Biodegradation of microcystins and nodularin in freshwaters

Microcystin-LR (MC-LR) was readily biodegraded on addition to six different water samples irrespective of their previous exposure to microcystins. Subsequent studies with water from three of these water bodies confirmed the degradation of MC-LR and also demonstrated the biodegradation of MC-LF, nodularin and mixture of microcystins and nodularin. Rates of degradation of MC-LR, MC-LF and NOD in individual water samples ranged from a half-life of 4 to 18d. Analysis by HPLC-PDA-ESI+ and MALDI MS/MS revealed novel intermediate degradation products of MC-LF and nodularin which included demethylation, hydrolysis, decarboxylation and condensation of the parent compound(s). Our study suggests a possible diversity of micro-organisms and/or pathways which has not been previously observed.     

Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia, Germany).

The presence of microcystins in cyanobacterial samples collected from the Bleiloch reservoir, formerly an important drinking-water supply in Thuringia, Germany, was proven by application of a combination of recently developed analytical methods. The raw extracts were cleaned by size-exclusion chromatography (SEC) or solid-phase extraction (SPE). The determination of microcystins was achieved by different HPLC separation followed by the application of alternative detection methods (UV, diode array detection (DAD), and mass spectrometry (MS), respectively). Furthermore, the different results of clean-up by SPE and SEC are demonstrated. The identity of microcystins was verified by MS/MS measurements. In the cyanobacterial sample from 1998, microcystin-RR, -LR and -YR were found, whereas in 1999 only microcystin-LR and -YR were detectable. In addition to detection of cell-bound microcystins, in 1999 traces of dissolved microcystins in water from the Bleiloch reservoir were detected. It can be assumed that not only the Bleiloch reservoir is contaminated with hepatotoxins but also many similar lakes still used for drinking water supply.     

微囊藻毒素的酶联免疫法分析影响因素探讨

考察样品与抗微囊藻毒素(MC)-LR的单克隆抗体(单抗)加入间隔时间、温育温度、检测时间、pH、H2O2、Fe2+等不同因素对酶联免疫法(ELISA)测定MC-LR的影响。结果表明:(1)随着样品与单抗加入间隔时间延长,MC-LR测定值逐渐降低。(2)25、37℃下,MC-LR测定值几乎没有差别,测定结果与取用标准样品具有很好的一致性。(3)不同检测时间下,无论是低浓度还是高浓度标准样品,MC-LR测定值均有一定变化,虽然幅度均不大,但仍然建议终止反应后尽快比色。(4)当pH为7～9时,MC-LR测定值变化不大。(5)当H2O2摩尔浓度为0.1、1.0、10.0mmol/L时,均会对ELISA产生干扰。通过水浴去除H2O2后,H2O2初始摩尔浓度分别为0.1、1.0mmol/L时,MC-LR回收率分别达到87%及81%。(6)Fe2+对酶有显著的抑制作用,可通过沉淀过滤的方式消除干扰。     

Optimization of an effective extraction procedure for the analysis of microcystins in soils and lake sediments

Microcystin analysis in sediments and soils is considered very difficult due to low recovery for extraction. This is the primary limiting factor for understanding the fate of toxins in the interface between water and sediment in both the aquatic ecosystem as well as in soils. In the present study, a wide range of extraction solvents were evaluated over a wide range of pH, extraction approaches and equilibration time to optimize an effective extraction procedure for the analysis of microcystins in soils and lake sediments. The number of extractions required and acids in extraction solutions were also studied. In this procedure, EDTA-sodium pyrophosphate solution was selected as an extraction solvent based on the adsorption mechanism study. The optimized procedure proved to be highly efficient and achieved over 90% recovery. Finally, the developed procedure was applied to field soil and sediment sample collected from Chinese lakes during bloom seasons and microcystins were determined in six of ten samples.     

阳澄湖和滆湖微囊藻毒素分布及其与富营养化因子的关系

2013年6—10月进行了阳澄湖和滆湖的水样采集及分析,对水体中胞内和胞外3种微囊藻毒素(MC-LR,MCRR,MC-YR)和TN、TP、Chl-a等富营养化指标在两湖的分布情况及关系进行了研究。结果表明,阳澄湖的微囊藻毒素及富营养化因子浓度在不同点位的差异小,而滆湖从北部到南部呈下降趋势,两湖相比,滆湖的浓度远远高于阳澄湖;富营养化因子影响微囊藻毒素的浓度分布和变化;相关性分析表明MC-LR、MC-RR和MC-YR与CODMn、TN、TP、PO3-4-P、Chl-a分别呈极显著正相关性(P0.01),MC-LR、MC-YR与NH+4-N呈显著负相关(P0.05);逐步回归性分析显示Chl-a是影响3种微囊藻毒素浓度的关键因子,可以通过Chl-a对水体中MCs的浓度进行预测,为微囊藻水华和微囊藻毒素污染的预警提供重要的科学参考。     

