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Effect-directed analysis of toxicants in sediment with combined passive dosing and in vivo toxicity testing Hongxue Qi, Huizhen Li, Yanli Wei, W. Tyler Mehler, Eddy Y. Zeng, and Jing You Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017
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Environmental Science & Technology
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Effect-directed analysis of toxicants in sediment with combined passive
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dosing and in vivo toxicity testing
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Hongxue Qia,c,e, Huizhen Lib, Yanli Weia,b, W. Tyler Mehlerd, Eddy Y. Zengb, Jing
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Youb,*
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a
State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
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b
School of Environment, Guangzhou Key Laboratory of Environmental Exposure and Health,
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and Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University,
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Guangzhou 510632, China
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c
College of Chemistry and Chemical Engineering, Jinzhong University, Jinzhong 030619, China
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d
School of Biosciences, Centre for Aquatic Pollution Identification and Management, The
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University of Melbourne, Parkville, Victoria, 3010, Australia e
University of Chinese Academy of Sciences, Beijing 10049, China
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ABSTRACT
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Identifying key toxicants in sediment is a great challenge, particularly if non-target
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toxicants are involved. To identify the contaminants responsible for sediment toxicity to
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Chironomus dilutus in Guangzhou reach of the Pearl River in South China, passive dosing and in
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vivo toxicity testing were incorporated into effect-directed analysis (EDA) to account for
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bioavailability. Fractionation of sediment extracts was performed with gel permeation
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chromatography and reverse phase liquid chromatography sequentially. Polydimethylsiloxane
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served as passive dosing matrix for midge bioassays. The fractions showing abnormal enzymatic
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response were subject to a non-target analysis, which screened out 15 candidate toxicants. The
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concentrations of the screened contaminants (log-based organic carbon normalized) in sediments
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of 10 sites were compared to sediment toxicity (10-d and 20-d mortality and 10-d enzymatic
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response) to C. dilutus using correlation analyses. The results suggested that oxidative stress
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induced by cypermethrin, dimethomorph, pebulate and thenylchlor may have in part caused the
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observed toxicity to C. dilutus. The present study shows that EDA procedures coupled with
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passive dosing and in vivo toxicity testing can be effective in identifying sediment-bound
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toxicants, which may pose high risk to benthic organisms but are not routinely monitored and/or
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regulated. The findings of the present study highlight the importance of incorporating
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environmentally relevant approaches in assessing sediment heavily impacted by a multitude of
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contaminants, which is often the case in many developing countries.
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Environmental Science & Technology
INTRODUCTION
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Sediment serves as a sink for a battery of hydrophobic contaminants in aquatic
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environment and deteriorated sediment quality due to multiple stressors has been reported
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worldwide, especially in rivers draining through large cities.1,2 In the case of complex mixtures,
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the identification of toxicants causing ecotoxicological effects is critical for effectively selecting
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sediment management measures. Toxicity identification evaluation (TIE) and effect-directed
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analysis (EDA) are the two most widely used approaches for diagnosing causes of sediment
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toxicity.3,4
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In the TIE work, researchers mainly focused on characterizing the contaminant classes
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causing toxicity, i.e., organics, metals and ammonia, then estimating the toxicity contribution of
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individual contaminants on the target lists (i.e., a pre-chosen list of chemicals to measure), and
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eventually identifying the toxicants causing adverse effects.4,5 This approach, however, often
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fails to find the main toxicants when the toxicants are not present in the list of target analytes.6
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Alternatively, EDA techniques have been proposed to find organic toxicants by using analytical
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techniques to fractionate test samples for both chemical analyses and biological tests and confirm
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the identity of key toxicants in the toxic fractions without solely relying on the target lists.3,6 As
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such, the EDA method is not limited to analyzing chemicals in specified target lists, but rather
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screens for contaminants of unknown identity under the guidance of the bioassay, providing a
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way to discover toxicants which are not monitored and/or regulated.3,6-8
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Traditional EDA is mainly guided by cell-based in vitro bioassays, which are easy to
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achieve high-throughput analysis, however it lacks ecological relevance and ignores the
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bioavailability and toxicokinetic process of the contaminants.8-10 The use of in vivo toxicity
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testing with benthic organisms is more environmentally realistic than cell-based assays for 3 ACS Paragon Plus Environment
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identifying sediment-bound toxicants. In addition, ignoring bioavailability may bias the
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estimation of toxicity of HOCs in sediment and provide false conclusions as to the suspected
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toxicants, which calls for the development of bioavailability-based EDA methods.8-12 Passive
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dosing techniques with polydimethylsiloxane (PDMS) have been shown to successfully maintain
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constant concentrations of hydrophobic contaminants in water.13-16 During the bioassays, PDMS
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serve as a partitioning delivery system to transfer chemical mixtures into water, acting as a
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surrogate for sediment organic carbon (OC).17,18 Therefore, a EDA procedure combined with
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passive dosing and in vivo bioassays can take chemical bioavailability into account, improving
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the accuracy in diagnosing causes of sediment toxicity in aquatic system containing complex
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mixtures, such as urban rivers.
