Responses of soil ammonia oxidizers to a short-term severe mercury stress

JES-00450; No of Pages 6 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 5 ) XX X–XXX

Available online at www.sciencedirect.com

ScienceDirect www.journals.elsevier.com/journal-of-environmental-sciences

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Zhi-Feng Zhou1 , Yu-Rong Liu2 , Guo-Xin Sun2 , Yuan-Ming Zheng2,⁎

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1. College of Resources and Environment, Southwest University, Chongqing 400716, China. E-mail: [email protected] 2. State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

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Responses of soil ammonia oxidizers to a short-term severe mercury stress

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ABSTR ACT

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Article history:

The responses of soil ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria 16

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Received 22 January 2015

(AOB) to mercury (Hg) stress were investigated through a short-term incubation 17

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Revised 30 March 2015

experiment. Treated with four different concentrations of Hg (CK, Hg25, Hg50, and Hg100, 18

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Accepted 2 April 2015

denoting 0, 25, 50, and 100 mg Hg/kg dry soil, respectively), samples were harvested after 3, 19

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Available online xxxx

7, and 28 day incubation. Results showed that the soil potential nitrification rate (PNR) was 20

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Keywords:

AOA (the highest in CK: 9.20 × 107 copies/g dry soil; the lowest in Hg50: 2.68 × 107 copies/g 22

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Mercury (Hg)

dry soil) and AOB (the highest in CK: 2.68 × 107 copies/g dry soil; the lowest in Hg50: 23

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Potential nitrification rate (PNR)

7.49 × 106 copies/g dry soil) were observed only at day 28 of incubation (P < 0.05). Moreover, 24

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Amonia oxidizers

only the community structure of soil AOB obviously shifted under Hg stress as seen through 25

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Abundance

DGGE profiles, which revealed that 2–3 distinct AOB bands emerged in the Hg treatments at 26 Q2

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Community structure

day 28. In summary, soil PNR might be a very useful parameter to assess acute Hg stress on 27

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significantly inhibited by Hg stress during the incubation. However, lower abundances of 21

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soil ecosystems, and the community structure of soil AOB might be a realistic biological 28 indicator for the assessment of heavy metal stress on soil ecosystems in the future.

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© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 30

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Introduction

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The ammonia oxidation process, as the first and key step in nitrification, plays a very important role in the global nitrogen (N) cycle (De-Boer et al., 1991). This process in soil is driven by both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) (Leininger, 2006; Adair and Schwartz, 2008; Shen et al., 2008). The abundance and community structure of soil ammonia oxidizers could be affected by many soil properties and environmental stresses (Nicol et al., 2008; He et al., 2007; Li et al., 2009; Mertens et al., 2009). As the crucial functional microbes involved in the soil N-cycle, AOA and AOB also are generating increasing interest

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Published by Elsevier B.V.

concerning their suitability as indicators to assess the environmental stresses on soil ecosystems (Ritz et al., 2009; Wessen and Hallin, 2011), especially with the assistance of molecular techniques (Liu et al., 2010; Ruyters et al., 2010; Subrahmanyam et al., 2014). Heavy metal contamination in soil has been a public concern for a long time due to their toxicity and persistency. Since the traditional risk assessment for soil heavy metals based on the total quantities was insufficient because it lacked measurement of bioavailability, soil microorganisms have been considered as important indicators for bioavailability measurement (Harms et al., 2006; Hinojosa et al., 2005; Stefanowicz et al., 2008). Previously, the study of the influence

⁎ Corresponding author. E-mail: [email protected] (Yuan-Ming Zheng).

http://dx.doi.org/10.1016/j.jes.2015.04.022 1001-0742/© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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1. Materials and methods

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1.1. Microcosms and experimental design

