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Zooremediation of leachates from municipal waste using Eisenia fetida (SAV.)
Environmental Pollution 254 (2019) 112871
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Zooremediation of leachates from municipal waste using Eisenia fetida (SAV.)* b _ Małgorzata Anna Jo zwiak a, *, Marek Jo zwiak b, Rafał Kozłowski b, Monika Zelezik a b
Integrated Monitoring Base Station “Lysogory”, Kielce, Poland The Jan Kochanowski University, Department of Environment Protection and Modeling, Kielce, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2019 Received in revised form 16 June 2019 Accepted 8 July 2019 Available online 17 July 2019
Leachates from municipal landfills are formed as infiltration waters flowing through the landfill. They contain toxic, dissolved products of biochemical reactions taking place in the deposit. They cause soil and groundwater pollution. It is necessary to take them out of the landfill cover and utilize toxins contained therein, in particular heavy metals. Such processes are conducted with the use of microorganisms. Due to the content of toxic compounds, introducing leachates into the process of biological purification poses a threat to the microorganisms used in these processes. An alternative to microbial co-treatment of sludge and leachate as well as soil contaminated with communal leachate is to use red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy metals. The aim of the conducted research is to demonstrate the possibility of using red hybrid of California in leachate bioutilization as a complementary or alternative method to the process of leachate utilization with the use of microorganisms. The obtained results led to the conclusion that Eisenia fetida accumulates environmental toxins well. By collecting and processing them in the tissues, it remedies the substrate and retains long life and fertility, and the ability to reproduce. The research demonstrated high dynamics of population growth (from 25 individuals in the initial deposit to 298 individuals after six months of research). These properties are related to the presence of enzyme proteins from the metallothionein group in the gastrointestinal tract cells. Packing heavy metals found in leachates into the metallothionein coat limits their toxic effect on earthworm tissues, which confirms the possibility of using earthworms in the processes of detoxification of municipal leachate. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Leachates Earthworm/Eisenia fetida Bioremediation Heavy metals
1. Introduction In Council Directive 1999/31/EC on the landfill of waste, leachates were defined as ‘any liquid leaking through landfilled waste and emitted from or contained in the landfill’. About 70% of landfill waste is subject to microbiological decomposition. A part of it (10%) leaves the landfill in the form of leachates. Therefore, leachates constitute infiltration water flowing through the landfill containing dissolved components formed as products of biochemical reactions taking place in the deposit. A small amount of about only 0.4%. is discharged to the treatment
* This paper has been recommended for acceptance by Wei Shi * Corresponding author. E-mail address: [email protected] (M.A. Jo zwiak).
https://doi.org/10.1016/j.envpol.2019.07.039 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
plant and managed safely (Cecen and Aktas, 2004). The remaining part gets to the soil and groundwater as a result of penetration through the landfill deposit. The amount of leachates moving into the soil, the rate of penetration and the composition thereof might differ. The amount depends mainly on the age of the landfill, whereas the rate of penetration depends on the structure of the substrate, and the chemical composition of the type of waste deposited in the landfill. The methods used in sewage treatment plants receiving leachates include microbiological and physical-chemical treatment by mixing leachates with sewage, i.e. co-purification (Fueyo et al., 2002). The leachate composition is dynamic, and the load and concentration of pollutants are changing. Dynamics of leachate composition concerns mainly nitrogen and phosphorus compounds (Robinson, 2002; Robinson et al., 2005). Slack et al. (2005) pointed out that due to the content of toxic compounds,
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
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the following heavy metals: Pb, Cd, Cr, Cu, Mn, Ni, Zn, Al, Fe, Co were determined by the conducted analyses. For the purpose of the analysis, earthworms were randomly chosen from experimental deposits. Chemical composition of the leachates collected from four landfills, tissues of earthworms and soils was analyzed (Table 2). IL 550 TOC-TN analyzer and ICS 3000 DIONEX ion chromatograph were used for this purpose. The analyses regarding heavy metal content were performed using ICP-MS-TOF spectrometer. In order to study the influence of leachates on reproductive processes in the analyzed populations, R birth ratio was applied, which specifies the total number of born individuals in relation to the total population size. It was calculated on the basis of the
introducing leachates into the process of biological purification poses a threat to microorganisms participating in the immobilization of toxins. Low participation of microbiomes in the processes of using toxins included in leachates results from the high content of ammoniacal nitrogen in leachates. A high level of ammoniacal nitrogen reduces the intensity of nitrification processes. As a result of these dependences, concentration of nitrite nitrogen in the substrate remains high. An alternative for microbial co-purification of sludge and leachates, as well as soils contaminated with communal leachate is the use of red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy ski, 2009; https:// metals (Suthar et al., 2008; Jo zwiak and Rybin www.sciencedirect.com/science/article/pii/S026974911830349X Andrade et al., 2014). The aim of the conducted research is to demonstrate the possibility of using red hybrid of California in leachate bioutilization as a complementary or alternative method to the process of leachate utilization with the use of microorganisms.
following formula: R ¼ Nb where: b e number of young forms (individuals not stained), N e average population size (individuals stained and not stained). In a three-month cycle, N value was t calculated on the basis of the following formula: N ¼ N0 þN where: 2 N0- initial number, Nt e final number (after the lapse of t time). The degree of metal accumulation by earthworms was calculated on the basis of the following formula:
2. Materials and methods The tests were used culture substrate, leachates and 100 individuals earthworms (Eisenia fetida). The substrate used in the study contained 3 kg of vermicompost, 0.5 kg of wood sawdust and 250 ml of leachates from four different landfills. The reference sample which made it possible to assess the changes in the population size was culture clean vermicompost, not contaminated with leachates. The temperature, pH using potentiometric method, humidity using graviometric method, C and N content using CHNS-O Flash 2000 analyzer were determined. Leachates taken from four landfills (Table 1). Landfills varied in age. The oldest was 47 years old, the youngest was 18 years old. Earthworm were taken from own breeding. Earthworms were acclimated for two weeks in the test soil before experiment. Worms were washed with sterile water and placed on wet filter paper in the dark at 25 C for 24 h to defecate before use. Eisenia fetida was used in 25 each sample. Before the beginning of the experiment the chemical composition of three elements were examined: firstly, that of applied leachates, secondly, earthworm tissue before the introduction into experimental deposits, and thirdly, the chemical composition of the culture substrate. The values of pH, SEC, TC (Total Carbon), TOC (Total Organic 2þ þ Carbon), Naþ NHþ K Ca2þ Cl- SO2 4 Mg 4 NO3 and the presence of
