. Michalowicz and Duda (2007) reviewed sources and toxicity of phenols and reported a broad distribution of these compounds in the environment. According to the authors, chlorophenols form naturally in the environment by the chlorination of mono- and poly-aromatic compounds present in soil and water. The germination index as well as the average mitotic indexes of A. cepa did not differ significantly among the four treatments (Table 2). These results demonstrated that the treatments lacked any toxic or cytotoxic activity that would prevent the division of A. cepa meristematic cells. Microscopic analysis of the meristematic cells of the exposed seeds showed alterations such as binucleated cells, nuclear budding, micronuclei, anaphase bridges, polyploid metaphase, metaphase with losses and telophase with chromosome loss and delay. The frequency of cells with micronuclei in the negative control was significantly fewer than the observed in meristems exposed to the polishing waste (Table 3). The onion seeds exposed to the lake water and resins did not show differences from the other experimental groups. The quantification of micronucleated cells is considered to be an assessment of mutagenic damage, and it is related to the presence of chromosome fragments or whole chromosomes that were lost during mitosis. These fragments and lost chromosomes enter the cytoplasm where they assume the form of micronucleus in the following interphase (Ribeiro et al., 2003). According to Leme and Marin-Morales (2009), micronuclei can be formed from chromosomal aberrations such as breaks and Table 2 Germination index (GI) and average and standard deviation of the mitotic index (MI) found in the root meristems of Allium cepa exposed to the different treatments. Treatment
GI (%)
MI (%)
Control Lake Waste Resin
96.6 80.0 86.6 85.0
94.4 7 5.3 92.17 8.9 95.0 7 4.2 97.17 3.0
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Table 3 Average frequency (%) and standard deviation of cells with micronuclei (MN), nuclear budding (NB) and chromosomal aberrations (CA) observed in Allium cepa root meristems from the different treatments. Treatments
MN
NB
CA
Control Lake Waste Resin
0.6 7 0.9A 3.8 7 3.9 3.8 7 1.9A 1.8 7 2.2
0.2 7 0.4a,B 1.4 7 2.1C 4.4 7 1.7a,C,D 2.0 7 2.2B,D
0.2 70.4A,B 0.8 70.8 1.8 71.3A 1.2 70.8B
The same UPPERCASE letters represent treatments that differ from each other with p o 0.05. The same LOWERCASE letters represent treatments that differ from each other with p o 0.01.
chromosomal losses. Additionally, Fernandes et al. (2007) stated that micronuclei may result from polyploidisation processes in which the nucleus of the cell expels fragments or whole chromosomes from its interior through the initial formation of a bud that detaches from the nucleus, forming a micronucleus. The analyses conducted here showed that onion seeds exposed to the polishing waste and to the dilution of the resins showed significantly higher numbers of nuclear buds compared to the negative control seeds. The number of cells with nuclear buds in the seeds exposed to the polishing waste was also significantly higher than the numbers found in the other two experimental groups (Table 3). This increase in the number of buds may be indicative of a process of elimination of excess genetic material from the nucleus of the cell as suggested by Fernandes et al. (2007). Leme and Marin-Morales (2009) suggest that micronuclei in F1 cells (nonmeristematic) should be counted to confirm the frequency of micronucleated cells that remain after the cellular division event. Leme and Marin-Morales (2009) observed a high frequency of micronucleated F1 cells and related this increase to events originating from other chromosomal aberrations that occur in meristematic cells. Other chromosomal aberrations observed in the root meristems of A. cepa were grouped to facilitate the analysis between the experimental groups. The number of these aberrations was shown to be significantly higher in seeds exposed to the polishing waste and to the dilution of the resins compared to the control group (Table 3). Thus, it was observed that A. cepa seeds exposed to the liquid phase of the waste from the polishing of marble and granite plates displayed a significant increase in chromosomal aberrations, which may have led to an increase in the frequency of nuclear buds and micronuclei in meristematic cells. Considering the characterisation of the liquid phase of the wastewater performed in the present study, the occurrence of this cellular damage can be associated with the presence of phenolic compounds.