微囊藻毒素测定中产生的干扰及排除

采用固相萃取-高效液相色谱法定性、定量测定水中微囊藻毒素,对水样富集管(观赏鱼用氧气塑料管、橡胶管、医用乳胶管和硅胶管)、塑料材质(聚丙烯离心管等)、针式过滤器(水系、有机系针式过滤器)产生的干扰和吸附现象进行了深入探索.结果表明,观赏鱼用氧气塑料管对微囊藻毒索测定会产生严重的正干扰,在流动相甲醇:磷酸盐缓冲溶液(体积比)为50:50时,干扰得以排除;聚丙烯离心管在微囊藻毒素投加量较低时,对微囊藻毒索吸附率较高,造成回收率偏低;不同的针式过滤器对溶解在不同体积分数的甲醇溶液中的微囊藻毒素有明显不同的吸附能力.     

Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry

Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.     

Production of a monoclonal antibody and development of an immunoassay for detection of Cr(III) in water samples

In this study we report the production of a monoclonal antibody (Mab) specific for Cr(III)-chelate and the development of a competitive immunoassay for detection of Cr(III) in water samples. In the assay, the complete antigen (Cr(III)-ITCBE-BSA) was used as coating antigen, and Cr(III)-ITCBE as competitor competes with coating antigen to bind with Mab. Using this approach, the spiked water samples with Cr(III) were detected. The linear range of the detection was 0.7–12.4 ng mL⁻¹. The limit of the detection (LOD) was 0.51 ng mL⁻¹. The spiked results were also confirmed by ICP-MS, which showed a good correlation (R² = 0.997) between the two methods. The results indicated that the developed assay was reliable and suitable for the detection of Cr(III) in water samples.     

Colloidal gold probe-based immunochromatographic assay for the rapid detection of lead ions in water samples

One-step immunochromatographic assay (ICA) has been developed using colloidal gold-labeled monoclonal antibody probe for the rapid detection of lead ions in water samples. The ICA was based on the theory of competitive reactivity, and the results can be easily judged based on the presence or absence of a red colored test line with visual detection. Under optimal conditions, this method shows high detecting sensitivity with a LOD (limit of detection) of 50 ng/ml. Stability test indicates that the immunochromatographic strips are stable for 8 weeks at room temperature. During practical application, nanometer TiO₂ is used to enrich the lead ions in water samples. The ICA is successfully applied in the measurement of lead ion concentrations in local water samples, and the results are highly consistent with that of ICP-MS. Detecting lead ions with ICA can be done within 4 min and is very useful for the rapid onsite testing.     

Monitoring of triazine and phenylurea herbicides in the surface waters of Greece

A large-scale study was implemented to monitor triazine and phenylurea herbicides in the main surface water bodies of continental Greece from October 1998 to September 1999. Samples from 10 rivers and 7 lakes were analyzed for the presence of five triazine (atrazine, cyanazine, prometryne, simazine, terbuthylazine) and five phenylurea (chlorotoluron, diuron, linuron, metobromuron, monolinuron) herbicides. The samples were extracted with C18 cartridges and analyzed by high-performance liquid chromatography-diode array detection (HPLC-DAD). The most frequently detected herbicides were atrazine, followed by prometryne, cyanazine, and simazine. The concentrations of the compounds were generally low (< 0.78 micro g/L) and are not considered harmful for the freshwater ecosystem. Most of the positive samples were taken from the water bodies of northern Greece where agricultural activity is more intense.     

Residues of organochlorine and organophosphorus pesticides in sugarcane crop soils and river water

The presence of residual organochlorine and organophosphorus pesticides was evaluated at different periods of sugarcane cultivation in agricultural soil and water samples from the town of Tlaltizapan, which is located in the state of Morelos in Mexico, to determine the presence and persistence of these compounds and their possible effects on the region. The compounds p,p′-DDE, p,p′-DDD (metabolites of p,p′-DDT), γ-HCH and heptachlor were found in more of 95% of the sampling zones in the three monitoring periods performed along 2 years. The highest concentration detected (129.6 μg/kg _{dry soil}) was for α-HCH, but its frequency of detection was ～5%. The low detection frequency of α-HCH and the high concentration values of γ-HCH indicate the repeated use of technical-grade HCH and Lindane (γ-HCH) in the region. Among the organophosphorus pesticides, ethyl parathion was the compound with the highest soil concentration, at ～2000 μg/kg_{dry soil}, during the initial monitoring. However, this compound was detected in the second monitoring with a concentration of ～4 μg/kg_{dry soil}, but it was not detected in the third, indicating that is was not accumulated in the environment. The heptachlor was the compound most commonly found in all water samples, within a range of 0.45–1.25 ng/L. The presence of this organochlorine compound in the water samples indicated a possible migration from the soil to water bodies due to soil erosion. The presence of organophosphorus compounds was not detected in the water samples, which could be attributed to the moderate persistence of these compounds and their consequent degradation before arriving at the water bodies.     