ACCUMULATION ¼
Cworms ½mg kg 1 h i Ccoprolite mg kg 1
where: Accumulation e degree of metal accumulation by earthworms in relation to the substrate Cworms e content of metal in the body of earthworms, mg kg-1 Ccoprolite e content of metal in the coprolite, mg kg-1
3. Results The analysis of chemical composition of leachates with regard to heavy metals and such parameters as pH, BOD5, COD, organic carbon, ammoniacal nitrogen and chloride must consider the age of the landfill because the time of organic matter decomposition affects the composition and level of toxin concentrations. This is due to dynamics of chemical processes taking place in the deposit. In older landfills (I and IV) the level of concentration of analytes is lower than the level of the same parameters in younger landfills. The leachates from young landfills are in the so-called acidogenesis,
Table 1 Information on municipal waste landfills. Landfill number
Landfill area [m2]
Exploitation from - to year
Geographic coordinates
I II III IV
20 000 42 700
1992 1984e2000 2001 1972e1983
E E E E
longitude
22 200
latitude
19 58‘32.53„ 20 25‘37.93„ 20 25‘47.85„ 20 42‘48.83„
N N N N
50 52‘18.07„ 50 54‘33.31„ 50 54‘33.81„ 50 57‘23.86„
Table 2 The chemical composition of used leachate. Landfill number
pH
I II III IV
7.66 8.7 7.8 8.25
SEC
TC
mS cm-1
Mg L-1
8.88 9.38 21.41 9.02
791.432 838.524 2723.12 828.311
TOC
Naþ
NHþ 4
Mg2þ
Kþ
Ca2þ
CI
SO2þ 4
NO 3
321.614 465 332 1844.734 187.221
689.468 939.378 1427.772 966.776
550.260 325.540 1442.004 152.618
37.248 67.882 123.972 126.062
363.964 615.808 1099.488 678.826
37.200 70.928 122.854 307.560
788.732 1232.544 2121.300 1309.658
0.722 82.792 24.784 1179.394
105.882 131.426 17.224 49.088
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and from old landfills in the so-called methanogenesis. The approximate dates of their establishment are presented in Table 1. The analysis of the remaining parameters demonstrates the highest pH values in the leachate from the so-called old quarter in landfill III with the amount equivalent to pH 8.70, and the lowest in landfill I - pH 7.7. The SEC values expressed in mScm1 are similar and amount to 21 for the landfill III and 8.9 for the landfill I (Table 2). The proportion of heavy metals in the chemical composition of the leachates used in the research indicated the highest content of these analyses from the landfills II and III (Table 3). These landfills are the youngest (established in 2000). The amount of each analyte is expressed in mg kg-1 d.m. for younger landfills, i.e. landfill II and III are many times higher than in the case of the landfills created much earlier. With reference to particular elements, they are presented in the following way: Pb e 14x, Cd e 0.4x, Cr- 32x, Co- 3x, Cu e 4x, Mn - 500x, Ni e 11x, Zn e 47x. The analysis of the concentration level of heavy metals in leachates shows the highest values for all metals in the landfill number II. They are 500 times higher with regard to Mn in landfill II (700 mg kg-1 d.m.) in relation to the content of the same metal in the landfill IV (1.4 mg kg-1 d.m.), and 32 times higher with regard to Cr in landfill II (850 mg kg-1 d.m.) in relation to the same metal in landfill I (26 mg kg-1 d.m.). The lowest concentrations of Pb, Mn, and Zn were recorded in the landfill IV. The chemical composition of the leachates used in the research 3 with regard to SO2 anions indicates the lowest values of 4 and NO
3
the analytes mentioned in landfill II. The remaining analyzed elements and chemical compounds significantly exceed the values of the same analytes from other landfills (Table 2). Assessment of the possibility of applying red hybrid of California in remediation processes of soils contaminated with leachates was made by analyzing the concentration of indicated analytes in VMW indicator tissues and the soil before starting the test compared to the same parameters after three, six and nine months of bioremediation in the earthworm tissues (Table 4 and in the soil (Table 3). The comparison of heavy metal content in earthworm tissues in the subsequent months of research in relation to the reference sample indicates a constant increase in their concentration for the analyzed analytes. The content of Pb in earthworm tissues is characterized by high variability in time and location of the landfill from which the leachate was collected, and varies between 0.11 and 9.8. A steady increase in concentrations over the nine months of the study was noted with reference to Cd and Co, while the level of Mn concentration in the tissues of the studied earthworms continued decreasing over the course of the experiment from maximum values of 17 to minimum values of 2.2. A similar pattern was found with respect to Ni, except for the tissues taken from earthworms from cultures on the leachates from the landfill number II, where the content of this metal continued growing. Accumulation of Zn, Cu and Cr showed specific tendencies. During the first half of the year, increases in concentrations of these metals were reported, and after nine months of the experiment a decrease was reported. It
Table 3 The content of heavy metals in the culture substrate. Landfill number
Exposure time
Pb
I
sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months
10.723 0.538 2.067 1.325 17.112 0.529 1.592 1.544 15.211 0.952 1.792 1.610 1.234 0.589 3.114 1.312
Cd
Cr
Co
Cu
Mn
Ni
Zn
0.000 0.000 0.169 0.124 0.431 0.077 0.322 0.130 0.144 0.000 0.138 0.125 0.000 0.011 0.219 0.159
26.142 0.963 3.718 3.979 846.234 0.861 3.837 4.009 85.714 0.978 3.949 4.390 27.431 1.257 3.741 4.185
0.233 0.112 0.407 0.337 3.411 0.143 0.553 0.318 1.121 0.103 0.459 0.344 1.321 0.127 0.476 0.283
0.000 2.065 8.738 4.386 3.941 1.916 8.326 4.869 1.723 1.929 7.708 7.131 2.431 2.533 15.429 4.758
7.321 17.281 84.066 59.948 699.714 22.580 71.039 58.432 177.611 17.797 105.592 74.089 1.434 24.317 253.648 53.595
3.614 1.457 5.134 3.565 39.121 1.131 4.328 3.943 16.522 0.482 6.435 5.860 9.541 3.402 3.893 3.277
2.933 17.092 50.262 43.215 27.914 17.183 48.506 46.475 4.224 14.791 48.359 46.467 0.611 17.243 85.550 56.362
Cd
Cr
Co
Cu
Mn
Ni
Zn
0.123 0.511 0.870 0.759 3.640 1.281 3.370 0.247 0.477 0.831 1.725 2.082 1.950
0.212 0.928 1.376 1.222 0.560 1.537 1.362 0.458 1.797 0.963 0.714 0.942 1.382
0.331 0.543 0.682 2.458 0.551 1.808 1.599 0.245 1.259 2.484 0.587 1.419 1.605
1.843 5.445 5.473 2.292 2.617 3.221 38.331 7.103 6.627 3.576 2.806 1.640 1.761
2.131 17.274 6.911 4.001 6.820 4.759 3.286 10.139 8.579 2.204 10.021 6.996 4.684
0.622 2.919 1.157 0.620 0.991 0.981 30.373 2.220 1.183 1.352 1.640 0.310 1.009
31.911 87.194 123.470 95.935 56.958 74.167 169.523 60.713 119.693 87.656 66.954 68.153 90.179
Mg Kg-1 d.m.
II
III
IV
Table 4 The content of heavy metals in the tissues of earthworms. Landfill number
Exposure time
Pb Mg*kg-1 d.m.
