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Environmental Risk Implications of Metals in Sludges from Waste Water Treatment Plants: The Discovery of Vast Stores of Metal-containing Nanoparticles Feiyun Tou, Yi Yang, Jingnan Feng, Zuoshun Niu, Hui Pan, Yukun Qin, Xingpan Guo, Xiang-Zhou Meng, Min Liu, and Michael F. Hochella Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05931 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017
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Environmental Risk Implications of Metals in Sludges from Waste
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Water Treatment Plants: The Discovery of Vast Stores of
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Metal-containing Nanoparticles
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Feiyun Tou †, Yi Yang *, †, ‡, Jingnan Feng †, Zuoshun Niu †, Hui Pan †, Yukun Qin †,
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Xingpan Guo†, Xiangzhou Meng , Min Liu †, Michael F. Hochella, Jr.§, ||
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†
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Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai,
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200241, China.
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‡
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⊥
Key Laboratory of Geographic Information Science of the Ministry of Education, School of
Shanghai Key lab for Urban Ecological Processes and Eco-Restoration, East China Normal
University, 500 Dongchuan Road, Shanghai 200241, China ⊥
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental
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Science and Engineering, Tongji University, Shanghai 200092, China
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§
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24061, USA
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||
The Center for NanoBioEarth, Department of Geosciences, Virginia Tech, Blacksburg, VA
Geosciences Group, Energy and Environment Directorate, Pacific Northwest National
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Laboratory, Richland, WA 99352, USA
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Corresponding author:
[email protected] 18 19
Table of Content Art
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ABSTRACT
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Nanoparticle (NP) assessment in sludge materials, although of growing importance in eco-
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and bio-toxicity studies, is commonly overlooked and, at best, understudied. In the present
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study, sewage sludge samples from across the mega-city of Shanghai, China were investigated
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for the first time using a sequential extraction method coupled with single particle inductively
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coupled plasma mass spectrometry (SP-ICP-MS) in order to quantify the abundance of
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metal-containing NPs in the extraction fractions, and transmission electron microscopy to
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specifically identify the nanophases present. In general, most sludges observed showed high
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concentrations of Cr, Cu, Cd, Ni, Zn and Pb, exceeding the maximum permitted values in the
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national application standard of acid soil in China. NPs in these sludges contribute little to the
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volume/mass, but account for about half of the total particle number. Based on electron
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microscopy techniques, various NPs were further identified, including Ti-, Fe-, Zn-, Sn-,
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Pb-containing NPs. All NPs, ignored by traditional metal risk evaluation methods, were
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observed at a concentration of 107 -1011 particles/g within the bioavailable fraction of metals.
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These results indicate the underestimate or mis-estimation in evaluating the environmental
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risks of metals based on traditional sequential extraction methods. A new approach for
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environmental risk assessment of metals, including NPs, is urgently needed.
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INTRODUCTION
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The most recent estimation of the production of sewage sludge in China is 40 million tons in
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2014 (calculated using the estimate that 10,000 tons of wastewater produces 5.6 tons of
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sewage sludge1), and this production is increasing at an annual average rate of 4.75 percent.2
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Of this tonnage, 45% is applied to agricultural land, 31% is landfilled, 3% of the sewage
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sludge is incinerated, and the remaining 21% is used for greening non-agricultural lands
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and/or discharged directly into rivers.3 Among these destinations, agricultural application and
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landfilling are most economical.4 However, the land application of sludge is limited by its
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high concentrations of various metals and other toxic organic pollutants, which in many cases
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has led to severe degradation of soils and therefore serious secondary environmental pollution.