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The Pearl River flows through Guangzhou, which is the largest city in South China.
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Various sediment-bound contaminants have been detected in Guangzhou reach of the Pearl
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River, including polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, pesticides
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and metals.2,5,19 Current-use pesticides in sediment, particularly pyrethroid insecticides, were
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deemed as the principal causes of the mortality to benthic invertebrates in urban tributaries of the
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Pearl River based on TIE methods.2,5 It was noted that urban sediments, such as those in
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Guangzhou reach of the Pearl River were quite complex with the presence of various pollutants
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many of which were of unknown identity. Similar to its tributaries, sediment-bound pyrethroids
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played a role in the toxicity to benthic invertebrates in Guangzhou reach of the Pearl River, yet
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their toxicity contribution was relatively small.20 Meanwhile, the concentrations of other
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routinely monitored contaminants (e. g., metals) appeared to not contributing to sediment toxicity
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in this river.2 These results suggested the need for exploration of non-target contaminants, which
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would contribute to the observed adverse effects in benthic organisms. 4 ACS Paragon Plus Environment
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Environmental Science & Technology
The aims of the present study were to develop and validate an EDA method in combination
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with passive dosing and in vivo testing using Chironomus dilutus (abnormal enzymatic response
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as the endpoint) for diagnosing causes of sediment toxicity, particularly for identifying organic
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toxicants which are not targeted in common chemical analyses. The applicability of the method
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was validated by a thorough evaluation of sediment toxicity and identification of key toxicants in
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a complex aquatic system (using Guangzhou reach of the Pearl River as an example).
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MATERIALS AND METHODS Experimental Design. An environmentally relevant EDA method for sediment-bound
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toxicants was developed and the stepwise procedures are shown in Figure 1. The experiments
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were separated into two parts: screening bioassays and EDA development. The screening
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bioassays evaluated sediment toxicity to C. dilutus collected from various sites along Guangzhou
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reach of the Pearl River. The EDA method was first developed with sediment from a
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representative site (P4), including sediment extraction, fractionation, bioassays and chemical
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screening, and the confirmation of potential toxicants was conducted with all sediments.
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In the screening bioassays, 10 sediment samples were collected in Guangzhou Reach of the
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Pearl River (Figure S1; “S” represents figures and tables in the Supporting information
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thereafter) and sediment toxicity was evaluated using 1st (20-d chronic testing) and 3rd instar C.
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dilutus (10-d acute testing) as part of the screening bioassays (more information can be found in
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the Supporting information and Cheng et al.20). In short, five of the 10 sediments exhibited acute
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lethality to the midges compared with the controls, and all of the sediments caused significant
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chronic toxicity to C. dilutus (Table S1). In addition, enzymatic activities in the surviving midges
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were significantly altered compared with the control, indicating ubiquitous sublethal toxicity in 5 ACS Paragon Plus Environment
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the study area. A previous study measured current-use pesticides (pyrethroids, organophosphates
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and fipronil) and heavy metals in the sediments and evaluated their toxicity contributions20. Only
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pyrethroids (mostly cypermethrin) were correlated to sediment toxicity, which, however, can
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explain only a small portion of the observed toxicity.
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To better explain the observed sediment toxicity, an EDA method using passive dosing and
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in vivo testing with C. dilutus was developed and used to diagnose other potential causes of
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toxicity besides the target analytes. The EDA procedure includes sediment extraction,
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fractionation, passive dosing, in vivo toxicity testing and non-target chemical analysis and is
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complex and laborious. Thus, only the sediments from site P4, which exhibited moderate
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mortality (37 ± 4%) to the midges, was chosen to test the EDA procedure. This site is adjacent to
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Chebei Creek, which is a well-studied urban tributary of the Pearl River and polluted by a variety
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of contaminants including pyrethroids and fipronils.5,12,19 After the candidate toxicants in
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sediment P4 were screened by the EDA procedure, their concentrations were determined in the
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remaining nine sediments and the toxicity contributions of these contaminants were assessed
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using linear correlation and canonical correlation analyses. Accordingly, key toxicants were
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identified in the sediments. Further information for each step of the EDA procedure is detailed
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below.