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Table 4 Results from NBR 10005 (ABNT, 2004a) leaching tests and NBR 10006 (ABNT, 2004b) solubilisation tests of the fine waste generated in the polishing of ornamental rocks. Parameter LQ (mg L 1) Leaching test
Pb Cr PHEN Cd Ag Al Fe Na FLU CHLO
0.001 0.001 0.00042 0.0002 0.001 0.001 0.001 0.003 0.01 0.01
Solubilisation test
MPL (mg L 1)
Value (mg L 1)
MPL (mg L 1)
Value (mg L 1)
1.0 5.0 – 0.5 5.0 – – – 150.0 –
0.007 0.0280 0.0040 0.009 0.005 0.260 38.600 16.70 0.160 81.20
0.01 0.05 0.01 0.005 0.05 0.20 0.30 200.00 1.50 250.00
0.01 0.004 0.007 0.002 0.002 0.110 0.070 65.300 0.080 67.500
OBS: sample pH ¼11.0. LQ¼ limit of quantification; MPL ¼ maximum permitted limit; VALUE¼ concentration measured in the sample; PHEN ¼total phenol; FLU ¼ fluoride; CHLO¼ chloride.
their intrinsic characteristics, do not present risks to health or to the environment. Table 4 shows the results from leaching and solubilisation tests for the solid waste used in the experiment, showing that none of the analysed parameters exceeded the maximum permitted limit. Among these parameters, only lead (Pb) in the solubilised extract was exactly at the limit of the maximum permitted value. This waste can, therefore, be classified as Inert (Class II-B). 3.3. Effects of the solid settled waste on fish erythrocytes The results obtained from fish exposed to the different treatments are shown in Table 5. Fishes in contact with the dilution of resins or the polishing waste showed significantly higher numbers of micronucleated cells when compared to those of the negative control group. Fishes exposed to the dilution of resins also showed significantly higher numbers of micronucleated cells when compared to fishes exposed to lake water. Fishes from the control group showed a significantly lower number of nuclear abnormalities than all other treatments. Fishes exposed to material from the lake also showed significantly lower numbers than those found for fishes exposed to the polishing waste or to the dilution of resins (Table 5). In a recent study, Seriani et al. (2015) quantified nuclear abnormalities and micronuclei in O. niloticus collected in the Ecological Park of Tietê (Parque Ecológico do Tietê) in São Paulo, Brazil. The authors observed a frequency of nuclear alterations and micronuclei five times higher in fish from the impacted region compared to the control group, and they related these alterations to the presence of metals in the water. The authors further discuss
3.2. Study of the solid settled wast According to the Brazilian Association of Technical Standards (Associação Brasileira de Normas Técnicas-ABNT), solid wastes can be classified as Class I or Class II. Class I solid wastes (Dangerous) are those that, as a function of their intrinsic characteristics, present risks to public health through an increase in mortality or morbidity, or that have adverse effects on the environment when handled or disposed improperly. Class II solid wastes (Non-Dangerous) can be divided into subclasses A and B. Class II-A (NonInert) includes wastes that show characteristics of combustibility, biodegradability or solubility with the possibility of resulting in risk to health or to the environment and can not be classified as other wastes. Class II-B (Inert) wastes include those that, due to
Table 5 Average and standard deviation for cells with micronuclei (MN) and nuclear abnormalities (NA) among erythrocytes from Oreochromis niloticus exposed to the different treatments. Treatment
MN (%)
NA (%)
Control Lake Waste Resin
0.007 0.01A,b 0.05 7 0.1C 0.047 0.04A 0.09 7 0.09 b,C
0.25 7 0.20a,b,c 1.88 7 1.20a,D,e 3.79 7 1.74 b,D 4.617 2.31c,e
The same UPPERCASE letters represent treatments that differ from each other with p o0.05. The same LOWERCASE letters represent treatments that differ from each other with p o0.01.
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that the nuclear abnormalities are related to cytotoxic damage, but they could also be considered alterations that preceded micronucleus formation. In the present study, a correlation was identified between the presence of nuclear abnormalities and micronuclei, with the nuclear abnormalities observed showing a much higher frequency compared to micronuclei. The higher number of abnormalities found in the present study corroborates the results of Seriani et al. (2015). If the abnormalities are alterations that precede micronucleus formation, they will probably be observed in greater quantity, to be accumulated or corrected, giving rise to a lower number of micronuclei. The presence of lead in the solid phase of the settled waste may be responsible for the formation of these genomic changes. However, another factor that may have affected the micronucleus formation and nuclear abnormalities is the presence of phenols in the waste, because phenolic resins are used in the polishing process. This procedure is aimed at improving the performance and durability of the ornamental rocks over time and at eliminating certain imperfections, cracks and fissures present in the rocks. Gad and Saad (2008) studied the effects of phenol on certain physiological parameters in O. niloticus using sub-lethal doses in a static system for 16 weeks, and they observed significantly higher frequencies of micronuclei even at the lowest phenol concentration (0.7 mg L 1), which corresponds to 1/40th of the LC50 for this species. The phenol concentration in the solubilisation test of the studied waste was equal to 0.007 mg L 1, 100 times lower than that studied in Gad and Saad (2008). However, it must be taken into consideration that this wastewater may be a complex mixture of metals and phenolic resins that, even at low concentrations, evokes different biological responses than those expected from the action of individual contaminants due to synergistic interactions with potentiation or additive effects.
