Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation

GEXPLO-05332; No of Pages 8 Journal of Geochemical Exploration xxx (2014) xxx–xxx

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Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation Plamen Georgiev ⁎, Stoyan Groudev, Irena Spasova, Marina Nicolova University of Mining and Geology “Saint Ivan Rilski”, Department of Engineering Geoecology, Sofia 1700, Bulgaria

a r t i c l e

i n f o

Article history: Received 2 October 2013 Revised 18 February 2014 Accepted 23 February 2014 Available online xxxx Keywords: Radionuclides Heavy metals Soil remediation Lysimeters Toxicity

a b s t r a c t Cinnamonic soils from southeastern Bulgaria are heavily polluted with radionuclides (uranium, radium) and toxic heavy metals (copper and lead) due to the aerial transportation of fine particles from flotation tailing dumps to the soil surface. As a result of this, the polluted soils are characterized by a slightly alkaline pH (7.82) and positive net neutralization potential (+136.8 kg CaCO3⁄t). A fresh sample of cinnamonic soil was subjected to remediation under laboratory conditions in four lysimeters each containing 70 kg of soil. The preliminary study revealed that most of the pollutants were presented as carbonate, reducible and oxidizable fractions, i.e. pollutant ions were specifically adsorbed by carbonate and ferric iron minerals or were capsulated in sulfides. The applied soil treatment was connected with the leaching of the pollutants located mainly in the horizon A, their transportation through the soil profile as soluble forms, and their precipitation in the rich-in-clay subhorizon B3. The efficiency of leaching depended on the activity of the indigenous microflora and on the chemical processes connected with the solubilization of pollutants and formation of stable complexes with some organic compounds and chloride and hydrocarbonate ions. These processes were considerably enhanced by adding hay to the horizon A and irrigating the soil with water solutions containing the above-mentioned ions and some nutrients. After 18 months of treatment, each of the soil profiles in the different lysimeters was divided into five sections reflecting the different soil layers. The soil in these sections was subjected to a detailed chemical analysis and the data obtained were compared with the relevant data obtained before the start of the experiment. The best leaching of pollutants from horizon A was measured in the variants where soil mulching was applied. For example, the best leaching of lead (54.5%) was found in the variant combining this technique and irrigation with solutions containing only nutrients. The best leaching of uranium (66.3%), radium (62.5%), and copper (15.1%) was measured in the variant in which the soil was subjected to mulching and irrigation with alkaline solutions containing hydrocarbonate ions. Despite the removal of higher amounts of these pollutants from the soil, the acute soil toxicity towards earthworms (Lumbricus terrestris) was higher in comparison to the toxicity of soil that had been treated in other variants. Furthermore, the highly alkaline soil pH (10.47) that was determined through the applied alkaline leaching resulted in an acute soil toxicity to oats (Avena sativa) and clover (Trifolium repens) that was even higher in comparison to the toxicity of the non-treated soil. These data revealed that the soil detoxification was depended not only on the decrease of the total concentration and on the bioavailable forms of the above-mentioned pollutants but also on the changes that had taken place in chemical and geotechnical properties of the treated soil. © 2014 Elsevier B.V. All rights reserved.

Introduction Remediation of soils polluted by radioactive elements and nonferrous metals could be divided into two main groups with respect to a pollutant's behavior during the remediation process. The first group includes methods where the main goal is to stop/prevent pollutant migration into the environment. It could be achieved by means of their transformation into solid phases with higher stability due to addition of some sorbents (Castaldi et al., 2005; Chen et al., 2003), suitable ⁎ Corresponding author. Tel./fax: +359 2806 0263 E-mail address: [email protected] (P. Georgiev).

change of soil acidity (Alva et al., 2000; Clemente et al., 2006) or redox conditions (Abdelous et al., 2000; Groudev et al., 2010) as well as establishment of suitable covers on the heavily contaminated sites (Komnitsas et al., 1999; Lu et al., 2013). The second group includes methods where the main goal is completely opposite to the previous group — to create and maintain conditions which enhance pollutant leaching from soil horizon and their downward migration by means of draining soil solutions (Kumar and Nagendran, 2009; Toth, 2005) or pollutant migration in upward direction by means of their uptake and accumulation into the plant biomass (Bhargava et al., 2012; Ebbs et al., 1998). Regardless of the chosen method, there are several key factors which preliminary evaluations determine the process efficiency at a

http://dx.doi.org/10.1016/j.gexplo.2014.02.024 0375-6742/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

