Environ Sci Pollut Res DOI 10.1007/s11356-014-3484-5
DANIO RERIO AS A MODEL IN AQUATIC TOXICOLOGY AND SEDIMENT RESEARCH
Toxicity of sediment cores from Yangtze River estuary to zebrafish (Danio rerio) embryos Peipei Wang & Lili Zhang & Li Liu & Ling Chen & Hongwen Gao & Lingling Wu
Received: 31 May 2014 / Accepted: 18 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10∼40 cm), which indicated that the pollution is more serious in recent decades. Keywords Sediment cores . Whole sediments . Sediment organic extracts . Toxicity . Zebrafish embryos . Yangtze River estuary
Introduction With the developing of industry and agriculture, more and more pollutants have been discharged into marine ecosystems Responsible editor: Philippe Garrigues P. Wang : L. Zhang : L. Liu : L. Chen : H. Gao : L. Wu (*) Key Laboratory of Yangtze River Water environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai, China 200092 e-mail: [email protected]
over the last century (Wu et al. 2010). As a coastal transitional system, an estuary is considered the most threatened aquatic ecosystem in the world because it is the final receptor of environmental contaminants derived from land or carried by river and atmosphere (Blaber et al. 2000). As their low water solubility, most hydrophobic compounds always accumulate in sediments and particulate matters (Tuikka et al. 2011). Sediments may serve as secondary sources of many heavy metals and persistent organic chemicals (Barjhoux et al. 2012) due to the complicated hydrodynamic force in an estuary, which poses a risk to benthic organisms (Tuikka et al. 2011). It is well established that the characterization of sediment quality should comprise of chemical analysis and toxicity bioassays (Chapman et al. 2002; Eklund et al. 2010). Chemical analysis determines the concentration of pollutants and the magnitude of anthropogenic pollution. Toxicity bioassays measure the complex effects of sediment-bound chemicals on the aquatic organisms (Fu et al. 2013; Tuikka et al. 2011). As an alternative tool for sediment assessment, sediment contact tests (SCTs) directly represent bioaccessibility (Feiler et al. 2013; Pane et al. 2008; Zielke et al. 2011). SCTs have been well developed in Europe using various species, such as algae, bacteria, yeast, nematodes, and fish (Feiler et al. 2013). Zebrafish (Danio rerio) is a small freshwater fish and has become one of the most popular model organisms in toxicity assessment (Scholz et al. 2008). However, in recent years, the embryo test has gained growing interest as a refinement or even a replacement for the acute fish test because of the new European regulation on the protection of animals used for scientific purpose (Barjhoux et al. 2012; Strecker et al. 2011). Owing to the transparency and rapid development of the eggs, and several observable lethal and sub-lethal end points during the exposure, zebrafish embryos have become the promising models not only for testing chemicals but also for investigations on environmental samples (Rocha et al.
Environ Sci Pollut Res
2011), e.g., sediments (Wu et al. 2010) or water samples (Keiter et al. 2006). The Yangtze River estuary is one of the largest estuaries worldwide with the population of over 15 million (Chen et al. 2001). With the development of industry and agriculture in Yangtze regions, the estuary suffers large amounts of industrial wastewater, urban sewage discharge, and oil-containing wastewater discharge from ships (Floehr et al. 2013), which contain huge amounts of chemical compounds. In recent years, 206 hazardous organic chemicals were detected in waters, and 106 were detected in sediments, 17 of them belonging to the priority controlled pollutants of America (Wang and Peng 2002). Typical heavy metals arsenic, mercury, cadmium, copper, chromium, zinc, and lead were also detected in the Yangtze River estuary (An et al. 2009). However, little has been reported about the toxicity of sediments from the Yangtze River estuary to aquatic organisms. In our previous study, we assessed the potential toxicity of the surface sediments from the Yangtze River estuary to zebrafish embryos and found that the sediment exposure increased the lethal rates and induced morphological abnormalities of zebrafish embryos (Wu et al. 2010). Study of core sediments can provide information about the events that occurred in past time. Changes in pollutant concentration with depth in sediment cores can show long-term trends of pollutant input and thereby imply pollution (Atalar et al. 2013). In this study, the toxicity of the sediment cores from the Yangtze River estuary will be assessed using zebrafish embryos. The purposes are to (1) assess the adverse effects of sediment cores from the Yangtze River estuary, (2) compare the potential risks of whole sediments to those of organic extracts in order to express the bioaccessibility of the sediment-bound pollutants, and (3) screen the historic sediment quality of the Yangtze River estuary.