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Sediment Extraction and Fractionation. To obtain sediment extracts for EDA analysis,
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ultrasound-assisted microwave extraction (UAME) was performed using a CW-2000 UAME
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extractor (Xintuo Company, Shanghai, China).21 In brief, 40 g of freeze-dried sediment was
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extracted twice with 100 mL of a mixture of hexane and acetone (1꞉1, v/v). The extraction was
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carried out for 6 min with ultrasound and microwave power at 50 and 100 W, respectively. After
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decanting the extract, the extraction was repeated with an additional 50 mL of fresh extraction 6 ACS Paragon Plus Environment
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solution. The extracts were combined, filtered through a Whatman 0.45 µm filter, evaporated to
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near dryness, and solvent exchanged to 2 mL of dichloromethane.
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The sediment extract was first fractionated using gel permeation chromatography (GPC)
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with a Bio-beads S-X3 column (300 mm × 20 mm) and dichloromethane as the mobile phase at a
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flow rate of 5 mL min-1.22 Four fractions were collected at the time intervals of 0–4–8–18–27
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min, and were solvent-exchanged to methanol for midge toxicity testing using the passive dosing
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method.
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Two of the GPC fractions exhibited toxicity (G3 and G4) (Figure 2), which were
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combined, solvent exchanged to methanol and further fractionated for the second round of EDA
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analysis. Reverse phase liquid chromatography (RPLC; Lab-Tech Corporation, China) with a
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C18 semi-preparative column (150 mm × 10 mm, 10 µm) was used for the fractionation and the
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mobile phase was consisted of methanol and water (with an initial composition of 50꞉50 and then
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increasing to 100 % methanol at a flow rate of 3 mL min-1).23 A total of nine RPLC fractions
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were collected (F1–F9) over a period of 45 min at 5-min intervals.
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On the basis of the toxicity results of these nine fractions, the third round of fractionations
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was carried out for the toxic F5 and F6 fractions (Figure 2). Again, the two fractions were
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combined and further separated using RPLC, resulting in five fractions that were collected every
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2 min (2F1–2F5). The individual fractions were then solvent-exchanged to hexane and methanol
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for chemical analysis and toxicity testing, respectively. Passive dosing method was applied for all
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toxicity tests in the EDA procedure.
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Passive Dosing and Midge Toxicity Testing. The PDMS films used for passive dosing
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procedures were made from a MDX4-4210 Bio-Medical grade elastomer kit (Dow Corning
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(China) Holding Company Limited, Shanghai, China), in accordance to the manufacturer's 7 ACS Paragon Plus Environment
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instruction. The method to prepare PDMS films24 was modified from a previously developed
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method by Mayer and Holmstrup.25 In brief, PDMS pre-polymer and catalyst (10꞉1) were
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thoroughly mixed to cast into a film and cured at 23 °C for 72 h. The thickness of PDMS film
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was 0.25 mm (± 0.1 mm) with a density of 1.11 g cm-3. The impurities and oligomers in the
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cured films were removed using three sequential ultrasonic extractions with methanol and three
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rinses using Milli-Q water. The films were then cut into small pieces (2 × 4 cm) before use.
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The fractions after GPC and RPLC separations were individually loaded onto the PDMS
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films. The loading of chemical mixture to the films was achieved by shaking methanol-water
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solutions of the extracts in beakers containing the films at 220 rpm for 48 h. Reconstituted water
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was gradually added to the solution to drive the contaminants into the PDMS film.24,26 For
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dosing sediment extracts containing the mixtures with unknown identity into water, it was crucial
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to normalize the concentrations of contaminants in PDMS to sediment OC-based concentrations.
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Li et al.27 suggested that the partitioning coefficient of a chemical between OC and PDMS was
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independent of its hydrophobicity and was a constant at approximately 2.1. Accordingly, the
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mass of PDMS used in the passive dosing was 2.1 times the mass of sediment OC and detailed
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calculations are presented in the Supporting information. After loading, the PDMS films were
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placed into the reconstituted water and equilibrated for 48 h by stirring at 660 rpm to release the
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contaminants in PDMS into the aqueous phase. This passive dosing procedure has been validated
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with a series of polychlorinated biphenyls (PCBs) with log Kow values ranging from 5.35–7.42
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and the results showed that the equilibrium time for all test PCBs was