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The soil sample for the experiment was collected from the topsoil (0–20 cm) of a wheat–maize rotation field in Shunyi District of Beijing (China) in July 2010. The soil, classified as Cinnamon soil (Semi-Luvisols), was air-dried and ground to pass through a 2 mm sieve for the incubation experiment. Some basic properties were pH, 7.8; organic matter, 22.8 g/kg; NO−3, 21.3 mg/kg; NH+4, 11.3 mg/kg; and Hg, 0.02 mg/kg. The effect of Hg on soil ammonia oxidizers was studied using four different Hg contents (0, 25, 50, and 100 mg Hg/kg dry soil, represented as CK, Hg25, Hg50, and Hg100, respectively) with three replicates. The soil samples were spiked with Hg by application of HgCl2 solutions to dry soil. Each soil sample (60 g) was placed in a tube (8 cm diameter, 15 cm

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1.2. Measurement of soil potential nitrification rate (PNR)

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The chlorate inhibition method (Kurola et al., 2005) was used to measure soil PNR. Briefly, 5.0 g of fresh soil, 20 mL of phosphate buffer solution (PBS) (g/L: NaCl, 8.0; KCl, 0.2; Na2HPO4, 0.2; NaH2PO4, 0.2) with pH 7.4, and 1 mmol/L (NH4)2SO4 were added into a 50 mL centrifuge tube successively, and then Potassium chlorate (10 mol/L) was added to inhibit nitrite oxidation. The suspension was incubated in a dark incubator at 25°C for 24 hr, after which the nitrite was extracted with 5 mL of 2 mol/L KCl and determined spectrophotometrically at 540 nm with N-(1-naphthy1) ethylenediamine dihydrochloride.

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1.3. Soil DNA extraction and real-time PCR assay

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height) in triplicate for each Hg concentration and each harvest time. In each tube, the water content in soil was adjusted to 35% of the maximum water holding capacity and maintained throughout the experiment. Each tube was weighed weekly and the loss of weight was supplemented with sterile distilled water if needed. Finally, each tube was loosely covered with a plastic cap allowing air-exchange and incubated in a dark incubator at 25°C for 28 days. Soil samples were collected from the tubes at 3, 7 and 28 days for PNR, real-time PCR and DGGE analysis.

Soil DNA was extracted from 0.5 g fresh soil using MoBio UltraClean™ soil DNA isolation kits (San Diego, CA, USA) according to the manufacturer's protocol with previously described modifications (He et al., 2007). Real-time PCR was performed on an iCycler iQ5 thermocycler (Bio-Rad, USA). Amplification was performed in 25 μL reaction mixtures by using SYBR® Premix Ex Taq™ as described by the suppliers (Takara Bio, Otsu, Shiga, Japan). The DNA extracts were diluted 5-fold and used as template, with a final content of 1–10 ng in each reaction mixture. The primer sets (AOA: Arch-amoAF/Arch-amoAR; AOB: amoA-1F/amoA-2R) and thermal profiles used to amplify each target gene with real-time PCR are listed in Table 1. Following the thermal profile, determined by measuring the fluorescence continuously as the temperature increased from 55 to 95°C, a melting curve analysis was performed to confirm the specificity of the PCR product for each real-time PCR amplification. Data analysis was carried out with iCycler software (version 1.0.1384.0 CR). The parameter Ct (threshold cycle) was determined as the cycle number at which a statistically significant increase in the reporter fluorescence was detected. Standard curves for real-time PCR assays were developed as described previously (He et al., 2007). Briefly, the AOA and AOB amoA genes were PCR-amplified from extracted DNA with the primer sets (AOA: Arch-amoAF/Arch-amoAR; AOB: amoA-1F/ amoA-2R) listed in Table 1, and then the PCR products were cloned into the pGEM-T Easy Vector (Promega Madison, USA). Plasmids used as standards for quantitative analyses were extracted from the correct insert clones of each target gene. The concentration of plasmid DNA was determined on a Nanodrop® ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, USA), then the copy numbers of the target genes were calculated. To generate an external standard curve, ten-fold serial dilutions of a known copy number of the plasmid DNA were subjected to real-time PCR assay in triplicate.