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For example, metals in sewage sludge are of significant concern and have been known to pose
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severe risks to the ecological environment and humans via agricultural application.5-10
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Besides various metals in sludge, metal-containing nanoparticles (NPs) have a number of
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origins, morphologies and sizes, atomic structures, and compositions, all of which play an
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important role in their precise behavior, and as a result, they have gained more and more
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attention11-28. NPs typically show distinct chemical and physical properties, especially when
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they are particularly small, relative to the corresponding bulk material, and can induce
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cytotoxicity, resulting in long term environmental and health risks.11-14 In addition, due to their
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inherent reactivity with other contaminants, NPs can serve as a carrier and may release toxins
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through transformation under certain environmental or biological conditions.15-18 It is worth
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noting that metal containing NPs, such as titanium oxides, iron oxides, and silver and zinc
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sulfides, have already been identified in sewage sludge and sludge-amended soils.19-23 These
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NPs can also be taken up by organisms from sludge-amended soil, posing eco-environmental
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risks.24-28 However, the occurrence and environmental function of NPs in soils and sediments
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has been challenging to assess.29-31
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As an important traditional method commonly applied to assess the environmental risk of metals, a chemical speciation analysis can indicate the mobility, bioavailability and
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potential eco-toxicity of metal-containing NPs and help predict their release in soil/sediment
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environments.32-34 Tessier sequential extraction, known as a sequential extraction method
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proposed by the European Community Bureau of Reference (BCR sequential extraction)35-36
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and its variations are the most widely applied extraction methods. For this study, we have
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chosen a modified BCR sequential extraction method due to its stability for extraction
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efficiency and ease of application. For this method, metals are divided into an
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acid-exchangeable fraction, a reducible fraction, an oxidizable fraction, and a residual fraction.
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The acid-exchangeable fraction, mainly consisting of water soluble, exchangeable and
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carbonate-bound metals, is readily released into the environment and represents the
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bioavailable fraction.32 However, until this present study, metal-containing NPs have never
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been considered in each fraction according to the existing citable literature.
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It is a great challenge to directly quantify metal-containing NPs in complex
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environmental samples and further differentiate them from their ionic species. Electron
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microscopy (EM) techniques, such as scanning transmission electron microscopy (S/TEM)
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and scanning electron microscopy (SEM), coupled with accessory capabilities such as
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energy-dispersive X-ray spectroscopy (EDX) and selected area electron diffraction (SAED),
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have been recognized by the scientific community as powerful tools to provide detailed
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information including particle size, morphology, chemical composition, and crystal structure
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on a single-particle basis.19-23 However, application of all of the above mentioned techniques
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in complicated environmental samples is somewhat limited by the lack of a way to quantify
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the number of NPs, that is there general abundance, which cannot be provided by the methods
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described above. Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS)
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is a relatively new technique to quantify the number of NPs even in complex environmental
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samples, and also to make an independent estimate of their size distribution. In addition, it can
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determine both metal concentrations in the dissolved and particulate forms simultaneously.
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It can also determine the size of NPs to compare with other commercially available
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techniques such as dynamic light scattering.37 The development of SP-ICP-MS techniques has
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been well described in other studies,38-39 and it has been successfully applied to directly 4
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determine the particle sizes and concentrations of NPs in drinking water, biological tissues,
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and suncreen37, 40-43, but none to date in sewage sludge materials.
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This study is designed to estimate the environmental implications of metals in sludge
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samples, specifically metal-containing NPs. To this end, twenty-six sewage sludge samples
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were taken from wastewater treatment plants (WWTPs) as a representative sampling
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throughout the mega-city of Shanghai, China. The feasibility of using traditional risk
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assessment methods for metals in sludge samples was evaluated, but in this case considering
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specific NP quantities, sizes, and compositions for the time. The specific objectives of this
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study are (1) to assess the risk of metals in sewage sludge based on analysis via modified
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BCR sequential extraction, (2) to reveal the occurrence of metal-NPs (such as Ti-NPs, Fe-NPs
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and Zn-NPs) in different metal chemical fractions, and to determine the concentration and
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size distribution of metal-NPs in each chemical fraction, especially in the bioavailable
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fraction of metals using SP-ICP-MS, and (3) to identify the dominant metal-NPs based on EM
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techniques.
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MATERIALS AND METHODS
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Sample Collection and Pretreatment.
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Shanghai, with an area of 6340km2 and a population of more than 24 million, is a sprawling
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megacity in a developing country. With increasing population, motorization, urbanization and
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industrial activities, the annual wastewater discharge has climbed to 2212 million tons in 2014,
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with 53 wastewater treatment plants scattered throughout the city.44 Shanghai is suffering
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tremendous stress from wastewater treatment and its main by-products, including sewage
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sludge. For this study, sewage sludge samples were collected from 26 WWTPs in Shanghai.
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Table S1 summarizes the details of the WWTPs. About 2 kg wet weight sewage sludge
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samples were collected, immediately transported to our Shanghai laboratory, stored at -20℃,
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freeze-dried and homogenized until further processing.
