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Occurrence of endocrine disrupting compounds in mariculture sediment of Pulau Kukup, Johor, Malaysia
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Occurrence of endocrine disrupting compounds in mariculture sediment of Pulau Kukup, Johor, Malaysia Nur Afifah Hanun Ismaila, Sze Yee Weea, Didi Erwandi Mohamad Haronb, ⁎ Nitty Hirawaty Kamarulzamanc, Ahmad Zaharin Arisa, a
Department of Environmental Sciences, Faculty of Environmental Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Shimadzu-UM Centre of Xenobiotic Studies, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia c Department of Agribusiness and Bioresource Economics, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b
ARTICLE INFO
ABSTRACT
Keywords: Endocrine disrupting compounds (EDCs) Sediment Soxhlet extraction Pulau Kukup, Malaysia Mariculture zone
Endocrine-disrupting compounds (EDCs) such as hormones, pesticides, phenolic compounds, and pharmaceuticals compounds can cause adverse effects on humans, animals, and other living organisms. One of the largest mariculture areas situated in Pulau Kukup, Johor, Malaysia, is actively involved in exporting marine fish to other countries worldwide. This paper aims to provide baseline data on the level of EDC pollutants found in mariculture sediments in Malaysia since no reports have investigated this issue. Calculated samples recovered are between 50.39 and 129.10% at 100 ng/g spiking level. The highest concentration in the sediment samples was bisphenol A (0.072–0.389 ng/g dry weight) followed by diethylstilbestrol (< 0.208–0.331 ng/g dry weight) and propranolol (< 0.250–0.275 ng/g dry weight). Even though the concentrations of the targeted compounds obtained were low, their effects could become more evident longer term, which raises not only environmental health concerns but the potential risk to humans.
Endocrine-disrupting compounds (EDCs) comprise of natural and synthetic compounds that are readily discharged into the environment through household, municipal, hospital, industrial, and livestock waste (Wang et al., 2013; Aris et al., 2014; Salgueiro-González et al., 2015; Tan et al., 2018). Both natural and artificial EDCs commonly exist in the environment in the range of pg/L to ng/L (Beck et al., 2005; Hibberd et al., 2009) in the form of active ingredients found in food products and other consumer products comprising of natural and artificial hormones, medicines and pharmaceuticals, industrial and household chemicals, pesticides, alkylphenol, and plasticiser (Bartelt-Hunt et al., 2009; Kabir et al., 2015; Grześkowiak et al., 2016; Niu and Zhang, 2018; De Solla et al., 2016). Not to mention, EDCs have the ability to mimic and block the endocrine system in humans causing severe effects such as cancer, abnormal reproductive growth, and metabolic disorders (i.e. diabetes, obesity, and endometriosis), and a wide variety of problems associated with human well-being (Esteban et al., 2014; Legler et al., 2015; Giulivo et al., 2016; Ismail et al., 2018). EDCs can also disrupt various bodily functions with different pathways and mechanisms. However, the study of EDCs in the context of the marine ecosystem is relatively scarce compared to the study of EDCs in humans, inland animals, ecological health, riverine and estuarine ecosystems (Robinson ⁎
et al., 2009; Bayen et al., 2013; Aris et al., 2014; Bayen et al., 2014; Omar et al., 2018; Wee et al., 2019). Notably, the exposure of EDCs can cause both bioaccumulation and biomagnification in the marine ecosystem, associated with potential health effects such as intersex, skewed sex ratios and reduced gonadal development and viability (Gaspare et al., 2009; Martin and Grant, 2019). The scarcity of data relating to EDCs in the marine ecosystem is mainly due to the analytical problems associated with low-quality assurance and quality control in detecting and quantifying EDCs (Beck et al., 2005). As such, given these difficulties, the knowledge regarding the occurrence, distribution, fate, and effects of these pollutants in marine environments remains a persistent issue that requires further research and investigation. On the other hand, while there are numerous studies on EDCs found in environmental matrices, a suitable method for quantifying multiresidues of EDCs in one single execution remains inadequate (Kim and Carlson, 2005). Moreover, given sedimentation, resuspension of the bed sediment, high salinity, and high matrix effect which contains lots of various organic matter, analysis of sediment matrices from the marine ecosystem is difficult to achieve compared to that of other matrices. Even though numerous analytical procedures have been developed and optimised to measure the presence of EDCs in the environment, prior studies have only focused on analysing water samples, with less interest
Corresponding author. E-mail address: [email protected] (A.Z. Aris).
https://doi.org/10.1016/j.marpolbul.2019.110735 Received 15 November 2018; Received in revised form 12 November 2019; Accepted 14 November 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Nur Afifah Hanun Ismail, et al., Marine Pollution Bulletin, https://doi.org/10.1016/j.marpolbul.2019.110735
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or concern shown regarding sediment analysis. According to Zhou and Broodbank (2014), in order to understand the long-term occurrence of EDC pollutants, sediment analysis is vastly needed instead of merely focusing on analysing water. Sediment is one of the crucial environmental matrices in the surrounding ecosystem, having numerous types of EDCs (chemicals and pollutants) remaining and/or residing in the sediment matrix of the marine environment (Zhou and Broodbank, 2014; Pintado-Herrera et al., 2016; Omar et al., 2017). Various types of pollutants that are deposited and accumulated in the sediment may disrupt the ecosystem directly or indirectly, impacting on the surrounding environment since sediment plays an extremely important role in maintaining the food web and thus acts as a pool of pollutants for bioaccumulation and trophic transfer (Burton Burton Jr, 2002). As such, information concerning the concentration levels of EDCs could be seen as a crucial tool in evaluating the impact of human activities on the aquatic ecosystem. Although no data as yet has been published in Directive 2008/105/EC (Pintado-Herrera et al., 2016). The total fishery production in Malaysia during 2017 was 1.7 million tonnes, with close to 1.5 million tonnes from capture and 0.2 million tonnes from aquaculture (excluding seaweeds) (FAO, 2019). The aquaculture production for brackish water, freshwater, and marine fish in Malaysia was 105,281, 103,096, and 16,171 t, respectively (FAO, 2019). The fishing industry in Malaysia has flourished with 70 breeders, and acquiring over 7500 cages in the Pulau Kukup area of Johor (The Star, 2006), representing the world's second-largest mangrove island actively involved in mariculture, industrial and commercial activities (Ismail et al., 2018). Covering a total area of 6.47 km2 the island offers physical protection of its shoreline, which acts as a barrier against strong winds and tides for the low-density coastal settlement (Barau and Stringer, 2015). Fish and other marine products are exported to neighbouring countries like Hong Kong, Taiwan, and Singapore (Barau and Stringer, 2015). Situated near the mariculture cage, there is a national park (Pulau Kukup National Park) which is protected by the state of Johor. In 1997, the Johor State Government gazetted Pulau Kukup as a state park for tourism and conservation purposes (Johor National Park, 2019). Therefore, based on the above discussion, this paper aims to firstly, investigate the occurrence of multi-residues of EDCs (pharmaceutical, phenolic compounds, hormone, and pesticides) in marine sediment, and secondly, elucidate the distribution pattern of EDCs in the collection of marine sediment from Pulau Kukup, Johor. The present study targets multiclass EDCs (pharmaceuticals, hormones, pesticides, and plasticisers) based on monitored occurrences in the ecosystem in the context of Malaysia and other countries. Most of the targeted compounds such as steroids hormone (Ismail et al., 2019; Omar et al., 2018; Omar et al., 2019; Wee et al., 2019), pharmaceuticals (Meierjohann et al., 2016; Omar et al., 2018; Ismail et al., 2019; Omar et al., 2019; Wee et al., 2019), pesticides (Wee et al., 2016; Omar et al., 2018; Wee et al., 2019), and phenolics compounds (Chang et al., 2011; Niu and Zhang, 2018; Omar et al., 2018; Ismail et al., 2019; Omar et al., 2019; Wee et al., 2019) have been detected in most matrices samples (water, sediment, or biota) worldwide. In fact, EDCs have been chosen to study based on their broad and growing application. For example, globally, the use of BPA has increased over time having an annual production exceeding 10 million tonnes (Giulivo et al., 2016). Whereas, pesticides used globally in agriculture and aquaculture activities are utilised to prevent disease and improve the stock quality (Ali et al., 2016). Similarly, steroid hormone and pharmaceutical products are used in the medical sector, such as 17α-ethynylestradiol used as female contraceptive pills (Ismail et al., 2017). In this study, seventeen EDC multi-residues consisting of pesticides (quinalphos, diazinon, and chlorpyrifos), pharmaceuticals and medical drugs (propranolol, atorvastatin, diethylstilbestrol, nitrofurazone, dexamethasone, primidone, sulfamethoxazole, and diclofenac), steroid hormone (testosterone, progesterone, 17β-estradiol, estrone, and 17α-ethynylestradiol), and phenolic compounds (bisphenol A) were selected.