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P. Georgiev et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx

later stage. First of all, solid phase pollutant characterization by means of suitable extraction tests (Pagnanelli et al., 2004; Tessier et al., 1979), the results of which throw light on the main mobile fractions as well as stimulation of which biogeochemical process will lead to their redistribution in a suitable direction. The second key factor is acid–base soil property which reflects on soil pH. Its value determines in large extent the size and charge, existing on the surface of soil grains (Dube et al., 2001) and if there is a need to manipulate it during application of the relevant remediation method (Harter and Naidu, 2001; Sakurai et al., 1989). The third factor concerns the chemistry of a pollutant itself — valence state(s), speciation and stability of its soluble forms, etc., which depend on the soil condition. Exact evaluation of all that information allows elaborating a suitable strategy for soil remediation. Regardless of the decreasing of the total concentration of a pollutant and/or its leaching in soil horizon, most methods for soil remediation are connected with changes of some basic parameters of the soil biotope. Assessing their effect towards soil biota is carried out by ecotoxicological tests which determine if there is a need for additional steps before the cleaned soil is used in agriculture. The main aim of this study was to evaluate possibilities for in situ remediation of heavily polluted soils by means of leaching of soil contaminants from topsoil and their precipitation into the soil depth. The approaches for soil remediation applied in this study to enhance the leaching of the soil contaminants from horizon A were transport of the released ions through the soil profile by means of soil solutions and contaminant precipitation in rich-in-clay soil horizon B3. Four different variants of soil treatment were tested in this study by means of large scale lysimeters. Materials and methods Soil characteristic and remediation The soil sample used in the experiment belonged to the cinnamonic soil type. The soil profile was consisted of: horizon A (0–30 cm), horizon B (31–70 cm), horizon C (71–90 cm), and a clay horizon (91–110 cm). The soil pH was determined at 1:2.5 ratio with distilled water. Humus content, cation exchange capacity (CEC) and carbonate content were determined by means of suitable methods (Pansu and Gautheyrou, 2006). The net neutralization potential was determined by a static acid–base accounting test (Sobek et al., 1978). Elemental analysis of the digested soil sample was determined by atomic absorption spectrometry (AAS) and induced plasma spectrometry (ICP). The specific activity of Ra-226 was measured by means of a gamma-spectrometer (ORTEC-USA). The mobile forms of heavy metals and uranium were determined by means of a sequential extraction method (Tessier et al., 1979) and a bioavailability test (Lindsay and Norvell, 1978). The soil permeability was determined by means of a double ring infiltrometer method (U.S.EPA, 1991). The soil sample was treated in zero suction type lysimeters. Each lysimeter was charged with 70 kg of soil keeping the natural soil structure. A sand layer was located beneath the soil profile which enhanced the soil solutions to drain easily. The soil in Lysimeter 1 was irrigated with solutions containing 0.10 g/l NH4Cl and 0.02 g/l K2HPO4. The soil in Lysimeter 2 was irrigated with the above-mentioned solution plus 0.05 N NaHCO3. Plant biomass (as a finely cut hay) was added to and mixed with the soil horizon A in Lysimeter 3 and Lysimeter 4 to a final content of 4%. The hay consisted of 36% cellulose, 24% hemicellulose, 18% lignin and 6.1% ash. The soils in Lysimeter 3 and Lysimeter 4 were irrigated with the same solutions as those used in Lysimeter 1 and Lysimeter 2, respectively. The irrigation rate was 50 l/t soil per week per lysimeter. Each week the pregnant effluents from the lysimeters were replaced with fresh solutions with the relevant initial composition. The leaching was carried out at temperatures varying in the range of about 15–23 °C for a period of 18 months.

A nutrient solution containing equimolar concentration of acetic and lactic acids (total organic carbon of 200–220 mg/l), preliminary neutralized to pH 6.1–6.3, was injected weekly at a depth of 75 cm during the soil treatment. Chemical analyses The heavy metal transport through the soil profile was monitored regularly by means of sampling of the drainage soil solutions. The collected solutions were characterized by measurement of pH, Eh, alkalinity, and dissolved organic carbon (APHA, 1995). The concentrations of heavy metals and uranium were determined after the preliminary digestion of dissolved organic compounds by means of 705 UV Digester (Metrohm). The heavy metals were analyzed by means of ICP spectrophotometry. Uranium concentration was measured photometrically using the Arsenazo III reagent (Savvin, 1961). Ecotoxicity analyses The ecotoxicity analyses were carried out with the non-treated soil sample of the horizon A as well as samples of the topsoil which have been remediated at relevant conditions. The soil toxicity towards oats (Avena sativa) and clover (Trifolium repens) was determined in accordance to a range-finding test (OECD, 1984) with a purpose to establish dose–response relationship for the plant species towards the tested soil sample. The test concentrations of the soil sample were in the range of 10–100% (weight) and the rest milieu for plant growth was composted biomass. Each pot was sown with ten seeds of one of the two plant species. Three replicates were used for each test concentration as well as for controls of each species. In the control the seeds were sown in composted biomass (pH (H2O) 5.9–6.1). The test was carried out in a greenhouse at temperatures 16–22 °C, and precise control on the duration of photoperiod (16 h) and the soil humidity (maintained by means of distilled water). The test's duration was 30 days. The soil toxicity towards earthworm (Lumbricus terrestris) was carried out with a synchronized population which was cultivated preliminary for 1 year at laboratory conditions in brown forest soil. The toxicity of the soil sample was determined by range-finding and definitive tests (U.S.EPA, 1996) which were carried out in plastic boxes with a volume of 1.0 l. The test concentrations of the soil sample were in the range of 10–100% (weight) and the rest milieu was brown forest soil. Three replicates were used for each test concentration with ten worms with similar lengths added to each. Ten worms were added to the control too which consisted of brown forest soil only. The duration of the test was 30 days. The worms' survival and marks of their activity were determined at the end of the toxicity test. The data from all replicates of each test concentration of the relevant soil sample to the relevant species were statistically assessed by means of determination of the average values and standard deviation. The main ecotoxicity parameters—No Observed Effect Concentration (NOEC), Lowest Observed Effect Concentration (LOEC), LC50 and LC100 were determined by processing experimental data by means of Shapiro–Wilk's test and the Probit method (U.S.EPA, 1994). Study site Some soils from the Southeast Bulgaria are heavily polluted with radioactive elements as well as non-ferrous metals. In that area, one of the facilities for mining and processing of copper ores in Bulgaria — Burgas Copper Mine is situated. The flotation tailings generated during the ore's processing had been deposited on a flotation dump for decades. That dump was a point source for the soil pollution with those contaminants due to the aerial transportation of mineral particles and their deposition on the soil surface. So, a soil plot with a surface area of almost a decare was excluded from agricultural activity due to the concentration of the

