Endocrine disruption of water and sediment extracts in a non-radioactive dot blot/RNAse protection-assay using isolated hepatocytes of rainbow trout

Goal, Scope and Background In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. Methods The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Results and Discussion Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17?-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80 %, 17?-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14 % of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti-estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15 % of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197 %) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2 - 2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Conclusions and Outlook Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.     

Toxicity of silver nanoparticles to rainbow trout: A toxicogenomic approach

Silver (Ag) nanoparticles are used as antimicrobial adjuvant in various products such as clothes and medical devices where the release of nano-Ag could contaminate the environment and harm wildlife. The purpose of this study was to examine the sublethal effects of nano-Ag and dissolved Ag on Oncorhynchus mykiss rainbow trout. Hepatic Ag contents and changes in gene expression were monitored to provide insights on bioavailability and mode of action of both forms of silver. Fish were exposed to increasing concentrations (0.06, 0.6 and 6 μg L⁻¹) of nano-Ag (20 nm) and silver nitrate (AgNO₃) for 96 h at 15 °C. A gene expression analysis was performed in the liver using a DNA microarray of 207 stress-related genes followed by a quantitative polymerase chain reaction on a selection of genes for validation. The biochemical markers consisted of the determination of labile zinc, metallothioneins, DNA strand breaks, lipid peroxidation (LPO) and vitellogenin-like proteins. The analysis of total Ag in the aquarium water revealed that nano-Ag was mostly aggregated, with 1% of the total Ag being dissolved. Nevertheless, hepatic Ag content was significantly increased in exposed fish. Indeed, dissolved Ag was significantly more bioavailable than nano-Ag only at the highest concentration with 38 ± 10 and 11 ± 3 ng Ag mg⁻¹ proteins for dissolved and nano-Ag respectively. Exposure to both forms of Ag led to significant changes in gene expression for 13% of tested gene targets. About 12% of genes responded specifically to nano-Ag, while 10% of total gene targets responded specifically to dissolved Ag. The levels of vitellogenin-like proteins and DNA strand breaks were significantly reduced by both forms of Ag, but DNA break levels were lower with nano-Ag and could not be explained by the presence of ionic Ag. Labile zinc and the oxidized fraction of metallothioneins were increased by both forms of Ag, but LPO was significantly induced by nano-Ag only. A discriminant function analysis revealed that the responses obtained by biochemical markers and a selection of ten target genes were able to discriminate completely (100%) the effects of both forms of Ag. Exposure to nano-Ag involved genes in inflammation and dissolved Ag involved oxidative stress and protein stability. Hence, the toxicity of Ag will differ depending on the presence of Ag nanoparticles and aggregates.     

Transformations of dissolved organic matter in a landfill leachate--a size exclusion chromatography/mass spectrometric approach

The aim of the present study was to search for qualitative changes in the landfill leachate DOM along a groundwater gradient. The study was focused on DOM characteristics of importance for its interaction with pollutants, such as molecular weight distribution and aromaticity. It was concluded that the leachate DOM underwent substantial qualitative changes along the investigated gradient at the Vejen landfill, Denmark. The molecular weight decreased, the polydispersity increased, and the aromaticity varied with the lowest values found in the middle of the gradient. The high aromaticity in the end of the gradient may explain the higher DOM binding capacity towards hydrophobic compounds seen earlier in these samples. The relative abundance of ions with mass to charge ratio (m/z) of 600-1200 seemed to be very stable along the gradient, indicating that the observed qualitative changes of the DOM is mostly attributed to changes in the m/z 100-600 range. The DOM seemed to become more similar to fulvic acids present in uncontaminated groundwater with respect to molecular weight and polydispersity along the gradient.     

Comparison of gas chromatography and immunoassay methods in measuring the distribution of dieldrin in rainbow trout tissues.