I
II
III
IV
sample"0" after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months
0.344 0.721 0.465 0.156 0.651 0.128 7.342 1.161 9.777 0.310 0.810 0.113 1.013
4
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
Fig. 1. The coefficient of determination R2 for four landfills.
should be explained by the physiological role of metallothionein maintaining homeostasis within the body by constant control of their level. One of the control methods is to excerpt heavy metals together with coprolites coated with metallothioneins which demonstrate different properties, as indicated by the studies conducted by Mills (2002) with the use of Diamond Light Source synchrotron. The content of heavy metals in the soil in subsequent measurements (3, 6 and 9 months) is characterized by large discrepancies. Upward trends may result from an increase in the population size and, consequently, an increased amount of excreted coprolites. These increases were recorded during the second measurement, i.e. after six months of the study. In subsequent measurements there were decreases in the accumulation of such elements in the soil. The coefficient of determination R2, which indicates how well the model fits, was analyzed for populations cultured on leachates for each landfill, and is presented as a normal distribution curve which assumes the highest value for landfill IV (0.9716), and then respectively, for the landfill II (0.887), landfill III (0.8477) and landfill IV (0.8284) - Fig. 1. Model fitting for the analyzed dependencies of the population size for level of toxin concentration falls within the range of 0.8 and 1, which indicates a proper fit of the listed dependencies in the model for the landfills II, III and I, and a very good fit for the landfill number IV. Simultaneously, the obtained result clearly indicates the influence of the level of toxins on the size of the grown population (Table 4). In order to compare the concentration level of heavy metals, three groups of elements were created in the analysis of standard deviation based on the maximum and minimum values of each element expressed in mg kg 1 d.m. A scale from 0 to 8 mg kg-1 d.m. was used for Pb, Cd, Cr, Co and a scale from 0 to 40 mg kg-1 d.m. for Cu, Mn and Ni, and a scale from 0 to 180 mg kg-1 d.m. for Zn and Al. In the first group, the maximum values were recorded for Pb (8.5 mg kg-1 d.m.), in the second group for Cu (38 mg kg-1 d.m.), and in the third group for Zn (170 mg kg-1 d.m.) (Fig. 2). The growing concentration level of heavy metals is presented in the following series Cr
The number of particular stages of development for each landfill is subject to changes. Earthworms grown on the leachate obtained from the landfill number IV are characterized by a decrease in the number of cocoons in the third measurement (after 9 months of zooremediation) and a simultaneous decrease in the number of juveniles. In subsequent measurements, the number in relation to both stages of developmental is constant. The first measurement (after 3 months of zooremediation) is characterized by an increase in the number of cocoons with a simultaneous increase of adult forms in the first and second measurement (Table 5). Changes in the number of particular stages of development of earthworms grown in the soil specified by the leachate from the landfill number I indicate a minimal decrease in the number of adult forms throughout the measurement period and a dynamic increase in the number of juvenile forms in the first, second and third measurements. A decline in the number of adult forms in the fourth and fifth measurements is followed by a decrease in the number of cocoons and juvenile forms, which is a natural consequence of the development cycle of red hybrid of California. The analysis of the number of each stage of the development cycle in the experimental deposit from the landfill II indicates the largest increase in the number of juvenile forms in the second measurement, and a small increase in the number of cocoons and adult forms in the same research period. A decrease in the number of cocoons in the fourth measurement does not translate into a simultaneous dynamic growth of sexually mature forms, which are fully able to perform the development cycle. The leachates from the landfill III used in the experimental deposit did not significantly affect changes in the number of adult forms. The number of folded cocoons was maintained at a relative level with regard to sexually mature forms. After the third and sixth month of bioremediation, a very dynamic growth of juvenile forms was recorded, which, after reaching the reproduction capacity, will increase the number of each stage of development. An increase in the number of juvenile forms in the second and third measurements does not result in an increase in the number of cocoons, which is a consequence of the earthworm development cycle (Table 5). 4. Discussion Introduction of leachate from municipal waste landfills containing biotoxic substances of heavy metals in the ecosystem is one of the most important civilization challenges. There is a need for the development, eco-friendly, and costeffective approach for the remediation of heavy metals (Pb, Cd, Cr, Co, Cu, Mn, Ni and Zn) released into the environment and to safeguard the ecosystem. Until now, in addition to conventional soil remediation techniques, bioremediation based on microorganisms has been used. However, not all heavy metals are biodegradable and may be toxic to microorganisms. Heavy metal toxicity involves several mechanisms, for example - reacting as redox catalysts in the production of reactive oxygen species (ROS), destructing ion regulation, and directly affecting the formation of DNA and breaking fatal enzymatic functions (Gauthier et al., 2014). Heavy metals could stop vital enzymatic functions by competitive or noncompetitive interactions with substrates that will cause configurational changes in enzymes (Hildebrandt et al., 2007). The physiological and biochemical properties of microorganisms can be altered by the presence of heavy metals. Chromium (Cr) and cadmium (Cd) are capable of inducing oxidative damage and denaturation of microorganisms as well as weakening the bioremediation capacity of microbes (Cervantes et al., 2001).
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Fig. 2. Analysis of standard deviation based on the maximum and minimum values.
Table 5 Spearman’s rank correlation between the content of metals in particular tissues and the content of the faeces (red p < 0.05).
Pb Cd Cr Co Cu Mn Ni Zn Al Fe
Pb
Cd
Cr
Co
Cu
Mn
Ni
Zn
Al
Fe
0,222 0,070 0,053 0,228 0,037 0,390 0,150 0,016 0,005 0,027
0,017 0,615 0,127 0,456 0,516 0,546 0,560 0,054 0,158 0,132
0,004 0,123 0,582 0,694 0,171 0,735 0,275 0,607 0,426 0,615
0,079 0,247 0,000 0,262 0,168 0,380 0,292 0,022 0,034 0,022
0,086 0,223 0,096 0,299 0,081 0,353 0,140 0,078 0,155 0,000
0,039 0,173 0,035 0,359 0,138 0,483 0,333 0,004 0,025 0,011
0,002 0,090 0,078 0,320 0,062 0,419 0,098 0,118 0,037 0,129
0,140 0,456 0,193 0,418 0,372 0,471 0,372 0,081 0,283 0,046
0,186 0,049 0,106 0,151 0,035 0,205 0,018 0,162 0,176 0,148
0,050 0,163 0,078 0,295 0,049 0,346 0,135 0,092 0,141 0,035
Chromium Cr (III) may change the structure and activity of enzymes by reacting with their carboxyl and thiol groups. An alternative for microbial co-purification of sludge and leachates, as well as soils contaminated with communal leachate is the use of red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy metals ski, 2009; https://www. (Suthar et al., 2008; Jo zwiak and Rybin sciencedirect.com/science/article/pii/S026974911830349X
Andrade et al., 2014). Resistance of red hybrid of California results from the presence of enzyme proteins in the gastrointestinal tract separating metals such as arsenic, lead, copper, zinc, cadmium, iron, mercury and nickel in the tissues (Blindauer et al. 2002, https:// www.sciencedirect.com/science/article/pii/S026974911830349X Kuzyakov and Blagodatskaya, 2015). Metallothioneins (MT) play a € ckner et al. 2015)). They are low key role in these processes (Ho molecular mass proteins (6000e7000 Da), but with a high content
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
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Table 6 Results of subsequent counts earthworm populations of Eisenia fetida in three attempts.
