This study aimed to use 7-ethoxyresorufin-o-deethylase (EROD) assay to investigate dioxin contamination in aquatic environments. Oysters were deployed at multiple sites along the coast of the New South Wales, Australia, between November 2009 and March 2010, and obtained from several oyster breeding pools within the Pearl River Delta, South China, between October and November 2010.
In the New South Wales, the highest dioxin concentration was detected in the inner zone of Port Kembla (range: 0.61-0.78 pg EROD-TEQ/g wet weight (ww), mean: 0.72 pg EROD-TEQ/g ww). In the Pearl River Delta, elevated levels of dioxins were observed in Zhuhai, Hengqin, Xinken and Nan'ao. The Pearl River Delta (1.11Â±0.40 pg EROD-TEQ/g ww) was more contaminated with dioxins than the New South Wales (0.43Â±0.16 pg EROD-TEQ/g ww). The weekly dietary intake (WDI) of dioxins from shellfish was estimated for local populations in these two regions, with 0.51 pg EROD-TEQ/kg body weight(bw)/week(wk) in the New South Wales and 3.07 pg EROD-TEQ/kg bw/wk in the Pearl River Delta. The dioxin concentrations in these two regions were below the maximum levels set by the European Commission (8 pg WHO-TEQ/g ww) and the NSW Food Authority (6 pg WHO-TEQ/g ww). The estimated WDIs did not exceed the benchmarks set by the WHO, the Scientific Committee on Food of European Commission (EC-SCF), and Joint FAO/WHO Expert Committee on Food Additives (JECFA) as well.
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Dioxins usually have several chlorine atoms substituted on different positions of benzene rings (Figure 1). Dioxins are highly soluble in lipids and able to accumulate in fatty tissues of living organisms. Furthermore, they are highly stable in the environment and resist to biological degradation. Typically, it takes 7 to 12 years for a human to remove half of the dioxins from the body. Dioxins can also be transported for a long distance in the air to reach remote areas (Agency for Toxic Substances and Disease Registry [ATSDR], 1998; USEPA, 2003). Because of these properties, dioxins tend to bind to particles, sink into sediments or soils, be taken up by living organisms, and undergo bioaccumulation all along the food chain (French Institute of Health and Medical Research [INSERM] Collective Expertise Centre, 2000; Otte et al., 2008).
Dioxins are ubiquitous in the environment (Safe, 1993). PCDDs and PCDFs (PCDD/Fs) are mainly produced as unintentional by-products of industrial processes which involve combustions (USEPA, 2003). Natural combustions, such as volcanoes and frost fires, also produce a small amount of dioxins (Srogi, 2008). Major sources of PCDD/Fs to the environment include waste incineration, chlorine bleaching and metallurgical processing (INSERM Collective Expertise Centre, 2000; Zhang et al., 2007). On the other hand, PCBs were commercially manufactured before their production was banned worldwide in the 1970s. PCBs were used in a wide range of applications, such as electronic appliances, heat-transfer systems, adhesives, paints, and surface coatings. The past PCB production predominantly contributes to the occurrence of PCBs in the environment (ATSDR, 2000).
The environmental release of dioxins have decreased in the past few decades in the developed countries, but increased exposure of dioxins have been reported in the developing countries (USEPA, 2003; WHO, 2010). In 2001, the Stockholm Convention on Persistent Organic Pollutants (POPs), an international treaty aimed to protect human and environmental health from the POPs, was adopted. Both China and Australia ratified and became a party in 2004. All participating countries are now taking actions to reduce production and release of POPs, which include PCDD/Fs and PCBs (United Nations Environment Programme [UNEP], 2008).
Over 90% of human exposure to dioxins is through consumption of food, especially animal products (Liem, Furst, & Rappe, 2000; Lee et al., 2007). Fish and shellfish were reported to contribute a large fraction of the total dietary exposure for dioxins (Jensen & Bolger, 2001; FSANZ, 2004). This may due to their direct ingestion of dioxins absorbed on sediment particles (Otte et al., 2008).
Dioxins can cause a multitude of toxic effects on human. Short-term exposure to a large amount of dioxins may lead to chloracne (skin lesions), whereas long-term exposure may cause a series of effects, which include hepatotoxicity, immunotoxicity, carcinogenicity, endocrine disruption, altered metabolism and birth defects (ATSDR, 1998; Safe, 1998). Noticeably, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF), and 3,4,5,3',4'-pentachlorobiphenyl (PCB 126) were classified as carcinogenic to humans by International Agency for Research on Cancer (IARC) (IARC, 2009). It is also suggested that there is no margin of dioxin exposure for humans with regards to the developmental adverse effects (White & Birnbaum, 2009).
