Determination of phenolic and steroid endocrine disrupting compounds in environmental matrices

BACKGROUND, AIM AND SCOPE: Many pollutants have received significant attention due to their potential estrogenic effect and are classified as endocrine disrupting compounds (EDCs). EDCs comprise many classes of organic compounds. The development or optimization of analytical protocols for the simultaneous determination of EDCs in environmental samples is an analytical challenge because these compounds exhibit different physicochemical characteristics, they occur in the aquatic environment in relatively low concentrations and, furthermore, environmental samples are considered as complex matrices. The aim of this study is the development of analytical methods for the simultaneous determination of phenolic and steroid EDCs in aqueous and solid samples. The target compounds are 4-nonylphenol, 4-octylphenol, their ethoxylate oligomers (mono- and di-ethoxylates of nonylphenol and octylphenol), bisphenol A, the estrogens (estriol, estrone, 17beta-estradiol, 17alpha-estradiol) and the synthetic steroids (mestranol and 17alpha-ethynylestradiol). MATERIALS AND METHODS: Solid phase extraction employing Oasis HLB cartridges and different elution solvents was used for the recovery studies of the target compounds from various types of water samples (ultrapure water, artificial seawater, river water and seawater). Ultrasonic assisted extraction was applied for the recovery of the target EDCs from the solid samples. The recoveries were assessed using various solvents for the extraction and the elution of EDCs from different SPE cartridges used for clean up. Gas chromatography-mass spectrometry after derivatization with N,O-bis(trimethylsilyl)-trifluoroacetamide was employed for the determination of these compounds. RESULTS AND DISCUSSION: The recovery rates of three elution solvents (methanol, acetone and ethylacetate) for the extraction of target EDCs from artificial seawater were assessed after preconcentration on SPE cartridges. Acetone showed better recoveries and was further tested for its extraction efficiency in different water types (river water, seawater). Ultrasonic assisted extraction was used for the recovery of target EDCs from solid matrices. Acetone, methanol, mixture of acetone-methanol (1:1) and ethylacetate were used as extraction solvents. Ethylacetate and the mixture of acetone-methanol (1:1) exhibited better extraction efficiencies. An additional clean up step was necessary for sediment samples. Different SPE cartridges were employed for clean up of the extracts (Oasis HLB, C18, Florisil, silica, combination of silica and alumina). Florisil cartridges were finally used. The proposed methods were further validated on the determination of target EDCs in field collected samples (river water, seawater, wastewater, total suspended solids and sediments) from the major area of Thessaloniki, Greece. CONCLUSIONS: Efficient and accurate integrated methods for the simultaneous determination of alkylphenols (nonylphenol, octylphenol), their ethoxylate oligomers (mono- and di-ethoxylate of nonylphenol and octylphenol), bisphenol A and steroids (estriol, estrone, 17beta-estradiol, 17alpha-estradiol, mestranol and 17alpha-ethynylestradiol) in aqueous and solid samples were developed. The proposed methods were applied for the determination of the target compounds in representative environmental samples in the area of Thessaloniki, Northern Greece. RECOMMENDATIONS AND PERSPECTIVES: This study confirms the occurrence of selected EDCs in inland and marine waters in the area of Thessaloniki, Northern Greece. Since there is no previous data on the occurrence of the target EDCs in the major area, an extended survey is in progress to evaluate the occurrence and fate of these compounds.     

Environmental and biological monitoring of endocrine disrupting chemicals.

Trends toward an increase of adverse health effects on reproductive organs have been reviewed. An urgent need has been recognised to establish validated in vivo and in vitro screening assays to test for hormonal activities of chemicals. Relevant existing OECD guidelines have been reviewed and enhancements of some of these have been identified, mainly to test for estrogenic and androgenic activity of chemicals. The problems connected to monitoring activities are outlined, particularly for ambient and biological monitoring. Indeed, the problem of assessing human exposure to endocrine disrupting chemicals through environmental chemical analysis tends to a very high level of complexity. This has been illustrated through the example of one single subclass of endocrine disrupting compounds (EDCs), the organohalogen compounds. Valid biological markers are also needed to be effectively used in epidemiological studies and risk assessment. A multidisciplinary approach and the collaboration among experts in the field of clinical biochemistry, toxicology, and epidemiology is required.     