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of heavy metals on soil microorganisms had been mainly focused on the community structure of bacteria and fungi (Gremion et al., 2004; Frey et al., 2006; Gomes et al., 2010). Recently, the responses of soil microbes involved in the N-cycle to heavy metal stress have been investigated (Li et al., 2009; Mertens et al., 2009; Subrahmanyam et al., 2014; Zhou et al., 2012). For example, using four different Chinese soils, Li et al. (2009) observed that copper (Cu) intensively inhibited the soil potential nitrification rate (PNR) and decreased the abundances of soil AOB and AOA within 20 days. Meanwhile, AOB were found to be more sensitive to heavy metals than AOA. Mertens et al. (2009) reported that the abundances of AOB and AOA decreased in Zn-spiked soil after 1 week, while only the community structure of AOB changed significantly after 2 years. It appeared that the abundances of soil AOB and AOA were sensitive to heavy metal stresses. Mercury (Hg) as a typical heavy metal has pronounced negative effects on all living organisms. During the past two decades, almost 50% of Hg production worldwide was consumed in China (Jiang et al., 2006). It was also reported that the total Hg ranged from 5.1 to 790 mg/kg in agricultural fields near a mining area in Guizhou Province, Southwest of China (Qiu et al., 2004). It has been recognized that mercury can change the community structure of soil microorganisms and inhibit soil enzymatic activities (Ranjard et al., 1997; Muller et al., 2001; Rasmussen and Sorensen, 2001; Harris-Hellal et al., 2009). Liu et al. (2010) tested the responses of soil AOB and AOA to Hg at up to 35 mg/kg and no variation in the abundance of either AOA or AOB was exhibited. Considering the heavy contamination of Hg in farm fields (up to 790 mg/kg), it is essential to investigate the effect of more severe Hg stress on soil AOB and AOA, which also could provide some useful information for the application of ammonia oxidizers in the risk assessment of soil Hg pollution in the future. In this study, the effect of Hg on the activity of soil ammonia oxidizers was evaluated for a series of Hg concentrations (0–100 mg/kg), as well as the abundance and community structures of soil AOA and AOB under Hg stress. The objective was to explore the response of the ammonia oxidizing activity and different ammonia oxidizers to severe Hg stress.

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Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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Table 1 – Primer sets and thermal profiles used in PCR amplification.

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Target group

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Sequence (5′–3′)

Thermal profile

Reference

amoA-1F amoA-2R a Arch-amoAF Arch-amoAR a

GGGGTTTCTACTGGTGGT CCCCTCKGSAAAGCCTTCTTC STAATGGTCTGGCTTAGACG GCGGCCATCCATCTGTATGT

5 min at 94°C, followed by 35 cycles of 30 sec at 94°C, 30 sec at 55°C and 1 min at 72°C, and 5 min at 72°C for the last cycle 5 min at 94°C, followed by 35 cycles of 30 sec at 94°C, 30 sec at 53°C and 1 min at 72°C, and 5 min at 72°C for the last cycle

Rotthauwe et al. (1997) Francis et al. (2005)

A 40 bp GC-clamp (cgcccgccgcgccccgcgcccggcccgccgcccccgcccc) was attached to the 5′ end of the primers used of DGGE.

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One-way analysis of variance (ANOVA) followed by the S-N-K-test was performed using SPSS 11.5 (SPSS Inc., Chicago, USA) to check for quantitative differences between samples. P < 0.05 was considered to be statistically significant. The PNR values in the soil treatments with different Hg concentration at day 28 were used to calculate EC50 by a logistic response model (Vanewijk and Hoekstra, 1993):

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k y¼ 1 þ ðx=x50 Þs

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where, y is the response of measured endpoint (PNR), k is the response of measured endpoint at x = 0, s is the slope parameter, x is the concentration (on a liner scale), and x50 = median effective concentration (EC50).