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Metal Analysis. 5
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In order to determine the total metal concentrations in sewage sludge, all samples were
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digested according to the EPA-approved, microwave assisted nitric acid digestion method
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3051A45 in duplicate and then analyzed by a Thermo Electron X-Series ICP-MS
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(Massachusetts, United States) via Standard Method 3125-B,46 and the calibration standards
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were prepared in a matrix of 2% nitric acid by volume. In detail, approximately 0.1g
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freeze-dried samples were pretreated with 2mL of 67% nitric acid (Trace Metal Grade, Fisher)
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at 90℃ for half an hour and 1mL of 30% hydrogen peroxide (Ultrapure Reagent, Fisher)
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overnight at 50℃ in 100mL bottles. Then all the reactants were transferred to microwave
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digestion vessels, with 7mL of 67% nitric acid. After cooling down to room temperature, the
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vessels were rinsed 3 times with Mill-Q water and the volume was set to 100mLbefore
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analysis. This method was appropriate for most metals, but not ideal for especially titanium
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oxides which are exceptionally insoluble.47 Analyzed metals include: Ti, V, Cr, Fe, Mn, Co, Ni,
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Cu, Zn, As, Se, Sr, Mo, Ag, Cd, Sn, Ba, Ce and Pb.
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Particle Size Analysis.
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Laser diffraction particle size analyzer LS13320 (Beckman Counter, California, United
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States), particle size analysis ranging from 0.017 to 2000µm, was applied for the particle size
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analysis of the sludge samples. The particle size of blank samples (Milli-Q water) ranged
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from 17nm to 40 nm, and the actual particle size detection limit was therefore set as 40 nm.
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All samples were pretreated with 10 mL of 10 % hydrogen peroxide at 100℃ until no
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bubbles were generated (more hydrogen peroxide solution was added if 10 mL was not
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sufficient) to reduce the particle aggregation caused by organic compounds. After cooling
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down to room temperature, 10mL of 36.1g/L sodium hexametaphosphate was added as a
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stabilizer and the mixture was dispersed by an ultrasonic probe (KQ5200E, Shumei, Kunshan,
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China) for 10 min.
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Sequential Extraction Procedure.
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Sequential extraction was performed with the modified BCR sequential extraction
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procedure.48 This procedure is described in detail in the supplemental information (Fig. S1).
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According to the industrial wastewater ratios (IWRs, the volume ratios of the industrial
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wastewater to the total inflow wastewater), fourteen of the 26 sludge samples (S4, S5, S10,
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S11, S12, S14, S15, S17, S18, S19, S20, S21, S23 and S26) were chosen to process the
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sequential extraction procedure in duplicate. Blanks were measured in parallel for each set of
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BCR-prepared samples. Negligible metal concentrations were detected in all the blanks.
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SP-ICP-MS Data Acquisition and Processing.
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To screen for the occurrence of metal containing-NPs in the bioavailable fraction of sludge
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and further quantify their particle size and concentrations, a PerkinElmer NexION 350D
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SP-ICP-MS (Massachusetts, United States) was used to analyze the acid-exchangeable
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extraction solutions (pH 3, acetic acid) of 14 sludge samples (S4, S5, S10, S11, S12, S14, S15,
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S17, S18, S19, S20, S21, S23 and S26). The instrument was set to detect Zn, Fe and
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Ti-containing NPs. In addition, as Ti oxide-NPs would not react with extraction reagents,
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these particles in S26 were further analyzed for the occurrence of NPs in the reducible
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fraction and oxidizable fraction.
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Masses of 47Ti (7.3% abundance, Standard Mode), 66Zn (27.9% abundance, Standard
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Mode) and 56Fe (91.7% abundance, Dynamic Reaction Cell (DRC) Mode) were monitored by
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SP-ICP-MS, with a dwell time of 100µs and a scan time of no less than 100s. Dissolved
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element calibrations for titanium, zinc and iron were prepared in a matrix of 2% nitric acid by
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volume, and Au-containing NPs stabilized with citrate in the size of 30 nm and 60 nm from
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the National Institute of Standards and Technology (NIST) were used as particle calibration
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standards. Particle size and dissolved element detection limits (according to the results of
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Milli-Q water) were determined to be 15-20 nm and 0.13 µg/L for Ti, 15-16 nm and 0.30 µg/L
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for Zn, and 12-17 nm and 0.10 µg/L for Fe, respectively. To get appropriate particle
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concentration, tested samples were diluted 100~10000 times and the results are summarized
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in Table S6.