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Figure 1 Chemical Structures of PCDDs (polychlorinated dibenzo-p-dioxins), PCDFs (polychlorinated dibenzofurans) and PCBs (polychlorinated biphenyls) (Baars et al., 2004)
Toxic equivalency factor (TEF) schemes for dioxins
Environmental samples usually contain dioxins as mixtures of congeners (Larsen, Farland, & Winters, 2000). A toxic equivalency factor (TEF) defines the toxic potential of an individual congener relative the most toxic one, 2,3,7,8-TCDD. The total toxic equivalency (TEQ) for a mixture is defined by the equation: TEQ = âˆ‘ [PCDDi] âˆ™ TEFi + âˆ‘ [PCDFi] âˆ™ TEFi + âˆ‘ [PCBi] âˆ™ TEFi , which measures the total 2,3,7,8-TCDD-like responses (Safe, 1998; Van den Berg et al., 2006). There mainly exist two TEF schemes for calculation of the TEQ: the international TEF scheme (NATO/CCMS, 1988) with I-TEQ prior to 1998, and the WHO-TEF scheme (Van Den Berg et al., 1998) with WHO-TEQ after 1998. Overall, the result of I-TEQ calculation is slightly higher than the WHO-TEQ's (Liem et al., 2000).
Risk assessment of dioxins
International or government guidelines usually use the term Tolerable Daily Intake (TDI) for risk assessment of dioxins. It indicates the amount of a substance that can be consumed on average per day over a lifetime with no adverse health effect (Pompa, Caloni, & Fracchiolla, 2003). Since the concept of TDI is based on a long-term exposure to dioxins, calculation of weekly intake or monthly intake of dioxins may be more appropriate to demonstrate the amount of substances that accumulate in body.
Current TDI standards vary between different guidelines (Table 1), with the general range of 1 - 4 pg WHO-TEQ/kg body weight (bw)/day (WHO, 1998; European Commission, 2001; Joint FAO/WHO Expert Committee on Food Additives [JECFA], 2001; Australian Government NHMRC, 2002; UK Food Standards Agency, 2004; Lee et al., 2007). The National Health and Medical Research Council (NHMRC) of Australia set the Tolerable Monthly Intake (TMI) of 70 pg WHO-TEQ/kg bw/month for dioxins, whereas there is no TDI standard set by the Chinese government so far (Australian Government NHMRC, 2002).
It was reported that the dioxin intakes in industrialized countries range from 2 - 6 pg WHO-TEQ/kg bw/day, and infants and toddlers' intake is 1 - 2 order higher than other age groups, due to their high intake of milk and low bodyweight (Liem et al., 2000; WHO, 1998).
Table 1 Comparison between the standards for human tolerable dioxin intake adopted by international organizations and governments (WHO, 1998; European Commission, 2001; Joint FAO/WHO Expert Committee on Food Additives [JECFA], 2001; Australian Government NHMRC, 2002; UK Food Standards Agency, 2004; Lee et al., 2007)
pg WHO-TEQ/kg bw/day
pg WHO-TEQ/kg bw/week
pg WHO-TEQ/kg bw/month
1 - 4
7 - 28
30 - 120
Scientific Committee on Food of European Commission (EC-SCF) (2001)
New South Wales, Australia
The New South Wales is a highly urbanized and industrialized region in Australia, with most of its population living in the coastal zone (Birch, 2000). Its capital, Sydney, has the highest population density of the country (Australian Bureau of Statistics, 2011). According to the results of the National Dioxins Program, which was the largest dioxin survey ever conducted in the country, urban or industrial areas showed more dioxin contaminations than the remote or agriculture areas. Furthermore, elevated levels of dioxins were found in the estuarine waters in Sydney, with the highest level detected from both sediments and biota in the Port Jackson area (Müeller et al., 2004; Ying et al., 2009). These sites in Sydney used to or currently undergo remediation, due to their unacceptable dioxin levels (Birch & Taylor, 2000; Birch, Harrington, Symons, & Hunt, 2007).