A multimedia environmental model of chemical distribution: fate, transport, and uncertainty analysis

This paper presented a framework for analysis of chemical concentration in the environment and evaluation of variance propagation within the model. This framework was illustrated through a case study of selected organic compounds of benzo[alpha]pyrene (BAP) and hexachlorobenzene (HCB) in the Great Lakes region. A multimedia environmental fate model was applied to perform stochastic simulations of chemical concentrations in various media. Both uncertainty in chemical properties and variability in hydrometeorological parameters were included in the Monte Carlo simulation, resulting in a distribution of concentrations in each medium. Parameters of compartmental dimensions, densities, emissions, and background concentrations were assumed to be constant in this study. The predicted concentrations in air, surface water and sediment were compared to reported data for validation purpose. Based on rank correlations, a sensitivity analysis was conducted to determine the influence of individual input parameters on the output variance for concentration in each environmental medium and for the basin-wide total mass inventory. Results of model validation indicated that the model predictions were in reasonable agreement with spatial distribution patterns, among the five lake basins, of reported data in the literature. For the chemical and environmental parameters given in this study, parameters associated to air-ground partitioning (such as moisture in surface soil, vapor pressure, and deposition velocity) and chemical distribution in soil solid (such as organic carbon partition coefficient and organic carbon content in root-zone soil) were targeted to reduce the uncertainty in basin-wide mass inventory. This results of sensitivity analysis in this study also indicated that the model sensitivity to an input parameter might be affected by the magnitudes of input parameters defined by the parameter settings in the simulation scenario. Therefore, uncertainty and sensitivity analyses for environmental fate models was suggested to be conducted after the model output was validated based on an appropriate input parameter settings.     

Biochemical and biological assays of endocrine disrupting compounds in various manure matrices

Wastes generated by animal agriculture have complex matrices and present a significant challenge for achieving accurate measurements of estrogens. The objective of this study was to compare two different extraction methods and two different biochemical and biological estrogen assays on two dairy manure matrices. The two extraction methods studied in this research were a two-step base-toluene extraction and a one-step cyclohexane extraction. The two assays assessed were an enzyme-linked immunosorbent assay (ELISA) and a yeast estrogen screen (YES) bioassay. Estrogenic activity was measured directly as 17beta-estradiol (E2) through the ELISA method or as E2 equivalents (E2-eq) through the YES bioassay. Both extraction methods yielded reasonable recoveries from distilled water in the absence of matrix interferences. In manure samples, ELISA resulted in recoveries slightly higher than 100% in two types of dairy manures, but YES recoveries varied. The YES bioassay detected much higher estrogenic activities in dairy manure compared to the E2 concentrations measured by ELISA. The base-solvent extraction yielded higher E2 concentrations in dairy manure compared to the one-step cyclohexane extraction. These results suggest that manure matrices vary sufficiently that extraction methods must be optimized for specific assays utilized to quantify estrogens in manures.     

Ecological risks of phenolic endocrine disrupting compounds in an urban tropical river

Environmental Science and Pollution Research - The distribution of emerging organic contaminants in drinking water sources in Africa is a subject with very scanty data and information. In order to...     