Landfill I
II
III
IV
Earthworms Number of individuals Number of individuals after on the day of 3 mounth 6 mounth 9 mounth establishment adults juveniles cocoones adults juveniles cocoones adults juveniles cocoones adults juveniles cocoones
25 0 0 25 0 0 25 0 0 25 0 0
25 36 68 25 32 27 26 88 67 23 15 103
27 248 95 32 130 33 23 260 60 26 20 105
17 262 88 33 119 35 19 298 72 41 21 73
of thiol eSH groups. There are no aromatic amino acids in their structure, but they have a tripeptide amino acid sequence composed of two molecules of cysteine and amino acid residue of amino acid other than cysteine (Cys-X-Cys). The role of metallothionein is to maintain homeostasis inside the body through k and constant control of the level of metal ions in tissues (Vasa Meloni, 2011). This control consists in distributing metals in the body, storing and excreting them. The process of formation itself is induced to a great extent by the presence of heavy metals in cells. The characteristic metabolism of earthworms constitutes a physiological adaptation to toxically changing biotope conditions. The enzymes from the group of metallothioneins separate heavy metals in tissues and control their level by storing and excreting them, which is reflected with variable content of heavy metals in experimental deposits. Separating metals with metallothioneins significantly affects their toxicity by protecting feeding earthworms. The obtained results confirm the research results of Blindauer et al. conducted in 2002. Earthworms are one of the most important soil animal’s which plays a role in bioremediation of soil for cleaner environment due to their biological, chemical and physical actions. Earthworms can be directly employed within bioremediation strategies to promote biodegradation of organic contaminants. Earthworms have been shown to aerate and bioturbate soils and improve their nutritional status and fertility, which are variables known to limit bioremediation. Prakash et al. (2017) showed that earthworms have retard the binding of organic contaminants in soils, release previously soilbound contaminants for subsequent degradation, and promote and disperse organic contaminant degrading microorganisms. Cheng and Wong (2008) reported that soil contaminated with Pb and Zn can be remedied using earthworms. Study Maity et al., (2008) showed that earthworm activity significantly increased the availability of phosphorous, potassium and decreased extractable Pb and Zn which implies the immobilization of metals in soils. 5. Conclusions The use of Californian earthworms for the immobilization of heavy metals contained in toxic leachates from landfills is justified. The arguments for their use include high resistance to contamination, which was confirmed by the studies of population dynamics in created experimental cultures. An increase in heavy metal accumulation in earthworm tissues did not significantly affect the reproductive and viability processes of adult and juvenile forms as well as the number of folded cocoons. Recorded decreases in numbers resulted from the development cycle in which an increase
in the number of sexually immature forms always results in a decrease of folded cocoons, but in subsequent analyses it demonstrates an increase in the number of mature forms which is a natural consequence of the development cycle of these animals. In few samples, despite the potential possibilities of accumulation increase, a downward trend was recorded in the metal concentration in earthworm tissues. This may have resulted from methodological imperfections of the undertaken research. The analysis of the accumulation level of metals in tissues was performed on randomly selected individuals from the deposit. This selection of research material does not provide information on the feeding time of the studied earthworm. Despite the fact that each time sexually mature individuals with clearly formed clitellum were acquired for the research, which was the criterion for tissue analysis, they could differ in time of active feeding. It should be assumed that accidental collection of biological material and, as a consequence, different age of the studied earthworms could affect the concentration of toxins in the tissues. Isolation of cocoons and observation of the metamorphosis, as well as an analysis of the content of toxins in the tissues of sexually immature individuals, constitute indication and methodological supplementation hereof. It would clarify the time of body exposure to the influence of toxins and make it possible to clarify the results. Acknowledgments This work was supported by the Ministry of Science and Higher Education (612495). References Andrade, N.A., Centofanti, T., McConnell, L.L., Hapeman, C.J., Torrents, A., Nguyen, A., Beyer, W.N., Chaney, R.L., Novak, J.M., Anderson, M.O., Cantre, K.B., 2014. Utilizing thin-film solid-phase extraction to assess the effect of organic carbon amendments on the bioavailability of DDT and dieldrin to earthworms. Environ. Pollut. 185, 307e313. https://doi.org/10.1016/j.envpol.2013.11.008. Blindauer, C.A., Harrison, M.D., Robinson, A.K., Parkinson, J.A., Bowness, P.W., Sadler, P.J., Robinson, N.J., 2002. Multiple bacteria encode metallothioneins and SmtA-like zinc fingers. Mol. Microbiol. 45, 1421e1432. https://doi.org/10.1046/j. 1365-2958.2002.03109.x. Cecen, F., Aktas, O., 2004. Aerobic Co-treatment of lanndfill leachate wit domestic wastewater. Environ. Eng. Sci. 21 (3), 303e312. https://doi.org/10.1089/ 109287504323066941. rrez-Corona, F., Loza-Tavera, H., Cervantes, C., Campos-García, J., Devars, S., Gutie n, J.C., Moreno-Sa nchez, R., 2001. Interactions of chromium with Torres-Guzma microorganisms and plants. FEMS Microbiol. Rev. 25 (3), 335e347. https:// doi.org/10.1111/j.1574-6976.2001.tb00581.x. Cheng, J., Wong, M.H., 2008. Effects of earthworm (Pheretima sp.) on three sequential ryegrass harvests for remediating lead/zinc mine tailings. Int. J. Phytoremediation 10 (3), 171e182. Fueyo, G., Gutierrez, A., Berrueta, J., 2002. Anaerobic degradation: The effect of the combined treatment of substrates on the refraction fraction. J. Chem. Technol. Biotechnol. 77, 910e916. Gauthier, P.T., Norwood, W.P., Prepas, E.E., Pyle, G.G., 2014. Metal-PAH mixtures in the aquatic environment: A review of co-toxic mechanisms leading to morethan-additive outcomes. Aquat. Toxicol. 154, 253e269. https://doi.org/10.1016/ j.aquatox.2014.05.026. Hildebrandt, U., Regvar, M., Bothe, H., 2007. Arbuscular mycorrhiza and heavy metal tolerance. Phytochemistry 68 (1), 139e146. https://doi.org/10.1016/ j.phytochem.2006.09.023. €ckner, R., Dallinger, R., Stürzenbaum, S.R., 2015. Metallothionein gene activation Ho in the earthworm (Lumbricus rubellus). Biochem Biophys Res Commun 460 (3), 537e542. https://doi.org/10.1016/j.bbrc.2015.03.065. ski, P., 2009. Possible uses of red hybrid of California (Eisenia Jo zwiak, M.A., Rybin foetida Sav.) for industrial organic wastes utilization. Nat. Environ. Mon. 10/09 (2009), 29e34 (In Polish, abstract in English). Kuzyakov, Y., Blagodatskaya, E., 2015. Microbial hotspots and hot moments in soil: Concept & review. Soil Biol. Biochem. 83, 184e199. https://doi.org/10.1016/j. soilbio.2015.01.025. Maity, S., Padhy, P.K., Chaudhury, S., 2008. The role of earthworm Lampito mauritii (Kinberg) in amending lead and zinc treated soil. Bioresour. Technol. 99 (15), 7291e7298. https://doi.org/10.1016/j.biortech.2007.12.079. Mills, D.M., 2002. Third Generation Hard X-Ray Synchrotron Radiation Sources: Source Properties, Optics and Experimental Techniques. John Wiley & Sons, Inc., New York.
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo Prakash, S., Selvaraju, M., Ravikumar, K., Punnagaiarasi, A., 2017. The Role of Decomposer Animals in Bioremediation of Soils. Bioremediation and Sustainable Technologies for Cleaner Environment. https://doi.org/10.1007/978-3-31948439-6_6. Robinson, N.J., 2002. Multiple bacteria encode metallothioneins and SmtA-like zinc fin-gers. Mol. Microbiol. 45, 1421e1432. Robinson, H.D., Knox, K., Bone, B.D., Picken, A., 2005. Leachate quality from landfilled MBT waste. Waste Manag. 25, 383e391. Slack, R.J., Gronow, J.R., Voulvoulisn, I., 2005. Household hazardous waste
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inmuncipial landfills: Contaminants in leachate. Sci. Total Environ. 337 (2005), 119e137. https://doi.org/10.1016/j.scitotenv.2004.07.002. Suthar, S., Singh, S., Dhawan, S., 2008. Earthworms as bioindicator of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: Is metal bioaccumulation affected by their ecological category? Ecol. Eng. 32, 99e107. https://doi.org/10.1016/ j.ecoleng.2007.10.003. k, M., Meloni, G., 2011. 2011 Chemistry and biology of mammalian metalVasa lothioneins. J Biol Inorg Chem. 16 (7), 1067e1078. https://doi.org/10.1007/ s00775-011-0799-2.