The pure standard of testosterone, progesterone, dexamethasone, primidone, propranolol, atorvastatin, sulfamethoxazole, diclofenac, diethylstilbestrol, quinalphos, diazinon, chlorpyrifos, nitrofurazone, 17β-estradiol, 17α-ethynylestradiol, estrone, and bisphenol A were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Isotopically labelled compounds, diazinon-d10, primidone-d9, diclofenac-d4, sulfamethoxazole-d4, 17β-estradiol-d4, and bisphenol A-d8 used as internal standards were obtained from Toronto Research Chemicals Inc. (Ontario, Canada) and Cambridge Isotope Laboratories (CIL) (Massachusetts, USA). All LCMS and high-performance liquid chromatography (HPLC) grade solvents like methanol (MeOH), acetone, acetonitrile (ACN) were obtained from Fisher Scientific (New Jersey, USA). Strata-X polymeric reversed-phase cartridges (C18, 200 mg/6 mL) were purchased from Phenomenex (California, USA), Soxhlet apparatus for sediment extraction was obtained from Witeg (Wertheim, Germany) and ammonium hydroxide (NH4OH) and methyl-tert-butyl ether (MTBE) were obtained from Fisher Scientific (Loughborough, UK). Aluminium oxide 90 active neutral and hydromatrix were obtained from Merck (Darmstadt, Germany) and Agilent Technologies (California, USA) respectively. Throughout the analyses, deionised water (water sensitivity > 18.2 MΩ cm at 25 °C) was purified from a Milli-Q water purification system (Millipore, Massachusetts, USA). The primary stock solutions were prepared and dissolved in pure MeOH and stored under −20 °C in the dark. Both the mixed external and internal standard was prepared from the individual standard for spiking during method recovery and in sample extraction. As mentioned earlier in this study, Pulau Kukup (refer to Fig. 1 and Table 1) is recognised as one of the most significant mariculture areas in Malaysia involved in a variety of marine fish culture. The sediment samples were collected along the mariculture cages where S1-S3 are located opposite north port and S4-S6 are located near to the Kukup Ferry Terminal. Sediment samples at a depth between 4.2 and 7.6 m from the surface water were collected from six different points of the mariculture cages (duplicate sample for each point, n = 12) using an Ekman grab sampler. Approximately 500 g of sediment as samples, were collected and stored in a pre-cleaned methanol-rinsed glass container. All samples were labelled correctly and kept in a cooler box before stored in the chiller at 4 °C. The samples collected were then left to dry under ambient air temperature to remove any moisture. The samples were then ground, sieved, and stored in a glass container before analysis. Total organic carbon (TOC) in the samples was analysed using a Total Carbon Analyser (TOC-VCSH, Shimadzu, Japan). The extraction and clean-up steps were adapted and modified based on the work of Omar et al. (2017) and Ismail et al. (2019) and were undertaken using Soxhlet extraction along with solid-phase extraction (SPE) as the clean-up step before analysis using liquid chromatographytandem mass spectrometry (LC-MS/MS). Sediment samples (approximately 5.00 g) were next mixed with pre-weighed comprising of hydromatrix (0.25 g) and aluminium oxide (0.25 g), followed by grounding and spiking of the internal standards. Approximately 200 mL of MeOH:acetone (50:50) was used in the Soxhlet extraction process (8 h), in which the apparatus was wrapped with aluminium foil to prevent any light penetrating the sample during the extraction process. Before the solid-phase clean-up extraction method, the samples were reduced to < 2 mL once going through a rotary evaporator and reconstituted with ACN:H2O (10:90) to 15–20 mL. Strata-X polymeric reversed-phase C18 cartridges were chosen for solid-phase extraction in which each cartridge was conditioned with 5 mL of ACN, 5 mL of ACN:MTBE:NH4OH (70:25:5), and 5 mL of ACN:H2O (10:90). Next, the sample was loaded into the cartridge and rinsed with 10 mL of ACN:H2O (10:90) to clean up any matrix interference. The cartridge was left to dry for about 3 to 5 min. Analytes were then eluted with 15 mL ACN:MTBE:NH4OH (70:25:5), pre-concentrating the extracts to 1–2 mL using a rotary evaporator. The final extracts were concentrated under a gentle nitrogen blow and reconstituted with ACN:H2O (30:70) 2
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Fig. 1. Sampling site at Pulau Kukup, Johor, Malaysia.