Ecological effects, transport, and fate of mercury: a general review

Mercury at low concentrations represents a major hazard to microorganisms. Inorganic mercury has been reported to produce harmful effects at 5 μg/l in a culture medium. Organomercury compounds can exert the same effect at concentrations 10 times lower than this. The organic forms of mercury are generally more toxic to aquatic organisms and birds than the inorganic forms. Aquatic plants are affected by mercury in water at concentrations of 1 mg/l for inorganic mercury and at much lower concentrations of organic mercury. Aquatic invertebrates widely vary in their susceptibility to mercury. In general, organisms in the larval stage are most sensitive. Methyl mercury in fish is caused by bacterial methylation of inorganic mercury, either in the environment or in bacteria associated with fish gills or gut. In aquatic matrices, mercury toxicity is affected by temperature, salinity, dissolved oxygen and water hardness. A wide variety of physiological, reproductive and biochemical abnormalities have been reported in fish exposed to sublethal concentrations of mercury. Birds fed inorganic mercury show a reduction in food intake and consequent poor growth. Other (more subtle) effects in avian receptors have been reported (i.e., increased enzyme production, decreased cardiovascular function, blood parameter changes, immune response, kidney function and structure, and behavioral changes). The form of retained mercury in birds is more variable and depends on species, target organ and geographical site. With few exceptions, terrestrial plants (woody plants in particular) are generally insensitive to the harmful effects of mercury compounds.     

Ecological risk assessments of endocrine disrupting organotin compounds using marine neogastropods in Hong Kong

As active ingredients of anti-fouling paints that are widely used on ship hulls, organotin compounds, in particular tributyltin (TBT), are well-known endocrine disruptors causing sex changes in marine organisms and widespread in coastal waters and sediments worldwide. In this study, a comprehensive ecological risk assessment (ERA) of organotins was conducted in Hong Kong waters through determining the imposex status, sex ratio and tissue burdens of these compounds in the neogastropods, Thais clavigera and Thais luteostoma collected from 29 coastal sites. We also investigated the historical trend of organotin effects on these gastropods, and performed a probabilistic ERA based on tissue burden of TBT in the animals. Our results demonstrated that imposex indices were positively correlated with the body burden of organotins in the gastropods. Across all sites, the sex ratio (female:male) decreased significantly with increasing imposex levels or tissue burden of organotins, implying that such pollutants can result in a male-biased population, potentially leading to local extinction in extreme cases. Based on the ERA, 5.4% of all populations of T. clavigera are at risk due to exposure to TBT; the risks include growth inhibition, impairment of immune functions and reduced fitness. Seriously impacted areas included Aberdeen, Repulse Bay, Butterfly Beach, Mui Wo and Ha Mei Wan. A comparison with historical data revealed that there had been some improvement in the areas with low marine traffic, and distant from the major harbour/port. This could partly be due to the restriction on the use of TBT on small vessels (<25m in length) since 1992. Nevertheless, the organotin contamination still remains severe in areas with high marine traffic or adjacent to large harbours/ports. In particular, the situation in the northeastern waters of Hong Kong has been getting worst since 1996 that is probably associated with the rapid development of the cargo container port at Yantian in China.     

Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe

INTRODUCTION: Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. DISCUSSION: The estrogenic effect of the organic sediment extract was determined using the yeast-estrogen-screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography-mass spectrometry and high-resolution LC-MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9?±?0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17β-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene (o-benzyl-p-chlorophenol)-a widely used antiseptic that was also identified in the sediment extract-has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4-n-nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.     

Geographic variation in tissue accumulation of endocrine disrupting compounds (EDCs) in grazing sheep

Muscle tissue was collected from ewes and lambs derived from farms throughout Scotland and sample concentrations of five endocrine disrupting compound groups were determined. Farms of origin were categorised according to geographic region. There were few statistically-significant differences with region or distance from cities. However, the magnitude of the difference between the highest and lowest mean values in ewe muscle from different regions exceeded 30% for 13 of the 15 compounds that were consistently detected in muscle, with animals derived from the industrialised region having the highest mean values for 11 of the 13 compounds. A less marked trend was apparent in the lamb muscle (8 of 13 highest were in the industrialised region). The physiological effects of such small differences in exposure to mixtures of pollutants remain to be determined.     