The Australian government has made a great effort in reducing dioxins in the environment for the past two decades. In 1990, the National Industrial Chemicals Notification and Assessment Scheme (NICNAS) was established to provide regulations on industrial chemicals (NICNAS, 2011). In 1996, the PCB Management Plan was published to deal with PCB waste treatment (Australian Government Environment Protection and Heritage Council, 2003). In 1999, the National Environment Protection (Assessment of Site Contamination) Measure 1999 was adopted to provide "a consistent method for assess site contamination across the country" (National Environment Protection Council Service Corporation, 1999). In 2001, a four-year National Dioxins Program (NDP) commenced to study dioxin levels in Australia and the associated risks to humans and the environment (Australian Government Department of the Environment and Heritage, 2005). In 2005, the National Action Plan for Addressing Dioxins (NAP) was adopted to outline detailed actions on dioxins for the Australian government (Australian Government Environment Protection and Heritage Council, 2005). Generally, the dioxin levels in the environment, food and the Australian population are low, when compared with other countries (Australian Government Department of the Environment and Heritage, 2005).
Pearl River Delta, South China
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The Pearl River Delta is a rapid-developing economic region in the Guangdong province of South China. The extensive industrial activities have caused serious environmental problems, especially adversely affecting the river water quality (Ouyang, Zhu, & Kuang, 2005). According to China's national implementation plan for the Stockholm Convention on POPs, the Pearl River Delta is within the second highest dioxin release region in China, the southern part of central China, and the major emission sources indentified within the whole region are metal production and waste incineration (UNEP, 2007). It was also found that the use of pentachlorophenol (PCP) and sodium pentachlorophenol (Na-PCP) in agriculture in this area resulted in contaminations of PCDD/Fs (Wen, Hui, Yang, Liu, & Xu, 2008). Besides, open burning of e-waste in some areas within the Guangdong province was reported to generate PCDD/Fs in atmosphere (Zheng, Leung, Jiao, & Wong, 2008). Scarce research information on dioxin contamination in the Pearl River Delta is available, with most of them measuring dioxin levels in sediments (Fu et al., 2003; Zhang, Peng, Huang, Li, & Zhang, 2009). The sediments in the Pearl River Delta were reported to be more contaminated with dioxins than those in Australia and New Zealand (Zhang et al., 2009).
Although China has ratified the Stockholm Convention on POPs, dioxins have yet been monitored in this country by now, due to lack of research capacity, as well as related government regulations (Zhang, Wang, Li, & Jiang, 2007).
7-Ethoxyresorufin-o-deethylase (EROD) Assay
At current stage, high-resolution gas chromatography and mass spectrometry (HRGC/HRMS) is the standard method for determining dioxins in environmental samples, which provides accurate and detailed information on individual dioxin congeners (Behnisch, Hosoe, & Sakai, 2001). However, when it comes to a lot of samples to be analyzed, the HRGC/HRMS method can cost a large amount of money and time (Li et al., 2002). In contrast, the 7-ethoxyresorufin-o-deethylase (EROD) assay is sensitive, cost-effective and faster than the HRGC/HRMS method. It provides a summation of the overall dioxin levels in the sample (Otte et al., 2008; Tsang et al., 2009).
The principle of the EROD assay is based on the toxic mechanism of dioxins in cells (Figure 2) (USEPA, 2003). Briefly, when dioxins enter the responsive cell, they bind to the aryl hydrocarbon receptor (AhR), which exists as a part of the aryl hydrocarbon receptor complex (AhRC). After binding, the AhR dissociates and translocates into the nucleus. The AhR then forms a heterodimer with the Ah receptor nuclear translocator (Arnt). The heterodimer then binds on the dioxin-responsive element (DRE) of target genes, leading to the expression of CYP1A1 (EROD). The enzyme EROD catalyzes the deethylation of 7-ethoxyresorufin substrate to resorufin, which can be detected by spectrofluorometer (Behnisch et al., 2001; Denison & Nagy, 2003).
Figure 2 Cellular mechanisms of aryl hydrocarbon receptor (AhR). AIP, associated immunophilin-like protein; hsp90, 90 kilodalton heat shock protein; p, sites of phosphorylization; Arnt, AhR nuclear translocator protein; RB, retinoblastoma protein; NF-kB, nuclear transcription factor; HIF, hypoxia inducible factor; DRE, dioxin-responsive element; BTFs, basal transcription factors; TATA, DNA recognition sequence (USEPA, 2003).
This study was based on the following hypothesis: (1) all biological and environmental factors from the two studied regions that affect the dioxin accumulation in oysters are negligible, (2) the toxic responses of individual substances in the oyster extracts are additive, (3) the TEQ values gotten from the EROD assay is comparable to the WHO-TEQ values, (4) human exposure to dioxins by consumption of shellfish can be calculated by determination of its concentration in the oysters in this study.
