Reactive Oxygen Species in MeHg

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The role of free radicals/reactive oxygen species in MeHg photodegradation-importance of utilizing appropriate scavengers Xiaoxiao Han, Yanbin Li, Dan Li, and Chang Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00205 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Environmental Science & Technology

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The role of free radicals/reactive oxygen species in MeHg photodegradation-importance

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of utilizing appropriate scavengers

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Xiaoxiao Han‡, Yanbin Li†,‡,*, Dan Li‡, and Chang Liu‡

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†

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University of China, Qingdao 266100, China

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‡

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China

Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean

College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100,

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*Corresponding Author:

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Yanbin Li

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Tel: +86-0532-66786355

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Fax: +86-0532-66782301

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Email address: [email protected]

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ACS Paragon Plus Environment


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TOC

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Abstract

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A variety of free radicals (FR)/reactive oxygen species (ROS) have been proposed to

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dominate methylmercury (MeHg) photodegradation, primarily based on the results of

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FR/ROS scavenger addition experiments. However, in addition to eliminating FR/ROS, the

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added scavengers may also affect the experimental results by altering some water chemical

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properties, resulting in a misleading assessment of the importance of FR/ROS. In this study,

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20 common FR/ROS scavengers were evaluated in terms of their influence on light

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absorbance, pH, MeHg analysis, MeHg-dissolved organic matter (DOM) complexation, and

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the scavenger-induced degradation of MeHg. Only nine scavengers were identified to be

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appropriate for investigating MeHg photodegradation. By utilizing these appropriate

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scavengers, direct photodegradation of MeHg-DOM complexes was found to be the major

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pathway of MeHg photodegradation in Laoshan Reservoir water and Stone Old Beach

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seawater. In contrast, MeHg photodegradation in Ink River water primarily occurs through

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both ·OH and

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MeHg-DOM complexes. The diverse pathways of MeHg photodegradation in the tested water

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may be due to differences in water chemical properties. A severe overestimation of the role of

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FR/ROS was observed when several improper but commonly used scavengers were adopted,

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highlighting the necessity of utilizing appropriate scavengers.

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DOM* mediated indirect pathways and direct photodegradation of

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3



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Introduction

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Methylmercury (MeHg), a neurotoxin and the most toxic form of mercury (Hg) in the

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environment, has drawn extensive public attention because of its bioaccumulation through

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food chains and high risk to human health. Photodegradation is a major sink of MeHg in

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aquatic environments,1-8 and great efforts have been made to elucidate the mechanisms

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governing this process.9-12 The chemical reactions underlying this process have been reported

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to be system-specific.4,

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proposed in previous studies. Indirect pathways are primarily mediated by photo-mediated

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produced free radicals (FR) or reactive oxygen species (ROS) and they have been reported to

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play crucial roles in MeHg photodegradation.2, 10, 17 Singlet oxygen (1O2) and hydroxyl radical

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(·OH) are the two species mostly reported to induce the photodegradation of MeHg,13, 16, 19-21

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whereas the involvement of triplet excited state of dissolved organic matters (3DOM*) was

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also observed.12, 22 Direct photodegradation of MeHg-inorganic ligand complexes such as

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MeHgCl or MeHgOH under sunlight is theoretically difficult because of the low energy of

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sunlight that reaches to the earth.23 This premise is supported by experimental data.11, 17 In

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contrast, direct photodegradation of MeHg-DOM complexes has been proposed to dominate

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MeHg photodegradation in some aquatic systems.11, 22 Due to the difficulty of establishing

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experimental conditions that would allow for the examination of the direct photodegradation

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of MeHg-DOM complexes, the approaches taken in current studies were mainly depending on

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validating or excluding the involvement of FR/ROS.11, 17 Therefore, accurately identifying the

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role of FR/ROS in MeHg photodegradation is important not only in systems in which

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FR/ROS dominate this process, but also in systems where FR/ROS are not involved.