to a final volume of 1 mL in an amber vial. Before the instrument analysis, the sample was filtered with 0.20 μm polytetrafluoroethylene (PTFE) membrane filter (25 mm, Phenomenex). For the entire analytical procedure, quality assurance and quality control measures were established. After washing the glassware apparatus using detergent (Decon 90) to remove any impurities and contaminants, it was then rinsed using deionised water, followed by an organic solvent. To check the interference plus eradicate cross-contamination, every batch of samples was monitored by performing one recovery procedure. Several parameters, including recovery, linearity, limits of detection, stability and robustness were assessed for SPE-LCMS/MS development and optimisation. The method detection limit (MDL) and method quantification limit (MQL) for all targeted multiresidues of EDCs were performed by calculating the signal-to-noise ratio (SNR) based on 3:1 and 10:1 ratio, respectively. To identify and determine the retention time (Rt), each multi-residue of the EDCs in the samples using LC-MS/MS, and Rt for authentic standard solution was verified. The extracted samples were next analysed using LC-MS/MS (Shimadzu 8030, Japan). Chromatographic separation for the targeted EDCs in the sediment samples were carried out by employing two different instrument methods; method A was acidic based while method B was basic based. For the acidic method, mobile phase A (water) was prepared by mixing 0.2% acetic acid in ultrapure water, and mobile phase B (organic) was prepared by using 100% pure MeOH. The determination of the column used was based on the suitability of the column to suppress the compound peak that appeared during the first 10 min of running the instrument. The column used in method A was a
Luna 5 μm PFP (2) 100A, 150 × 2.00 mm column. In the basic based method, mobile phase A (water) was prepared by mixing 0.01% NH4OH in ultrapure water, while mobile phase B (organic) was prepared using 100% pure MeOH. Column used in this method was a Kinetex® 5 μm EVO C18 100A, 150 × 2.1 mm column. A different mobile phase and column were prepared for method optimisation. The flow rate used for method A was fixed at 0.30 mL/min and 0.35 mL/min for method B. Whereas, the column temperature and run time was fixed at 40 °C and 10 min respectively. The injection volume for running the samples was set at 10 μL. The optimised method was validated for various validation parameters such as linearity, method recovery (%), MDL and MQL (Thompson et al., 2002; Omar et al., 2017). The linearity of the multiresidues was evaluated at five different concentrations of standard solutions ranging from 5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, and 200 ng/mL respectively. A strong positive and significant linearity indicating the reliability and robustness of the method was obtained for all targeted compounds with the goodness of fit (r) ranging between 0.9566 and 0.9992. The range for MDL and MQL for the targeted compounds was calculated between 0.001 and 0.300 ng/g and 0.004 to 1.000 ng/g, respectively. Using the spiking the method at a concentration of 100 ng/g, the method recovery for all targeted compounds in the present study was in the range between 50.39% and 129.10% (refer to Table 2), representing acceptable accuracy for the analytical method. The targeted EDCs in the present study, consisting of multiclass residues, (i.e., steroid hormones, pharmaceutical and medical drugs, pesticides, and phenolic compounds), with method recovery were in the
Table 1 Coordinate and description of sampling site at Pulau Kukup, Johor, Malaysia. Station
Coordinate
Description
1 2 3 4 5 6
N N N N N N
Near mangrove area, opposite to the north port Opposite to the north port, near with residential area Near mangrove area, opposite to the north port Near Kukup Ferry Terminal Near Kukup Ferry Terminal Near Kukup Ferry Terminal
01°19.958′ E 103°26.209′ 01°20.158′ E 103°26.205′ 01°20.083′ E 103°26.357′ 01°19.0491′ E 102°26.336′ 01°19.182′ E 103°26.475′ 01°19.101′ E 103°25.679′
3
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Approximately, six compounds were detected at S1, with most of the compounds observed having the highest concentration compared to the other sampling sites. Testosterone (0.094 ng/g dry weight), dexamethasone (0.144 ng/g dry weight), propranolol (0.275 ng/g dry weight), and diclofenac (0.228 ng/g dry weight) displayed the highest concentration detected at sampling point S1. While other EDC contaminants were detected at different sampling points indicating that the anthropogenic and mariculture activities in the ocean significantly influence the concentration of EDCs in marine sediment. Furthermore, there were eight multi-residues of EDCs (testosterone, estrone, dexamethasone, primidone, propranolol, diclofenac,
Table 2 Linearity, method recovery percentage, MDL and MQL for targeted compounds in sediment sample. Compounds Testosterone Progesterone Dexamethasone Primidone Propranolol Atorvastatin Sulfamethoxazole Diclofenac Diethylstilbestrol Quinalphos Diazinon Chlorpyrifos Nitrofurazone 17β-estradiol 17α-eEthynylestradiol Estrone Bisphenol A
Linearity (r)
Recovery sediment (%) (RSD)
MDL (ng/g)
MQL (ng/g)
0.9982 0.9978 0.9894 0.9982 0.9966 0.9965 0.9960 0.9566 0.9959 0.9992 0.9954 0.9948 0.9903 0.9977 0.9957 0.9974 0.9980
129.10 (29.50) 124.05 (13.71) 91.67 (4.67) 100.17 (8.17) 77.91 (9.05) 53.25 (0.65) 101.18 (1.70) 93.94 (14.11) 53.76 (8.05) 57.62 (5.18) 93.94 (14.11) 63.59 (12.01) 62.62 (14.60) 82.32 (11.18) 50.39 (23.09) 58.18 (9.48) 102.65 (2.00)
0.015 0.016 0.040 0.015 0.250 0.001 0.002 0.093 0.208 0.037 0.006 0.093 0.043 0.049 0.300 0.013 0.004
0.051 0.055 0.134 0.051 0.033 0.004 0.007 0.062 0.694 0.123 0.020 0.310 0.144 0.163 1.000 0.046 0.012
RSD: relative standard deviation; MDL: method detection limit; MQL: method quantification limit.
range between 50 and 129% at the spiking concentration of 100 ng/g (Table 2). Notably, the performance was even more accurate and sensitive compared to previous studies such as US EPA (2007), which reported a range between 5 and 180% for pharmaceutical analysis in sediments. Although, the present study involved the analysis of multiclass EDCs. It is both difficult and challenging to achieve a high recovery percentage for each compound during examining multi-class EDCs in a single analytical run (Wee et al., 2016). Besides, the solubility for every EDC compound was different, which can be a further limitation in acquiring a high percentage of method recovery (> 90%). Also, some of the targeted compounds such as atorvastatin, sulfamethoxazole, and bisphenol A achieved a very low MDL < 0.010 ng/g, and most of the other compounds had lower MDL compared to previous studies. Table 2 displays the details of linearity, percentage of method recovery with relative standard deviation (RSD), MDL, and MQL for the multi-residues of EDCs. All data were analysed using statistical software, IBM SPSS (Statistical Package for Social Science) version 23. Spatial variation and distribution were evaluated using descriptive statistics in order to observe the significant association of targeted emerging organic contaminants with physico-chemical characteristic. The concentration and spatial distribution of EDCs in the sediments collected from Pulau Kukup, are depicted in Fig. 2 and are further detailed in Table 3. All detected compounds showed a coefficient of variation > 30%, indicating a high variation of EDCs concentration among the sampling points. Sediment analysis revealed the presence of multi-residues of EDCs in all sampling sites (S1-S6). Testosterone, estrone, dexamethasone, primidone, propranolol, diclofenac, diethylstilbestrol, and bisphenol A were detected in the sample, except for other residues which were below MDL at all sampling stations. Primidone and bisphenol A were detected in all samples (S1-S6) and other contaminants like testosterone, dexamethasone, propranolol, and diclofenac were ubiquitously detected in most of the sampling stations. The results obtained showed that regularly monitoring needs to be performed in the marine ecosystem at Pulau Kukup. Testosterone, estrone, and dexamethasone were the most prominent compounds detected in the sediment samples, which were collected at 87.39%, 64.41%, and 53.76% respectively (Table 3) whereas propranolol was less detected in the sediment samples at 18.03%. The highest concentration was observed for bisphenol A (0.072–0.389 ng/g dry weight) followed by diethylstilbestrol (< 0.208–0.331 ng/g dry weight) and propranolol (< 0.250–0.275 ng/g dry weight).
