Environ Sci Pollut Res DOI 10.1007/s11356-013-2415-1

RESEARCH ARTICLE

Combined use of DGT and transplanted shrimp (Litopenaeus vannamei ) to assess the bioavailable metals of complex contamination: implications for implementing bioavailability-based water quality criteria Zaosheng Wang & Peihong Zhao & Changzhou Yan & Vulpe D. Chris & Yijun Yan & Qiaoqiao Chi

Received: 30 September 2013 / Accepted: 27 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The diffusive gradients in thin films (DGT) were field deployed alongside the shrimp Litopenaeus vannamei at seven sites with different levels of contamination to assess the potentially bioavailable and toxic fraction of metal contaminants. After 7 days of exposure, several antioxidant biomarkers were quantified in hepatopancreas of exposed shrimps, and tissue levels as well as the total, dissolved, and DGT-labile concentrations of metal contaminants were determined in the pooled site samples. The results showed that the caged shrimps had high tissue contaminant concentrations and significantly inhibited antioxidant responses at the more contaminated sites. DGT-labile metal concentrations provided better spatial resolution of differences in metal contamination when compared with traditional bottle sampling and transplanted shrimp. The total, dissolved, and DGT-labile metal fractions were used to evaluate the potential bioavailability of metal contaminants, comparing with metal accumulation and further linking to antioxidant biomarker responses in tissues of exposed shrimps. Regression analysis showed the significant correlations between DGT-Cu concentrations and tissue-Cu and activities of some biomarker responses in the Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-2415-1) contains supplementary material, which is available to authorized users. Z. Wang (*) : P. Zhao : C. Yan (*) : Y. Yan : Q. Chi Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Boulevard, Xiamen City 361021, China e-mail: [email protected] e-mail: [email protected] Z. Wang : V. D. Chris Department of Nutritional Sciences and Toxicology, University of California, 317 Morgan hall, Berkeley, CA 94720, USA

shrimp hepatopancreas. This indicated that DGT-labile Cu concentrations provided the better prediction of produced biological effects and of the bioavailability than the total or dissolved concentrations. The study supports the use of methods combining transplanted organisms and passive sampling for assessing the chemical and ecotoxicological status of aqueous environments and demonstrates the capability of the DGT technique as a powerful tool for measuring the bioavailability-based water quality in variable coastal environments. Keywords DGT . Litopenaeus vannamei . Bioavailable metals . Complex contamination . Biomarker responses . Bioavailability-based WQC

Introduction In natural waters, a variety of physico-chemical forms or species of trace metals may exist, which play a critical role in metal distribution and fate, and influence metal reactivity, mobility, bioavailability, and toxicity to aquatic organisms (Slaveykova et al. 2009; Guéguen et al. 2011). Because metal species are not all equally bioavailable, speciation analyses provide an important complement to traditional dissolved or total metal measures. This results in a more comprehensive picture of metal fate, improving the understanding of the processes that govern their ultimate impacts in aquatic systems. In this respect, recent publications suggest that passive sampling methods such as diffusive gradients in thin films (DGT) are useful for measuring labile species of aqueous metal concentrations, which may represent the bioavailable fraction (Buzier et al. 2006; Allan et al. 2007; Schintu et al. 2008). However, to date only few studies have compared