Studies have been conducted to determine the distribution of dieldrin in various tissues of rainbow trout when exposed to several dieldrin concentrations. Medium sized fish with an average weight and length of 195.4 +/- 30.5 g and 25.7 +/- 1.4 cm, respectively, were placed in groups of 6 in 300 L tanks containing purified and aerated water and maintained at 10 degrees C. Following an acclimatization period of 10 days, each group of fish was exposed to one of four dieldrin concentrations ranging from 50 to 80 ppb. After 24 hours, the fish were taken out of the tanks and sacrificed. The brain, gills, liver, muscles and skin were collected from each fish. Dieldrin was extracted from each tissue using SPE techniques and analyzed by both gas chromatography (GC) and enzyme linked immunosorbent assay (ELISA). Results of analyses by the two techniques were highly correlated. The results also showed that liver and skin tissues had the highest level of dieldrin residues. In comparing the means of the six fish samples, it was found that liver or skin contained about 1.5-fold the level in brain, about 4.0 fold the level in muscles and about 6.5 fold the level in gills. Immunoassay proved to be as reliable an analytical tool as gas chromatography in this case.     

Characterization of dissolved organic matter using high-performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) with a multiple wavelength absorbance detector

High-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) coupled with a multiple wavelength absorbance detector (200-445 nm) was used in this study to investigate the apparent molecular weight (AMW) distributions of dissolved organic matter (DOM). Standard DOM, namely humic acid, fulvic acid and hydrophilic acid, from the Suwannee River were tested to ascertain the performance and sensitivity of the method. In addition to four compounds groups: humic substances (Peak 1, AMW 16 kD), fulvic acids (Peak 2, AMW 11 kD), low AMW acids (Peak 3, AMW 5 kD), and low AMW neutral and amphiphilic molecules, proteins and their amino acid building blocks (Peak 4, AMW 3 kD), an new group that appears to include low AMW, 6-10 kD, humic substances was found based on investigating the spectra at various elution times. The spectroscopic parameter S_>365 (slope at wavelengths >365 nm) was determined to be a good predictor of the AMW of the DOM. The detector wavelength played an important role in evaluating the AMW distribution. For some fractions, such as the humic and low AMW non-aromatic substances, the error in measurement was ±30% as determined by two-dimensional chromatograms detected at an artificially selected wavelength. HPLC-SEC with multiple wavelength absorbance detection was found to be a useful technique for DOM characterization. It characterized the AMW distributions of DOM more accurately and provided additional, potentially important information concerning the properties of DOM with varying AMWs.     

Water quality and fish size affect toxicity of endosulfan, an organochlorine pesticide, to rainbow trout

The acute toxicity of endosulfan in juvenile rainbow trout (Oncorhynchus mykiss, 10.61+/-1.69 g) was evaluated in glass aquaria under static conditions. Nominal concentrations of endosulfan in the toxicity test ranged from 1.3 microg l(-1) to 29 microg l(-1). The concentrations of endosulfan that killed 50% of the rainbow trout within 24-h (24-h LC50), 48-h LC50, 72-h LC50, and 96-h LC50 were 19.78, 8.89, 5.28, and 1.75 microg l(-1), respectively. None of the unexposed control fish died, and the first fish died 4 h after exposure to 26.3 microg l(-1) of endosulfan. Survival of fish was significantly increased with increasing fish size and decreased with decreased fish size at the same temperature (p<0.001). Temperature also had a significant effect on survival of fish. Alkalinity at levels above 20 mg l(-1) as CaCO3 significantly increased survival of fish at 19.78 microg l(-1) of endosulfan. Increasing alkalinity from 20 mg l(-1) as CaCO3 to 42 or higher concentrations tested in this study (121 mg l(-1) as CaCO3) significantly increased survival of fish (p<0.01). Total hardness ranging from 55 mg l(-1) as CaCO3 to 126 mg l(-1) as CaCO3 did not affect survival of fish exposed to endosulfan. Endosulfan toxicity was found to be irreversible when fish were exposed to minimum concentrations of endosulfan tested. Histologically, fish gills had lamellar edema, separation of epithelium from lamellae, lamellar fusion, and swelling of the epithelial cells. Melanomacrophage centers were scattered throughout the trunk kidney, head kidney, and spleen. The liver of endosulfan-exposed fish had severe focal necrosis. None of these lesions were seen in unexposed control fish. Results indicate that alkalinity, temperature, and fish size affect endosulfan toxicity of rainbow trout.     