Fig. 2. Distribution pattern of multi-residue for endocrine disrupting compounds in sediment of Pulau Kukup, Johor. 4
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potential lack of efficient regulation of discharges from residential areas and livestock activities. Bisphenol A, commonly known as an important industrial chemical, was detected in all sampling sites, indicating the widespread application of bisphenol A in those industries located along with the mariculture cages (Ismail et al., 2018; Wang et al., 2019). Plasticiser is the most produced and consumed substance globally (Pintado-Herrera et al., 2016), whereas bisphenol A is a chemical involved in the production of polycarbonate plastics and epoxy resins. In addition to bisphenol A, primidone was also detected in every site of the sampling points (S1–S6), revealing the possible improper disposal of unused medications (Verlicchi and Zambello, 2016; Omar et al., 2018). Previous studies conducted in the Asia region reported that diclofenac and propranolol had been detected in the effluent of wastewater treatment plants (WWTP) (Pal et al., 2010) and polyaromatic hydrocarbons (PAHs), persistent organic pollutants (POPs), alkylphenol ethoxylates (APEs) likewise, have been detected along the Singapore Straits, located close to the Straits of Malacca (Sin et al., 2016). The occurrence of emerging organic pollutants along the Singapore Straits also indicates that these pollutants are potentially penetrating the Straits of Malacca through the movement of water currents. The relationship between physico-chemical properties (total organic carbon and pH) and the concentration of multi-residues of EDCs in the marine sediment samples collected from Pulau Kukup was described using Pearson's analysis correlation coefficient (r). The pH range for the sediment collected ranged between 6.66 and 7.95, while the total organic compound ranged between 1.41 and 1.83%. Also, significant correlation of targeted compounds with the total organic compounds, ranged from a weak positive correlation, diclofenac and propranolol (r = 0.332, p < 0.05), to a strong positive correlation estrone (r = 0.692, p < 0.05) and testosterone (r = 0.649, p < 0.05) as shown in Table 4. The occurrence and distribution of EDCs concentrations in the sediment samples were influenced by total organic carbon (Omar et al., 2018) and hormone group (estrone and testosterone) showing a strong positive correlation with total organic carbon compared to the other group of EDCs. Aside from total organic carbon, some compounds also showed a significant association with pH testosterone (r = 0.653, p < 0.05), dexamethasone (r = 0.684, p < 0.05), propranolol (r = 0.760, p < 0.05), diclofenac (r = 0.760, p < 0.05), and bisphenol A (r = 0.183, p < 0.05). The data also showed that the pH values were influenced by the sorption of EDCs in sediment matrices. The present study presented the data and information on EDC contaminants in marine sediment matrices collected from Pulau Kukup, Johor. Three groups of EDCs were observed in the samples namely, steroid hormone (estrone), phenolic compounds (bisphenol A), and pharmaceutical (testosterone, dexamethasone, primidone, propranolol, diclofenac, and diethylstilbestrol). The presence of EDCs in the sediment samples signified potential unregulated discharges that originated from anthropogenic activities and influenced by naturogenic factors surrounding the mariculture sites, including those from upstream. The contamination is of great concern given these contaminants could lead to having a severe impact on the environment, thus causing an imbalance to the ecosystem. Also, given the adverse effects of exposure to these contaminants, humans, being on top of the food chain/food web, could also suffer as a result since long-term exposure to EDCs will cause health problems, such as a reduction in the human reproduction system, disease such as cancer, and alteration of the body's functions. In gaining a better understanding of the occurrence, distribution, pathways and fate of EDCs contaminants in the surrounding ecosystem, further study should include other diverse matrices such as surface water, aquatic animals, plants, and humans. The baseline data would be extremely beneficial in the future to profile the current trend of EDCs pollutants and consequential pollution in the marine ecosystem, especially in the mariculture area. Also, future research should investigate the potentially toxic effects of such contaminants towards humans via dietary consumption and the elimination process of emerging pollutants in the marine ecosystem.
Fig. 2. (continued)
diethylstilbestrol, and bisphenol A) that were present in the marine sediment collected from Pulau Kukup. Based on the literature from other studies, the sources of EDCs are believed to originate from wastewater treatment plants, domestic/medical waste discharge, livestock activities, industrial waste discharge, and agricultural activities. The presence of testosterone and estrone in the environment are typically observed from sewage treatment facility waste, uniquely human and animal wastes (Lorenzen et al., 2004; Raman et al., 2004; Zhang et al., 2013; Li, 2014; Pessoa et al., 2014). Estrone was also detected in the marine sediment, while 17β-estradiol and 17α-ethynylestradiol were found to be relatively less abundant in the samples. Indeed, the occurrence of steroid hormones along the coastal area indicates the 5
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Table 3 Concentration of multi-residue EDCs contaminants in sediment samples from Pulau Kukup, Johor. Compounds
Concentration in sediment (ng/g dry weight) (n = 12)
Testosterone Progesterone Estrone 17β-estradiol 17α-ethynylestradiol Dexamethasone Primidone Propranolol Atorvastatin Sulfamethoxazole Diclofenac Diethylstilbestrol Nitrofurazone Quinalphos Diazinon Chlorphyrifos Bisphenol A
ST1
ST2
ST3
ST4
ST5
ST6
Range
% CV
Percentage of detection (%)
0.094 < MDL < MDL < MDL < MDL 0.144 0.024 0.275 < MDL < MDL 0.228 < MDL < MDL < MDL < MDL < MDL 0.238
0.021 < MDL < MDL < MDL < MDL < MDL 0.030 < MDL < MDL < MDL < MDL 0.331 < MDL < MDL < MDL < MDL 0.389
0.068 < MDL 0.038 < MDL < MDL 0.042 0.097 < MDL < MDL < MDL < MDL 0.226 < MDL < MDL < MDL < MDL 0.139
< MDL < MDL 0.020 < MDL < MDL < MDL 0.077 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.367
0.025 < MDL < MDL < MDL < MDL < MDL 0.048 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.160
< MDL < MDL 0.018 < MDL < MDL < MDL 0.020 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.072
< 0.015–0.094 NA < 0.014–0.038 NA NA < 0.040–0.144 0.020–0.097 < 0.250–0.275 NA NA < 0.093–0.228 < 0.208–0.331 NA NA NA NA 0.072–0.389
133.82 NA 123.61 NA NA 186.50 63.41 244.95 NA NA 244.95 159.05 NA NA NA NA 56.45
87.39 NA 64.41 NA NA 53.76 100.00 18.03 NA NA 32.90 40.10 NA NA NA NA 100.00
MDL: Method detection limit, NA: Not available, CV: Coefficient of variation.
Table 4 Statistical analysis for correlation coefficient, TOC, pH, and concentration of targeted EDCs in marine sediment based on Pearson analysis.
TOC pH Testosterone Estrone Dexamethasone Primidone Propranolol Diclofenac Diethylstilbestrol Bisphenol A ⁎ ⁎⁎
TOC
pH
Testosterone
Estrone
Dexamethasone
Primidone
Propranolol
Diclofenac
Diethylstilbestrol
Bisphenol A
1.000 −0.079 0.649 0.692 0.561 0.473 0.332 0.332 −0.090 −0.500
1.000 0.653 −0.729 0.684 −0.368 0.760 0.760 −0.138 0.183
1.000 −0.017 0.899⁎ 0.070 0.760 0.760 0.107 −0.136
1.000 −0.169 0.755 −0.400 −0.400 0.134 −0.339
1.000 −0.181 0.957⁎⁎ 0.957⁎⁎ −0.182 −0.059
1.000 −0.396 −0.396 0.189 0.045
1.000 1.000⁎⁎ −0.308 0.040
1.000 −0.308 0.040
1.000 0.354
1.000
Correlation is significant at the 0.05 level (2-tailed). Correlation is significant at the 0.01 level (2-tailed).
Author contributions section
References
Nur Afifah Hanun Ismail: Method Development, Laboratory Analysis, Data Analysis, Writing-Original draft. Sze Yee Wee: Data Analysis, Writing-Reviewing and Editing. Didi Erwandi Mohamad Haron: Method Development. Nitty Hirawaty Kamarulzaman: Writing-Reviewing and Editing. Ahmad Zaharin Aris: Supervision, Writing- Reviewing and Editing, Research Design, and Funding acquisition.