4. Conclusion The liquid phase of the dimension stone polishing effluent showed high values of total dissolved solids, chloride and phenol. The experiment with A. cepa demonstrated that this effluent presents mutagenic potential at studied conditions. Although the solid phase of the waste has shown parameters below the limits allowed by Brazilian legislation, the experiment with O. niloticus showed that this waste has mutagenic potential. Given these results, this paper warns that better attention should be given to the treatment and disposal of dimension stone polishing wastes in order to reduce soil and water pollution, as well as major damage to aquatic life.
Acknowledgements The authors would like to thank the Universidade Federal do Espírito Santo for supporting this research and the fish farming department of the Instituto Federal do Espírito Santo (Campus de Alegre) for providing the fishes used in the study.
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Mutagenic potential of fine wastes from dimension stone industry Luara Louzada Aguiar a, Camila Bruschi Tonon a, Erika Takagi Nunes a, Adriane Cristina Araújo Braga a, Mirna Aparecida Neves b, José Augusto de Oliveira David a,n a b
Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, Alegre, Espírito Santo CEP 29500-000, Brasil Departamento de Geologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, Alegre, Espírito Santo CEP 29500-000, Brasil
art ic l e i nf o
a b s t r a c t
Article history: Received 10 July 2015 Received in revised form 26 November 2015 Accepted 27 November 2015
The industrial treatment of dimension stones, such as marbles and granites, includes a stage of plate polishing, in which resins and abrasives are used, producing a fine grained waste with high moisture content. These wastes pass through decantation tanks in order to separate the solid and liquid phases. Until now, there is no knowledge about the mutagenic effects that this effluent can cause to organisms exposed to it. Thus, this study evaluated the mutagenic potential of dimension stone polishing wastes in onion root cells and fish erythrocytes. The onion seeds were germinated in Petri dishes with filter paper moistened in the liquid phase of the effluent. After germination, the onion roots were prepared for analysis of chromosomal aberrations in meristematic cells. The fishes were exposed during 72 h to the solid phase of the effluent diluted in pure groundwater. Blood samples were used for counting of micronucleus and nuclear abnormalities. The onion seeds had similar germination and mitotic index in all treatments. However, it was observed in the seeds exposed to the polishing waste, numbers significantly higher of micronucleus, nuclear buds and other chromosomal aberrations when compared with the negative control. The fishes exposed to the waste showed numbers significantly higher of micronucleus when compared with the negative control. The fishes from all treatments showed significant increase in nuclear abnormalities when compared to the negative control. We concluded that the analysed wastes have mutagenic potential at the studied conditions; this effect can be related to the high content of phenolic compounds identified in the samples. & 2015 Elsevier Inc. All rights reserved.
Keywords: Allium cepa Oreochromis niloticus Mutagenesis Phenolic compounds
1. Introduction Brazil is between the main dimension stone producer countries, exporting blocks and plates of marble and granite for use as surface coverings. From January to October 2014, Brazil exported approximately $1 billion worth of rock plates (Abirochas, 2014) with 75% produced in the state of Espírito Santo (ES). According to Villaschi Filho and Sabadini (2000), the oldest production centre in Brazil is located at Cachoeiro de Itapemirim, a municipality in the southern portion of ES, which is the source of the majority of Brazilian production; however, it has been receiving attention due to the large quantity of waste generated. After sawing of blocks, the produced plates are polished to confer gloss to the final material. This process involves the use of abrasives that are based on magnesium, diamond or phenolic resins. The generated waste is taken to settling tanks where the liquid and solid phases of the wastewater are separated. The liquid phase returns to the processing plant, and the solid phase n
Corresponding author. E-mail address: [email protected] (J.A. de Oliveira David).