Inhibition of rainbow trout acetylcholinesterase by aqueous and suspended particle-associated organophosphorous insecticides

Cadmium (Cd) adsorption on 14 non-calcareous New Jersey soils was investigated with a batch method. Both adsorption edge and isotherm experiments were conducted covering a wide range of soil composition, e.g. soil organic carbon (SOC) concentration ranging from 0.18% to 7.15%, and varying Cd concentrations and solution pH. The SOC and solution pH were the most important parameters controlling Cd partition equilibrium between soils and solutions in our experimental conditions. The Windermere humic aqueous model (WHAM) was used to calculate Cd adsorption on soils. The effect of solution chemistry (various pH and Cd concentrations) on Cd adsorption can be well accounted for by WHAM. For different soil compositions, SOC concentration is the most important parameter for Cd binding. Only a fraction of SOC, the so-called active organic carbon (AOC), is responsible for Cd binding. We found a linear relationship between SOC and AOC based on the adsorption edge data. The linear relationship was validated by the independent data sets: adsorption isotherm data, which presumably can be used to predict Cd partition equilibrium across a wide range of soil compositions. The modeling approach presented in this study helps to quantitatively predict Cd behavior in the environment.     

Toxicity evaluation of new antifouling compounds using suspension-cultured fish cells

A simple, rapid toxicity test was developed using the suspension-cultured fish cell line CHSE-sp derived from chinook salmon Oncorhynchus tshawytscha embryos in order to assess the toxicity of new marine antifouling compounds. The compounds tested were copper pyrithione, Diuron, Irgarol 1051, KH101, Sea-Nine 211, and zinc pyrithione, all of which have been nominated in Japan as possible replacements for organotin compounds. The in vitro acute toxicity (24-h EC50) of the six compounds to these fish cells was evaluated using the dye Alamar Blue to determine cell viability, and then correlated with the results of in vivo chronic toxicities (28-day LC50) to juvenile rainbow trout Oncorhynchus mykiss. The suspension-cultured fish cells were found to be suitable for the screening of such chemicals before performing an in vivo test. The toxicities of the test compounds obtained from both tests, shown in decreasing order, were as follows: copper pyrithione > zinc pyrithione > KH101 > or = Sea-Nine 211 > Diuron > Irgarol 1051. The herbicides Diuron and Irgarol 1051 showed the least toxicity, while the pyrithiones had the greatest toxicity.     

Toxicity characterisation of sediment porewaters collected from UK estuaries using a Tisbe battagliai bioassay

The lethal toxicity of sediment porewater isolated from samples collected from six United Kingdom estuaries was tested using a Tisbe battagliai bioassay. A selection of Phase 1 toxicity identification evaluation (TIE) procedures was then used to characterise the substances responsible for the measured toxicity. In samples collected in 2000, cationic metals, ammonia, organic compounds were identified as the cause of toxicity in the most toxic sample collected. Some of the toxicity remained uncharacterised. It was shown that the toxicity in samples collected from the same locations in 2001 was due to ammonia. The successful application of the T. battagliai bioassay demonstrates that it is a suitable assay for marine sediment porewater TIE and that combined with simple characterisation procedures allow an initial assessment to be made on the type of substances responsible for the measured toxicity.     

Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment.

Pyrene biodegradation in a freshwater sediment without fungi supply, or inoculated with two sediment micromycetes, Mucor racemosus var. sphaerosporus and Phialophora alba was studied after 0, 5, 13, 28, 60 and 90 days. The influence of glucose addition was estimated, and a liquid chromatographic method for simultaneous quantitative determination of residual anthracene, fluoranthene and pyrene in the sediment was developed. Samples with PAHs were extracted in Soxhlet with ethyl acetate, and LC analysis was performed on a 5 microm Supelcosil column (150 x 4.6 mm I.D.) with gradient elution (2 ml min(-1)) of acetonitrile-water and UV detection at 254 nm. Recoveries of anthracene, fluoranthene and pyrene were 90.3%+/-1.1%, 93.2%+/-0.9% and 90.42%+/-1.9%, respectively, without interference. The native sediment microorganisms (with or without glucose added) have shown 35% pyrene degradation and sediment with glucose inoculated by the strains revealed 40%.     