In situ studies on the distribution patterns and dynamics of microcystins in a biomanipulation fish--bighead carp (Aristichthys nobilis)

The distribution and dynamics of microcystins in various organs of the phytoplanktivorous bighead carp were studied monthly in Lake Taihu, which is dominated by toxic cyanobacteria. There was a good agreement between LC-MS and HPLC-UV determinations. Average recoveries of spiked fish samples were 63% for MC-RR and 71% for MC-LR. The highest MC contents in intestine, liver, kidney and spleen were 85.67, 2.83, 1.70 and 1.57 microg g-1 DW, respectively. MCs were much higher in mid-gut walls (1.22 microg g-1 DW) than in hind- and fore-gut walls (0.31 and 0.18 microg g-1 DW, respectively), suggesting the importance of mid-gut wall as major site for MC absorption. A cysteine conjugate of MC-LR was detected frequently in kidney. Among the muscle samples analyzed, 25% were above the provisional tolerable daily intake level by WHO. Bighead is strongly resistant to microcystins and can be used as biomanipulation fish to counteract cyanotoxin contamination in eutrophic waters.     

Degradation of microcystins using immobilized microorganism isolated in an eutrophic lake

The final purpose of our series of studies is to establish a biological removal method of cyanobacteria and their toxic products using immobilized microorganisms that can lyse cyanobacteria and decompose microcystins. To establish the biological removal method in non-point areas and water purification plants, as the first step, we explored bacteria active against the cyanobacterial hepatotoxin microcystin in the present study. Eleven active bacteria were isolated from samples taken from Lakes Tsukui and Sagami, Japan. Among 3 strains (B-9 to B-11) with degradative activity, strain B-9 exhibited the strongest activity. The 16S rDNA sequence of the strain B-9 showed the highest similarity to that of Sphingomonas sp. Y2 (AB084247, 99% similarity). Microcystins-RR and -LR were completely degraded by strain B-9 (SC16) within 1d, which led to an immobilized microorganism with a polyester resin. The degradation of microcystin-RR in a bioreactor using the immobilized strain B-9 was observed and microcystin-RR (> 90%) was completely degraded after 24 h. Microcystin-RR was added to the lake water at regular intervals and the degradation after 24 h was observed in the bioreactor over a 72-d period. An over 80% removal efficiency continued for 2 months, showing that the life of the immobilized B-9 in terms of activity was at least 2 months under the optimized conditions. From these results, this immobilized B-9 is feasible for the practical treatment of microcystins in non-point areas and water purification plants.     

Monitoring of Triazine and Phenylurea Herbicides in the Surface Waters of Greece

A large-scale study was implemented to monitor triazine and phenylurea herbicides in the main surface water bodies of continental Greece from October 1998 to September 1999. Samples from 10 rivers and 7 lakes were analyzed for the presence of five triazine (atrazine, cyanazine, prometryne, simazine, terbuthylazine) and five phenylurea (chlorotoluron, diuron, linuron, metobromuron, monolinuron) herbicides. The samples were extracted with C18 cartridges and analyzed by high-performance liquid chromatography–diode array detection (HPLC-DAD). The most frequently detected herbicides were atrazine, followed by prometryne, cyanazine, and simazine. The concentrations of the compounds were generally low (< 0.78 μ g/L) and are not considered harmful for the freshwater ecosystem. Most of the positive samples were taken from the water bodies of northern Greece where agricultural activity is more intense.     

A review of neurotoxicity of microcystins

Cyanobacterial blooms-produced microcystins are secondary metabolites which can accumulate in the food chain and contaminate water, thus posing a potential threat to the health of aquatic animals and even humans. Microcystin toxicity affects not only the liver but also the other organs, i.e., the brain. The serious neurotoxicity effects caused by microcystins then lead to various symptoms. This review focuses on the neurotoxicity of microcystins. Microcystins can cross blood-brain barrier with the transport of Oatps/OATPs, causing neurostructural, functional, and behavioral changes. In this review, potential uptake mechanisms and neurotoxicity mechanisms are summarized, including neurotransmissions, neurochannels, signal transduction, oxidative stress, and cytoskeleton disruption. However, further researches are needed for detailed studies on signaling pathways and the downstream pathways of neurotoxicity of microcystins.     

