Environ Sci Pollut Res (2016) 23:5644–5653 DOI 10.1007/s11356-015-5788-5
RESEARCH ARTICLE
Effects of long-term radionuclide and heavy metal contamination on the activity of microbial communities, inhabiting uranium mining impacted soils Silvena Boteva 1 & Galina Radeva 2 & Ivan Traykov 1 & Anelia Kenarova 1
Received: 21 July 2015 / Accepted: 9 November 2015 / Published online: 18 November 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Ore mining and processing have greatly altered ecosystems, often limiting their capacity to provide ecosystem services critical to our survival. The soil environments of two abandoned uranium mines were chosen to analyze the effects of long-term uranium and heavy metal contamination on soil microbial communities using dehydrogenase and phosphatase activities as indicators of metal stress. The levels of soil contamination were low, ranging from ‘precaution’ to ‘moderate’, calculated as Nemerow index. Multivariate analyses of enzyme activities revealed the following: (i) spatial pattern of microbial endpoints where the more contaminated soils had higher dehydrogenase and phosphatase activities, (ii) biological grouping of soils depended on both the level of soil contamination and management practice, (iii) significant correlations between both dehydrogenase and alkaline phosphatase activities and soil organic matter and metals (Cd, Co, Cr, and Zn, but not U), and (iv) multiple relationships between the alkaline than the acid phosphatase and the environmental factors. The results showed an evidence of microbial tolerance and adaptation to the soil contamination established during the long-term metal exposure and the key role of soil organic matter in maintaining high microbial enzyme activities and mitigating the metal toxicity. Additionally, the results suggested that the soil microbial communities are able to reduce the metal stress by intensive phosphatase synthesis, benefiting Responsible editor: Robert Duran * Anelia Kenarova [email protected] 1
Faculty of Biology, Sofia University BSt. Kl. Ohridski^, 8 Dragan Tsankov Blvd, Sofia 1164, Bulgaria
2
Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, Sofia 1113, Bulgaria
a passive environmental remediation and provision of vital ecosystem services. Keywords Soil contamination . Uranium and heavy metals . Dehydrogenase . Phosphatase . Soil organic matter
Introduction The concept of ecosystem services has become an important model for linking the functioning of ecosystems to human welfare benefits (Fisher et al. 2007). In the humandominated biosphere, human activities have altered the ecosystems (Vitousek and Harold 1997), so that in many cases, their capacity to provide necessary services has been either overexploited or eroded (Palmer et al. 2004; Kremen and Ostfeld 2005). Ore mining and processing are one of the widespread human activities deteriorating the terrestrial ecosystems, causing physical disturbance and chemical/radiological contamination of soil and water. Soil is a critical ecosystem component functioning in primary productivity and maintaining the local, regional, and global environmental quality. The soil functioning is driven by the activity of soil biota, particularly microorganisms taking part in metabolic processes like respiration and productivity, nutrient cycling and fluxes, trophic links with secondary consumers, and numerous biogeochemical processes. The selective power of contaminants modifies the composition and activity of soil microbial communities, altering their capacity to support the production of ecosystem services. Since enzymes catalyze all biochemical transformations, including detoxification of contaminants, the measurement of soil enzyme activities is a useful indicator of biological activity and how it is influenced by the contaminants and natural environmental fluctuations. In nature, the most important are enzymes which
Environ Sci Pollut Res (2016) 23:5644–5653
belong to oxidoreductases (dehydrogenases and catalase) and hydrolases (acid and alkaline phosphatases, urease, arylsulphatase, and β-glucosidase), taking part in the degradation of plant residues and transformation of nitrogen, phosphorus, and sulfur compounds (Gu et al. 2009; Wyszkowska et al. 2013). In many studies, the researchers have tied the health of soil microbial communities to the activities of dehydrogenases (Kenarova and Radeva 2010; Antunes et al. 2011; Pan and Yu 2011) and phosphatases (Wang et al. 2007; Khan et al. 2010) as sensitive indicators of metal stress, highlighting the dependence of heavy metal contamination effects on local geographic and climatic factors, soil characteristics (Tan et al. 2008; Yuan and Yue 2012; Chodak et al. 2013), and management practices (Madejón et al. 2001). This research was focused to determine if (1) long-term, (2) low level, and (3) multi-radionuclide and heavy metal contamination of soil can provoke different bacterial responses depending on the site specific level of contamination and soil management practices. We tested this assumption using soil dehydrogenase and phosphatase (acid and alkaline) activities as indicators of soil microbial health, comparing different locations of two abandoned uranium mines from Bulgaria. Some of our results confirmed the microbial adaptive responses to both site specific and low level of long-term multi-U and heavy metal contamination (Diaz-Raviña et al. 1994; Caetano et al. 2014), whereas other results from the study for the first time provided evidence of different relationships between the soil acid and alkaline phosphatases and the environmental factors including the contaminants. This study established the basis for future development of advanced remediation strategy to provide high ecosystem services even in metal and radionuclide impacted ecosystems.