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

P. Georgiev et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx

contaminants several times above the relevant permissible levels in the soil horizon A. Cinnamonic soil is the main soil type in the area which is characterized by neutral pH, higher sorption capacity, and relatively high humus content (3.8%). The soil profile is well structured; soil porosity as well as soil permeability is relatively low. Because of the deposition of fine particles with higher content of carbonates and net positive neutralization potential, soil pH changed to a slightly alkaline value (Table 2). A Bulgarian guideline for soil pollution with heavy metals is based on the value of soil pH and the total concentration of an element in the topsoil of a relevant soil type (Guideline No. 3, 2008). As the standard applied in other countries (CCME, 2006; Swartjes, 1999), it considers different degrees of pollution and regulates different thresholds-background, target, maximum admissible, and intervention concentrations, which values depend on the relevant land use (agricultural, residential, or industrial). The studied soil plot was heavily polluted with radioactive elements as well as non-ferrous metals. For example, uranium content and the specific activity of radium-226 in the topsoil were 3.4 and 6.2 times higher than the relevant permissible levels for soils in Bulgaria (Table 1). In accordance to copper and lead, their concentrations in the horizon A were both 3.4 times higher than the relevant permissible concentrations for soils with neutral soil pH. The studied soil plot was characterized as highly risky towards the environment and human health and for that reason the present study was carried out. The sequential extraction and bioavailable test revealed the distribution of the soil contaminants amongst the main mobile fractions. For example, more than 50% of uranium in the soil horizon A was bioavailable to plants and microorganisms due to its presence as easily leachable fraction (exchangeable and carbonate mobile fractions) (Table 5). At slightly alkaline pH, apart from the permanent, negative pHinterdependent surface charge which existed on the basal surface of soil clay minerals, an additional negative pH-dependent surface charge emerged due to the deprotonation of some functional groups of the soil constituents. As a result, hexa-valent uranium complexes with bicarbonate as well as organic compounds with net negative charge were repulsed by the soil surface with the same charge. In opposite, copper and lead ions hydrolyze quickly and form hydroxy-complexes (as Me(OH)+) which were adsorbed preferably on the negative charged soil surface in comparison with other bivalent cations. As a result, a higher number of the non-ferrous metals were detected as carbonate, reducible and oxidizable mobile fractions and less than 0.5% were presented as an exchangeable fraction in the topsoil. The carbonate and reducible fractions presented mainly ions which were specifically sorbed on or entrapped within the surface of the carbonates and ferric and manganese hydroxide minerals, respectively. It is well-known that particles enriched with both heavy metals and carbonates present high risk for human health through different exposure routes such as ingestion, inhalation, direct soil contact, and secondary soil contamination (Xenidis et al., 2003). A significant part of the contaminants were presented as an oxidizable fraction. It presented minerals which would be leached at oxidizing conditions. For example, some uranium was in tetra-valency state and it could be leached after its preliminary oxidation by means of some strong oxidants as H2O2, Cl0, and Fe3 + in

dependence on soil pH (Barbusinsky, 2009). In accordance to nonferrous metals, that fraction presented sulfides which could be leached efficiently by means of bacterial or chemical oxidation. An inert fraction presented the part of contaminants which were capsulated in the lattice of silicate minerals. For that reason, the amounts of soil pollutants which were detected as an inert mobile fraction were practically not bioavailable and couldn't be leached out of the soil horizon A for a short period of time. Radium is a lithophile element, chemically similar to calcium and the results of other researchers (Edsfeldt, 1999; Uchida and Tagami, 2007) show that a higher number of ions of the element are presented as carbonate and reducible fractions in soils. Data about microflora of the non-treated soil revealed that the number of microbes sensitive to the presence of heavy metals was low, while the microbes applying different mechanisms for pollutant detoxification dominated (Table 4). For example, the number of nitrifying as well as aerobic heterotrophic bacteria which play a crucial role in the biogeochemical cycles of nitrogen and carbon was lower in the topsoil. The main reason was the concentrations of dissolved copper and uranium in the soil solutions (0.13 and 0.15 mg/l respectively (Table 3)). Copper hydrolyses easily due to the slightly alkaline soil pH and probably it was presented in soil solutions as CuOH+ and [Cu2(OH)2]2+ complexes. Both complexes are easily bioavailable and for that reason they are characterized as highly toxic to organisms (Flemming and Trevors, 1989). However, a few groups of microorganisms as fungi and actinomycetes amongst the heterotrophic microorganisms as well as sulfur-oxidizing bacteria amongst chemolithotrophic bacteria apply different mechanisms to detoxify copper and other heavy metals which explained their dominance (Ferreira et al., 2010; Joner et al., 2007). The main processes of leaching of heavy metals in the environment are biologically controlled or at least dependable on the rate of the biological processes (Gadd, 1999; Suzuki and Suko, 2006). For that reason, the main goal during the soil remediation was to create and maintain such conditions which enhanced proliferation of soil microflora and the leaching of heavy metals from the soil horizon A to carry out with higher rate by this way.