http://dx.doi.org/10.1016/j.ecoenv.2015.11.035 0147-6513/& 2015 Elsevier Inc. All rights reserved.
continues to be deposited until the maximum capacity of the tank is reached. When this capacity is reached, the liquid phase is released in water bodies and the solid phase is deposited directly onto soil without proper sealing. Several substances disposed of by industrial processes in water bodies can cause genomic mutation. These substances show binding affinity to the genetic material and can potentially cause DNA damage. They are known as genotoxic or mutagenic agents (Boer And Hoeijmakers, 2000). Among the organisms used to monitor aquatic pollution, the most commonly used are fish, molluscs and plants. Onion, Allium cepa, has stood out among higher plants as a model in tests to detect the mutagenic action of chemical substances or contaminated water (Matsumoto et al., 2006). The efficiency of onion as a test organism is due to the knowledge regarding the duration of its cell cycle, its rapid root growth, high tolerance to various cultivation conditions and large chromosomes in reduced number, characteristics that are essential for genotoxicity studies (Evseeva et al., 2003; Egito et al., 2007). The use of fish as test organisms in toxicological monitoring has also been shown to be efficient because it precisely evaluates agents that potentially cause genetic damage in the aquatic
L.L. Aguiar et al. / Ecotoxicology and Environmental Safety 125 (2016) 116–120
environment. Fish have nucleated erythrocytes, which allows easy visualisation of micronuclei. According to Vijayan et al. (1996), tilapia, Oreochromis niloticus, is a species that is recognised to have high sensitivity for the detection of pollutants, rapidly responding to environmental alterations. The economic relevance of the dimension stone industry and the volume of wastes generated emphasise the necessity of knowing the potential impacts that these materials can cause to the environment. Thus, the present study aimed to analyse the mutagenic potential of the waste generated during processing of dimension stones in onion root meristems and fish erythrocytes.
2. Materials and methods The experiments were performed with wastes collected at a dimension stone processing company located in the municipality of Cachoeiro do Itapemirim, in the southern portion of the state of Espírito Santo (ES), Brazil. The water that supplies the processing plant comes from a lake located near the property. After polishing marble and granite plates, the water that circulates through the polisher is directed to the settling tanks to decrease the quantity of suspended sediment, after which the water is reused in the process. The settling tanks are emptied when their storage capacity is exhausted, and the settled solid waste is removed for disposal. Filling of the settling tanks is then reinitiated. 2.1. Study of the liquid phase of the waste Two sampling points were selected for the liquid phase of the polishing waste, one at the inlet of the settling tank (TI) and another at the outlet (TF). One sampling point was in the supply lake of the company (LA). Water temperature (t), pH, electrical conductivity (EC), turbidity (T) and dissolved oxygen (DO) were measured in situ using portable equipment. Sample collection was performed according to NBR 9898 (ABNT, 1987), and the samples were sent to the Hydrogeology Laboratory of the Centre for Agrarian Sciences of the Federal University of Espírito Santo (Laboratório de Hidrogeologia do Centro de Ciências Agrárias da Universidade Federal do Espírito Santo) for analyses of alkalinity (ALK), chloride (CHLO) and total phenols (PHEN). Alkalinity was determined by titration with a base according to NBR 13736 (ABNT, 1996), and the analyses of chloride and total phenols were performed by colorimetry using a scanning UV–vis spectrophotometer. 2.2. Experiment with Allium cepa Seeds of Allium cepa from the same lot were germinated on Petri dishes lined with filter paper moistened in a solution according to the following treatments: (1) Control–negative control group containing distilled water; (2) Lake-water collected from a lake that supply the company; (3) Waste-liquid phase of the polishing waste collected at the inlet of the settling tank; and (4) Resin-dilution of the polishing resins with distilled water (2.5 mL L 1). In each plate were placed 60 seeds. The plates were kept under a 12 h light/dark cycle and controlled temperature (24 °C) in incubator. After reaching approximately 2.0 cm, the total number of germinated seeds was counted and the germination index (GI) was calculated, which represents the percentage of germinated seeds in each treatment, represented by the formula:
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The roots were fixed in alcohol:acetic acid (3:1) for 24 h. Next, they underwent hydrolysis in 1 N HCl for 5 min at room temperature. The meristematic region (the first three millimetres of the root) from each of five roots was cut off, stained with a drop of acetic orcein and covered with a coverslip. The material was crushed, providing better spreading of the cells, and was analysed with a light microscope at 400 magnification. To assess the cytotoxic potential a total of 2000 cells were counted per slide, differentiating cells at interphase and at different stages of mitosis to calculate the mitotic index (MI), which corresponds to the ratio between the number of dividing cells (DC) and the total number of cells observed (CO). During counts the micronucleated cells, nuclear budding and chromosomal aberrations were quantified, in order to determine mutagenicity. The data obtained with the onion root meristems were subjected to statistical analysis using Bioestat 5.0 software and the non-parametric Mann–Whitney test for comparisons between pairs of treatments. 2.3. Study of the solid settled waste The solid phase of the waste was collected according to the procedures of NBR 9898 (ABNT, 1987), the concentrations of lead, chromium, cadmium, silver, aluminium, iron, sodium, phenol, fluoride and chloride were measured by leaching test according to NBR 10005 (ABNT, 2004a), and a solubilisation test was performed according to NBR 10006 (ABNT, 2004b). The results were used for classification of the wastes according to NBR 10004 (ABNT, 2004c). 2.4. Experiment with Oreochromis niloticus The experiment with O. niloticus was conducted with animals approximately 10.0 cm in length obtained from the fish farming department of the Federal Institute of Espírito Santo-Alegre Campus (Instituto Federal do Espírito Santo-Campus de Alegre). Fishes were exposed for 72 hours in 50 L tanks, in four treatments with 10 individuals each: (1) Control-containing pure groundwater; (2) Lake-containing 50 L of pure groundwater mixed with 2.0 Kg of sediment collected in the lake that supplies the marble factory; (3) Waste-containing 50 L of pure groundwater mixed with 2.0 Kg of polishing waste collected from the inlet of the settling tank; and (4) Resin-containing 50 L of pure groundwater and 10 mL of the resin used in the polishing process. The tanks were kept under constant aeration. After 72 h of exposure, a 0.1 mL blood sample from each fish was used for the preparation of two slides with a blood smear per individual. The slides were stained with panoptic dye and were analysed under a light microscope with 1000 magnification. A total of 6000 erythrocytes were counted per fish (3000 per slide). Micronuclei that were smaller than the nucleus, clearly separated, not refringent and that showed the same staining as the nucleus were quantified. Cells bearing other nuclear alterations were also analysed according to Carrasco et al. (1990). The data obtained with the fish erythrocytes were subjected to statistical analysis using Bioestat 5.0 software and the non-parametric Mann–Whitney test for comparisons between pairs of treatments.
3. Results and discussion
GI=TN x 100/PN
3.1. Effects of the liquid phase of the waste on germination of A. cepa
where TN is the total number of germinated seeds and PN the total number of seeds in each plate.
Results from the analyses of the liquid phase of the waste from the processing plant and the supply water are shown in Table 1. In
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Table 1 Analysis of the wastewater liquid phase from the inlet of the settling tank (TI), the outlet of the settling tank (TF) and the supply lake water (LA). Sample
t (°C)
pH
T (FTU)
EC (mS)
TDS (mg L 1)
CLO (mg L 1)
ALK (mg L 1CaCO3)
DO (%)
PHEN (mg L 1)
TI TF LA
30 30 32
10.0 10.1 8.6
707.0 78.0 4.9
4.44 4.49 1.07
2352.4 2254.4 473.6
1544.9 1739.6 415.3
702.80 700.93 55.07
84.0 74.2 70.3
1.976 0.811 0.265
(t¼ temperature, T ¼ turbidity, EC ¼electrical conductivity, TDS¼ total dissolved solids, CLO ¼chlorides, ALK ¼alkalinity, DO ¼dissolved oxygen, PHEN¼ total phenol).