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Zooremediation of leachates from municipal waste using Eisenia fetida (SAV.)* b _ Małgorzata Anna Jo zwiak a, *, Marek Jo zwiak b, Rafał Kozłowski b, Monika Zelezik a b
Integrated Monitoring Base Station “Lysogory”, Kielce, Poland The Jan Kochanowski University, Department of Environment Protection and Modeling, Kielce, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2019 Received in revised form 16 June 2019 Accepted 8 July 2019 Available online 17 July 2019
Leachates from municipal landfills are formed as infiltration waters flowing through the landfill. They contain toxic, dissolved products of biochemical reactions taking place in the deposit. They cause soil and groundwater pollution. It is necessary to take them out of the landfill cover and utilize toxins contained therein, in particular heavy metals. Such processes are conducted with the use of microorganisms. Due to the content of toxic compounds, introducing leachates into the process of biological purification poses a threat to the microorganisms used in these processes. An alternative to microbial co-treatment of sludge and leachate as well as soil contaminated with communal leachate is to use red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy metals. The aim of the conducted research is to demonstrate the possibility of using red hybrid of California in leachate bioutilization as a complementary or alternative method to the process of leachate utilization with the use of microorganisms. The obtained results led to the conclusion that Eisenia fetida accumulates environmental toxins well. By collecting and processing them in the tissues, it remedies the substrate and retains long life and fertility, and the ability to reproduce. The research demonstrated high dynamics of population growth (from 25 individuals in the initial deposit to 298 individuals after six months of research). These properties are related to the presence of enzyme proteins from the metallothionein group in the gastrointestinal tract cells. Packing heavy metals found in leachates into the metallothionein coat limits their toxic effect on earthworm tissues, which confirms the possibility of using earthworms in the processes of detoxification of municipal leachate. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Leachates Earthworm/Eisenia fetida Bioremediation Heavy metals
1. Introduction In Council Directive 1999/31/EC on the landfill of waste, leachates were defined as ‘any liquid leaking through landfilled waste and emitted from or contained in the landfill’. About 70% of landfill waste is subject to microbiological decomposition. A part of it (10%) leaves the landfill in the form of leachates. Therefore, leachates constitute infiltration water flowing through the landfill containing dissolved components formed as products of biochemical reactions taking place in the deposit. A small amount of about only 0.4%. is discharged to the treatment
* This paper has been recommended for acceptance by Wei Shi * Corresponding author. E-mail address: [email protected] (M.A. Jo zwiak).
https://doi.org/10.1016/j.envpol.2019.07.039 0269-7491/© 2019 Elsevier Ltd. All rights reserved.
plant and managed safely (Cecen and Aktas, 2004). The remaining part gets to the soil and groundwater as a result of penetration through the landfill deposit. The amount of leachates moving into the soil, the rate of penetration and the composition thereof might differ. The amount depends mainly on the age of the landfill, whereas the rate of penetration depends on the structure of the substrate, and the chemical composition of the type of waste deposited in the landfill. The methods used in sewage treatment plants receiving leachates include microbiological and physical-chemical treatment by mixing leachates with sewage, i.e. co-purification (Fueyo et al., 2002). The leachate composition is dynamic, and the load and concentration of pollutants are changing. Dynamics of leachate composition concerns mainly nitrogen and phosphorus compounds (Robinson, 2002; Robinson et al., 2005). Slack et al. (2005) pointed out that due to the content of toxic compounds,
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
2
the following heavy metals: Pb, Cd, Cr, Cu, Mn, Ni, Zn, Al, Fe, Co were determined by the conducted analyses. For the purpose of the analysis, earthworms were randomly chosen from experimental deposits. Chemical composition of the leachates collected from four landfills, tissues of earthworms and soils was analyzed (Table 2). IL 550 TOC-TN analyzer and ICS 3000 DIONEX ion chromatograph were used for this purpose. The analyses regarding heavy metal content were performed using ICP-MS-TOF spectrometer. In order to study the influence of leachates on reproductive processes in the analyzed populations, R birth ratio was applied, which specifies the total number of born individuals in relation to the total population size. It was calculated on the basis of the
introducing leachates into the process of biological purification poses a threat to microorganisms participating in the immobilization of toxins. Low participation of microbiomes in the processes of using toxins included in leachates results from the high content of ammoniacal nitrogen in leachates. A high level of ammoniacal nitrogen reduces the intensity of nitrification processes. As a result of these dependences, concentration of nitrite nitrogen in the substrate remains high. An alternative for microbial co-purification of sludge and leachates, as well as soils contaminated with communal leachate is the use of red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy ski, 2009; https:// metals (Suthar et al., 2008; Jo zwiak and Rybin www.sciencedirect.com/science/article/pii/S026974911830349X Andrade et al., 2014). The aim of the conducted research is to demonstrate the possibility of using red hybrid of California in leachate bioutilization as a complementary or alternative method to the process of leachate utilization with the use of microorganisms.
following formula: R ¼ Nb where: b e number of young forms (individuals not stained), N e average population size (individuals stained and not stained). In a three-month cycle, N value was t calculated on the basis of the following formula: N ¼ N0 þN where: 2 N0- initial number, Nt e final number (after the lapse of t time). The degree of metal accumulation by earthworms was calculated on the basis of the following formula:
2. Materials and methods The tests were used culture substrate, leachates and 100 individuals earthworms (Eisenia fetida). The substrate used in the study contained 3 kg of vermicompost, 0.5 kg of wood sawdust and 250 ml of leachates from four different landfills. The reference sample which made it possible to assess the changes in the population size was culture clean vermicompost, not contaminated with leachates. The temperature, pH using potentiometric method, humidity using graviometric method, C and N content using CHNS-O Flash 2000 analyzer were determined. Leachates taken from four landfills (Table 1). Landfills varied in age. The oldest was 47 years old, the youngest was 18 years old. Earthworm were taken from own breeding. Earthworms were acclimated for two weeks in the test soil before experiment. Worms were washed with sterile water and placed on wet filter paper in the dark at 25 C for 24 h to defecate before use. Eisenia fetida was used in 25 each sample. Before the beginning of the experiment the chemical composition of three elements were examined: firstly, that of applied leachates, secondly, earthworm tissue before the introduction into experimental deposits, and thirdly, the chemical composition of the culture substrate. The values of pH, SEC, TC (Total Carbon), TOC (Total Organic 2þ þ Carbon), Naþ NHþ K Ca2þ Cl- SO2 4 Mg 4 NO3 and the presence of