Results and discussion In Lysimeter 1, the soil was irrigated with solutions enriched with nitrogen and phosphorous presented in their bioavailable ions for plants and soil microflora. However, due to the still low content of suitable donors of electrons during the soil remediation, the number of the main microbial groups didn't increase significantly in comparison with microflora of the non-treated soil (Table 4). As a result of this, a negligible part of the soil contaminants were leached. Apart from the lower microbial activity, other reasons were lower content of suitable complexing ions into the solutions, which could form stable complexes with soil pollutants, as well as insignificant alteration in the acid–base properties of the soil horizon A (Tables 2, 3). The effect of those factors on pollutant mobility is well-studied (Harter and Naidu, 2001; Temminghoff et al., 1997).

Table 1 Data about the total content of heavy metals and radionuclides in soil horizon A before and after the soil remediation. Index

Pb, mg/kg Zn, mg/kg Cu, mg/kg Ni, mg/kg Co, mg/kg U, mg/kg Ra-226, Bq/kg

Before treatment

272 241 649 71 98 34.5 400

3

Maximum admissible concentration for agricultural soil with pH N7.4

After treatment Lysimeter 1

Lysimeter 2

Lysimeter 3

Lysimeter 4

184 235 637 70 96 29.7 370

202 187 616 65 92 18.6 370

123 173 584 66 86 28.8 250

149 166 551 65 88 11.6 250

80 370 280 10

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

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P. Georgiev et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx

Table 2 Data about the main properties of the soil horizon A before and after the soil remediation. Index

Before treatment

pH (in H2O) Carbonate content, % Content of sulfidic sulfur, g/kg Net neutralization potential, kg CaCO3/t Humus content, %

After treatment

7.82 8.4 3.36 +136.8 3.8

Lysimeter 1

Lysimeter 2

Lysimeter 3

Lysimeter 4

7.88 6.4 2.74 +104 3.6

10.54 5.7 3.11 +88.5 2.15

7.93 4.6 2.32 +66.8 3.70

10.47 4.8 2.63 +66.6 3.0

The characteristic of the soil treated in Lysimeter 1 was quite similar to that of the non-treated soil. For that reason, soil with such characteristic still presents a high risk to the environment and human health. So, the best way to deal with it was to apply a suitable method, based on the prevention of pollutant migration from soil to the environment, which would decrease the risk significantly. A classical method applied in hydrometallurgy for uranium recovery from raw materials as well as for remediation of uranium contaminated sites, characterized by net positive neutralization potential, is the alkaline leaching with solutions containing bicarbonate ions (HCO− 3 ) (Phillips et al., 2005). For that reason, a classical scheme for alkaline leaching of uranium from soil with slightly alkaline pH was applied in Lysimeter 2 and bicarbonate ions were added to the same irrigation solution that was used in Lysimeter 1. The presence of bicarbonate ions enhanced the extraction of hexa-valent uranium considerably by means of formation of stable uranyl carbonate complexes (UO2(CO3)2− 2 , 0 2+ UO2(CO3)4− 3 , CaUO2(CO3)3) as well as UO2 –humate complexes with net negative charge (Kohler et al., 2004; Langmuir, 1978). The main sources for uranium complexolysis were carbonate, reducible as well as oxidizable mobile fractions (Table 5). At alkaline pH, oxidation of tetravalent uranium by means of molecular oxygen was enhanced significantly which allowed 46.1% of uranium to be leached from the horizon A for 18 months of soil remediation (Table 1). However, the residual concentration of uranium was still higher than the relevant permissible level. Amongst the soil microflora, the number of sulfur-oxidizing bacteria growing at neutral pH was increased significantly (Table 4). Their proliferation was determined by the alkaline soil pH which enhanced the chemical oxidation of sulfides and formation of polysulfides as a passivation film on their surface (Gonzalez-Sanchez and Revah, 2009). Chemolithotrophic sulfur-oxidizing bacteria (similar to Thiobacillus thioparus and Thiobacillus neapolitanus) oxidized that sulfur to sulfate ions, acidity in the pore waters was generated and the passivated film was removed. However, the rate of the process wasn't high; for example, 7.4% of sulfide sulfur was oxidized during the remediation period (Table 2). The lower leaching of sulfide sulfur could be explained by two processes. On one hand, ferrous iron complexes (FeOHCO03,