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DGT-measured metals to accumulation in organisms. Previous works has focused on bivalve and microalgae (Jordan et al. 2008; Webb and Keough 2002; Topperwien et al. 2007) or on acute toxicity to Daphnia magna under controlled laboratory conditions (Tusseau-Vuillemin et al. 2004). However, the correspondence between DGTmeasured metals, and bioavailability and the toxicological significance of contaminants in waters has not yet been adequately confirmed. Further, metals usually exist in the environment as complex mixtures with different routes of uptake by organisms including directly from water or through suspended particulate materials, complicating the experimental interpretation (Dragun et al. 2009). As a consequence, there is a lack of information about the relationship between the different trace metal forms as well as the link between metal speciation and bioavailability and ecotoxicological responses of organisms in seawater. Reliable information on the speciation and bioavailability of trace metals is necessary to better understand metal impacts on biota, as well as for an improved understanding of the biological and dynamical processes in the surface waters (Bowles et al. 2006). This information is best obtained by concurrent in situ experiments, which offer a more ecologically realistic approach than laboratory studies. This approach encompasses all of the factors that occur in the field and may possibly interfere with or influence relevant dynamic processes (Hédouin et al. 2011). Accordingly, DGT probes were deployed in parallel with in situ bioassays to investigate the speciation and bioavailability of trace metals present at the contaminated field locations. Specifically, the marine shrimp Litopenaeus vannamei was selected as a bioindicator organism. The main endpoints were metal accumulation and the effects in the hepatopancreas, which is a very important organ for the metabolism and storage of xenobiotics in crustaceans. Previous work on the hepatopancreas of this shrimp species has shown excellent exposure concentration–response relationships of biomarkers in relation to pollution stress and metal accumulation in monitoring studies (Wu et al. 2008). The species is widely distributed in marine environments and is commercially important in aquaculture (Liu et al. 2007). Tissue concentrations of contaminants are excellent indicators of bioavailability in the environment, but numerous studies suggest tissue concentration do not provide adequate information to assess biological effects in organisms (Pereira et al. 2010). In this study, both environmental concentrations and tissue burdens were measured, together with biomarkers representing early signs of biological responses to various xenobiotics. Several biomarkers of exposure and/or effect were selected, including antioxidant enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). These biomarkers represent the possible oxidative stress in shrimp resulting from disturbing antioxidant efficiency and the enhancement of the intracellular reactive oxygen species (ROS) induced by corresponding pollutants. Additionally, the activity

of phase two bio-transformation enzyme glutathione-S-transferase (GST) was determined. In the present investigation, L. vannamei and DGT devices were co-deployed in situ at seven sites within a metalcontaminated coastal ecosystem. After 7-day field exposure, the concentrations of Cd, Pb, Ni, Cu, and Zn in surface waters were measured by restricted spot sampling and DGT, and compared to metal accumulation and biomarker responses in the hepatopancreas of exposed shrimps explored. The objectives were to investigate the relationship between the different approaches for metal measurement and the biological effects in organisms. To the best of our knowledge, this is the first in situ study that combines simultaneous use of DGT and transplanted shrimp bioassays to assess bioavailability and effects of metals in coastal waters.

Materials and methods Study area and deployment sites Maluan Bay (24°32′47″N, 118°00′38″E) is a sub-tropical, coastal lagoon in the northwest of Xiamen promontory in southeastern China, while this area has strong anthropogenic influence with significant and diverse sources of aquatic contaminants due to urbanization, and industrial and shipping activities (Wang et al. 2011). As the Xinlin industrial complex has increased over the past 30 years, human-induced activities, especially metal industries such as metallurgy, coking, and plating factories, which now line most of the eastern shorelines, are discharging metal contaminants directly into the nearshore environments. Harbor activities in the West Sea lead to significant maritime traffic, which influences coastal waters during ingoing tide. Furthermore, an artificial seawall was constructed in 1960s restricting the exchange of seawater. These various anthropogenic influences mean that circulation and hydrodynamic patterns are likely to be complex leading to a particularly unique aquatic environment. Seven deployment sites were selected based on previous field surveys (Wang et al. 2011), the information provided by local authorities, the predicted tidal influence, accessibility, and risk of vandalism. These sites covered a wide range of metal exposures, a variety of different coastal conditions, and anthropogenic influences and contaminant gradients representative of much of Maluan Bay. ML1 and ML7 were placed at the innermost point and the outermost part of tidal influence, respectively. More detailed information about locations of deployment sites is shown in Fig. 1. Experimental design The fieldwork was carried out during November 2010. L. vannamei were obtained from the aquaculture farm of