order to evaluate water and wastewater quality, the data were compared with thresholds fixed by the National Council of Environment (Conselho Nacional de Meio Ambiente–CONAMA). Such limits are in the Resolution no. 375/2005 (Brasil, 2005), which addresses the classification of water bodies and gives environmental guidelines for their classification and Resolution no. 430/ 2011 (Brasil, 2011), which addresses the conditions and standards for wastewater disposal. The acceptable pH range for this standards is between 5.0 and 9.0 (Brasil, 2011). The water collected in the supply lake is within this range, but the samples of the wastewater showed pH values higher than permitted limits. The pH of the processing dimension stone wastes is typically elevated due to the use of lime in the process of sawing the rock. CONAMA resolution no. 357/2005 for Class 2 rivers, that is, waters that can be used to supply human consumption (among other purposes) after conventional treatment, recommends turbidity values below 100 formalin turbidity units (FTU). The water at the tank inlet has values around seven times higher than that expected for natural water. At the tank outlet, after sedimentation of the majority of the particulate matter, the turbidity falls to acceptable but still high levels, contrasting with the values measured in the lake water. Thus, it is necessary to recommend that before water from the tank inlet is released to bodies of water, it must remain in the settling tank for sufficient time to reach levels below 100 FTU. Although there is no limit defined for electrical conductivity, it can be considered an indirect measure of water quality. Generally, values above 100 mS cm 1 indicate a polluted environment with a high concentration of dissolved salts (CETESB, 2010). Thus, all of the analysed mixtures indicate a saline environment, which is corroborated by the concentrations of total dissolved solids (TDS) and chlorides. The TDS values in the samples from the settling tank are above the maximum value permitted (MPV) by CONAMA resolution no. 357/2005 (MPV¼500 mg L 1), and the values are close to this limit in the supply lake. The TDS concentration reflects the quantity of dissolved salts in the water, and the high values observed are corroborated by the high concentrations of chlorides measured, including in the lake water. The inputs used in the process of finishing the plates are magnesium-based and have chloride in their composition. Although the MPV of chlorides is 250 mg L 1, the chlorides recorded in the lake water reached almost the double of this value, which may be explained by the use of magnesia compounds in the finishing process of the plates, these compounds present chloride in its composition (Roxo et al.,2006). CONAMA resolution no. 357/2005 does not impose limits on the alkalinity of water, but the values commonly found in surface waters are between 30 and 500 mg L 1 CaCO3. Higher values can be found in domestic and industrial wastewater. In the studied area, the high alkalinity of the liquid phase of the wastewater, similar to its pH, is due to the lime used in the process of sawing the rocky blocks, and it can alter the alkalinity of any body of water that receives the wastewater. In contrast, the dissolved oxygen concentrations were found to be adequate at all points studied (minimum value permitted by
CONAMA, 2011 is 70%), including in the waste settling tank, where the constant movement of the liquid phase causes aeration of the environment, favouring oxygenation. Total phenols concentration was very high in all samples when compared to the established by CONAMA resolutions no. 430/2011 (MPV of 0.5 mg L 1 for wastewater) and no. 357/2005 (MPV of 0.003 mg L 1 for Class 2 bodies of water). At the inlet of the settling tank, this value reached 1.976 mg L 1, and it dropped to 0.811 mg L 1 at the tank outlet, probably due to movement of the wastewater and exposure to the oxidising conditions of the fluid. The total phenol content present in the liquid phase of the wastewater originates from the phenolic resins used to seal small fissures and orifices commonly present in the rocks. However, there must be a small contribution from the water that supplies the facility, as a phenol concentration of 0.265 mg L 1 was also found in the lake samples. The presence of phenols in the lake water may be from natural processes considering that there is no entry of industrial or domestic wastes into the lake. Michalowicz and Duda (2007) reviewed sources and toxicity of phenols and reported a broad distribution of these compounds in the environment. According to the authors, chlorophenols form naturally in the environment by the chlorination of mono- and poly-aromatic compounds present in soil and water. The germination index as well as the average mitotic indexes of A. cepa did not differ significantly among the four treatments (Table 2). These results demonstrated that the treatments lacked any toxic or cytotoxic activity that would prevent the division of A. cepa meristematic cells. Microscopic analysis of the meristematic cells of the exposed seeds showed alterations such as binucleated cells, nuclear budding, micronuclei, anaphase bridges, polyploid metaphase, metaphase with losses and telophase with chromosome loss and delay. The frequency of cells with micronuclei in the negative control was significantly fewer than the observed in meristems exposed to the polishing waste (Table 3). The onion seeds exposed to the lake water and resins did not show differences from the other experimental groups. The quantification of micronucleated cells is considered to be an assessment of mutagenic damage, and it is related to the presence of chromosome fragments or whole chromosomes that were lost during mitosis. These fragments and lost chromosomes enter the cytoplasm where they assume the form of micronucleus in the following interphase (Ribeiro et al., 2003). According to Leme and Marin-Morales (2009), micronuclei can be formed from chromosomal aberrations such as breaks and Table 2 Germination index (GI) and average and standard deviation of the mitotic index (MI) found in the root meristems of Allium cepa exposed to the different treatments. Treatment
GI (%)
MI (%)
Control Lake Waste Resin
96.6 80.0 86.6 85.0
94.4 7 5.3 92.17 8.9 95.0 7 4.2 97.17 3.0
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Table 3 Average frequency (%) and standard deviation of cells with micronuclei (MN), nuclear budding (NB) and chromosomal aberrations (CA) observed in Allium cepa root meristems from the different treatments. Treatments
MN
NB
CA
Control Lake Waste Resin
0.6 7 0.9A 3.8 7 3.9 3.8 7 1.9A 1.8 7 2.2
0.2 7 0.4a,B 1.4 7 2.1C 4.4 7 1.7a,C,D 2.0 7 2.2B,D
0.2 70.4A,B 0.8 70.8 1.8 71.3A 1.2 70.8B
The same UPPERCASE letters represent treatments that differ from each other with p o 0.05. The same LOWERCASE letters represent treatments that differ from each other with p o 0.01.
