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Bioassay-directed identification of organic toxicants in water and sediment of Tai Lake, China Xinxin Hu a,b, Wei Shi a,*, Nanyang Yu a, Xia Jiang c, Shuhang Wang a, John P. Giesy a,d,e,f,g, Xiaowei Zhang a, Si Wei a, Hongxia Yu a,* a

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, People's Republic of China b Shandong Academy of Environmental Science, Jinan, People's Republic of China c Key Laboratory of Environmental Protection of Lake Pollution Control, Chinese Research Academy of Environmental Science, Beijing, People's Republic of China d Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada e Department of Zoology, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA f School of Biological Sciences, University of Hong Kong, Hong Kong, China g Department of Biology and Chemistry and State Key Laboratory for Marine Pollution, City University of Hong Kong, Hong Kong, China

article info

abstract

Article history:

The government of China has invested large amounts of money and manpower into

Received 10 June 2014

revision of water quality standards (WQS). Priority organic pollutants have been screened

Received in revised form

for WQS establishment using the potential hazard index method, however, some unsus-

6 January 2015

pected chemicals that could cause adverse effects might have been ignored. A large

Accepted 22 January 2015

number of chemicals exist in environment and there might be interactions between or

Available online 31 January 2015

among chemicals especially those with the same mode of action. Therefore, a toxicitydirected analysis, based on acute toxicity to Daphnia magna, was conducted for organic

Keywords:

extracts of water and sediment from Tai Lake (Ch: Taihu) to determine toxicants respon-

Tai Lake

sible for adverse effects. Extracts of five of twelve samples of water and all extracts of

Organic extract

sediment were acutely toxic. Based on toxic units, water from location L1 in July and

Toxicity-directed analysis

sediments from locations L1 and L4 during several months would be expected to result in

Chlorpyrifos

some toxicity. Twenty one (21) organophosphorus pesticides, 25 organophosphorus pes-

Cyfluthrin

ticides and 10 pyrethroids were detected in samples, extracts of which caused toxicity to D.

Predominant pollutants

magna. Chlorpyrifos and cyfluthrin were identified as predominant pollutants in organic extracts of sediments, accounting for up to 71% and 57% of bioassay-derived toxicity equivalents (BEQs), respectively. Chlorpyrifos was identified as the major contributor to toxicity of organic extracts of surface water, accounting for 71% to 83 % of BEQs. The putative causative agents were confirmed by use of three lines of evidence, including statistical correlation, addition of key pollutants or synergists. Greater attention should

* Corresponding authors. Tel./fax: þ86 25 8968 0356. E-mail addresses: [email protected] (W. Shi), [email protected] (H. Yu). http://dx.doi.org/10.1016/j.watres.2015.01.033 0043-1354/© 2015 Elsevier Ltd. All rights reserved.

232

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be paid to chlorpyrifos and cyfluthrin, neither of which is currently on the list of priority pollutants in China. Bioassay-directed analysis should be added for screening for the presence of priority organic pollutants in environmental media. © 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Chinese surface water quality standards (WQS) include criteria mainly for inorganic contaminants, while few criteria have been established for organic pollutants (SEPA, 2002). However, contamination of some bodies of water in China with organic pollutants is serious and, because of the complexity, persistence, bioaccumulation and toxic effects of organic pollutants, has been attracting more and more attention (Spearow et al., 2011; Kidd et al., 2007). Hydrophobic, organic pollutants can be adsorbed by sediments (Liu et al., 2009; Kuivila et al., 2012), where they can persist and cause toxic effects to benthic organisms. The government of China is adding criteria for organic pollutants for various basins, and several hundred million RMB has been invested to select model organisms, screen priority pollutants, and revise the present water quality standards (WQS). In this long-term program to revise WQS, Tai Lake (Ch: Taihu) has been chosen as the first demonstration area because it is important for production of rice and also because it has a commercial fishery. It is also used as a major source of drinking water in one of the most populous and economically developed regions of China. Organochlorine pesticides (OCPs), organophosphorus pesticides (OPs), polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), all of which can be toxic to aquatic organisms (USEPA, 2007), have been detected in water and sediments of Tai Lake (Shi et al., 2011; Ta et al., 2006; Wang et al., 2003). Decreases in populations of fishes and richness of the zooplankton community in Tai Lake have attracted increasing attention (Zhu, 2004; Fan, 1996). The organic pollutants can cause decreases in populations of aquatic organisms (Spearow et al., 2011; Kidd et al., 2007). However, the existing surface water quality standards include only three classes of organic contaminants, including volatile phenols, petroleum compounds and anionic surfactants (SEPA, 2002). Addition of classes of organic contaminants to the WQS for protection of aquatic organisms in Tai Lake is necessary. Screening and evaluation of listed priority organic toxicants in Tai Lake has been undertaken to develop a baseline information on the current status of concentrations of these toxicants against which future trends can be compared. Using potential hazard index method, five chemicals, including pyrene, dimethyl phthalate (DMP), di-n-butyl phthalate (DNBP), di(2-ethylhexyl) phthalate (DEHP) and 1,4-dichlorobenzene, have been identified as priority organic pollutants in Tai Lake. However, potential effects of un-listed pollutants that might be present were unknown and if only those on the priority list are considered in the monitoring program, some toxicants which could cause toxicity to organisms might be ignored. Because