Comparison of the removal efficiency of endocrine disrupting compounds in pilot scale sewage treatment processes

The removal efficiency of endocrine disrupting compounds from effluents using pilot scale sewage treatment processes, including various treatment technologies, such as membrane bioreactors (MBR), nanofiltration (NF) and reverse osmosis (RO) for the purpose of water reuse, were estimated and compared. The calculated estrogenic activity, expressed in ng-EEQ/l, based on the concentration detected by GC/MS, and relative potencies for each target compound were compared to those measured using the E-screen assay. The removal efficiencies for nonylphenol, was within the range of 55-83% in effluents. High removal efficiencies of approximately >70% based on the detection limits were obtained for bisphenol A, E1, EE2 and genistein with each treatment processes, with the exception of E1 ( approximately 64%) using the MBR process. The measured EEQ values for the effluents from the MBR, NF and RO processes also indicated low estrogenic activities of 0.65, 0.23 and 0.05 ng-EEQ/l, respectively. These were markedly reduced values compared with the value of 1.2 ng-EEQ/l in influent. Consequently, the removals of EDCs in terms of the EEQ value from the biological and chemical determinations were sufficiently achieved by the treatment process applied in this study, especially in the cases of the NF and RO treatments.     

环境内分泌干扰物诱发小鼠睾丸细胞DNA损伤的研究

应用单细胞凝胶电泳技术技术，研究了烷基酚类化合物对离体小鼠睾丸细胞DNA的损伤作用。实验结果表明，9种烷基酚类化合物对小鼠睾丸细胞均能引起不同程度的DNA损伤，并呈现剂量效应关系，其高剂量组与对照组相比，均有极显著性差异（P＜0．01）。实验结果亦表明，其DNA的损伤程序与化学结构有一定的关系。     

Oryzalin fate and transport in runoff water in Mediterranean vineyards

An experimental study was conducted in a 91.4-ha Mediterranean vineyard catchment in southern France to characterize the fate and transport of oryzalin in runoff water and thus to assess the risk of contamination of surface waters. Oryzalin concentrations in soil were monitored on two fields, one no-till and one tilled from March 1998 to March 2000. Concentrations in solution and on solid phase of runoff water were measured at the outlets of both fields and the catchment. The droughts in the two summer periods reduced the dissipation of oryzalin and increased its field half-life up to 35 days. Consequently, oryzalin was detected throughout the year in runoff water, with maximum dissolved concentrations > 600 microg l(-1) at the field scale. Oryzalin transport essentially occurred in solution. At the no-till field, seasonal losses were 2.29% and 1.89% of the applied amount in 1998 and 1999, respectively. The corresponding values at the tilled field were 1.56% and 0.29%, since tillage reduced total losses by reducing surface runoff. At the catchment scale, oryzalin concentrations were smaller than those at the field scale, due to dilution effects and staggering of application. Large part of the overland flow from the fields reinfiltrated in the ditches before reaching the outlet of the catchment. As a result, seasonal oryzalin losses were <0.2% of the applied amount.     

Partitioning of endocrine disrupting compounds in inland waters and wastewaters discharged into the coastal area of Thessaloniki,Northern Greece

Background, aim, and scope  

In the Water Framework Directive 2000/60/EC, environmental objectives for the proper quality of inland, surface, transitional, coastal, and ground waters have been set. Member states are required to identify chemical pollutants of significance in the water bodies, to establish emission control measures, and to achieve quality standards. A specific category of pollutants are the compounds that may possess endocrine-related functions known as endocrine disrupting compounds (EDCs). This means that member states have the obligation to take action in order to prevent human exposure to these compounds via aquatic environment. The objective of this research was to study the occurrence and distribution of phenolic and steroid EDCs in inland waters and wastewaters discharged in the area of Thermaikos Gulf, Thessaloniki, Northern Greece.     