chromosomal losses. Additionally, Fernandes et al. (2007) stated that micronuclei may result from polyploidisation processes in which the nucleus of the cell expels fragments or whole chromosomes from its interior through the initial formation of a bud that detaches from the nucleus, forming a micronucleus. The analyses conducted here showed that onion seeds exposed to the polishing waste and to the dilution of the resins showed significantly higher numbers of nuclear buds compared to the negative control seeds. The number of cells with nuclear buds in the seeds exposed to the polishing waste was also significantly higher than the numbers found in the other two experimental groups (Table 3). This increase in the number of buds may be indicative of a process of elimination of excess genetic material from the nucleus of the cell as suggested by Fernandes et al. (2007). Leme and Marin-Morales (2009) suggest that micronuclei in F1 cells (nonmeristematic) should be counted to confirm the frequency of micronucleated cells that remain after the cellular division event. Leme and Marin-Morales (2009) observed a high frequency of micronucleated F1 cells and related this increase to events originating from other chromosomal aberrations that occur in meristematic cells. Other chromosomal aberrations observed in the root meristems of A. cepa were grouped to facilitate the analysis between the experimental groups. The number of these aberrations was shown to be significantly higher in seeds exposed to the polishing waste and to the dilution of the resins compared to the control group (Table 3). Thus, it was observed that A. cepa seeds exposed to the liquid phase of the waste from the polishing of marble and granite plates displayed a significant increase in chromosomal aberrations, which may have led to an increase in the frequency of nuclear buds and micronuclei in meristematic cells. Considering the characterisation of the liquid phase of the wastewater performed in the present study, the occurrence of this cellular damage can be associated with the presence of phenolic compounds.
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Table 4 Results from NBR 10005 (ABNT, 2004a) leaching tests and NBR 10006 (ABNT, 2004b) solubilisation tests of the fine waste generated in the polishing of ornamental rocks. Parameter LQ (mg L 1) Leaching test
Pb Cr PHEN Cd Ag Al Fe Na FLU CHLO
0.001 0.001 0.00042 0.0002 0.001 0.001 0.001 0.003 0.01 0.01
Solubilisation test
MPL (mg L 1)
Value (mg L 1)
MPL (mg L 1)
Value (mg L 1)
1.0 5.0 – 0.5 5.0 – – – 150.0 –
0.007 0.0280 0.0040 0.009 0.005 0.260 38.600 16.70 0.160 81.20
0.01 0.05 0.01 0.005 0.05 0.20 0.30 200.00 1.50 250.00
0.01 0.004 0.007 0.002 0.002 0.110 0.070 65.300 0.080 67.500
OBS: sample pH ¼11.0. LQ¼ limit of quantification; MPL ¼ maximum permitted limit; VALUE¼ concentration measured in the sample; PHEN ¼total phenol; FLU ¼ fluoride; CHLO¼ chloride.