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2.2. Effects of Hg on the abundance of soil AOA and AOB

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The abundance of AOA and AOB showed the same response to Hg stress during the incubation (Fig. 2). In detail, at days 3 and 7, no difference was exhibited among the treatments for AOA and AOB. At the end of the experiment (day 28), the abundance of AOA and AOB in CK was significantly higher than that in all the Hg-amended soils, suggesting that the numbers of AOA and AOB were inhibited in the presence of Hg. In addition, at day 28, the AOA abundance in all treatments was of the same order of magnitude ((.68–9.20) × 107 copies/g dry soil), but the AOB abundance in the CK (2.68 × 107 copies/g dry soil) was an order of magnitude higher than those in the Hg50 (7.49 × 106 copies/g dry soil) and Hg100 (8.35 × 106 copies/g dry soil) treatments.

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For DGGE analysis, the amplification was performed in 50 μL reaction mixtures including 1 × PCR buffer, 400 μmol/L each dNTP, 2.5 U hot star Taq DNA polymerase (Takara Bio, Otsu, Shiga, Japan) plus primers (AOA: Arch-amoAF/Arch-amoAR*; AOB: amoA-1F/amoA-2R*) (Table 1). DGGE analysis of each PCR product was performed with the DCode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, USA). The PCR product of each target gene was loaded onto 6% (W/V) polyacrylamide (37.5:1, acrylamide:bisacrylamide) gels with denaturing gradients of 20%–45% and 40%–65% (100% denaturant contained 7 mol/L urea and 40% (V/V) formamide), respectively. Electrophoreses were run at 60°C and 100 V for 12 hr and 15 hr for AOA and AOB amoA genes, respectively. The gels were stained for 30 min according to the manufacturer's (Invitrogen 20 Molecular Probes, Eugene, USA) instructions, and scanned by a GBOX/HR-E-M (Gene Company Limited, Syngene, UK). The obtained results were analyzed using Quantity One Software 4.5.2 (Bio-Rad Laboratories, Hercules, USA).

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in PNR value was found between the CK and the Hg treatments. But at day 28, the soil PNR in some treatments was restored, especially for low Hg treatment such as Hg25, and showed a decreasing tendency with increased soil Hg content, with an order of CK > Hg25 > Hg50, Hg100. The PNRs of day 28 could be well fitted with the logistic dose–response model (P < 0.05), from which the EC50 of PNRs was found to be 22.6 mg/kg.

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2. Results

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2.1. Effects of Hg on soil potential nitrification rate (PNR)

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The PNR value of CK at day 3 was significantly higher than that of all the Hg amendments, by approximately a factor of 4 (Fig. 1), indicating the intensive inhibition of soil PNR by Hg. No significant differences were observed among the Hg treatments. At day 7, the PNR of CK decreased and no significant difference

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During the incubation, the community structures of soil AOA and 245 AOB at days 7 and 28 were quite different (Fig. 3). No obvious 246 difference was found in the AOA community structure among all 247

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Potential nitrification rate (µg NO2-N/(g dry soil.hr))

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Fig. 1 – Soil potential nitrification rate (PNR) in the treatments with different concentrations of Hg (CK, Hg25, Hg50, and Hg100, representing 0, 25, 50, and 100 mg Hg/kg dry soil, respectively) during the incubation. Error bars indicate standard deviations (n = 3). The different letters above bars indicate significant differences (P < 0.05) among the treatments for the same sampling day.

Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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3. Discussion

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The inhibiting effect of heavy metals on soil PNR had been reported previously (Rother et al., 1982; Smolders et al., 2001; Broos et al., 2005; Yang et al., 2005). For example, Broos et al. (2005) demonstrated that soil PNR was a more sensitive endpoint than SIR (substrate-induced respiration) or basal respiration (BR) under cadmium (Cd) and Zn stress. In this study, the intensive inhibition of soil PNR by Hg after 3-day

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CK Hg 25 Hg 50 Hg 100 CK Hg 25 Hg 50 Hg 100 1 2 3 1 2 3 1 2 3 1 2 3 1 2 31 2 31 2 3 1 2 3