Toxicity of copper in an oxic stream sediment receiving aquaculture effluent.

Sediments were collected from a stream (upstream, outfall and downstream) receiving copper laden catfish pond effluent to assess toxicity to non-target biota. No significant reduction in Hyalella azteca survival or growth (10 d), or Typha latifolia germination and root and shoot growth (7 d) were observed after exposure to upstream and outfall sediments. A significant reduction in H. azteca survival was observed after exposure to the downstream sediment sample; however, no reduction in T. latifolia germination or seedling growth was detected. Bulk sediment copper concentrations in the upstream, outfall and downstream samples were 29, 31, and 25 mg Cu/kg dry weight, respectively. Interstitial water (IW) concentrations ranged from 0.053 to 0.14 mg Cu/l with 10 d IW toxicity units > or = 0.7. Outfall samples were amended with additional concentrations of copper sulfate so that bulk sediment measured concentrations in the amended samples were 172, 663, 1245, and 1515 mg Cu/kg dry weight. Survival was the most sensitive endpoint examined with respect to H. azteca with a no observed effects concentration (NOEC) and lowest observed effects concentration (LOEC) of 1245 and 1515 mg Cu/kg, respectively. NOEC and LOEC for T. latifolia root growth were 663 and 1245 mg Cu/kg, respectively. IW copper concentrations were > or = 0.86 mg Cu/l with H. azteca intersitial water toxicity unit (IWTU) concentrations > or = 1.2. Sequential extraction qualitatively revealed the carbonate and iron oxide fractions which accounted for a majority of the copper binding. In this instance, the copper which was applied to catfish ponds does not appear to be adversely impacting the receiving stream system.     

Separation of PCBs and PAHs in sediment samples using silica gel fractionation chromatography

The aim of this research is to develop a silica gel fractionation procedure for sediment sample extracts, which separates polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) into two groups, while simultaneously eliminating most interfering substances for subsequent instrumental analysis. This is achieved by optimizing the fraction cut-off volume of eluting solvent and the deactivation level of the silica gel. Using fully activated silica gel and cutting off PCB collection after passing 60-65 ml eluting solvent (pentane or hexane) through the column resulted in satisfactory separation of PCBs and PAHs. This procedure tends to have a higher reliability for PCBs and PAHs with higher molecular mass. This approach deviates only slightly from the standard methods of the USEPA, and it is less expensive due to reduced sample pre-treatment time and solvent consumption.     

Wastewater analysis for volatile organic sulfides using purge-and-trap with gas chromatography/mass spectrometry.

This paper presented a modified method for the analysis of volatile organic sulfides (VOS) simultaneously with volatile organic compounds (VOC) in wastewater using purge-and-trap with gas chromatography/ mass spectrometry. Calibration standards were prepared using filtered and nitrogen-purged VOS-free wastewater, acidified to pH 1.4. Samples were also acidified to pH 1.4. This approach minimized the oxidation of methanethiol to dimethyl disulfide (DMDS), which hampered the liquid-phase analysis of VOS. Compounds were concentrated from the liquid phase, and automated analyses were performed without additional equipment, other than that required for routine wastewater VOC analysis. The linear range was 5 to 500 microg/L, with r2 > or = 0.99. The average recovery from replicate analyses of spiked samples was 81 +/- 0.5% for methanethiol, 100 +/- 1.5% fordimethyl sulfide (DMS), and 92 +/- 1.5% for DMDS. Method detection limits were 4.8, 2.8, and 1.2 microg/L for methanethiol, DMS, and DMDS, respectively. The relative percent differences were between 0 and 8%.     

Photoinductive properties of soil humic acids and their fractions obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis.