Material and methods
5645 Fig. 1 Map of Bulgaria with the locations of a studied sites and sampling points of b Eleshnitsa (EU) and c Senokos (SU) mines
Sampling strategy Total U and HM concentrations were assessed at different distances from the Senokos mine (SU) and the entrances to the Eleshnitsa mine (EU), and the highest metal contents were recorded within 1 km of the sources of contamination (preliminary study). According to the preliminary results, the study sites were selected in the most impacted area of Senokos (SU 1, SU 2, SU 5) and Eleshnitsa (EU 1, EU 2, EU 3) mines and on further located agriculture lands (SU 24 and EU 5), where the secondary pollution was caused by contaminated irrigation water (Stoyanova et al. 2010, 2011) and manure (unpublished data: U—4.12±7.2, Sr—85.4±34, Zn—1807±551, Cu—168 ±46, Pb—30.6±25, Cr—83.6±78, in mg/kg) for land treatment. The sampling sites, except SU 24 and EU 5, were secondary grass lands, dominated by Poaceae, Caryophyllaceae, Fabaceae, Brassicaceae, and used for livestock grazing. At each study site, three sampling plots (3×3 m) were selected, and three subsamples from each plot were randomly taken (500 g for chemical and 50 g for microbiological analyses) and mixed in a representative sample for further analysis. In the study, reference soil was not used due to our (Radeva et al. 2013, Kenarova et al. 2014) and Giller et al. (1998) experience connected to the difficulties in finding a Breference^ soil which differed only in the term of contamination from the studied ones. This challenge was overcome using multivariate analysis (see Statistical analysis) and interpreting the results on cause–effect relationships. The survey was conducted in May and October 2012, and May 2013, and a total of 72 soil samples were collected for analyses.
Study areas Sample collection and pretreatment Abandoned uranium mines Senokos (41°49′53.0″ N; 23°13′ 11.8″ E) and Eleshnitsa (41°51′18.0′′ N; 23°38′13.7′′ E), located in the southwestern part of Bulgaria, were selected for the study of the impact of U and heavy metal (HM) contamination on soil bacterial communities (Fig. 1). Senokos was an open-cast mine, whereas the mining operations in Eleshnitsa had been conducted in a conventional underground manner. The uranium production in Bulgaria was ceased by Government decree, and since 1992, the U mines and tailings were technically liquidated and rehabilitated. Nevertheless, during the mine operations and later as a result of compromised mines’ rehabilitation, large amounts of mine wastes have been dispersed on mine surroundings by both surface erosion and wind action (Senokos mine) and by water effluents draining the mine galleries (Eleshnitsa mine).
Samples for microbiological analysis were collected from the soil surface layer (5–10 cm in depth) by soil probe under sterile conditions, transported in cold box, sieved (2 mm), stored at 4 °C, and analyzed within 3 days after sampling. Samples for chemical analyses were collected and transported in plastic bags, dried, sieved (2 mm), and stored at room temperature. Physico-chemical analysis The pHH2O was measured potentiometrically (HANNA pHmeter) in distilled water solution (1:5; soil:water). The gravimetric water content was determined by drying soils at 105 °C for 24 h. The soils were then combusted at 550 °C for a
5646
Environ Sci Pollut Res (2016) 23:5644–5653
Environ Sci Pollut Res (2016) 23:5644–5653 Table 1 Physical and chemical characteristics of Senokos (SU) and Eleshnitsa (EU) uranium mining impacted soils. The data are presented as mean and (standard deviation) (n=3)
Parameter
Temp.