there are criteria for only a limited set of organic contaminants that might occur in surface waters and sediments, thus it was deemed necessary to screen and evaluate toxicity of contaminants in water or sediment. This was undertaken by use of a screening-level bioassay in the context of a bioassaydirected identification scheme. Because organic compounds occur in mixtures, it is difficult to evaluate the potential hazards of organic pollutants in surface water and sediment based on measurements of their individual concentrations. Also, there might be contaminants present that are not identified because they are not on the list of priority pollutants and for which there might not be methods or authentic standards available. Effect-directed analysis (EDA) is one useful method for evaluating potential toxicity and identifying the responsible organic toxicants in extracts of environmental samples, which has been used in identifying major toxicants in samples of surface water, sediment and effluents (Bandow et al., 2009; Grung et al., 2007) and have been described and reviewed in detail (Hecker and Giesy, 2011). However, the volume of environmental samples that is available for fractionation and identification of the major pollutants by use of EDA is often limited. Mass balance (or potency balance) analysis can be used to assess potential risks of chemicals detected in samples and calculate their contributions to the observed toxicity of extracts of environmental samples. Once suspect key toxicants have been identified, confirmation of putative causative agents is essential. Spiking of suspect toxicants has been used as a approach for confirming the suspect toxicants (Bandow et al., 2009; Schwab et al., 2009). Moreover, the presence of synergists or antagonists can modulate toxic potencies of some classes of pollutants and also needs to be considered as a confirmation approach. Since some compounds with similar properties could react similarly, confirmation of specific causative agents using only one approach may not be definitive. Therefore, use of multiple techniques and lines of evidence for confirmation is suggested. Daphnia magna is a resident aquatic crustacean and used as one of the six families of aquatic species for which data are required in development of WQS in China (Yang et al., 2012; Yan et al., 2012). D. magna has been used to evaluate toxicity of a range of types of samples (Ribe et al., 2012; Diamantino et al., 1998). In the present study, acute toxicity was determined by use of D. magna exposed to organic extracts of water or sediment of Tai Lake collected from several locations, during several months. The objectives of this study were to: 1) examine toxicity of organic extracts of water and sediment from Tai Lake in different months and different locations; 2) determine concentrations of organic pollutants in water and sediment from

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Tai Lake; 3) identify organic pollutants that contributed to toxicity of organic extracts of water and sediment and 4) determine potential additional contaminants to be added to the priority pollutants list for which WQS need to be derived.

2.

Materials and methods

2.1.

Chemicals and materials

Chemicals used for bioassays included chlorpyrifos (CPY, >98.5% purity), cyfluthrin (>99.4% purity) and triphenyl phosphate (TPP, >99% purity). Chlorpyrifos was purchased € fers (Augsburg, Germany). from Labor Dr. Ehrenstorfer-Scha Cyfluthrin and TPP were purchased from AccuStandard (New Haven, CT, USA). Descriptions of OPs, pyrethroids, OCPs and nitro-anilines used for instrumental analysis are given in Table S1 in the supporting information (SI).

2.2.