Toxicity monitoring and classification of endocrine disrupting chemicals (EDCs) using recombinant bioluminescent bacteria

A recombinant bioluminescent Escherichia coli, DPD2794, containing the recA promoter region fused to luxCDABE originating from Vibrio fischeri was used for detecting genotoxicity caused by endocrine disrupting chemicals (EDCs) to cells. As well, several other recombinant bioluminescent bacteria, including TV1061, which is sensitive to protein damage (grpE::luxCDA BE), DPD2511, sensitive to oxidative damage (katG::luxCDABE), and DPD2540, sensitive to membrane damage (fabA::luxCDABE), were used for evaluating other possible modes of toxicity. It was found that the recombinant bacteria could monitor the toxic effect, not estrogenic effect, due to the presence of various EDCs through the measurement of bioluminescence (BL) and cell growth rate, which depend upon the type of toxicity occurring and which of the four strains was used. In addition, it was found that the damage caused by EDCs can be classified into several groups upon their mechanisms of toxic action.     

Occurrence and removal of endocrine disrupting chemicals in wastewater

This paper discusses the occurrence and removal of endocrine disrupting chemicals (EDCs) in Horsham sewage treatment plant (STP), West Sussex, UK. Photodegradation aided by a catalyst (TiO(2)) was studied for its efficiency of removing EDCs from wastewater. The degradation of estrone and 17beta-estradiol under natural sunlight followed a pseudo-first-order kinetics, with a rate constant of 0.01h(-1). The degradation by sunlight is also independent of initial chemical concentration, suggesting some potential of using this environmentally friendly method for waste minimisation. In comparison, more efficient degradation was observed with UV irradiation (253nm), with the rate constants being increased to 2.7 and 2.5h(-1) for the two compounds. Subsequent application of the photocatalysis method to real effluent treatment at Horsham STP was successful, as demonstrated by the non-detection of the target compounds following treatment.     

The fate and importance of organics in drinking water treatment: a review

In the pioneer days, the main driving forces for research of organics in drinking water treatment (DWT) were human health risks and optimisation of technology. The focus was on natural organic matter (NOM) structure, disinfection by-products (DBPs) formation, NOM removal by means of coagulation, adsorption, and oxidation, and development of the most efficient water treatment trains. Surprisingly, after decades of research, rapid development of analytical techniques and progress in risk assessment, the same driving forces are still in the limelight — although the topics have changed slightly. The attention switched from trihalomethanes to a new generation of DBPs. The definition of hydrophilic/hydrophobic NOM depends on the technique used for characterisation. It has become evident that numerous organic compounds can threaten water supply sources. Some of them had been ignored or overlooked in the past, but have recently been detected by advanced analytical tools even in drinking water. Prioritisation becomes priority per se. As far as processes are concerned, mainstream research has been following three lines: fouling mechanisms, application of hybrid processes and interactions between synthetic organic chemicals, other water constituents and materials used in DWT. Significant development has been made in membrane technology. This paper presents a broad overview of the recent organics research. Although the state-of-the-art technologies seem to have an answer to each and every question raised, it is still necessary to deal with specific problems on a case-by-case basis mainly due to the unique nature of NOM and different xenobiotics that may appear in various types of waters. In the end, human health risk, which derives from the presence/absence of organics, is only the tip of the iceberg — underneath lies a whole new universe — the socio-economic aspect of water treatment and quality that deserves much more attention.     