5647
D
°C
Sampling plots SU 1
SU 2
SU 5
SU 24
EU 1
EU 2
EU 3
EU 5
13.8
12.3
14.7
15.7
12.7
14.3
13.7
12.0
(2.9)
(4.4)
(0.6)
(6.6)
(1.2)
(0.6)
(2.3)
(2.0)
10.7
19.80
22.1
13.3
19.4
16.7
33.4
Moisture
%
26.4 (5.4)
(0.9)
(8.2)
(6.0)
(5.6)
(7.0)
(7.1)
(5.0)
pH
-
6.6
6.6
6.8
5.8
5.8
6.1
5.9
6.1
(0.1)
(0.1)
(0.4)
(0.1)
(0.0)
(0.1)
(0.0)
(0.1)
OM
%
12.4
4.0
10.2
5.8
2.0
3.7
3.4
6.8
NO3-N
mg/kg
(3.6) 12.7
(1.7) 0.3
(2.5) 16.4
(1.1) 11.7
(0.5) 4.8
(0.8) 17.1
(0.5) 8.8
(1.0) 12.5
NH4-N
mg/kg
(13.7) 7.7
(0.4) 6.5
(23.7) 13.6
(13.3) 7.4
(6.6) 5.0
(7.2) 3.0
(7.9) 2.5
(10.9) 5.5
PO4-P
mg/kg
(6.4) 16.7
(3.4) 12.7
(8.1) 23.2
(3.0) 3.9
(6.2) 7.5
(4.2) 4.9
(4.3) 4.7
(2.0) 4.0
(7.9)
(6.0)
(12.1)
(1.9)
(7.1)
(3.6)
(3.7)
(1.0)
Ptot
mg/kg
22 287
1 215
20 591
1 383
1 209
1 376
1 259
1 393
As
mg/kg
(2 229) 3.5
(422)
RESEARCH ARTICLE
Effects of long-term radionuclide and heavy metal contamination on the activity of microbial communities, inhabiting uranium mining impacted soils Silvena Boteva 1 & Galina Radeva 2 & Ivan Traykov 1 & Anelia Kenarova 1
Received: 21 July 2015 / Accepted: 9 November 2015 / Published online: 18 November 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Ore mining and processing have greatly altered ecosystems, often limiting their capacity to provide ecosystem services critical to our survival. The soil environments of two abandoned uranium mines were chosen to analyze the effects of long-term uranium and heavy metal contamination on soil microbial communities using dehydrogenase and phosphatase activities as indicators of metal stress. The levels of soil contamination were low, ranging from ‘precaution’ to ‘moderate’, calculated as Nemerow index. Multivariate analyses of enzyme activities revealed the following: (i) spatial pattern of microbial endpoints where the more contaminated soils had higher dehydrogenase and phosphatase activities, (ii) biological grouping of soils depended on both the level of soil contamination and management practice, (iii) significant correlations between both dehydrogenase and alkaline phosphatase activities and soil organic matter and metals (Cd, Co, Cr, and Zn, but not U), and (iv) multiple relationships between the alkaline than the acid phosphatase and the environmental factors. The results showed an evidence of microbial tolerance and adaptation to the soil contamination established during the long-term metal exposure and the key role of soil organic matter in maintaining high microbial enzyme activities and mitigating the metal toxicity. Additionally, the results suggested that the soil microbial communities are able to reduce the metal stress by intensive phosphatase synthesis, benefiting Responsible editor: Robert Duran * Anelia Kenarova [email protected] 1
Faculty of Biology, Sofia University BSt. Kl. Ohridski^, 8 Dragan Tsankov Blvd, Sofia 1164, Bulgaria
2
Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 21, Sofia 1113, Bulgaria
a passive environmental remediation and provision of vital ecosystem services. Keywords Soil contamination . Uranium and heavy metals . Dehydrogenase . Phosphatase . Soil organic matter
Introduction The concept of ecosystem services has become an important model for linking the functioning of ecosystems to human welfare benefits (Fisher et al. 2007). In the humandominated biosphere, human activities have altered the ecosystems (Vitousek and Harold 1997), so that in many cases, their capacity to provide necessary services has been either overexploited or eroded (Palmer et al. 2004; Kremen and Ostfeld 2005). Ore mining and processing are one of the widespread human activities deteriorating the terrestrial ecosystems, causing physical disturbance and chemical/radiological contamination of soil and water. Soil is a critical ecosystem component functioning in primary productivity and maintaining the local, regional, and global environmental quality. The soil functioning is driven by the activity of soil biota, particularly microorganisms taking part in metabolic processes like respiration and productivity, nutrient cycling and fluxes, trophic links with secondary consumers, and numerous biogeochemical processes. The selective power of contaminants modifies the composition and activity of soil microbial communities, altering their capacity to support the production of ecosystem services. Since enzymes catalyze all biochemical transformations, including detoxification of contaminants, the measurement of soil enzyme activities is a useful indicator of biological activity and how it is influenced by the contaminants and natural environmental fluctuations. In nature, the most important are enzymes which