Antibiotic contamination in a typical water-rich city in southeast China: a concern for drinking water resource safety

Abstract

The occurrence and distribution in the aquatic environment of Nanjing of 49 antibiotics from seven therapeutic classes were investigated using an improved analytical method developed for multiclass target analysis. The results showed that these antibiotics are widely present in the water bodies of this city, with a total concentration of up to 1.958?μg L^?1. The most abundant class was tetracyclines, contributing 43.7% to the total antibiotic burden. Lincomycin was detected in all samples, and the detection rate of clindamycin was 90.5%. An exploratory analysis of the data points was performed by unsupervised pattern recognition (hierarchical cluster analysis, HCA) in an attempt to clarify the pollution level in different sampling areas, and robust cluster solutions grouped the data according to their different antibiotic contaminant profiles. The safety of drinking water resources was emphasized, and the rivers, as the main receiving water body for treated and untreated wastewater in this city, were more seriously contaminated than the surrounding lakes and reservoir, not only in concentration but also in detection frequency, in our study as well as in similar research studies. A correlation analysis between the presence of antibiotics and the environmental factors was conducted, and it was found that antibiotic contamination and water quality were closely connected; the better the water quality, the lower the antibiotic contamination. Positive correlations existed between the antibiotics and tested heavy metals, as well as between antibiotics and boron and arsenic. However, whether these correlations resulted from their reaction or a common source was difficult to determine, and the mechanism requires further exploration.     

An immunoassay for metolachlor detection in river water and soil.

An indirect enzyme-linked immunosorbent assay (EIA) for metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamid e) detection in river water and soil was developed using serum obtained from rabbits immunized against the acid of metalaxyl ((N-(2,6-dimethylphenyl)-N-(methoxy-acetyl)-DL-alanine methyl ester) conjugated to bovine serum albumin. The assay had a linear working range from 1 to 50 ng/ml with a mean I50 value of 13.6 ng/ml and a lower detection limit of 2.0 ng/ml. Both the mean interwell and interassay coefficients of variation were less than 4% over the range of the standard curves for samples which had been prepared in phosphate buffered saline (PBS), river water, or soil extract. Assay cross-reactivity to the following four structurally related chloro-acetanilide pesticides were: propachlor (0%), metazachlor (0%), alachlor (23%), and metalaxyl (5,000%). Mean recoveries of metolachlor in spiked (2.0 to 32.0 ng/ml range) PBS, river water, and soil extract were 102%, 103%, and 110%, respectively. Soil samples were taken over a 56-d period from field plots treated with metolachlor and analyzed by GC and EIA. The correlation coefficient for comparison of the two methods was 0.96 with the slope of the linear regression line being 0.78. Furthermore, no statistical difference (P less than 0.05) was found between the dissipation curves of metolachlor derived from GC data versus EIA data.     

The impact of the waterborne transmission of Toxoplasma gondii and analysis efforts for water detection: an overview and update

The ubiquitous protozoa Toxoplasma gondii is now the subject of renewed interest, due to the spread of oocysts via water causing waterborne outbreaks of toxoplasmosis in different parts of the world. This overview discusses the different methods for detection of Toxoplasma in drinking and environmental water. It includes a combination of conventional and molecular tools for effective oocyst recovery and detection in water sources as well as factors hindering the detection of this parasite and shedding light on a promising new molecular assay for the diagnosis of Toxoplasma in environmental samples. Hopefully, this attempt will facilitate future approaches for better recovery, concentration, and detection of Toxoplasma oocysts in environmental waters.     

Development of IC-ELISA for detection of organophosphorus pesticides in water

Diethyl (carboxymethyl) phosphonate (DECP) was used as the hapten to develop an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for detecting organophosphorus pesticides (OPs). Conjugator of DECP with bovin serum albumin (BSA) was used as the immunogen for producing the polyclonal antibodies (PcAbs). Three antisera were obtained after the immune procedure. Characterization studies of the PcAbs indicated that the titer of antiserum-1 was highest in 3 antisera, and antiserum-1 had high affinity and specificity to the parathion, dichlorvos and pirimiphos. The IC-ELISA showed an IC50 of 0.428 micro g/mL with a detection limit of 0.0125 micro g/mL to parathion. The assay also indicated that the IC50 values of pirimiphos and dichlorvos were 0.331 micro g/mL and 1.25 micro g/mL respectively, and the detection limits of pirimiphos and dichlorvos were 0.0116 micro g/mL and 0.048 micro g/mL respectively. Recoveries of parathion, pirimiphos and dichlorvos spiked into water samples ranged from 90% to 160%. The results indicated that the ELISA could be a convenient and supplemental analytical tool for monitoring OPs residues in environmental water samples.     

Development of an immunochromatographic assay for the rapid detection of bromoxynil in water

A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01–10 mg L⁻¹ in water were tested by this method and the visual limit was 0.06 mg L⁻¹. The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water.