ACCUMULATION ¼
Cworms ½mg kg 1 h i Ccoprolite mg kg 1
where: Accumulation e degree of metal accumulation by earthworms in relation to the substrate Cworms e content of metal in the body of earthworms, mg kg-1 Ccoprolite e content of metal in the coprolite, mg kg-1
3. Results The analysis of chemical composition of leachates with regard to heavy metals and such parameters as pH, BOD5, COD, organic carbon, ammoniacal nitrogen and chloride must consider the age of the landfill because the time of organic matter decomposition affects the composition and level of toxin concentrations. This is due to dynamics of chemical processes taking place in the deposit. In older landfills (I and IV) the level of concentration of analytes is lower than the level of the same parameters in younger landfills. The leachates from young landfills are in the so-called acidogenesis,
Table 1 Information on municipal waste landfills. Landfill number
Landfill area [m2]
Exploitation from - to year
Geographic coordinates
I II III IV
20 000 42 700
1992 1984e2000 2001 1972e1983
E E E E
longitude
22 200
latitude
19 58‘32.53„ 20 25‘37.93„ 20 25‘47.85„ 20 42‘48.83„
N N N N
50 52‘18.07„ 50 54‘33.31„ 50 54‘33.81„ 50 57‘23.86„
Table 2 The chemical composition of used leachate. Landfill number
pH
I II III IV
7.66 8.7 7.8 8.25
SEC
TC
mS cm-1
Mg L-1
8.88 9.38 21.41 9.02
791.432 838.524 2723.12 828.311
TOC
Naþ
NHþ 4
Mg2þ
Kþ
Ca2þ
CI
SO2þ 4
NO 3
321.614 465 332 1844.734 187.221
689.468 939.378 1427.772 966.776
550.260 325.540 1442.004 152.618
37.248 67.882 123.972 126.062
363.964 615.808 1099.488 678.826
37.200 70.928 122.854 307.560
788.732 1232.544 2121.300 1309.658
0.722 82.792 24.784 1179.394
105.882 131.426 17.224 49.088
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and from old landfills in the so-called methanogenesis. The approximate dates of their establishment are presented in Table 1. The analysis of the remaining parameters demonstrates the highest pH values in the leachate from the so-called old quarter in landfill III with the amount equivalent to pH 8.70, and the lowest in landfill I - pH 7.7. The SEC values expressed in mScm1 are similar and amount to 21 for the landfill III and 8.9 for the landfill I (Table 2). The proportion of heavy metals in the chemical composition of the leachates used in the research indicated the highest content of these analyses from the landfills II and III (Table 3). These landfills are the youngest (established in 2000). The amount of each analyte is expressed in mg kg-1 d.m. for younger landfills, i.e. landfill II and III are many times higher than in the case of the landfills created much earlier. With reference to particular elements, they are presented in the following way: Pb e 14x, Cd e 0.4x, Cr- 32x, Co- 3x, Cu e 4x, Mn - 500x, Ni e 11x, Zn e 47x. The analysis of the concentration level of heavy metals in leachates shows the highest values for all metals in the landfill number II. They are 500 times higher with regard to Mn in landfill II (700 mg kg-1 d.m.) in relation to the content of the same metal in the landfill IV (1.4 mg kg-1 d.m.), and 32 times higher with regard to Cr in landfill II (850 mg kg-1 d.m.) in relation to the same metal in landfill I (26 mg kg-1 d.m.). The lowest concentrations of Pb, Mn, and Zn were recorded in the landfill IV. The chemical composition of the leachates used in the research 3 with regard to SO2 anions indicates the lowest values of 4 and NO
3
the analytes mentioned in landfill II. The remaining analyzed elements and chemical compounds significantly exceed the values of the same analytes from other landfills (Table 2). Assessment of the possibility of applying red hybrid of California in remediation processes of soils contaminated with leachates was made by analyzing the concentration of indicated analytes in VMW indicator tissues and the soil before starting the test compared to the same parameters after three, six and nine months of bioremediation in the earthworm tissues (Table 4 and in the soil (Table 3). The comparison of heavy metal content in earthworm tissues in the subsequent months of research in relation to the reference sample indicates a constant increase in their concentration for the analyzed analytes. The content of Pb in earthworm tissues is characterized by high variability in time and location of the landfill from which the leachate was collected, and varies between 0.11 and 9.8. A steady increase in concentrations over the nine months of the study was noted with reference to Cd and Co, while the level of Mn concentration in the tissues of the studied earthworms continued decreasing over the course of the experiment from maximum values of 17 to minimum values of 2.2. A similar pattern was found with respect to Ni, except for the tissues taken from earthworms from cultures on the leachates from the landfill number II, where the content of this metal continued growing. Accumulation of Zn, Cu and Cr showed specific tendencies. During the first half of the year, increases in concentrations of these metals were reported, and after nine months of the experiment a decrease was reported. It
Table 3 The content of heavy metals in the culture substrate. Landfill number
Exposure time
Pb
I
sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months sample"0" after 3 months after 6 months after 9 months
10.723 0.538 2.067 1.325 17.112 0.529 1.592 1.544 15.211 0.952 1.792 1.610 1.234 0.589 3.114 1.312
Cd
Cr
Co
Cu
Mn
Ni
Zn
0.000 0.000 0.169 0.124 0.431 0.077 0.322 0.130 0.144 0.000 0.138 0.125 0.000 0.011 0.219 0.159
26.142 0.963 3.718 3.979 846.234 0.861 3.837 4.009 85.714 0.978 3.949 4.390 27.431 1.257 3.741 4.185
0.233 0.112 0.407 0.337 3.411 0.143 0.553 0.318 1.121 0.103 0.459 0.344 1.321 0.127 0.476 0.283
0.000 2.065 8.738 4.386 3.941 1.916 8.326 4.869 1.723 1.929 7.708 7.131 2.431 2.533 15.429 4.758
7.321 17.281 84.066 59.948 699.714 22.580 71.039 58.432 177.611 17.797 105.592 74.089 1.434 24.317 253.648 53.595
3.614 1.457 5.134 3.565 39.121 1.131 4.328 3.943 16.522 0.482 6.435 5.860 9.541 3.402 3.893 3.277
2.933 17.092 50.262 43.215 27.914 17.183 48.506 46.475 4.224 14.791 48.359 46.467 0.611 17.243 85.550 56.362
Cd
Cr
Co
Cu
Mn
Ni
Zn
0.123 0.511 0.870 0.759 3.640 1.281 3.370 0.247 0.477 0.831 1.725 2.082 1.950
0.212 0.928 1.376 1.222 0.560 1.537 1.362 0.458 1.797 0.963 0.714 0.942 1.382
0.331 0.543 0.682 2.458 0.551 1.808 1.599 0.245 1.259 2.484 0.587 1.419 1.605
1.843 5.445 5.473 2.292 2.617 3.221 38.331 7.103 6.627 3.576 2.806 1.640 1.761
2.131 17.274 6.911 4.001 6.820 4.759 3.286 10.139 8.579 2.204 10.021 6.996 4.684
0.622 2.919 1.157 0.620 0.991 0.981 30.373 2.220 1.183 1.352 1.640 0.310 1.009
31.911 87.194 123.470 95.935 56.958 74.167 169.523 60.713 119.693 87.656 66.954 68.153 90.179
Mg Kg-1 d.m.
II
III
IV
Table 4 The content of heavy metals in the tissues of earthworms. Landfill number
Exposure time
Pb Mg*kg-1 d.m.