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Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was fully supported by Geran Putra Malaysia (GP/2017/ 9574800) and International Environmental Research Institute, IERI Research Grant (6389800) from Gwangju Institute of Science and Technology, Gwangju, Korea. Ismail NAH want to thank Universiti Putra Malaysia, UPM for allowance through Graduate Research Fellowship (GRF) programme. 6
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Baseline
Occurrence of endocrine disrupting compounds in mariculture sediment of Pulau Kukup, Johor, Malaysia Nur Afifah Hanun Ismaila, Sze Yee Weea, Didi Erwandi Mohamad Haronb, ⁎ Nitty Hirawaty Kamarulzamanc, Ahmad Zaharin Arisa, a
Department of Environmental Sciences, Faculty of Environmental Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Shimadzu-UM Centre of Xenobiotic Studies, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia c Department of Agribusiness and Bioresource Economics, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b
ARTICLE INFO
ABSTRACT
Keywords: Endocrine disrupting compounds (EDCs) Sediment Soxhlet extraction Pulau Kukup, Malaysia Mariculture zone
Endocrine-disrupting compounds (EDCs) such as hormones, pesticides, phenolic compounds, and pharmaceuticals compounds can cause adverse effects on humans, animals, and other living organisms. One of the largest mariculture areas situated in Pulau Kukup, Johor, Malaysia, is actively involved in exporting marine fish to other countries worldwide. This paper aims to provide baseline data on the level of EDC pollutants found in mariculture sediments in Malaysia since no reports have investigated this issue. Calculated samples recovered are between 50.39 and 129.10% at 100 ng/g spiking level. The highest concentration in the sediment samples was bisphenol A (0.072–0.389 ng/g dry weight) followed by diethylstilbestrol (< 0.208–0.331 ng/g dry weight) and propranolol (< 0.250–0.275 ng/g dry weight). Even though the concentrations of the targeted compounds obtained were low, their effects could become more evident longer term, which raises not only environmental health concerns but the potential risk to humans.
Endocrine-disrupting compounds (EDCs) comprise of natural and synthetic compounds that are readily discharged into the environment through household, municipal, hospital, industrial, and livestock waste (Wang et al., 2013; Aris et al., 2014; Salgueiro-González et al., 2015; Tan et al., 2018). Both natural and artificial EDCs commonly exist in the environment in the range of pg/L to ng/L (Beck et al., 2005; Hibberd et al., 2009) in the form of active ingredients found in food products and other consumer products comprising of natural and artificial hormones, medicines and pharmaceuticals, industrial and household chemicals, pesticides, alkylphenol, and plasticiser (Bartelt-Hunt et al., 2009; Kabir et al., 2015; Grześkowiak et al., 2016; Niu and Zhang, 2018; De Solla et al., 2016). Not to mention, EDCs have the ability to mimic and block the endocrine system in humans causing severe effects such as cancer, abnormal reproductive growth, and metabolic disorders (i.e. diabetes, obesity, and endometriosis), and a wide variety of problems associated with human well-being (Esteban et al., 2014; Legler et al., 2015; Giulivo et al., 2016; Ismail et al., 2018). EDCs can also disrupt various bodily functions with different pathways and mechanisms. However, the study of EDCs in the context of the marine ecosystem is relatively scarce compared to the study of EDCs in humans, inland animals, ecological health, riverine and estuarine ecosystems (Robinson ⁎
et al., 2009; Bayen et al., 2013; Aris et al., 2014; Bayen et al., 2014; Omar et al., 2018; Wee et al., 2019). Notably, the exposure of EDCs can cause both bioaccumulation and biomagnification in the marine ecosystem, associated with potential health effects such as intersex, skewed sex ratios and reduced gonadal development and viability (Gaspare et al., 2009; Martin and Grant, 2019). The scarcity of data relating to EDCs in the marine ecosystem is mainly due to the analytical problems associated with low-quality assurance and quality control in detecting and quantifying EDCs (Beck et al., 2005). As such, given these difficulties, the knowledge regarding the occurrence, distribution, fate, and effects of these pollutants in marine environments remains a persistent issue that requires further research and investigation. On the other hand, while there are numerous studies on EDCs found in environmental matrices, a suitable method for quantifying multiresidues of EDCs in one single execution remains inadequate (Kim and Carlson, 2005). Moreover, given sedimentation, resuspension of the bed sediment, high salinity, and high matrix effect which contains lots of various organic matter, analysis of sediment matrices from the marine ecosystem is difficult to achieve compared to that of other matrices. Even though numerous analytical procedures have been developed and optimised to measure the presence of EDCs in the environment, prior studies have only focused on analysing water samples, with less interest
Corresponding author. E-mail address: [email protected] (A.Z. Aris).
https://doi.org/10.1016/j.marpolbul.2019.110735 Received 15 November 2018; Received in revised form 12 November 2019; Accepted 14 November 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Nur Afifah Hanun Ismail, et al., Marine Pollution Bulletin, https://doi.org/10.1016/j.marpolbul.2019.110735
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or concern shown regarding sediment analysis. According to Zhou and Broodbank (2014), in order to understand the long-term occurrence of EDC pollutants, sediment analysis is vastly needed instead of merely focusing on analysing water. Sediment is one of the crucial environmental matrices in the surrounding ecosystem, having numerous types of EDCs (chemicals and pollutants) remaining and/or residing in the sediment matrix of the marine environment (Zhou and Broodbank, 2014; Pintado-Herrera et al., 2016; Omar et al., 2017). Various types of pollutants that are deposited and accumulated in the sediment may disrupt the ecosystem directly or indirectly, impacting on the surrounding environment since sediment plays an extremely important role in maintaining the food web and thus acts as a pool of pollutants for bioaccumulation and trophic transfer (Burton Burton Jr, 2002). As such, information concerning the concentration levels of EDCs could be seen as a crucial tool in evaluating the impact of human activities on the aquatic ecosystem. Although no data as yet has been published in Directive 2008/105/EC (Pintado-Herrera et al., 2016). The total fishery production in Malaysia during 2017 was 1.7 million tonnes, with close to 1.5 million tonnes from capture and 0.2 million tonnes from aquaculture (excluding seaweeds) (FAO, 2019). The aquaculture production for brackish water, freshwater, and marine fish in Malaysia was 105,281, 103,096, and 16,171 t, respectively (FAO, 2019). The fishing industry in Malaysia has flourished with 70 breeders, and acquiring over 7500 cages in the Pulau Kukup area of Johor (The Star, 2006), representing the world's second-largest mangrove island actively involved in mariculture, industrial and commercial activities (Ismail et al., 2018). Covering a total area of 6.47 km2 the island offers physical protection of its shoreline, which acts as a barrier against strong winds and tides for the low-density coastal settlement (Barau and Stringer, 2015). Fish and other marine products are exported to neighbouring countries like Hong Kong, Taiwan, and Singapore (Barau and Stringer, 2015). Situated near the mariculture cage, there is a national park (Pulau Kukup National Park) which is protected by the state of Johor. In 1997, the Johor State Government gazetted Pulau Kukup as a state park for tourism and conservation purposes (Johor National Park, 2019). Therefore, based on the above discussion, this paper aims to firstly, investigate the occurrence of multi-residues of EDCs (pharmaceutical, phenolic compounds, hormone, and pesticides) in marine sediment, and secondly, elucidate the distribution pattern of EDCs in the collection of marine sediment from Pulau Kukup, Johor. The present study targets multiclass EDCs (pharmaceuticals, hormones, pesticides, and plasticisers) based on monitored occurrences in the ecosystem in the context of Malaysia and other countries. Most of the targeted compounds such as steroids hormone (Ismail et al., 2019; Omar et al., 2018; Omar et al., 2019; Wee et al., 2019), pharmaceuticals (Meierjohann et al., 2016; Omar et al., 2018; Ismail et al., 2019; Omar et al., 2019; Wee et al., 2019), pesticides (Wee et al., 2016; Omar et al., 2018; Wee et al., 2019), and phenolics compounds (Chang et al., 2011; Niu and Zhang, 2018; Omar et al., 2018; Ismail et al., 2019; Omar et al., 2019; Wee et al., 2019) have been detected in most matrices samples (water, sediment, or biota) worldwide. In fact, EDCs have been chosen to study based on their broad and growing application. For example, globally, the use of BPA has increased over time having an annual production exceeding 10 million tonnes (Giulivo et al., 2016). Whereas, pesticides used globally in agriculture and aquaculture activities are utilised to prevent disease and improve the stock quality (Ali et al., 2016). Similarly, steroid hormone and pharmaceutical products are used in the medical sector, such as 17α-ethynylestradiol used as female contraceptive pills (Ismail et al., 2017). In this study, seventeen EDC multi-residues consisting of pesticides (quinalphos, diazinon, and chlorpyrifos), pharmaceuticals and medical drugs (propranolol, atorvastatin, diethylstilbestrol, nitrofurazone, dexamethasone, primidone, sulfamethoxazole, and diclofenac), steroid hormone (testosterone, progesterone, 17β-estradiol, estrone, and 17α-ethynylestradiol), and phenolic compounds (bisphenol A) were selected.