11, 13-16

Both indirect

11, 13, 17, 18

and direct15 pathways have been

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Due to the high reactivity and short lifetime of FR/ROS,24 it is a challenge to

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quantitatively determine their levels and directly prove their involvement in a specific

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chemical process in natural environments. Instead, two indirect methods, i.e., FR/ROS

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scavenger addition6,

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utilized. For the chemical FR/ROS generator method, FR/ROS are generated via chemical

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reactions (e.g.MoO42-/H2O2 for 1O2,11 Fe2+/H2O2 for ·OH 25) and their involvement in a certain

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reaction is then tested. This method is useful for investigating the kinetics of FR/ROS

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involved reactions. However, there is usually a substantial difference between the simulated

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experimental system and the natural environment in FR/ROS concentrations and other crucial

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environmental parameters (e.g., pH, light absorbance).11 To solve this problem, the scavenger

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addition method has been more commonly used to explore the role of FR/ROS in natural

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environments.11,

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FR/ROS scavengers are compared to evaluate the importance of FR/ROS in a certain

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process.20 This technique has significant advantages over the chemical FR/ROS generator

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addition method in mimicking the natural environments. However, the added scavengers may

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also alter the water chemical characteristics in addition to eliminating the FR/ROS. Some of

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these changes can significantly promote or inhibit the concerned processes and produce

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misleading results regarding the importance of FR/ROS. The reliability of the scavenger

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addition method largely depends on the utilization of appropriate reagents as scavengers.

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However, little attention has so far been paid to the feasibility of scavengers adopted for

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investigating the role of FR/ROS in natural environments.

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11-13, 15, 17

12, 15, 17

and chemical FR/ROS generator addition,11,

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are widely

In this method, reaction rates with and without the addition of

A variety of scavengers (e.g., dimethyl sulfoxide (DMSO), isopropanol, ethyl alchol and 5



10,

11,

13,

15,

20

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benzoic acid (BA) for ·OH,6,

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1,4-diazabicyclooctane (DABCO) and methionine for 1O2,11, 12, 17 isoprene for 3DOM*,11, 12

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and isopropanol/N2O for hydrated electron (e-aq)11) have been used to identify the role of

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FR/ROS in MeHg photodegradation. MeHg photodegradation can be affected by a variety of

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environmental factors (e.g., pH,10, 15 irradiance intensity,9, 15 and DOM composition.11, 12, 22, 26).

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Some of these scavengers are expected to greatly alter the chemical characteristics of the

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tested water. For instance, BA and NaN3 have typical functional groups (unsaturated bonds,

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azide group and aromaticity groups27) that can absorb sunlight. Methionine, histidine and

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NaN3 have high affinity with Hg (logK=7.7-8.8)28 and they may affect the complexation of

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MeHg with DOM, which is thought to be necessary for MeHg photodegradation.11,

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Phenylurea, BA and 2-chlorophenol have carboxyl and aromaticity moieties27, and these

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moieties may induce the degradation of MeHg under sunlight.12,

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commonly used method for MeHg analysis (i.e., aqueous ethylation derivatization) is very

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sensitive to matrix interference,29, 30 and the added scavengers may interfere with the analysis

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of MeHg. The results obtained using scavenger addition experiments could be biased if any of

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these unwanted effects appear. Our previous study demonstrated that NaN3 can inhibit MeHg

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photodegradation by competing with DOM for complexation with MeHg, along with

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eliminating 1O2 and ·OH.11

β-carotene, sodium azide (NaN3),

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Besides, the most

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The major objectives of this study were to develop a protocol for screening and selecting

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appropriate FR/ROS scavengers for studying MeHg photodegradation, and to identify the role

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of FR/ROS in MeHg photodegradation in natural water. To achieve these aims, 20 commonly

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used scavengers for ·OH, 1O2, 3DOM*, O2•-, and e-aq were evaluated by testing their influence 6


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on the light absorbance and pH of water, MeHg analysis, MeHg-DOM complexation, and the

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possible degradation of MeHg induced by these scavengers. The appropriate scavengers were

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then applied to investigate the role of FR/ROS in MeHg photodegradation in seawater

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collected from Stone Old Beach and fresh water collected from Laoshan Reservoir and Ink

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River, near Qingdao, China.