Environ Sci Pollut Res (2016) 23:5644–5653
belong to oxidoreductases (dehydrogenases and catalase) and hydrolases (acid and alkaline phosphatases, urease, arylsulphatase, and β-glucosidase), taking part in the degradation of plant residues and transformation of nitrogen, phosphorus, and sulfur compounds (Gu et al. 2009; Wyszkowska et al. 2013). In many studies, the researchers have tied the health of soil microbial communities to the activities of dehydrogenases (Kenarova and Radeva 2010; Antunes et al. 2011; Pan and Yu 2011) and phosphatases (Wang et al. 2007; Khan et al. 2010) as sensitive indicators of metal stress, highlighting the dependence of heavy metal contamination effects on local geographic and climatic factors, soil characteristics (Tan et al. 2008; Yuan and Yue 2012; Chodak et al. 2013), and management practices (Madejón et al. 2001). This research was focused to determine if (1) long-term, (2) low level, and (3) multi-radionuclide and heavy metal contamination of soil can provoke different bacterial responses depending on the site specific level of contamination and soil management practices. We tested this assumption using soil dehydrogenase and phosphatase (acid and alkaline) activities as indicators of soil microbial health, comparing different locations of two abandoned uranium mines from Bulgaria. Some of our results confirmed the microbial adaptive responses to both site specific and low level of long-term multi-U and heavy metal contamination (Diaz-Raviña et al. 1994; Caetano et al. 2014), whereas other results from the study for the first time provided evidence of different relationships between the soil acid and alkaline phosphatases and the environmental factors including the contaminants. This study established the basis for future development of advanced remediation strategy to provide high ecosystem services even in metal and radionuclide impacted ecosystems.
Material and methods
5645 Fig. 1 Map of Bulgaria with the locations of a studied sites and sampling points of b Eleshnitsa (EU) and c Senokos (SU) mines
Sampling strategy Total U and HM concentrations were assessed at different distances from the Senokos mine (SU) and the entrances to the Eleshnitsa mine (EU), and the highest metal contents were recorded within 1 km of the sources of contamination (preliminary study). According to the preliminary results, the study sites were selected in the most impacted area of Senokos (SU 1, SU 2, SU 5) and Eleshnitsa (EU 1, EU 2, EU 3) mines and on further located agriculture lands (SU 24 and EU 5), where the secondary pollution was caused by contaminated irrigation water (Stoyanova et al. 2010, 2011) and manure (unpublished data: U—4.12±7.2, Sr—85.4±34, Zn—1807±551, Cu—168 ±46, Pb—30.6±25, Cr—83.6±78, in mg/kg) for land treatment. The sampling sites, except SU 24 and EU 5, were secondary grass lands, dominated by Poaceae, Caryophyllaceae, Fabaceae, Brassicaceae, and used for livestock grazing. At each study site, three sampling plots (3×3 m) were selected, and three subsamples from each plot were randomly taken (500 g for chemical and 50 g for microbiological analyses) and mixed in a representative sample for further analysis. In the study, reference soil was not used due to our (Radeva et al. 2013, Kenarova et al. 2014) and Giller et al. (1998) experience connected to the difficulties in finding a Breference^ soil which differed only in the term of contamination from the studied ones. This challenge was overcome using multivariate analysis (see Statistical analysis) and interpreting the results on cause–effect relationships. The survey was conducted in May and October 2012, and May 2013, and a total of 72 soil samples were collected for analyses.