The pure standard of testosterone, progesterone, dexamethasone, primidone, propranolol, atorvastatin, sulfamethoxazole, diclofenac, diethylstilbestrol, quinalphos, diazinon, chlorpyrifos, nitrofurazone, 17β-estradiol, 17α-ethynylestradiol, estrone, and bisphenol A were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Isotopically labelled compounds, diazinon-d10, primidone-d9, diclofenac-d4, sulfamethoxazole-d4, 17β-estradiol-d4, and bisphenol A-d8 used as internal standards were obtained from Toronto Research Chemicals Inc. (Ontario, Canada) and Cambridge Isotope Laboratories (CIL) (Massachusetts, USA). All LCMS and high-performance liquid chromatography (HPLC) grade solvents like methanol (MeOH), acetone, acetonitrile (ACN) were obtained from Fisher Scientific (New Jersey, USA). Strata-X polymeric reversed-phase cartridges (C18, 200 mg/6 mL) were purchased from Phenomenex (California, USA), Soxhlet apparatus for sediment extraction was obtained from Witeg (Wertheim, Germany) and ammonium hydroxide (NH4OH) and methyl-tert-butyl ether (MTBE) were obtained from Fisher Scientific (Loughborough, UK). Aluminium oxide 90 active neutral and hydromatrix were obtained from Merck (Darmstadt, Germany) and Agilent Technologies (California, USA) respectively. Throughout the analyses, deionised water (water sensitivity > 18.2 MΩ cm at 25 °C) was purified from a Milli-Q water purification system (Millipore, Massachusetts, USA). The primary stock solutions were prepared and dissolved in pure MeOH and stored under −20 °C in the dark. Both the mixed external and internal standard was prepared from the individual standard for spiking during method recovery and in sample extraction. As mentioned earlier in this study, Pulau Kukup (refer to Fig. 1 and Table 1) is recognised as one of the most significant mariculture areas in Malaysia involved in a variety of marine fish culture. The sediment samples were collected along the mariculture cages where S1-S3 are located opposite north port and S4-S6 are located near to the Kukup Ferry Terminal. Sediment samples at a depth between 4.2 and 7.6 m from the surface water were collected from six different points of the mariculture cages (duplicate sample for each point, n = 12) using an Ekman grab sampler. Approximately 500 g of sediment as samples, were collected and stored in a pre-cleaned methanol-rinsed glass container. All samples were labelled correctly and kept in a cooler box before stored in the chiller at 4 °C. The samples collected were then left to dry under ambient air temperature to remove any moisture. The samples were then ground, sieved, and stored in a glass container before analysis. Total organic carbon (TOC) in the samples was analysed using a Total Carbon Analyser (TOC-VCSH, Shimadzu, Japan). The extraction and clean-up steps were adapted and modified based on the work of Omar et al. (2017) and Ismail et al. (2019) and were undertaken using Soxhlet extraction along with solid-phase extraction (SPE) as the clean-up step before analysis using liquid chromatographytandem mass spectrometry (LC-MS/MS). Sediment samples (approximately 5.00 g) were next mixed with pre-weighed comprising of hydromatrix (0.25 g) and aluminium oxide (0.25 g), followed by grounding and spiking of the internal standards. Approximately 200 mL of MeOH:acetone (50:50) was used in the Soxhlet extraction process (8 h), in which the apparatus was wrapped with aluminium foil to prevent any light penetrating the sample during the extraction process. Before the solid-phase clean-up extraction method, the samples were reduced to < 2 mL once going through a rotary evaporator and reconstituted with ACN:H2O (10:90) to 15–20 mL. Strata-X polymeric reversed-phase C18 cartridges were chosen for solid-phase extraction in which each cartridge was conditioned with 5 mL of ACN, 5 mL of ACN:MTBE:NH4OH (70:25:5), and 5 mL of ACN:H2O (10:90). Next, the sample was loaded into the cartridge and rinsed with 10 mL of ACN:H2O (10:90) to clean up any matrix interference. The cartridge was left to dry for about 3 to 5 min. Analytes were then eluted with 15 mL ACN:MTBE:NH4OH (70:25:5), pre-concentrating the extracts to 1–2 mL using a rotary evaporator. The final extracts were concentrated under a gentle nitrogen blow and reconstituted with ACN:H2O (30:70) 2
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Fig. 1. Sampling site at Pulau Kukup, Johor, Malaysia.