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Electron Microscope Analysis. 7
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Selected samples (S11, S12, S18 and S21) were characterized using electron microscopy
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techniques. An Environmental Scanning Electron Microscope (ESEM, FEI Quanta 600 FEG,
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Oregon, United States), equipped with an energy dispersive X-ray spectrometer (EDS,
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QUANTAX 400, Bruker, Karlsruhe, Germany) system, and a Transmission Electron
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Microscope (TEM, JEOL 2100 TEM, Tokyo, Japan), coupled with EDS and selected area
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electron diffraction (SAED), were applied to characterize the morphology, composition and
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crystal structures of the NPs.
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RESULTS AND DISCUSSION
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Total Concentrations of Metals in Sludge Samples.
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Total concentrations of Ti, V, Cr, Fe, Mn, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Ag, Cd, Sn, Ba, Ce,
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and Pb in sludge samples are shown in Figure 1. Fe is the most abundant metal (8.1-54.5
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gkg-1), with an average concentration of 22.7 gkg-1 which is 1-5 orders of magnitude higher
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than all other metals. Ag exhibited the lowest abundance (0.25-29.6 mgkg-1), with an average
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of 5.4 mgkg-1 in all sludge samples. Zn and Cu, with an average concentration of 2091.6 and
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908.4 mgkg-1, respectively, are the second and third most abundant metal contaminants.
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Noticeably, the concentrations of lead in S21 (2007.1 mgkg-1) and S11 (446.2 mgkg-1) are
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much higher than those in other sludge samples, ranging from 21.3 to 198.1 mgkg-1. The
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elevated concentrations of lead in these two samples can likely be attributed to the occurrence
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of metallic material processing works and metal smelting factories in the S21 and S11 service
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areas, respectively. In addition, the concentrations of cadmium in S5 (170.6 mgkg-1) and
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cobalt in S18 (803.2 mgkg-1) are 1-2 orders of magnitude higher than those in other sludge
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samples, ranging from 0.67 to 4.4 mgkg-1 and 2.7 to 91.9 mgkg-1, respectively. It is likely
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that these elevated concentrations are due to the electroplating factories and petrochemical
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industries in both S5 and S18 service areas.
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It is noticeable that the concentrations of Cr, Ni, Cu, Zn in Shanghai sludge samples are
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2~3 times higher than those found in the samples collected from 193 WWTPs in 111 cities of
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China,49 which are likely reasonable averages of heavy metal pollution levels from WWTP
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sludges in urban China; likewise, the concentration of Pb across Shanghai is 32% higher than
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the average concentration in the China-wide study (Table S3). In addition, the concentrations
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of Cr, Ni, Cu, Zn, Cd, and Pb in this study are higher than the permissible values for acid soils
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(issued by the Ministry of Environmental Protection of the People’s Republic of China in
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2002) in 3.8 to 38.5% of the samples, depending on the metal.50 According to the results from
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a sewage sludge survey launched by the U.S. Environmental Protection Agency (EPA),51 the
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concentrations of Cd, Cr, and Pb in U.S. sludge samples are lower than those in Shanghai
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sludge according to the present study. However, the silver concentrations in U.S. sludge
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samples are more than ten times higher than those in Shanghai sludge samples.
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The correlation coefficient matrix among different metal elements as well as IWR (Table
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S1, analyzed by IBM SPSS Statistics 23.0) of WWPTs shows that there are significant
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correlations between Ti, V, Cr, Co, Cu, Se, Sr, Mo, Cd, Ce and IWR (Table S3), indicating
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that industrial wastewater are likely an important input of these heavy metal contaminants to
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sewage sludge. Meanwhile, in most samples, Ti, Cr, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Sn, and
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Pb are significantly correlated with each other at the 0.01 level, and the higher correlations (r >
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0.8) are observed between V and Co, Cr and Cu, and As and Pb, suggesting their similar or
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the same origins.52-54
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Particle Size Analysis of Sludge Samples.
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The particle size distributions of sludge samples, plotted as a function of volume percent for
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each size fraction, are plotted in Figure S2. It is obvious that the large particles (>1000 nm),
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with an average volume percent of 90.3 %, is the dominant fraction of total volume, while the
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nanoparticles (