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Fig. 1 Map of the study area located in Maluan Bay, showing the deployment sites of DGTs and shrimps in transplantation experiments

Xiamen coast (Fig. 1), which is far from the industrial area and is considered as a reference site since no pollution point sources were identified nearby. Metal pollutants are present in the whole Maluan Bay; consequently, a field negative control site in the bay itself was not available. All shrimp specimens selected and transported to the deployment sites were of a similar size and length, and were at the same stage of reproduction (mature; carapace width about 9.7±0.2 cm). Shrimps and commercially available standard DGT units (DGT Research Ltd. UK) were simultaneously deployed in situ by using custom-made nontoxic polyvinylchloride materials, designed for field exposure and placed in plastic cages (length×width×height = 60×42×24 cm; mesh size = approximately 8.0 cm2 allowing free circulation of seawater) in order to protect the exposure containers from possible mechanical stress. The dimensions of the exposure container were 50× 32×20 cm (length×width×height) with a rectangular window (10×10 cm) at each side, covered with 150-μm-mesh-size nylon netting. The aim was to create a regular flow and to allow the free exchange of seawater inside the apparatus (i.e., exposure concentrations outside and inside the equipment should remain equal) while preventing the entry of predatory organisms. The exposure equipment was separated by two sets of adjustable netting of 300-μm mesh size, which provided sufficient convection to minimize the thickness of the

diffusive boundary layer (DBL) for DGTs and produced three consecutive compartments. Since the study was designed for the measurement of metal bioaccumulation and multiple stress biomarkers, more organisms were required for post-exposure analysis. Thus, ten individuals of random groups were introduced at the two compartments to allow replication and deployed alongside the three DGT probes, which were deployed in the central compartment as shown in Fig. 2. DGTs consisted of polyacrylamide hydrogel diffusion layers (0.76 mm thick), Chelex-100 impregnated binding phases (0.40 mm thick), and nylon DGT holders. They were assembled with 0.45-μm-pore-size cellulose acetate filters, used as covering membranes. At each site, three DGT devices were positioned in the middle of the equipment and held vertically in the water column with a polypropylene rope. Furthermore, the upper surfaces of equipment were transparent to enable sunlight to enter, allowing organisms to be directly exposed to the contaminants under natural environmental conditions. Each cage was suspended at 30– 50 cm below the water surface at neap tide and attached by nylon ropes to four wooden pegs, which were fixed to the bottom by means of an anchor. Another batch of shrimps of similar characteristics and DGTs were kept at the reference site as the control group. The field deployment is illustrated in Fig. 1.

Environ Sci Pollut Res Fig. 2 Schematic representation of DGT and L. vannamei field exposure

At the start and end of deployment, water samples were collected from approximately 50 cm below the water surface for later measurements of the total and dissolved metal concentrations and immediately transported to the laboratory for analysis. Physicochemical parameters were measured regularly in situ using a portable multiprobe YSI 6600V2 meter (YSI, Yellow Spring, OH, USA), which showed that pH and dissolved oxygen ranged from 8.02 to 8.15 and 6.8 to 7.8 mg/L, respectively, and the surface water temperature ranged between 15.7 and 20.3 °C with a mean value of 18.0 °C. Climatological or oceanographic level recorded indicated that water quality conditions were relatively constant. Chemical analysis Determination of total and dissolved metal concentrations In the laboratory, water samples were agitated to maintain homogeneity during sub-sampling that occurred within a few hours from collection. Aliquots were transferred to a polyethylene bottle, acidified with concentrated HNO3 (to pH < 2), and then filtered using a filtration device and 0.45-μm filters (Xinya membrane; Millipore) for the measurement of total metal concentrations. Further triplicate samples (50 mL) from each site were filtered through 0.45-μm filters under clean-bench conditions before being acidified with Suprapur HNO3 (to pH