to a final volume of 1 mL in an amber vial. Before the instrument analysis, the sample was filtered with 0.20 μm polytetrafluoroethylene (PTFE) membrane filter (25 mm, Phenomenex). For the entire analytical procedure, quality assurance and quality control measures were established. After washing the glassware apparatus using detergent (Decon 90) to remove any impurities and contaminants, it was then rinsed using deionised water, followed by an organic solvent. To check the interference plus eradicate cross-contamination, every batch of samples was monitored by performing one recovery procedure. Several parameters, including recovery, linearity, limits of detection, stability and robustness were assessed for SPE-LCMS/MS development and optimisation. The method detection limit (MDL) and method quantification limit (MQL) for all targeted multiresidues of EDCs were performed by calculating the signal-to-noise ratio (SNR) based on 3:1 and 10:1 ratio, respectively. To identify and determine the retention time (Rt), each multi-residue of the EDCs in the samples using LC-MS/MS, and Rt for authentic standard solution was verified. The extracted samples were next analysed using LC-MS/MS (Shimadzu 8030, Japan). Chromatographic separation for the targeted EDCs in the sediment samples were carried out by employing two different instrument methods; method A was acidic based while method B was basic based. For the acidic method, mobile phase A (water) was prepared by mixing 0.2% acetic acid in ultrapure water, and mobile phase B (organic) was prepared by using 100% pure MeOH. The determination of the column used was based on the suitability of the column to suppress the compound peak that appeared during the first 10 min of running the instrument. The column used in method A was a
Luna 5 μm PFP (2) 100A, 150 × 2.00 mm column. In the basic based method, mobile phase A (water) was prepared by mixing 0.01% NH4OH in ultrapure water, while mobile phase B (organic) was prepared using 100% pure MeOH. Column used in this method was a Kinetex® 5 μm EVO C18 100A, 150 × 2.1 mm column. A different mobile phase and column were prepared for method optimisation. The flow rate used for method A was fixed at 0.30 mL/min and 0.35 mL/min for method B. Whereas, the column temperature and run time was fixed at 40 °C and 10 min respectively. The injection volume for running the samples was set at 10 μL. The optimised method was validated for various validation parameters such as linearity, method recovery (%), MDL and MQL (Thompson et al., 2002; Omar et al., 2017). The linearity of the multiresidues was evaluated at five different concentrations of standard solutions ranging from 5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, and 200 ng/mL respectively. A strong positive and significant linearity indicating the reliability and robustness of the method was obtained for all targeted compounds with the goodness of fit (r) ranging between 0.9566 and 0.9992. The range for MDL and MQL for the targeted compounds was calculated between 0.001 and 0.300 ng/g and 0.004 to 1.000 ng/g, respectively. Using the spiking the method at a concentration of 100 ng/g, the method recovery for all targeted compounds in the present study was in the range between 50.39% and 129.10% (refer to Table 2), representing acceptable accuracy for the analytical method. The targeted EDCs in the present study, consisting of multiclass residues, (i.e., steroid hormones, pharmaceutical and medical drugs, pesticides, and phenolic compounds), with method recovery were in the
Table 1 Coordinate and description of sampling site at Pulau Kukup, Johor, Malaysia. Station
Coordinate
Description
1 2 3 4 5 6
N N N N N N
Near mangrove area, opposite to the north port Opposite to the north port, near with residential area Near mangrove area, opposite to the north port Near Kukup Ferry Terminal Near Kukup Ferry Terminal Near Kukup Ferry Terminal
01°19.958′ E 103°26.209′ 01°20.158′ E 103°26.205′ 01°20.083′ E 103°26.357′ 01°19.0491′ E 102°26.336′ 01°19.182′ E 103°26.475′ 01°19.101′ E 103°25.679′
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Approximately, six compounds were detected at S1, with most of the compounds observed having the highest concentration compared to the other sampling sites. Testosterone (0.094 ng/g dry weight), dexamethasone (0.144 ng/g dry weight), propranolol (0.275 ng/g dry weight), and diclofenac (0.228 ng/g dry weight) displayed the highest concentration detected at sampling point S1. While other EDC contaminants were detected at different sampling points indicating that the anthropogenic and mariculture activities in the ocean significantly influence the concentration of EDCs in marine sediment. Furthermore, there were eight multi-residues of EDCs (testosterone, estrone, dexamethasone, primidone, propranolol, diclofenac,
Table 2 Linearity, method recovery percentage, MDL and MQL for targeted compounds in sediment sample. Compounds Testosterone Progesterone Dexamethasone Primidone Propranolol Atorvastatin Sulfamethoxazole Diclofenac Diethylstilbestrol Quinalphos Diazinon Chlorpyrifos Nitrofurazone 17β-estradiol 17α-eEthynylestradiol Estrone Bisphenol A
Linearity (r)
Recovery sediment (%) (RSD)
MDL (ng/g)
MQL (ng/g)
0.9982 0.9978 0.9894 0.9982 0.9966 0.9965 0.9960 0.9566 0.9959 0.9992 0.9954 0.9948 0.9903 0.9977 0.9957 0.9974 0.9980
129.10 (29.50) 124.05 (13.71) 91.67 (4.67) 100.17 (8.17) 77.91 (9.05) 53.25 (0.65) 101.18 (1.70) 93.94 (14.11) 53.76 (8.05) 57.62 (5.18) 93.94 (14.11) 63.59 (12.01) 62.62 (14.60) 82.32 (11.18) 50.39 (23.09) 58.18 (9.48) 102.65 (2.00)
0.015 0.016 0.040 0.015 0.250 0.001 0.002 0.093 0.208 0.037 0.006 0.093 0.043 0.049 0.300 0.013 0.004
0.051 0.055 0.134 0.051 0.033 0.004 0.007 0.062 0.694 0.123 0.020 0.310 0.144 0.163 1.000 0.046 0.012
RSD: relative standard deviation; MDL: method detection limit; MQL: method quantification limit.
range between 50 and 129% at the spiking concentration of 100 ng/g (Table 2). Notably, the performance was even more accurate and sensitive compared to previous studies such as US EPA (2007), which reported a range between 5 and 180% for pharmaceutical analysis in sediments. Although, the present study involved the analysis of multiclass EDCs. It is both difficult and challenging to achieve a high recovery percentage for each compound during examining multi-class EDCs in a single analytical run (Wee et al., 2016). Besides, the solubility for every EDC compound was different, which can be a further limitation in acquiring a high percentage of method recovery (> 90%). Also, some of the targeted compounds such as atorvastatin, sulfamethoxazole, and bisphenol A achieved a very low MDL < 0.010 ng/g, and most of the other compounds had lower MDL compared to previous studies. Table 2 displays the details of linearity, percentage of method recovery with relative standard deviation (RSD), MDL, and MQL for the multi-residues of EDCs. All data were analysed using statistical software, IBM SPSS (Statistical Package for Social Science) version 23. Spatial variation and distribution were evaluated using descriptive statistics in order to observe the significant association of targeted emerging organic contaminants with physico-chemical characteristic. The concentration and spatial distribution of EDCs in the sediments collected from Pulau Kukup, are depicted in Fig. 2 and are further detailed in Table 3. All detected compounds showed a coefficient of variation > 30%, indicating a high variation of EDCs concentration among the sampling points. Sediment analysis revealed the presence of multi-residues of EDCs in all sampling sites (S1-S6). Testosterone, estrone, dexamethasone, primidone, propranolol, diclofenac, diethylstilbestrol, and bisphenol A were detected in the sample, except for other residues which were below MDL at all sampling stations. Primidone and bisphenol A were detected in all samples (S1-S6) and other contaminants like testosterone, dexamethasone, propranolol, and diclofenac were ubiquitously detected in most of the sampling stations. The results obtained showed that regularly monitoring needs to be performed in the marine ecosystem at Pulau Kukup. Testosterone, estrone, and dexamethasone were the most prominent compounds detected in the sediment samples, which were collected at 87.39%, 64.41%, and 53.76% respectively (Table 3) whereas propranolol was less detected in the sediment samples at 18.03%. The highest concentration was observed for bisphenol A (0.072–0.389 ng/g dry weight) followed by diethylstilbestrol (< 0.208–0.331 ng/g dry weight) and propranolol (< 0.250–0.275 ng/g dry weight).