Fe(CO3)2−2) were formed during the chemical oxidation of pyritic sulfur at alkaline pH and their diffusion due to the alkaline soil pH was detained significantly (Descostes et al., 2002). On the other hand, those complexes were oxidized chemically by means of molecular oxygen to ferric iron state and a passivation film of iron hydroxides was formed as a final product. Iron hydroxides are stable at alkaline pH and aerobic conditions which restricted further leaching of the soil pollutants (Moses and Herman, 1991). The structure of the horizon A was worsened due to the conversion of insoluble complexes of humic acids into a soluble form as a result of displacement of iron and calcium with sodium ions. In spite of the presence of humic acids in the soil solutions, significant leaching of nonferrous metals wasn't detected (Table 3). The main reasons were the pH-dependable negative surface charge which was increased further due to enhanced deprotonation of functional groups of the soil constituents as well as the precipitation of some contaminants (lead and radium) as highly insoluble sulfates (Dong et al., 2000; Langmuir and Riese, 1985). Soil mulching as a remediation method for metal-contaminated soils was applied in Lysimeters 3 and 4. That treatment was connected with the addition of finely cut hay enriched in easily degradable biopolymers (cellulose and hemicellulose mainly) and mixed to the upper soil horizon. The main aim of mulching was to enrich the soil biotope with biomass and to stimulate the growth and activity of all heterotrophic groups of microorganisms that take part in the plant biomass biodegradation as well as to enhance pollutant leaching due to secretion of some microbial metabolites into the soil solutions. For example, it is well-known that approximately 38–43% of organic compounds released during hay degradation are hydrophilic acids which deprotonate easily its carboxylic group so that inner sphere complexes with heavy metals ions could be formed (Merrit and Erich, 2003; Zhou and Wong, 2003). The relative content of a hydrophilic base released from that process is significantly lower. Neutral pH retarded deprotonation of their hydroxyl group and formation of outer sphere complexes with heavy metal ions wasn't possible at such conditions. Soil mulching combined with regular plowing enhanced the maintenance of higher porosity and aerobic conditions in the soil horizon A. As

Table 3 Data about the content and properties of the soil solutions generated from soil horizon A at the relevant variants for soil remediation. Index

pH Eh, mV Alkalinity, mmol/l Reducible sugars, mg/l Dissolved organic carbon, mg/l Pb, mg/l Zn, mg/l Cu, mg/l U, mg/l Fe, mg/l Mn, mg/l Ca, mg/l Mg, mg/l

Before treatment

7.27 +70 0.9 – 4.2 0.22 0.07 0.13 0.15 0.2 0.6 365 58

Lysimeter 1

2

3

4

7.35–7.63 (+82)–(+116) 1.1–1.6 – 5.1–9.4 0.12–1.7 0.05–0.28 0.15–0.42 0.08–0.17 0.3–2.4 0.8–1.65 396–455 65–103

8.57–9.10 (+75)–(+103) 23–29 2–4 21.3–24.5 0.16–2.05 0.18–2.4 0.08–1.05 0.19–1.12 0.5–4.7 0.2–3.11 490–540 52–115

7.77–8.46 (+52)–(+88) 2.7–3.5 4.7–8.3 38.5–62.3 0.4–4.7 0.51–2.48 0.38–2.11 0.10–0.22 0.4–9.6 1.2–6.4 93–275 28.2–57

8.82–9.66 (+41)–(+72) 24–30.5 14.2–22.5 92.7–148.4 0.1–3.66 0.67–3.5 0.29–3.17 0.24–1.18 0.52–7.81 0.08–5.2 360–650 42–106

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

P. Georgiev et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx

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Table 4 Data about the microflora of soil horizon A (0–30 cm) before and during the soil remediation. Microorganisms

Before treatment

During soil treatment Lysimeter 1

S2O2− 3 -oxidizing

chemolithotrophs (at pH 7) Nitrifying bacteria 2+ Fe -oxidizing bacteria (at pH 7) Aerobic heterotrophic bacteria Fungi Anaerobic heterotrophic bacteria Cellulose-degrading bacteria Denitrifying bacteria Fe3+-reducing bacteria Sulfate-reducing bacteria