On-line solid-phase extraction and fluorescence detection of selected endocrine disrupting chemicals in water by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed to analyse selected endocrine disrupting chemicals in water by using automated on-line solid-phase extraction with a fluorescence detector. The excitation and emission wavelengths of the fluorescence detector were 230 nm and 290 nm, respectively. The selected endocrine disrupting chemicals include hormone steroids such as estradiol (E2), estriol (E3), ethynylestradiol (EE2), and ethynylestradiol 3-methyl ether (MeEE2) as well as nonylphenols (NP), octylphenols (OP), POE(1-2) nonyl phenol (NPE) and bisphenol A (BP). Three types of on-line cartridges (C18, PLRP-s and PRP-1) were tested to pre-concentrate the endocrine disruptors in deionised water. It was found that the recoveries of these chemicals at 1 microg/L were close to 100% except for 4-octyl phenol and 4-n-nonyl phenol, which had recoveries of about 40% to 80%. The two polymer cartridges (PLRP-s and PRP-1) gave higher recoveries than the C18 cartridges. The addition of methanol at 5% to 10% in water significantly improved the recovery of 4-octyl phenol and 4-n-nonyl phenol. The addition of methanol also led to an improvement in the recovery with C18 cartridges. With the addition of methanol in water samples, these three types of cartridges gave similar recoveries for the chemicals. The detection limits of this method ranged from 20 ng/L to 50 ng/L. A river water sample spiked with these chemicals was analysed using the above method and we found no interference with the peaks of the selected endocrine disrupting chemicals. The recoveries for these chemicals were more than 92% except for 4-NP with a recovery of 61%. This relatively simple method is useful for laboratory studies on the environmental fate of these endocrine disrupting chemicals in water.     

中国地表饮用水水源地有机类内分泌干扰物污染现况分析

有机类内分泌干扰物(EDCs)是一类对人类和生物内分泌系统产生干扰,并可造成其紊乱的特殊外来物质,对人类和生物健康有极大的危害.以与人类生活密切相关的地表饮用水水源地中的有机类EDCs为研究重点,对中国目前地表饮用水水源地EDCs污染状况、污染物种类及其来源及可能的污染途径进行了评述.目前,中国各地区地表饮用水水源地均有有机类EDCs检出.其中以有机氯农药六六六及其异构体、DDT及其代谢产物和多氯联苯检出率最高,且某些地区检测浓度相当高,主要来源是农药使用和污水排放.国内外对地表饮用水水源地EDCs突发污染事件的研究均较少,应引起高度重视.     

再生水补给河道中内分泌干扰物壬基酚变化特征分析

通过监测再生水补给河道中壬基酚同分异构体的含量和分布,分析壬基酚在河道中的变化过程并对其进行综合评价,为再生水补给河道的生态风险评价提供科学支撑。结果表明,再生水中NP同分异构体的含量存在差别,其中含量最大的是NP10、NP9、NP4和NP2,含量最高的NP10是含量最低的NP8的6倍左右。再生水补水河道中壬基酚同分异构体的含量波动较大,壬基酚在河道中降解现象明显,但是不存在明显的同分异构体间的转化。河道中壬基酚含量的变化与季节关联性较弱,与补水壬基酚的含量关联性较强。基于因子加权法得出的壬基酚浓度能够较客观地表征河道中壬基酚的危害性,河道断面壬基酚的综合评价结果远小于将同分异构体数值几何相加的结果,直接几何相加检测浓度难免夸大河道中壬基酚的危害。结果为再生水安全回用提供参考。     

Bioanalytical and instrumental analysis of thyroid hormone disrupting compounds in water sources along the Yangtze River

Thyroid hormone (TH) agonist and antagonist activities of water sources along the Yangtze River in China were surveyed by a green monkey kidney fibroblast (CV-1) cell-based TH reporter gene assay. Instrumental analysis was conducted to identify the responsible thyroid-active compounds. Instrumentally derived l-3,5,3′-triiodothyronine (T₃) equivalents (T₃-EQs) and thyroid receptor (TR) antagonist activity equivalents referring to dibutyl phthalate (DBP-EQs) were calculated from the concentrations of individual congeners. The reporter gene assay demonstrated that three out of eleven water sources contained TR agonist activity equivalents (TR-EQs), ranging from 286 to 293 ng T₃/L. Anti-thyroid hormone activities were found in all water sources with the TR antagonist activity equivalents referring to DBP (Ant-TR-EQs), ranging from 51.5 to 555.3 μg/L. Comparisons of the equivalents from instrumental and biological assays suggested that high concentrations of DBP and di-2-ethylhexyl phthalate (DEHP) were responsible for the observed TR antagonist activities at some locations along the Yangtze River.