Fig. 2. Distribution pattern of multi-residue for endocrine disrupting compounds in sediment of Pulau Kukup, Johor. 4
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potential lack of efficient regulation of discharges from residential areas and livestock activities. Bisphenol A, commonly known as an important industrial chemical, was detected in all sampling sites, indicating the widespread application of bisphenol A in those industries located along with the mariculture cages (Ismail et al., 2018; Wang et al., 2019). Plasticiser is the most produced and consumed substance globally (Pintado-Herrera et al., 2016), whereas bisphenol A is a chemical involved in the production of polycarbonate plastics and epoxy resins. In addition to bisphenol A, primidone was also detected in every site of the sampling points (S1–S6), revealing the possible improper disposal of unused medications (Verlicchi and Zambello, 2016; Omar et al., 2018). Previous studies conducted in the Asia region reported that diclofenac and propranolol had been detected in the effluent of wastewater treatment plants (WWTP) (Pal et al., 2010) and polyaromatic hydrocarbons (PAHs), persistent organic pollutants (POPs), alkylphenol ethoxylates (APEs) likewise, have been detected along the Singapore Straits, located close to the Straits of Malacca (Sin et al., 2016). The occurrence of emerging organic pollutants along the Singapore Straits also indicates that these pollutants are potentially penetrating the Straits of Malacca through the movement of water currents. The relationship between physico-chemical properties (total organic carbon and pH) and the concentration of multi-residues of EDCs in the marine sediment samples collected from Pulau Kukup was described using Pearson's analysis correlation coefficient (r). The pH range for the sediment collected ranged between 6.66 and 7.95, while the total organic compound ranged between 1.41 and 1.83%. Also, significant correlation of targeted compounds with the total organic compounds, ranged from a weak positive correlation, diclofenac and propranolol (r = 0.332, p < 0.05), to a strong positive correlation estrone (r = 0.692, p < 0.05) and testosterone (r = 0.649, p < 0.05) as shown in Table 4. The occurrence and distribution of EDCs concentrations in the sediment samples were influenced by total organic carbon (Omar et al., 2018) and hormone group (estrone and testosterone) showing a strong positive correlation with total organic carbon compared to the other group of EDCs. Aside from total organic carbon, some compounds also showed a significant association with pH testosterone (r = 0.653, p < 0.05), dexamethasone (r = 0.684, p < 0.05), propranolol (r = 0.760, p < 0.05), diclofenac (r = 0.760, p < 0.05), and bisphenol A (r = 0.183, p < 0.05). The data also showed that the pH values were influenced by the sorption of EDCs in sediment matrices. The present study presented the data and information on EDC contaminants in marine sediment matrices collected from Pulau Kukup, Johor. Three groups of EDCs were observed in the samples namely, steroid hormone (estrone), phenolic compounds (bisphenol A), and pharmaceutical (testosterone, dexamethasone, primidone, propranolol, diclofenac, and diethylstilbestrol). The presence of EDCs in the sediment samples signified potential unregulated discharges that originated from anthropogenic activities and influenced by naturogenic factors surrounding the mariculture sites, including those from upstream. The contamination is of great concern given these contaminants could lead to having a severe impact on the environment, thus causing an imbalance to the ecosystem. Also, given the adverse effects of exposure to these contaminants, humans, being on top of the food chain/food web, could also suffer as a result since long-term exposure to EDCs will cause health problems, such as a reduction in the human reproduction system, disease such as cancer, and alteration of the body's functions. In gaining a better understanding of the occurrence, distribution, pathways and fate of EDCs contaminants in the surrounding ecosystem, further study should include other diverse matrices such as surface water, aquatic animals, plants, and humans. The baseline data would be extremely beneficial in the future to profile the current trend of EDCs pollutants and consequential pollution in the marine ecosystem, especially in the mariculture area. Also, future research should investigate the potentially toxic effects of such contaminants towards humans via dietary consumption and the elimination process of emerging pollutants in the marine ecosystem.
Fig. 2. (continued)
diethylstilbestrol, and bisphenol A) that were present in the marine sediment collected from Pulau Kukup. Based on the literature from other studies, the sources of EDCs are believed to originate from wastewater treatment plants, domestic/medical waste discharge, livestock activities, industrial waste discharge, and agricultural activities. The presence of testosterone and estrone in the environment are typically observed from sewage treatment facility waste, uniquely human and animal wastes (Lorenzen et al., 2004; Raman et al., 2004; Zhang et al., 2013; Li, 2014; Pessoa et al., 2014). Estrone was also detected in the marine sediment, while 17β-estradiol and 17α-ethynylestradiol were found to be relatively less abundant in the samples. Indeed, the occurrence of steroid hormones along the coastal area indicates the 5
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Table 3 Concentration of multi-residue EDCs contaminants in sediment samples from Pulau Kukup, Johor. Compounds
Concentration in sediment (ng/g dry weight) (n = 12)
Testosterone Progesterone Estrone 17β-estradiol 17α-ethynylestradiol Dexamethasone Primidone Propranolol Atorvastatin Sulfamethoxazole Diclofenac Diethylstilbestrol Nitrofurazone Quinalphos Diazinon Chlorphyrifos Bisphenol A
ST1
ST2
ST3
ST4
ST5
ST6
Range
% CV
Percentage of detection (%)
0.094 < MDL < MDL < MDL < MDL 0.144 0.024 0.275 < MDL < MDL 0.228 < MDL < MDL < MDL < MDL < MDL 0.238
0.021 < MDL < MDL < MDL < MDL < MDL 0.030 < MDL < MDL < MDL < MDL 0.331 < MDL < MDL < MDL < MDL 0.389
0.068 < MDL 0.038 < MDL < MDL 0.042 0.097 < MDL < MDL < MDL < MDL 0.226 < MDL < MDL < MDL < MDL 0.139
< MDL < MDL 0.020 < MDL < MDL < MDL 0.077 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.367
0.025 < MDL < MDL < MDL < MDL < MDL 0.048 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.160
< MDL < MDL 0.018 < MDL < MDL < MDL 0.020 < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL < MDL 0.072
< 0.015–0.094 NA < 0.014–0.038 NA NA < 0.040–0.144 0.020–0.097 < 0.250–0.275 NA NA < 0.093–0.228 < 0.208–0.331 NA NA NA NA 0.072–0.389
133.82 NA 123.61 NA NA 186.50 63.41 244.95 NA NA 244.95 159.05 NA NA NA NA 56.45
87.39 NA 64.41 NA NA 53.76 100.00 18.03 NA NA 32.90 40.10 NA NA NA NA 100.00
MDL: Method detection limit, NA: Not available, CV: Coefficient of variation.
Table 4 Statistical analysis for correlation coefficient, TOC, pH, and concentration of targeted EDCs in marine sediment based on Pearson analysis.
TOC pH Testosterone Estrone Dexamethasone Primidone Propranolol Diclofenac Diethylstilbestrol Bisphenol A ⁎ ⁎⁎
TOC
pH
Testosterone
Estrone
Dexamethasone
Primidone
Propranolol
Diclofenac
Diethylstilbestrol
Bisphenol A
1.000 −0.079 0.649 0.692 0.561 0.473 0.332 0.332 −0.090 −0.500
1.000 0.653 −0.729 0.684 −0.368 0.760 0.760 −0.138 0.183
1.000 −0.017 0.899⁎ 0.070 0.760 0.760 0.107 −0.136
1.000 −0.169 0.755 −0.400 −0.400 0.134 −0.339
1.000 −0.181 0.957⁎⁎ 0.957⁎⁎ −0.182 −0.059
1.000 −0.396 −0.396 0.189 0.045
1.000 1.000⁎⁎ −0.308 0.040
1.000 −0.308 0.040
1.000 0.354
1.000
Correlation is significant at the 0.05 level (2-tailed). Correlation is significant at the 0.01 level (2-tailed).
Author contributions section
References
Nur Afifah Hanun Ismail: Method Development, Laboratory Analysis, Data Analysis, Writing-Original draft. Sze Yee Wee: Data Analysis, Writing-Reviewing and Editing. Didi Erwandi Mohamad Haron: Method Development. Nitty Hirawaty Kamarulzaman: Writing-Reviewing and Editing. Ahmad Zaharin Aris: Supervision, Writing- Reviewing and Editing, Research Design, and Funding acquisition.
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Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was fully supported by Geran Putra Malaysia (GP/2017/ 9574800) and International Environmental Research Institute, IERI Research Grant (6389800) from Gwangju Institute of Science and Technology, Gwangju, Korea. Ismail NAH want to thank Universiti Putra Malaysia, UPM for allowance through Graduate Research Fellowship (GRF) programme. 6
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