Sampling locations

Pollution of Tai Lake is greatest in the northern portion in areas such as Meiliang and Zhushan Bays (Guo et al., 2012; Wang et al., 2012; Lu et al., 2013). The surrounding terrestrial landscape of these regions is farmland and agricultural effluent is a primary source of contaminants. Both water and sediments were collected in May, July and October, 2009 from four locations including L1, L2, L3 and L4 in the northern part of Tai Lake (Fig. S1 in SI). At each location, 10 L of water and 2000 g wet mass (wm) of sediment were collected and placed into brown, glass bottles pre-cleaned with Milli-Q water, methanol (Tedia Co. Ltd, Fairfield, OH, USA), acetone (Tedia Co. Ltd, Fairfield, OH, USA), dichloromethane (Tedia Co. Ltd, Fairfield, OH, USA) and n-hexane (Merck, Darmstadt, Germany). Detailed information on sampling locations, sampling time, sediments collection and samples storage is shown in supporting information.

2.3.

Sample preparation

Samples of water and sediment were extracted by use of solid phase extraction (SPE, Oasis HLB cartridges, Waters, Milford, MA, USA) and accelerated solvent extraction (ASE, Dionex ASE 300, Dionex, Idstein, Germany), respectively. Extracts of water and sediment samples were separated into two aliquots for use in instrumental analysis or bioassays. The aliquot for use in instrumental analysis was concentrated, blown to dryness and reconstituted in 0.5 mL and 1 mL dichloromethane for water and sediments, respectively. The other aliquot for use in bioassays was concentrated, blown to dryness and reconstituted in 0.5 mL and 1 mL DMSO for water and sediments, respectively. Greater details of these methods are provided in the supporting information.

2.4.

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randomly selected to expose to the treatment or control conditions for 48 h. Seven concentrations were used for each organic extract. There were four replicates for each concentration and five animals in each breaker. Extracts of water samples in DMSO were diluted to provide concentrations equivalent to 40-, 20-, 10-, 5-, 2.5-, 1.25-, 0.625-fold relative to the original concentrations in water. Extracts of sediments in DMSO were diluted into concentrations equivalent to 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 mg/mL (mg-dry mass (dm) per mL of test solution). Tests were conducted in 10 mL of solution in 25 mL glass breakers. Blanks (no solvent) and solvent controls (0.5% DMSO) were also performed. Tests were conducted under a 14:10 lightedark cycle at 22 ± 1  C. Immobilization of D. magna after 48 h was used as the measurement to determine mortality. If D. magna did not move after gentle agitation of the solution, they were considered to be immobilized. Potassium dichromate was used as a positive control, reference toxicant. D. magna were treated with vehicle or various concentrations of potassium dichromate with three replications and repeated once a week. The EC50 of potassium dichromate was calculated in control charts by use of normal statistical procedures. If EC50s for potassium dichromate were between 0.9 and 1.7 mg/L, the test conditions and sensitivities of D. magna were similar among tests and the tests deemed valid (ISO, 1996).

2.5.

Instrumental analyses

Qualitative analysis of organic pollutants in water and sediment extracts was conducted by use of Thermo TSQ Quantum Discovery triple-quadrupole mass spectrometer (San Jose, CA, USA) in selected reaction monitoring (SRM) mode. Organic pollutants in the water or sediment extracts detected in the qualitative analysis were mainly pesticides, including OCPs, OPs, pyrethroids and nitro-anilines. Concentrations of OPs and OCPs in water and sediment were quantified by use of a Thermo Single Quadrupole GCeMS (San Jose, CA, USA) in selected ion monitoring (SIM) mode. Concentrations of pyrethroids and nitro-anilines were quantified using Thermo TSQ Quantum Discovery triple-quadrupole mass spectrometer (San Jose, CA, USA) in selected reaction monitoring (SRM) mode. Recoveries of pesticides were determined by use of external standards. Limits of detection (LODs) of pesticides were defined based on a signal three times the background noise (S/N ¼ 3). Internal standards parathion-d10, 13C-PCB 141 and PCB-189 from Sigma (St. Louis, MO, USA) were spiked into extracts before chemical analysis to quantify OPs, OCPs and pyrethroids according to previously published methods (Li et al., 2010; Wang et al., 2010; Tao et al., 2010). Average recovery, limit of quantification (LOQ) and LOD of each chemical are given in Table S2 in the supporting information. None of the procedural blanks contained detectable concentrations of target compounds. Detailed information about instrumental analysis is showed in supporting information.

Toxicity testing 2.6.

The water flea (D. magna) was used as the test organism in 48h tests that followed OECD protocols (OECD, 1984) with some modifications. To evaluate acute toxic potency of organic extracts of water or sediment, young D. magna (