I
II
III
IV
sample"0" after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months after 3 months after 6 months after 9 months
0.344 0.721 0.465 0.156 0.651 0.128 7.342 1.161 9.777 0.310 0.810 0.113 1.013
4
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
Fig. 1. The coefficient of determination R2 for four landfills.
should be explained by the physiological role of metallothionein maintaining homeostasis within the body by constant control of their level. One of the control methods is to excerpt heavy metals together with coprolites coated with metallothioneins which demonstrate different properties, as indicated by the studies conducted by Mills (2002) with the use of Diamond Light Source synchrotron. The content of heavy metals in the soil in subsequent measurements (3, 6 and 9 months) is characterized by large discrepancies. Upward trends may result from an increase in the population size and, consequently, an increased amount of excreted coprolites. These increases were recorded during the second measurement, i.e. after six months of the study. In subsequent measurements there were decreases in the accumulation of such elements in the soil. The coefficient of determination R2, which indicates how well the model fits, was analyzed for populations cultured on leachates for each landfill, and is presented as a normal distribution curve which assumes the highest value for landfill IV (0.9716), and then respectively, for the landfill II (0.887), landfill III (0.8477) and landfill IV (0.8284) - Fig. 1. Model fitting for the analyzed dependencies of the population size for level of toxin concentration falls within the range of 0.8 and 1, which indicates a proper fit of the listed dependencies in the model for the landfills II, III and I, and a very good fit for the landfill number IV. Simultaneously, the obtained result clearly indicates the influence of the level of toxins on the size of the grown population (Table 4). In order to compare the concentration level of heavy metals, three groups of elements were created in the analysis of standard deviation based on the maximum and minimum values of each element expressed in mg kg 1 d.m. A scale from 0 to 8 mg kg-1 d.m. was used for Pb, Cd, Cr, Co and a scale from 0 to 40 mg kg-1 d.m. for Cu, Mn and Ni, and a scale from 0 to 180 mg kg-1 d.m. for Zn and Al. In the first group, the maximum values were recorded for Pb (8.5 mg kg-1 d.m.), in the second group for Cu (38 mg kg-1 d.m.), and in the third group for Zn (170 mg kg-1 d.m.) (Fig. 2). The growing concentration level of heavy metals is presented in the following series Cr
The number of particular stages of development for each landfill is subject to changes. Earthworms grown on the leachate obtained from the landfill number IV are characterized by a decrease in the number of cocoons in the third measurement (after 9 months of zooremediation) and a simultaneous decrease in the number of juveniles. In subsequent measurements, the number in relation to both stages of developmental is constant. The first measurement (after 3 months of zooremediation) is characterized by an increase in the number of cocoons with a simultaneous increase of adult forms in the first and second measurement (Table 5). Changes in the number of particular stages of development of earthworms grown in the soil specified by the leachate from the landfill number I indicate a minimal decrease in the number of adult forms throughout the measurement period and a dynamic increase in the number of juvenile forms in the first, second and third measurements. A decline in the number of adult forms in the fourth and fifth measurements is followed by a decrease in the number of cocoons and juvenile forms, which is a natural consequence of the development cycle of red hybrid of California. The analysis of the number of each stage of the development cycle in the experimental deposit from the landfill II indicates the largest increase in the number of juvenile forms in the second measurement, and a small increase in the number of cocoons and adult forms in the same research period. A decrease in the number of cocoons in the fourth measurement does not translate into a simultaneous dynamic growth of sexually mature forms, which are fully able to perform the development cycle. The leachates from the landfill III used in the experimental deposit did not significantly affect changes in the number of adult forms. The number of folded cocoons was maintained at a relative level with regard to sexually mature forms. After the third and sixth month of bioremediation, a very dynamic growth of juvenile forms was recorded, which, after reaching the reproduction capacity, will increase the number of each stage of development. An increase in the number of juvenile forms in the second and third measurements does not result in an increase in the number of cocoons, which is a consequence of the earthworm development cycle (Table 5). 4. Discussion Introduction of leachate from municipal waste landfills containing biotoxic substances of heavy metals in the ecosystem is one of the most important civilization challenges. There is a need for the development, eco-friendly, and costeffective approach for the remediation of heavy metals (Pb, Cd, Cr, Co, Cu, Mn, Ni and Zn) released into the environment and to safeguard the ecosystem. Until now, in addition to conventional soil remediation techniques, bioremediation based on microorganisms has been used. However, not all heavy metals are biodegradable and may be toxic to microorganisms. Heavy metal toxicity involves several mechanisms, for example - reacting as redox catalysts in the production of reactive oxygen species (ROS), destructing ion regulation, and directly affecting the formation of DNA and breaking fatal enzymatic functions (Gauthier et al., 2014). Heavy metals could stop vital enzymatic functions by competitive or noncompetitive interactions with substrates that will cause configurational changes in enzymes (Hildebrandt et al., 2007). The physiological and biochemical properties of microorganisms can be altered by the presence of heavy metals. Chromium (Cr) and cadmium (Cd) are capable of inducing oxidative damage and denaturation of microorganisms as well as weakening the bioremediation capacity of microbes (Cervantes et al., 2001).
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
5
Fig. 2. Analysis of standard deviation based on the maximum and minimum values.
Table 5 Spearman’s rank correlation between the content of metals in particular tissues and the content of the faeces (red p < 0.05).
Pb Cd Cr Co Cu Mn Ni Zn Al Fe
Pb
Cd
Cr
Co
Cu
Mn
Ni
Zn
Al
Fe
0,222 0,070 0,053 0,228 0,037 0,390 0,150 0,016 0,005 0,027
0,017 0,615 0,127 0,456 0,516 0,546 0,560 0,054 0,158 0,132
0,004 0,123 0,582 0,694 0,171 0,735 0,275 0,607 0,426 0,615
0,079 0,247 0,000 0,262 0,168 0,380 0,292 0,022 0,034 0,022
0,086 0,223 0,096 0,299 0,081 0,353 0,140 0,078 0,155 0,000
0,039 0,173 0,035 0,359 0,138 0,483 0,333 0,004 0,025 0,011
0,002 0,090 0,078 0,320 0,062 0,419 0,098 0,118 0,037 0,129
0,140 0,456 0,193 0,418 0,372 0,471 0,372 0,081 0,283 0,046
0,186 0,049 0,106 0,151 0,035 0,205 0,018 0,162 0,176 0,148
0,050 0,163 0,078 0,295 0,049 0,346 0,135 0,092 0,141 0,035
Chromium Cr (III) may change the structure and activity of enzymes by reacting with their carboxyl and thiol groups. An alternative for microbial co-purification of sludge and leachates, as well as soils contaminated with communal leachate is the use of red hybrid of California (Eisenia fetida Sav.), an earthworm resistant to environmental toxins, in particular heavy metals ski, 2009; https://www. (Suthar et al., 2008; Jo zwiak and Rybin sciencedirect.com/science/article/pii/S026974911830349X
Andrade et al., 2014). Resistance of red hybrid of California results from the presence of enzyme proteins in the gastrointestinal tract separating metals such as arsenic, lead, copper, zinc, cadmium, iron, mercury and nickel in the tissues (Blindauer et al. 2002, https:// www.sciencedirect.com/science/article/pii/S026974911830349X Kuzyakov and Blagodatskaya, 2015). Metallothioneins (MT) play a € ckner et al. 2015)). They are low key role in these processes (Ho molecular mass proteins (6000e7000 Da), but with a high content
zwiak et al. / Environmental Pollution 254 (2019) 112871 M.A. Jo
6
Table 6 Results of subsequent counts earthworm populations of Eisenia fetida in three attempts.