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Materials and Methods

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Reagents

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MeHgCl standard was purchased from the National Institute of Metrology, China.

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Suwannee river natural organic matter (SRNOM) was purchased from the International

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Humic Substances Society. All solutions used were prepared in ultra-pure water (>18 MΩ

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cm-1), while β-carotene was prepared by dissolving it in tetrahydrofuran (THF) and then

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adding it to deionized (DI) water. SRNOM solution (>3 kDa) was obtained by ultra-filtrating

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10 mL of the SRNOM solution (~200 mg C L-1) using a 3 kDa centrifugal filter (Sartorius) at

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6000 rpm for 60 minutes and then cleaning the pallet ten times using 10 mL of DI water. The

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dissolved organic carbon (DOC) concentration of the obtained SRNOM DOM (>3 kDa) was

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determined by a total organic carbon analyzer (Analytikjena, Germany). All reagents used

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were of reagent grade or higher.

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The pH and light absorbance characteristics of candidate FR/ROS scavengers

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The tested candidate FR/ROS scavengers (β-carotene,15 2,5-dimethylfuran,19 THF,17

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DABCO,19 methionine19 and NaN311 for 1O2; histidine,15 DMSO,13 BA,10 isopropanol,11, 12

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ethyl alcohol,15, 20 mannitol31 and formic acid13 for .OH; isoprene11, 12, 17 and phenylurea32 for 7



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DOM*; isopropanol/N2O11, DMSO/N2O33 and 2-chlorophenol34 for eaq-; superoxide

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dismutase (SOD)20 and 1,4-benzoquinone35 for O2•-) were added to 30 mL DI water to form

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final concentrations of 1, 10 and 100 mmol L-1 (mg L-1 for SOD). The pH of these solutions

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was then measured using a pH meter (Sartorius, Germany). The tested candidate scavengers

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were added into 10 mg L-1 DOM solutions (>3 kDa SRNOM) containing 10 ng L-1 of MeHg

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to form a final scavenger concentration of 1, 10 and 100 mmol L-1. The light absorbance

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spectrum of each scavenger dissolved in DI water or DOM solutions was measured using a

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UV-2550 ultraviolet (UV)-visible spectrophotometer (SHIMADZU, Japan) at wavelengths

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between 280-700 nm, to mimic sunlight. The transmittance of UV-A, UV-B and visible light

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in the DI water and DOM solutions was calculated by the average transmittance of irradiance

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from 280 to 320 nm, 320 to 400 nm, and 400 to 700 nm, respectively.

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Effects of candidate FR/ROS scavengers on MeHg analysis

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To assess the effects of candidate FR/ROS scavengers on MeHg analysis using aqueous

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ethylation derivatization, MeHg was added to 1, 10 or 100 mmol L-1 (mg L-1 for SOD) of

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scavenger solutions to form a final MeHg concentration of 1 ng L-1. MeHg in the scavenger

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solutions was determined using the distillation-ethylation derivatization-purge and

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trap-GC/CV-AFS method, following the EPA Method 1630. Details of the method can be

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found in the Supporting Information. Recoveries of MeHg in the scavenger solutions were

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calculated to examine the effects of these scavengers on MeHg analysis. Triplicate analyses

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were employed for each scavenger.

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Effects of candidate FR/ROS scavengers on the complexation of MeHg with DOM

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To evaluate the effects of candidate scavengers on MeHg-DOM complexation, 8


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scavengers were added into the DI water containing 10 ng L-1 MeHg and 10 mg L-1 SRNOM

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solutions (>3 kDa), to form a final scavenger concentration of 1, 10 and 100 mmol L-1.

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Another trial without adding any scavengers was employed as a control. After equilibrating

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for 24 h, 10 mL of each solutions were ultra-filtrated using a 3 kDa centrifugal filter

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(Sartorius) at 6000 rpm for 25 min. MeHg concentrations in the two fractions (≥3 kDa and 10 kDa, the competitive complexation of SOD

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with DOM was investigated using

Reactive Oxygen Species in MeHg

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