This study aimed to use EROD assay to investigate dioxin contamination in the New South Wales, Australia, and the Pearl River Delta, China. Oysters were used as bioindicator to assess contamination levels, due to their wide geographical distributions in both studied regions and the ability to accumulate contaminants (Scanes, 1996; Abad, Pérez, Llerena, Caixach, & Rivera, 2003; Hedge, Knott, & Johnston, 2009). Furthermore, as oysters are served as seafood in the studied regions, this study also estimated the dietary intake of dioxins from shellfish for the local populations.
Materials and methods
In the New South Wales (Figure 3), Sydney Rock Oysters (Saccostrea glomerata) were deployed at eight areas along the coast between November 2009 and March 2010. The sampling areas were characterized as extensively modified ones (Botany Bay, Port Kembla, Port Jackson and Port Hacking N), or largely unmodified ones (The Clyde, Jervis Bay, Port Hacking S, and Wagonga Inlet), regarding to their levels of urbanization and industrialization. Port Hacking N and Port Hacking S referred to the north and south side of the port, respectively. In addition, all these areas were divided into inner zones and outer zones for study, based on morphology. About 10 sites, which were in close proximity to rocky reef, were sampled in each area. At each site, 15 oysters were deployed and collected after 12 weeks.
In the Pearl River Delta (Figure 4), Pacific Oysters (Crassostrea gigas), which had grown for two years, were obtained from oyster breeding pools which were located within seven different areas (Shajing, Xixiang, Xinken, Xiashan, Zhuhai, Hengqin and Nan'ao), between October and November 2010. Three sites were sampled within each area and about 6 - 8 oysters were collected at each site.
The weekly consumption amount for shellfish by the New South Wales local people was 80 g/person/wk and the mean population body weight was 67 kg, according to the study of Food Standards Australia New Zealand (FSANZ, 2007). The calculated WDI of dioxins from shellfish was 0.51 pg EROD-TEQ/kg bw/wk.
Due to the absence of shellfish consumption data in the Pearl River Delta, the shellfish consumption data of Taiwan residents was used for the estimation of WDI. This was because of the similar eating habits of residents in these two regions. The weekly consumption of shellfish was 165.9 g/person/wk and the mean population body weight was 60 kg (Lin, Wong, & Li, 2004). The calculated WDI of dioxins from shellfish was 3.07 pg EROD-TEQ/kg bw/wk.
Comparison with international and government standards
Overall, the mean dioxin levels of the analyzed oysters were below the maximum dioxin level for fish muscle and fishery products (8 pg WHO-TEQ/g ww) set in 2006 by the Commission of the European Communities under commission regulation (EC) No. 1881/2006, and the action TEQ level for dioxins in seafood (6 pg WHO-TEQ/g ww) set by the New South Wales (NSW) Food Authority in 2006 (European Commission, 2006; NSW Food Authority, 2006).
The WDIs of dioxins from shellfish in the New South Wales and the Pearl River Delta did not exceed the tolerable weekly intake (TWI) of 7-28 pg WHO-TEQ/kg bw/wk, which was set by WHO in 1998 (WHO, 1998). Furthermore, the WDI values were below the TWI of 14 pg WHO-TEQ/kg bw/wk, which was set by the EC-SCF, and 16.3 pg WHO-TEQ/kg bw/wk, which was proposed by the JECFA and the NHMRC of Australia (European Commission, 2001; JECFA, 2001; Australian Government NHMRC, 2002).
Comparison with international publications
Table 4 presents a summary of dioxin levels in shellfish from international publications. The dioxin concentrations of oysters from the New South Wales in this study were within the range (0.0068-3.4 pg WHO-TEQ/g ww) on bivalves, which was reported by the National Dioxins Program in Australia (Müeller et al., 2004). Also the results were close to the WHO-TEQ levels, 0.071 to 0.63 (mean: 0.37) pg/g ww, detected in the shellfish collected in the Moreton Bay, near the city of Brisbane, in Queensland, Australia (Matthews, Papke, & Gaus, 2008).
According to Kachenko and Singh (2006), and Hedge et al. (2009), heavy industry, which included copper smelter and steelworks, has been in operation in the Port Kembla for more than 70 years, and the inner zone of the port directly received urban and industrial waste water from the nearby areas. Moreover, sewage discharge and waste dumps were identified around the Sydney's coastal bays, like Botany Bay (Spooner, Maher, & Otway, 2003). Further, Port Jackson was reported to have highest recorded sediment and biota dioxin levels in Australia, and in February 2006, a permanent ban on fishing was issued in this area (Birch et al., 2007). It was also found that the organochlorine chemical industry in the upper estuary of Port Jackson caused the dioxin contamination in the downstream waters (Birch et al., 2007; Ying et al., 2009). These previous records may explain the increased dioxin levels that were detected in oysters from the New South Wales in this study. The elevated level of dioxins in The Clyde was unknown, since this area was generally classified as uncontaminated area (Robinson, Maher, Krikowa, Nell, & Hand, 2005). Further investigation on dioxin levels in sediments and other biota tissue samples, as well as identification of potential pollution sources are needed to validate this finding.