Study areas Sample collection and pretreatment Abandoned uranium mines Senokos (41°49′53.0″ N; 23°13′ 11.8″ E) and Eleshnitsa (41°51′18.0′′ N; 23°38′13.7′′ E), located in the southwestern part of Bulgaria, were selected for the study of the impact of U and heavy metal (HM) contamination on soil bacterial communities (Fig. 1). Senokos was an open-cast mine, whereas the mining operations in Eleshnitsa had been conducted in a conventional underground manner. The uranium production in Bulgaria was ceased by Government decree, and since 1992, the U mines and tailings were technically liquidated and rehabilitated. Nevertheless, during the mine operations and later as a result of compromised mines’ rehabilitation, large amounts of mine wastes have been dispersed on mine surroundings by both surface erosion and wind action (Senokos mine) and by water effluents draining the mine galleries (Eleshnitsa mine).
Samples for microbiological analysis were collected from the soil surface layer (5–10 cm in depth) by soil probe under sterile conditions, transported in cold box, sieved (2 mm), stored at 4 °C, and analyzed within 3 days after sampling. Samples for chemical analyses were collected and transported in plastic bags, dried, sieved (2 mm), and stored at room temperature. Physico-chemical analysis The pHH2O was measured potentiometrically (HANNA pHmeter) in distilled water solution (1:5; soil:water). The gravimetric water content was determined by drying soils at 105 °C for 24 h. The soils were then combusted at 550 °C for a
5646
Environ Sci Pollut Res (2016) 23:5644–5653
Environ Sci Pollut Res (2016) 23:5644–5653 Table 1 Physical and chemical characteristics of Senokos (SU) and Eleshnitsa (EU) uranium mining impacted soils. The data are presented as mean and (standard deviation) (n=3)
Parameter
Temp.
5647
D
°C
Sampling plots SU 1
SU 2
SU 5
SU 24
EU 1
EU 2
EU 3
EU 5
13.8
12.3
14.7
15.7
12.7
14.3
13.7
12.0
(2.9)
(4.4)
(0.6)
(6.6)
(1.2)
(0.6)
(2.3)
(2.0)
10.7
19.80
22.1
13.3
19.4
16.7
33.4
Moisture
%
26.4 (5.4)
(0.9)
(8.2)
(6.0)
(5.6)
(7.0)
(7.1)
(5.0)
pH
-
6.6
6.6
6.8
5.8
5.8
6.1
5.9
6.1
(0.1)
(0.1)
(0.4)
(0.1)
(0.0)
(0.1)
(0.0)
(0.1)
OM
%
12.4
4.0
10.2
5.8
2.0
3.7
3.4
6.8
NO3-N
mg/kg
(3.6) 12.7
(1.7) 0.3
(2.5) 16.4
(1.1) 11.7
(0.5) 4.8
(0.8) 17.1
(0.5) 8.8
(1.0) 12.5
NH4-N
mg/kg
(13.7) 7.7
(0.4) 6.5
(23.7) 13.6
(13.3) 7.4
(6.6) 5.0
(7.2) 3.0
(7.9) 2.5
(10.9) 5.5
PO4-P
mg/kg
(6.4) 16.7
(3.4) 12.7
(8.1) 23.2
(3.0) 3.9
(6.2) 7.5
(4.2) 4.9
(4.3) 4.7
(2.0) 4.0
(7.9)
(6.0)
(12.1)
(1.9)
(7.1)
(3.6)
(3.7)
(1.0)
Ptot
mg/kg
22 287
1 215
20 591
1 383
1 209
1 376
1 259
1 393
As
mg/kg
(2 229) 3.5
(422)