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exposure indicated the influence of Hg on the activity of soil ammonia oxidizers. The soil PNR showed a clear dose– response relationship with EC50 (22.59 mg/kg) at day 28, which further confirmed the negative effects of Hg on those soil key functional microbes. It seemed that soil PNR had different responses to severe and mild Hg stress. The PNR was partially restored at day 28 at low Hg concentration, but not for the treatment of Hg100 (Fig. 1). Liu et al. (2010) reported that soil PNR was inhibited by Hg (30 mg/kg) after 1 week, and subsequently recovered after 2 weeks. Additionally, only the PNRs at 1 week could be well fitted with the logistic dose– response model (EC50, 1.59 mg/kg). The mild Hg stress inhibited soil PNR with an obvious dose–response relationship at 1 week, but severe Hg stress could be evaluated after 4 weeks as the soil PNR began to recover. Other researchers also reported that Zn inhibited the soil PNR after 1 week, but that the soil PNR recovered gradually after 1 year (Ruyters et al., 2010). Li et al. (2009) found that soil PNR was inhibited by

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the treatments (Fig. 3 for soil AOA). However, distinct differences were exhibited in the community structure of soil AOB (Fig. 3B for soil AOB) between CK and all the Hg treatments. Several bands disappeared in the Hg-amended soil at days 7 and 28 (top rectangle in Fig. 3B), and some special bands were detected in some Hg treatments (Hg25, Hg50) at day 28 (bottom rectangle in Fig. 3 for soil AOB).

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Fig. 2 – Quantification of soil ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) amoA gene copy numbers in the treatments with different concentrations of Hg during the incubation. Error bars indicate standard deviations (n = 3). The different letters above bars indicate significant differences (P < 0.05) among the treatments for the same sampling day.

Soil AOA

Soil AOB

Fig. 3 – DGGE profiles of soil ammonia-oxidizing archaea (AOA) and ammonia-oxidizing archaea (AOB) amoA gene amplified from the treatments with different concentrations of Hg at days 7 and 28. The districts in the rectangles marked with dotted lines show the obviously different bands in the CK and Hg treatments. Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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4. Conclusions

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In conclusion, soil PNR was inhibited rapidly by Hg stress (day 3), and restored with the decrease of Hg levels. Significant inhibiting effects of Hg on the abundance of soil AOB and AOA were observed at the end of the incubation (day 28), which might be responsible for the reduced PNR. Only the community structure of soil AOB was changed, suggesting AOB are more sensitive than AOA to Hg stress.

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Acknowledgments

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This work was supported by the Natural Science Foundation of China (Nos. 41371477, 41071163), the National Key Technology R&D Program (No. 2014BAD14B01), and the Fundamental Research Funds for the Central Universities (No. XDJK2014B047).

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REFERENCES

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Adair, K.L., Schwartz, E., 2008. Evidence that ammonia-oxidizing archaea are more abundant than ammonia-oxidizing bacteria in semiarid soils of Northern Arizona, USA. Microb. Ecol. 56 (3), 420–426. Broos, K., Mertens, J., Smolders, E., 2005. Toxicity of heavy metals in soil assessed with various soil microbial and plant growth assays: a comparative study. Environ. Toxicol. Chem. 24 (3), 634–640. De-Boer, W., Gunnewiek, P.J.A.K., Veenhuis, M., Bock, E., Laanbroek, H.J., 1991. Nitrification at low pH by aggregated chemolithotrophic bacteria. Appl. Environ. Microbiol. 57 (12), 3600–3604. Di, H.J., Cameron, K.C., Shen, J.P., Winefield, C.S., O'Callaghan, M., Bowatte, S., et al., 2009. Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils. Nat. Geosci. 2 (9), 621–624. Erguder, T.H., Boon, N., Wittebolle, L., Marzorati, M., Verstraete, W., 2009. Environmental factors shaping the ecological niches