In China, Shen et al. (2009) analyzed dioxins in shellfish from the markets in six coastal provinces and found that the mean dioxin concentration was 0.066 pg WHO-TEQ/g ww, with the range of 0.003-0.28, which was much lower than the dioxin concentrations that were detected in the Pearl River Delta in this study. A few previous records indicated that several large waste incineration factories were in use since the end of 1980s in Shenzhen and Zhuhai. In addition, many polyvinyl chloride (PVC) industries occurred in this region (Fu et al., 2003). It was believed that e-waste recycling in this area was also a source of dioxins (Zhang et al., 2007). These factors may contribute to the elevated level of dioxins in the Pearl River Delta. As the monitoring and research data on dioxins in the Pearl River Delta was limited, it was impossible to precisely determine the emission factors of dioxins in this area.
Studies on oysters that were obtained in markets in South Korea reported the mean dioxin concentration of 0.306 pg WHO-TEQ/g ww (Lee et al., 2007). And oysters from markets in Spain were found to have mean dioxin concentration of 0.79 pg WHO-TEQ/g ww (Gomara et al., 2005). In France, bivalves from the coastal waters were measured to have mean WHO-TEQ level of 1.76 pg/g ww (Abarnou & Fraisse, 2002). Fernandes et al. (2008) investigated the dioxin levels in oysters collected in Scotland and reported that the WHO-TEQ value ranged from 0.28 - 0.69 pg/g ww (mean: 0.42, median: 0.35). The mean dioxin level in oysters obtained from a general study in UK was 0.5 pg WHO-TEQ/g ww (UK Food Standards Agency, 2006). Overall, the EROD-TEQ levels of oysters from the Pearl River Delta were ranked high within the WHO-TEQ levels from similar studies in developed countries. The EROD-TEQ levels of oysters from the New South Wales were relatively low when compared with the WHO-TEQ levels in other developed countries.
In addition, there were many studies worldwide which only measured the PCDD/Fs in shellfish. Wen et al. (2008) determined PCDD/Fs in mussels collected from the Changjiang Estuary, which is another most developed region in China, as 1.51 - 4.78 pg WHO-TEQ/g ww. Mussel samples from the Ya-Er Lake and the Dongting Lake in China showed relatively lower PCDD/F levels than those from the Changjiang Estuary (Wu, Schramm, & Kettrup, 2001; Gao, Zheng, Zhang, & Liu, 2006). Bivalves from the Bohai Sea coastline, China had the lowest PCDD/F levels of all Chinese data in the table (0.0001-0.08 pg WHO-TEQ/g ww) (Zhao et al., 2005). In New Zealand, the shellfish were showed to have I-TEQ levels (PCDD/Fs) ranged from 0.015 to 0.26 pg/g ww, whereas the two studies conducted in the US and Canada detected relatively higher levels of PCDD/Fs (Brochu, Moore, & Pelletier, 1995; Scobie, Buckland, Ellis, & Salter, 1999; Marvin, Howell, Kolic, & Reiner, 2002).
It should be noted that when doing comparisons between different studies, many biological variables, which include feeding habit, reproductive status, age and trophic level, and environmental variables, such as sampling location and season may influence the tissue contamination levels (Zhao et al., 2005; Matthews et al., 2008; Wen et al., 2008). Therefore, interpretations of data should be done with care.
A significant increase of dioxin concentration was observed in the inner zone of Port Kembla in the New South Wales, which followed by the outer zone of the Port Kembla and the inner zones of The Clyde, Botany Bay, and Port Jackson. In the Pearl River Delta, South China, Zhuhai, Hengqin, Xinken and Nan'ao were found to have relatively high levels of dioxins. The Pearl River Delta had higher levels of dioxins in oysters and higher estimated weekly dietary intake (WDI) than the New South Wales. None of the dioxin levels detected in these two regions was above the benchmarks set by the European Commission and the NSW Food Authority. The estimated WDIs did not exceed the range set by international guidelines. It was suggested that the Chinese government should take actions to monitor dioxins levels in general populations as well as the environment, and set up related regulations.