3

4

4

10 –10 101–102 104–105 103–104 103–104 103–105 101–102 103–104 103–104 102–104

Lysimeter 2

5

6

10 –10 102–103 104–105 104–105 104–105 103–105 102–103 103–105 103–105 103–104

a result, the number of aerobic chemolithotrophic bacteria as well as heterotrophic microorganisms increased significantly in comparison with the microflora of the non-treated soil and the soil treated in Lysimeter 1. All these groups had direct and indirect roles towards pollutant solubilization. For example, sulfur oxidizing bacteria, growing at neutral pH (Thiobacillus spp.) were the dominant group amongst chemolithotrophic bacteria (similarly as soil treated in Lysimeter 2) and they took part in bacterial oxidation and leaching of the oxidizable fraction of heavy metals. By this way, the total concentration of sulfide sulfur was decreased by 30.9% for the period of soil remediation, which was the best result amongst the tested lysimeters. Sulfuric acid which was formed was used in the acidolysis of carbonates in soil. The main difference in the remediation scheme in comparison with the previous lysimeters was the presence of dissolved organic carbon released into the soil solutions due to biomass degradation. Data (Table 3, 4) revealed the simultaneous existence of aerobic and anaerobic conditions in the topsoil. On one hand, these compounds were oxidized by aerobic heterotrophic bacteria in zones with higher concentration of molecular oxygen and the process was competing with chemolithotrophic bacteria towards molecular oxygen. On the other hand, the organic compounds were oxidized by anaerobic heterotrophic bacteria in zones with very low concentration of molecular oxygen, as the process of ferric iron hydroxide reduction was the dominant. H2O2 and Fe2+ were formed as a result of these processes, which at some soil depth could be oxidized uraninite (UO2) in a way similar to the Fenton reaction by means of generation of hydroxyl radicals (Barbusinsky, K., 2009; Amme et al., 2005).

7

Lysimeter 3 5

10 –10 102–103 104–105 103–104 103–104 103–105 102–104 102–103 103–104 102–104

6

Lysimeter 4 105–107 103–104 105–106 106–107 106–107 106–107 104–105 104–105 105–106 103–105

10 –10 104–105 103–105 106–107 107–108 106–107 105–107 104–105 106–107 104–105

Nitrifying bacteria was the next essential group amongst chemolithotrophic bacteria. Apart from additional production of hydrogen ions during bacterial oxidation of ammonia ions, the process enriched the soil solutions with nitrate ions which were suitable complexing agents for heavy metal cations as lead and radium, for example (Beneš et al., 1982; Doner, 1978). Amongst aerobe bacteria, species belonging to the genera Pseudomonas, Bacillus, and Arthrobacter dominated, which are typical inhabitants of soil ecosystems (Zul et al., 2007). The growth of soil fungi played a crucial role in cellulose and hemicellulose biodegradation by means of secretion of exo-enzymes. During that process a lot of low molecular organic compounds (such as organic and sugar acids) were released into the soil solutions. The positive role of these organic compounds in accordance to soil remediation was expressed in two ways. On one hand, it stimulated the growth and activity of all heterotrophic microorganisms. On the other hand, a lot of produced organic acids are characterized by noticeable complexing properties towards iron and heavy metal ions (Marchand and Silverstein, 2002). As a result, the concentration of dissolved pollutants in pregnant solutions increased significantly in comparison with their concentrations measured in soil solutions generated during the remediation applied in Lysimeters 1 and 2 (Table 3). At the end of soil remediation, the concentrations of copper and lead in the horizon A decreased by 10.0 and 54.8%, respectively. It meant that apart from the higher rates of acidolysis processes which took place during the soil remediation in comparison with the previous two

Table 5 Data about the main mobility fractions and bioavailability of lead, copper, and uranium in soil horizon A before and after the soil remediation. Index

Mobility fraction

Before treatment

After treatment Lysimeter 3

Pb

Exchangeable Carbonate Reducible Oxidizable Inert

Total content Bioavailability determined by DTPA Cu Exchangeable Carbonate Reducible Oxidizable Inert Total content Bioavailability determined by DTPA U Exchangeable Carbonate Reducible Oxidizable Inert Total content Bioavailability determined by DTPA

Lysimeter 4

mg/kg

%

mg/kg

%

mg/kg

%

1.3 19.7 34.3 132.6 84.1 272 28.7 1.0 65.6 16.0 312.0 254.4 649.0 22.7 1.7 13.8 7.9 4.7 6.4 34.5 17.8

0.5 7.2 12.6 48.8 30.9 100 10.5 0.2 10.1 2.5 48.0 39.2 100 3.5 4.9 40.0 22.9 13.6 18.6 100 51.6

0.6 11.8 48.1 42.6 20.5 123.6 20.3 10.6 34.1 9.3 286 244 584 13.2 1.9 11.9 10.2 2.7 5.9 32.6 13.5

0.5 9.5 38.9 34.5 16.6 100 16.4 1.8 5.9 1.6 49 41.7 100 2.3 5.8 36.5 31.2 8.3 18.1 100 40.5

2.5 14.3 59.5 47.7 25.7 149.7 22.5 6.4 34 13.6 259 240 551 14.5 0.2 0.6 4.0 1.9 4.9 11.6 1.3

1.7 9.6 39.7 31.9 17.1 100 15.0 1.1 6.1 2.5 46.8 43.5 100 2.6 1.7 5.2 34.5 16.4 42.2 100 11.2