Landfill I
II
III
IV
Earthworms Number of individuals Number of individuals after on the day of 3 mounth 6 mounth 9 mounth establishment adults juveniles cocoones adults juveniles cocoones adults juveniles cocoones adults juveniles cocoones
25 0 0 25 0 0 25 0 0 25 0 0
25 36 68 25 32 27 26 88 67 23 15 103
27 248 95 32 130 33 23 260 60 26 20 105
17 262 88 33 119 35 19 298 72 41 21 73
of thiol eSH groups. There are no aromatic amino acids in their structure, but they have a tripeptide amino acid sequence composed of two molecules of cysteine and amino acid residue of amino acid other than cysteine (Cys-X-Cys). The role of metallothionein is to maintain homeostasis inside the body through k and constant control of the level of metal ions in tissues (Vasa Meloni, 2011). This control consists in distributing metals in the body, storing and excreting them. The process of formation itself is induced to a great extent by the presence of heavy metals in cells. The characteristic metabolism of earthworms constitutes a physiological adaptation to toxically changing biotope conditions. The enzymes from the group of metallothioneins separate heavy metals in tissues and control their level by storing and excreting them, which is reflected with variable content of heavy metals in experimental deposits. Separating metals with metallothioneins significantly affects their toxicity by protecting feeding earthworms. The obtained results confirm the research results of Blindauer et al. conducted in 2002. Earthworms are one of the most important soil animal’s which plays a role in bioremediation of soil for cleaner environment due to their biological, chemical and physical actions. Earthworms can be directly employed within bioremediation strategies to promote biodegradation of organic contaminants. Earthworms have been shown to aerate and bioturbate soils and improve their nutritional status and fertility, which are variables known to limit bioremediation. Prakash et al. (2017) showed that earthworms have retard the binding of organic contaminants in soils, release previously soilbound contaminants for subsequent degradation, and promote and disperse organic contaminant degrading microorganisms. Cheng and Wong (2008) reported that soil contaminated with Pb and Zn can be remedied using earthworms. Study Maity et al., (2008) showed that earthworm activity significantly increased the availability of phosphorous, potassium and decreased extractable Pb and Zn which implies the immobilization of metals in soils. 5. Conclusions The use of Californian earthworms for the immobilization of heavy metals contained in toxic leachates from landfills is justified. The arguments for their use include high resistance to contamination, which was confirmed by the studies of population dynamics in created experimental cultures. An increase in heavy metal accumulation in earthworm tissues did not significantly affect the reproductive and viability processes of adult and juvenile forms as well as the number of folded cocoons. Recorded decreases in numbers resulted from the development cycle in which an increase
in the number of sexually immature forms always results in a decrease of folded cocoons, but in subsequent analyses it demonstrates an increase in the number of mature forms which is a natural consequence of the development cycle of these animals. In few samples, despite the potential possibilities of accumulation increase, a downward trend was recorded in the metal concentration in earthworm tissues. This may have resulted from methodological imperfections of the undertaken research. The analysis of the accumulation level of metals in tissues was performed on randomly selected individuals from the deposit. This selection of research material does not provide information on the feeding time of the studied earthworm. Despite the fact that each time sexually mature individuals with clearly formed clitellum were acquired for the research, which was the criterion for tissue analysis, they could differ in time of active feeding. It should be assumed that accidental collection of biological material and, as a consequence, different age of the studied earthworms could affect the concentration of toxins in the tissues. Isolation of cocoons and observation of the metamorphosis, as well as an analysis of the content of toxins in the tissues of sexually immature individuals, constitute indication and methodological supplementation hereof. It would clarify the time of body exposure to the influence of toxins and make it possible to clarify the results. Acknowledgments This work was supported by the Ministry of Science and Higher Education (612495). References Andrade, N.A., Centofanti, T., McConnell, L.L., Hapeman, C.J., Torrents, A., Nguyen, A., Beyer, W.N., Chaney, R.L., Novak, J.M., Anderson, M.O., Cantre, K.B., 2014. Utilizing thin-film solid-phase extraction to assess the effect of organic carbon amendments on the bioavailability of DDT and dieldrin to earthworms. Environ. Pollut. 185, 307e313. https://doi.org/10.1016/j.envpol.2013.11.008. Blindauer, C.A., Harrison, M.D., Robinson, A.K., Parkinson, J.A., Bowness, P.W., Sadler, P.J., Robinson, N.J., 2002. Multiple bacteria encode metallothioneins and SmtA-like zinc fingers. Mol. Microbiol. 45, 1421e1432. https://doi.org/10.1046/j. 1365-2958.2002.03109.x. Cecen, F., Aktas, O., 2004. Aerobic Co-treatment of lanndfill leachate wit domestic wastewater. Environ. Eng. Sci. 21 (3), 303e312. https://doi.org/10.1089/ 109287504323066941. rrez-Corona, F., Loza-Tavera, H., Cervantes, C., Campos-García, J., Devars, S., Gutie n, J.C., Moreno-Sa nchez, R., 2001. Interactions of chromium with Torres-Guzma microorganisms and plants. FEMS Microbiol. Rev. 25 (3), 335e347. https:// doi.org/10.1111/j.1574-6976.2001.tb00581.x. Cheng, J., Wong, M.H., 2008. Effects of earthworm (Pheretima sp.) on three sequential ryegrass harvests for remediating lead/zinc mine tailings. Int. J. Phytoremediation 10 (3), 171e182. Fueyo, G., Gutierrez, A., Berrueta, J., 2002. Anaerobic degradation: The effect of the combined treatment of substrates on the refraction fraction. J. Chem. Technol. Biotechnol. 77, 910e916. Gauthier, P.T., Norwood, W.P., Prepas, E.E., Pyle, G.G., 2014. Metal-PAH mixtures in the aquatic environment: A review of co-toxic mechanisms leading to morethan-additive outcomes. Aquat. Toxicol. 154, 253e269. https://doi.org/10.1016/ j.aquatox.2014.05.026. Hildebrandt, U., Regvar, M., Bothe, H., 2007. Arbuscular mycorrhiza and heavy metal tolerance. Phytochemistry 68 (1), 139e146. https://doi.org/10.1016/ j.phytochem.2006.09.023. €ckner, R., Dallinger, R., Stürzenbaum, S.R., 2015. Metallothionein gene activation Ho in the earthworm (Lumbricus rubellus). Biochem Biophys Res Commun 460 (3), 537e542. https://doi.org/10.1016/j.bbrc.2015.03.065. ski, P., 2009. Possible uses of red hybrid of California (Eisenia Jo zwiak, M.A., Rybin foetida Sav.) for industrial organic wastes utilization. Nat. Environ. Mon. 10/09 (2009), 29e34 (In Polish, abstract in English). Kuzyakov, Y., Blagodatskaya, E., 2015. Microbial hotspots and hot moments in soil: Concept & review. Soil Biol. Biochem. 83, 184e199. https://doi.org/10.1016/j. soilbio.2015.01.025. Maity, S., Padhy, P.K., Chaudhury, S., 2008. The role of earthworm Lampito mauritii (Kinberg) in amending lead and zinc treated soil. Bioresour. Technol. 99 (15), 7291e7298. https://doi.org/10.1016/j.biortech.2007.12.079. Mills, D.M., 2002. Third Generation Hard X-Ray Synchrotron Radiation Sources: Source Properties, Optics and Experimental Techniques. John Wiley & Sons, Inc., New York.
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inmuncipial landfills: Contaminants in leachate. Sci. Total Environ. 337 (2005), 119e137. https://doi.org/10.1016/j.scitotenv.2004.07.002. Suthar, S., Singh, S., Dhawan, S., 2008. Earthworms as bioindicator of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: Is metal bioaccumulation affected by their ecological category? Ecol. Eng. 32, 99e107. https://doi.org/10.1016/ j.ecoleng.2007.10.003. k, M., Meloni, G., 2011. 2011 Chemistry and biology of mammalian metalVasa lothioneins. J Biol Inorg Chem. 16 (7), 1067e1078. https://doi.org/10.1007/ s00775-011-0799-2.