Materials and methods Sediment collection and treatment Three sediment cores were collected from the Yangtze River estuary in March 2012. The sampling locations of the three sediment cores are shown in Fig. 1. The cores were collected using a gravity corer with a diameter of 8 cm. Only the central part was sampled to avoid possible contamination from the corer. The samples of sediment cores were sliced into 10-cm thickness to ensure sufficient sediment to make three replicates per section for toxicity testing. All these samples were well mixed separately and stored at −20 °C until analysis. Native sediment samples were freeze-dried at −30 °C under continuous rotation for 24 h and sieved with a 0.15-mm mesh sieve to remove coarse debris and stones, then stored at 4 °C in plastic jars.
Extraction of sediment Samples were extracted according to the methods established by Hollert et al. (2000). Briefly, extracts from 20-g dried sediments were separately extracted using a Soxhlet apparatus at 6 cycles per hour for 22 h with acetone. The acetonedissolved extracts were reduced in volume by means of vacuum evaporation. Extracts were concentrated close to dryness with N2, and the solvent was changed to dimethyl sulfoxide (DMSO). Then, extracts from 20-g dried sediments were dissolved in 1.0 ml DMSO (i.e., 20,000 mg/ml), and samples were stored at 4 °C until testing. The maximum concentration of DMSO was 0.3 % in the test assay. Chemical analysis of total organic carbon and heavy metals Total organic carbon (TOC) of the sieved sediment samples (≤0.15 mm) was determined by dry combustion with a TOCVCPN (Shimadzu, Japan). The sieved sediment samples (≤0.15 mm) were directly digested to determine the total metal contents. For analysis of the contents of Zn, Pb, Ni, Cr, and Cu, 0.125-g sediment samples were digested with HCl–HNO3–HClO4 mixture. The digested samples were analyzed by ICP/AES (Optima 2100 DV, PerkinElmer, USA) for element composition. Egg production Groups of nine sexually mature zebrafish with a ratio of 2:1 (males to females) were used for egg production. Spawning trays consisting of a translucent plastic box (12 cm×20 cm× 12 cm) and a mesh insert (3∼4-mm mesh size) which prevents the fish from feeding on their own spawn were placed into the aquaria. The control condition of the zebrafish was the following: temperature (26.0±1.0 °C), hardness (250 mg/l), pH (7.5±0.5), dissolved oxygen (10.5±0.5 mg/l=95 % saturation), and light/dark cycle (14:10 h). Spawning and fertilization took place within 30 min after the light had been turned on in the morning. The eggs were transferred into Petri dishes and rinsed several times with reconstituted water (ISO 7346/3: 294 mg/l CaCl2·2H2O, 123 mg/l MgSO4·7H2O, 123 mg/l NaHCO3, and 5.5 mg/l KCl). Then, healthy and normally developing eggs within 2-h postfertilization (hpf) were selected for the following exposure test. For valid experiments, eggs were obtained only from spawns with a fertilization rate higher than 90 %. Whole sediment testing The whole sediment exposure was carried out according to the test protocol of Hollert et al. (2003). Six-well plates were used as test chambers for the exposure. Sediment (1.5 g) and 7.0-ml saturated reconstituted water were mixed in each well (i.e., the
Environ Sci Pollut Res Fig. 1 Map showing the location of three sampling sites in the Yangtze River estuary
sediment concentration was 215 mg/ml H2O). Each well contained five eggs. The transfer of eggs (2 hpf) was performed rapidly to ensure that the exposure during early developmental stages was guaranteed. The sediment was settled for 1 h before adding the eggs to eliminate suspending clay particles, reducing the turbidity of the water to negligible levels (Kosmehl et al. 2006). Two types of negative controls were used: silica control (1.5 g silica+7 ml reconstituted water) and water control (7 ml of reconstituted water without sediment). Five replicates (wells) per sediment sample and controls were tested. The embryos were incubated at 26.0± 1.0 °C with a 14:10-h light/dark cycle. Embryo development was monitored at specified time points (24, 48, 72, and 96 hpf) for lethal and sub-lethal end points. Dead embryos were removed from the plate immediately. All tests were replicated three times.
26.0±1.0 °C with a 14:10-h light/dark cycle. The eggs were examined after 24, 48, 72, and 96-h exposure for lethal and sub-lethal end points. All tests were replicated three times. Statistical analysis The data were expressed as mean ± standard deviation (SD). All statistical analyses were conducted using SPSS 19.0, and the significance was p