Humic acids (HAs) from three soils of different origin (Chernozem, Ferralsol and Ranker) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE) on three fractions (fractions A, B, C + D) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EM). Fractions identically marked had similar EM and MS, independently of HA sources. The photoinductive properties of the whole HAs and their fractions were compared by studying the photoinduced transformation of fenuron at 365 nm. High MS fractions A and B appeared to exhibit poor photoinductive activities compared to the whole HAs, whereas low MS fraction C + D in Chernozem and Ranker were more efficient than the whole HAs. A fourth intermediary fraction containing a mixture of fractions B and C + D with small amount of D was shown to photoinduce poorly the transformation of fenuron. It was therefore concluded that the molecules capable of photoinducing the transformation of fenuron were mainly contained in fraction D. Fluorescence properties of Chernozem HA and its fractions have been tested. Fraction C + D exhibited a very similar fluorescence emission spectrum in comparison with the whole HA and in contrast, the fractions A and B emitted very weakly.     

Allelopathic potential and systematic evaluation of organic extracts from Canavalia ensiformis leaves (Jack beans)

This article describes the assessment of possible allelopathic potential of organic extracts obtained from leaves of Canavalia ensiformis under laboratory conditions. Furthermore, a systematic evaluation of these extracts was carried out using specific protocols developed in capillary electrophoresis (CE) to determine some groups of secondary metabolites. After the identification and quantification of compounds, the effects of compounds on germination of some common weeds was investigated, which are becoming a real problem in pastures in the state of Pará, Brazil.     

大型蚤标志酶用于城市二级出水毒性的评价

大型蚤是一种国际公认的标准实验生物,广泛应用于污水、地表水等水质毒性检测。毒性水平较低的城市二级出水,对大型蚤往往无急性毒性效应,而具有慢性毒性效应,但慢性毒性检测周期过长,因此探索一种更灵敏的指标,实现快速检测,对于控制二级出水的水质风险具有重要意义。本研究考察了大型蚤在短期暴露于城市二级出水条件下,其体内乙酰胆碱酯酶、超氧化物歧化酶、过氧化氢酶、ATP酶、羧酸酯酶和碱性磷酸酯酶的酶活变化特征,从中筛选出对二级出水毒性响应灵敏的标志酶指标。实验结果表明,碱性磷酸酯酶、过氧化氢酶对二级出水毒性响应相对较灵敏,具有成为标志酶的潜力,研究结果为城市二级出水生物毒性评价方法优化提供新的思路。     

Analysis of volatile and semivolatile organic compounds in municipal wastewater using headspace solid-phase microextraction and gas chromatography.

The aim of this work was to develop a simple and fast analytical method for the determination of a wide range of organic compounds (volatile and semivolatile compounds) in municipal wastewater. The headspace-solid-phase microextraction (HS-SPME) and gas chromatography (with mass spectroscopy) was used for determination of the organic compounds. In this study, 39 organic compounds were determined, including 3 sulfur compounds, 28 substituted benzenes, and 8 substituted phenols. The extraction parameters, such as types of SPME fiber, extraction temperature, extraction time, desorption time, salt effect, and magnetic stirring, were investigated. The method had very good repeatability, because the relative standard deviations ranged from 0.5 to 12%. The detection limit of each compound was at or below the microgram-per-liter level. This method was applied for determination of the organic compounds in raw wastewater, primary effluent, secondary effluent, and chlorinated secondary effluent samples from the Chania Municipal Wastewater Treatment Plant (Crete, Greece).     

兼性厌氧菌群对湖滨带底泥有机污染物的降解效应

研究了兼性厌氧菌群不同接种量(1%(体积分数,下同)、5%、10%、20%、50%)下对太湖湖滨带底泥有机污染物的降解效果及微生物群落结构的影响。结果表明,与对照组相比,接种兼性厌氧菌群能显著提高总有机碳(TOC)、TN的降解率且降解率高,但是TP的降解率波动较大。补充实验证明接种菌液中大量的有机碳、氮源会引起样品中初始浓度升高,建议在使用接种菌液前进行3次无菌水冲洗。综合考虑接种兼性厌氧菌群对湖滨带底泥有机污染物的降解效果,确定其最佳接种量为10%。另外,接种兼性厌氧菌群后其菌群数量和菌活性都有显著提高而严格好氧菌、严格厌氧菌数量均无显著变化;第0天,微生物在接种量为10%时菌活性电子传递体系(ETS)最高。