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organic inhibitors, such as allylthiourea and sulfadiazine, possibly because of the differences in cell walls and membranes (Hatzenpichler et al., 2008; Schauss et al., 2009). On the other hand, soil AOA might be the primary participant in the N-cycling under some harsh environmental conditions, such as low pH, extremely low or high temperature, high salinity, low oxygen, and low NH+4 concentration (Erguder et al., 2009; He et al., 2012). The soil used in this study had a rich NH+4 content with a neutral pH, which should be not suitable for the growth of AOA, and the soil AOA in a dormant state might be not sensitive to Hg stress. The band numbers in the Hg treatments were less than that in CK at day 7, but some new bands emerged in the Hg treatments (Hg20 and Hg50) at day 28 (Fig. 3B). So the results indicated that severe Hg stress might have a strong selectivity on the soil AOB species that might be related with the restoration of soil PNR. Severe and mild Hg stress had different effects on the community structure of soil AOB. In addition, the community structure of soil AOB might be more suitable for the assessment on soil Hg stress both in short or long-term than that of soil AOA.

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more than 50% at a Cu concentration of 600 mg/kg in four different Chinese soils during a incubation of 20 days, and different soil had different ED50 (50% effective doses) under Cu stress. Meanwhile, the heavy metal concentration and the soil type could affect the dose–response relationship between soil PNR and heavy metal concentration, especially the former, which showed that a clear dose–response relationship occurred in the inhibition or initial recovery period. Moreover, the PNR values of CK at days 3 and 7 were notably lower than that at day 28, indicating the recovery process of soil PNR during the incubation, which might be due to the influence of soil pretreatment (air-drying and sieving) on the activity of ammonia oxidizers and the low growth rate of ammonia oxidizers (obligate chemoautotrophic organisms). Significant inhibition effects of Hg on the abundance of soil AOB and AOA were only observed at the end of the incubation (at day 28). This is the probably the reason for PNR decrease. Li et al. (2009) studied the responses of soil AOA and AOB gene copies to Cu stress and observed much lower numbers of AOB amoA gene copies in the treatment with Cu concentration of 1000 mg/kg at day 21, indicating the inhibiting effect of Cu on the abundance of AOB. However, other researchers reported different results. The abundance of soil AOB and AOA had no changes under Hg (0–30 mg/kg soil) stress during an incubation experiment of 8 weeks (Liu et al., 2010). It is generally considered that there is a positive relationship between soil PNR and AOB amoA gene abundance (He et al., 2007; Di et al., 2009; Tian et al., 2014). By contrast, according to our results, soil PNR exhibited no obvious correlation with amoA gene abundance under short-term Hg stress at days 3 and 7, which might be explained by the following reason. Short-term Hg stress on the abundance of soil ammonia oxidizers is likely to be difficult to detect by real-time PCR based on soil DNA, since the DNA from the cells that were killed or inhibited under Hg stress might not be completely digested in a short period of time, and thus also might be extracted from the soil and amplified by real-time PCR. With the extension of incubation time, compared with CK, relatively lower PNRs and amoA gene abundances were simultaneously detected in the Hg treatments at day 28. The result of DGGE indicated that Hg had no effect on the community structure of soil AOA, but significantly changed the community structure of soil AOB, especially at day 28. This result suggested that soil AOA and AOB had different sensitivity to Hg stress, and AOB were more sensitive than AOA. The results were supported by the previous investigation (Liu et al., 2010), in which the community structure of soil AOB but not AOA changed dramatically under Hg stress. The percentage of AOB community in cluster 3a.1 decreased with ascending soil Hg concentration. Mertens et al. (2009) investigated the community structures of soil AOA and AOB under Zn stress, and suggested that only the community structure of soil AOB was intensively affected by 2-year exposure to Zn. These results confirmed that the community structure of soil AOB was more sensitive to heavy metals than that of soil AOA, which might be due to the distinctions of cell structure and physiology between bacterial and archaea, although the exact mechanism of the different responses of AOB and AOA to environmental stresses was still unclear. It was reported that AOB also were more susceptible than AOA to some

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Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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Please cite this article as: Zhou, Z.-F., et al., Responses of soil ammonia oxidizers to a short-term severe mercury stress, J. Environ. Sci. (2015), http://dx.doi.org/10.1016/j.jes.2015.04.022

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