their intrinsic characteristics, do not present risks to health or to the environment. Table 4 shows the results from leaching and solubilisation tests for the solid waste used in the experiment, showing that none of the analysed parameters exceeded the maximum permitted limit. Among these parameters, only lead (Pb) in the solubilised extract was exactly at the limit of the maximum permitted value. This waste can, therefore, be classified as Inert (Class II-B). 3.3. Effects of the solid settled waste on fish erythrocytes The results obtained from fish exposed to the different treatments are shown in Table 5. Fishes in contact with the dilution of resins or the polishing waste showed significantly higher numbers of micronucleated cells when compared to those of the negative control group. Fishes exposed to the dilution of resins also showed significantly higher numbers of micronucleated cells when compared to fishes exposed to lake water. Fishes from the control group showed a significantly lower number of nuclear abnormalities than all other treatments. Fishes exposed to material from the lake also showed significantly lower numbers than those found for fishes exposed to the polishing waste or to the dilution of resins (Table 5). In a recent study, Seriani et al. (2015) quantified nuclear abnormalities and micronuclei in O. niloticus collected in the Ecological Park of Tietê (Parque Ecológico do Tietê) in São Paulo, Brazil. The authors observed a frequency of nuclear alterations and micronuclei five times higher in fish from the impacted region compared to the control group, and they related these alterations to the presence of metals in the water. The authors further discuss
3.2. Study of the solid settled wast According to the Brazilian Association of Technical Standards (Associação Brasileira de Normas Técnicas-ABNT), solid wastes can be classified as Class I or Class II. Class I solid wastes (Dangerous) are those that, as a function of their intrinsic characteristics, present risks to public health through an increase in mortality or morbidity, or that have adverse effects on the environment when handled or disposed improperly. Class II solid wastes (Non-Dangerous) can be divided into subclasses A and B. Class II-A (NonInert) includes wastes that show characteristics of combustibility, biodegradability or solubility with the possibility of resulting in risk to health or to the environment and can not be classified as other wastes. Class II-B (Inert) wastes include those that, due to
Table 5 Average and standard deviation for cells with micronuclei (MN) and nuclear abnormalities (NA) among erythrocytes from Oreochromis niloticus exposed to the different treatments. Treatment
MN (%)
NA (%)
Control Lake Waste Resin
0.007 0.01A,b 0.05 7 0.1C 0.047 0.04A 0.09 7 0.09 b,C
0.25 7 0.20a,b,c 1.88 7 1.20a,D,e 3.79 7 1.74 b,D 4.617 2.31c,e
The same UPPERCASE letters represent treatments that differ from each other with p o0.05. The same LOWERCASE letters represent treatments that differ from each other with p o0.01.
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that the nuclear abnormalities are related to cytotoxic damage, but they could also be considered alterations that preceded micronucleus formation. In the present study, a correlation was identified between the presence of nuclear abnormalities and micronuclei, with the nuclear abnormalities observed showing a much higher frequency compared to micronuclei. The higher number of abnormalities found in the present study corroborates the results of Seriani et al. (2015). If the abnormalities are alterations that precede micronucleus formation, they will probably be observed in greater quantity, to be accumulated or corrected, giving rise to a lower number of micronuclei. The presence of lead in the solid phase of the settled waste may be responsible for the formation of these genomic changes. However, another factor that may have affected the micronucleus formation and nuclear abnormalities is the presence of phenols in the waste, because phenolic resins are used in the polishing process. This procedure is aimed at improving the performance and durability of the ornamental rocks over time and at eliminating certain imperfections, cracks and fissures present in the rocks. Gad and Saad (2008) studied the effects of phenol on certain physiological parameters in O. niloticus using sub-lethal doses in a static system for 16 weeks, and they observed significantly higher frequencies of micronuclei even at the lowest phenol concentration (0.7 mg L 1), which corresponds to 1/40th of the LC50 for this species. The phenol concentration in the solubilisation test of the studied waste was equal to 0.007 mg L 1, 100 times lower than that studied in Gad and Saad (2008). However, it must be taken into consideration that this wastewater may be a complex mixture of metals and phenolic resins that, even at low concentrations, evokes different biological responses than those expected from the action of individual contaminants due to synergistic interactions with potentiation or additive effects.
4. Conclusion The liquid phase of the dimension stone polishing effluent showed high values of total dissolved solids, chloride and phenol. The experiment with A. cepa demonstrated that this effluent presents mutagenic potential at studied conditions. Although the solid phase of the waste has shown parameters below the limits allowed by Brazilian legislation, the experiment with O. niloticus showed that this waste has mutagenic potential. Given these results, this paper warns that better attention should be given to the treatment and disposal of dimension stone polishing wastes in order to reduce soil and water pollution, as well as major damage to aquatic life.
Acknowledgements The authors would like to thank the Universidade Federal do Espírito Santo for supporting this research and the fish farming department of the Instituto Federal do Espírito Santo (Campus de Alegre) for providing the fishes used in the study.
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