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

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lysimeters, the sufficient leaching of non-ferrous metals from the soil horizon A within 18 months wasn't measured due to the still lower rate of complexolysis of the released soil contaminants. For example, the main phases for non-ferrous metal leaching were oxidizable, carbonate, as well as reducible fractions of copper. However, a surface with additional net negative charge was formed in the soil due to the partial biodegradation of the hay's biopolymers. That surface charge was compensated by means of attraction and sorption of cations, including those of non-ferrous metals. As a result, the exchangeable fraction of both pollutants was even increased in comparison to the values of the non-treated soil. The bioavailability of copper and lead was decreased by 29.3 and 41.8%, respectively, due to their better extraction from the carbonate fraction. Hydrogen peroxide and carbon dioxide were formed as final products of the aerobic oxidation of organic compounds in the horizon A. Hydrogen peroxide is a strong oxidant which was consumed for the oxidation of chemical elements presented in their reducing state, including tetra-valent uranium. By this way, the content of tetravalent uranium decreased by almost 50% at the end of soil remediation. In spite of the higher alkalinity of the soil solutions in Lysimeter 3 due to partial dissolution of CO2, the leaching of hexa-valent uranium was not sufficient. The specific activity of radium-226 was decreased by 37.5% and the residual value was approximately 250 Bq/kg soil. The results of the soil which has been treated in Lysimeter 3 were quite promising regarding the leaching of lead and copper, especially. On one hand, their residual concentrations as well as the concentration of uranium and the specific activity of Ra-226 were still higher than the relevant Maximum Admissible Concentrations. On the other hand, the bioavailability of heavy metals decreased significantly which would allow the cultivation and growing of suitable industrial crops at these soil conditions. The best leaching of soil contaminants from the horizon A within 18 months was measured in Lysimeter 4 where soil mulching was combined with soil irrigation with solutions containing nutrients and bicarbonate ions. For example, the concentrations of copper and lead decreased by 98 mg/kg and 123 mg/kg which were relevant to 15.1 and 45.2% of leaching, respectively (Table 1). The main donors of non-ferrous metal leaching were carbonate, reducible and oxidizable fractions. In accordance to exchangeable fraction, the higher content of organic anions in soil solutions allowed the sorption of non-ferrous metals to net negative charge soil surface to be depressed to a great extent. The main reasons for the higher leaching of the soil pollutants in comparison with other three lysimeters were suitable combinations between chemical and biological processes, which took part in the mobile fraction leaching, with efficient complexolysis of the released heavy metals ions. The soil remediation applied in Lysimeter 4 was connected with significant changes of the acid–base properties of the treated soil (Table 2). As a result, pH of soil changed to strongly alkaline which enhanced chemical transformations of biopolymers contained in the hay. For example, cellulose and hemicellulose were hydrolyzed chemically and shorter chains of oligomers were produced due to the peeling off reactions of the biopolymers (van Loon and Glaus, 1997) and the soil solutions were enriched with low molecular organic compounds as sugar acids. Those compounds formed chelate as well as bidentate complexes with heavy metal ions due to the deprotonation of both carboxylic and hydroxyl groups (Fisher and Bipp, 2002). The concentration of uranium in the soil horizon A decreased by 66.4% and it was determined by the greater variety of suitable oxidizing agents for tetra-valent uranium oxidation (as molecular oxygen, humic acids (Gu et al., 2005) and hydrogen peroxide (Rawlings and Silver, 1995)) which were presented in higher concentrations in comparison with conditions in other three lysimeters. The higher alkalinity of soil solutions enhanced considerably the faster leaching of hexa-valent uranium from its all mobile fractions.

The specific activity of radium-226 in soil was decreased by 37.5% at the end of the soil remediation. It demonstrated that radium leaching, as in the case of the way of remediation applied in Lysimeter 3, could be enhanced greatly if a higher concentration of organic compounds was presented in the soil solutions. Those anions suppressed the pollutant precipitation as RaSO4 probably as well as its sorption on the surface of iron hydroxides (Langmuir and Riese, 1985). The best leaching of copper and uranium was measured in the soil which has been treated at conditions of Lysimeter 4. The main disadvantages of that remediation were strongly alkaline soil pH which was measured at the end of the soil treatment as well as the higher content of water dissolved solids. That made such kind of soil quite similar to the solonchak soil type (a type of salt-affected soil). For that reason, the most suitable method to change these characteristics and to make the soil suitable for plant cultivation would be the addition of elemental sulfur and biodegradable biomass as a next stage of the soil reclamation. Due to the oxidation of S° to sulfuric acid by means of some sulfuroxidizing halophilic bacteria, soil pH would be changed to slightly acidic or neutral point. As a result of that, some amount of the soil contaminants will be leached additionally, mainly from their bioavailable fraction. Exactly the residual content of that fraction with respect to each pollutant will determine if that soil is suitable for agriculture cultivation or industrial cultures. The highest removal of radioactive elements and heavy metals from the soil horizon A as a result of the soil remediation was achieved in Lysimeter 3 and Lysimeter 4. The processes, which had been used, however had a significant effect not only on the pollutants' bioavailability but also on some basic soil characteristics. For that reason, it was important to assess their mutual effect on the survival of some typical soil inhabitants. Toxicity of the non-treated contaminated soil towards soil biota was determined by some specific characteristic of the soil which had been changed due to its contamination. First of all, as a result of deposition of particles enriched with carbonate and characterized by slightly alkaline pH, a misbalance of nutrients and macroelements to plants was established in the studied soil plot due to their lower bioavailability. That imbalance was determined by the higher content of exchangeable calcium in soil which decreased the bioavailability of orthophosphate ions due to their precipitation as calcium phosphate. At the next place, iron was less bioavailable due to the rapid chemical oxidation of ferrous ions and further precipitation of ferric iron as (oxy)hydroxide minerals. It is well-known that iron is a vital element needed in plant metabolism and its lower content is compensated with higher production of organic acid by plants, release of hydrogen ions by roots, acidolysis of irons oxide minerals and uptake of the leached iron ions. However, some ions of hydrogen were consumed in the acidolysis of the pollutant minerals. As a result, a zone with higher concentrations of heavy metal ions was created around plant roots which enhanced their uptake and accumulation into the plant biomass. So, the toxicity of the nontreated contaminated soil towards plants was determined by the accumulation of organic anions as well as heavy metal ions into their biomass. Visible signs of that imbalance were the lower growth rate of the plant species as well as chlorosis of the plant leaves, especially of oats. Clover (T. repens) and oats (A. sativa) were the most sensitive species amongst the tested plants and on their reaction the toxicity of the nontreated soil was 1.2 and 1.7 toxicity units, respectively. The acute toxicity of the soil horizon A towards the plants was not so high regardless of the fact that the contents of the non-ferrous metals as well as radioactive elements were several times higher than the relevant permissible levels. The pollutants' bioavailability in the horizon A was decreased by almost 50% due to the soil remediation applied in Lysimeter 3. As a result, the soil acute toxicity towards clover and oats was 16.7 and 23.5% lower in comparison with the toxicity of the non-treated soil (Table 6). Despite the better leaching of uranium and non-ferrous metals, the acute toxicity of the soil treated in Lysimeter 4 towards oats and clover

Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024

P. Georgiev et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx Table 6 Data about the toxicitya of soil horizon A to typical soil inhabitants before and after the soil remediation. Test organism

Before treatment

Oats (Avena sativa) NOEC 35 LOEC 40 LC50 55 Toxicity unit 1.8 Toxicity reduction, % – Clover (Trifolium repens) NOEC 5 LOEC 10.6 LC50 60 Toxicity unit 1.7 Toxicity reduction, % – Earthworms (Lumbricus terrestris) NOEC 25 LOEC 30 LC50 55 Toxicity unit 1.8 Toxicity reduction, % –

7

residual toxicity towards plants and earthworms will be reduced in a large extent. Conclusions

After treatment Lysimeter 3

Lysimeter 4

45 50 75 1.3 27.8

40 45 60 1.7 5.5

25 29.3 78 1.3 23.5

20 18.1 56.1 1.8 –

60 65 90 1.1 38.9

45 50 70 1.4 22.2

a Expressed as the sample's weight at which the relevant characteristic was determined.

was even higher in comparison to the toxicity of the non-treated soil. The main reason was the alkaline soil pH which determined the ion imbalance in the plants' nutrition to become stronger. It expressed lower bioavailability not only of iron (as was in non-treated soil) but also of zinc. It is well-known that at such conditions organic acids are extraaccumulated into the plant biomass, production of chlorophyll is insufficient and pH balance of the plant tissues is disturbed (McBride, 2001; Yang et al., 1994). The visible sign of these processes was deep chlorosis which was observed on the oat leaves. Earthworms play a crucial role in soil forming processes by means of uptake and processing of organic compounds in the soil ecosystem. The toxicity of the non-treated soil towards earthworm (L. terrestris) was 1.8 toxicity units. It was determined by the higher bioavailability of copper and uranium in the soil horizon A and their partially leaching from flotation particles during the digestion process. For example, Dai et al. (2004) and Lukkari et al. (2006) have established significant leaching of copper and zinc from their exchangeable, carbonate and reducible mobile fractions during tests with Aporrectodea caliginosa and Lumbricus rubellus. The toxicity of the soil in the horizon A treated in Lysimeter 3 towards earthworms was decreased to 1.1 toxicity units which was 35.3% lower than the toxicity of the non-treated soil. The residual toxicity of the soil was determined by means of secondary dissolution and accumulation of the heavy metals when soil particles passed through the digestive system of the earthworms. For example, the carbonate fraction of lead in the treated soil was increased which determined its leaching and accumulated into the worms' biomass. In comparison to copper and zinc which are released from worm biomass by means of secretion processes, lead is accumulated firmly to biomass which determined the higher toxicity of that non-ferrous metal (Spurgeon and Hopkin, 1999). The toxicity of the soil in the horizon A treated in Lysimeter 4 towards earthworms was 22.2% lower than the toxicity of the nontreated soil. However, alkaline soil pH had a strong negative effect on the earthworms' activity and survival and when the percent of the cleaned soil during the test was higher than 50%, the earthworms decreased their activity and became in inactive form (diapause) as a protective mechanism. The ecotoxicity tests revealed that by means of additional correction of soil pH to neutral point after the remediation process, the conditions in soil biotope will be improved significantly and by this way the

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Please cite this article as: Georgiev, P., et al., Ecotoxicological characteristic of a soil polluted by radioactive elements and heavy metals before and after its bioremediation, J. Geochem. Explor. (2014), http://dx.doi.org/10.1016/j.gexplo.2014.02.024