Total Analysis of Microcystins in Fish Tissue Using Laser Thermal Desorption-Atmospheric Pressure Chemical Ionization-High-Resolution Mass Spectrometry (LDTD-APCI-HRMS).

Microcystins (MCs) are cyanobacterial toxins encountered in aquatic environments worldwide. Over 100 MC variants have been identified and have the capacity to covalently bind to animal tissue. This study presents a new approach for cell-bound and free microcystin analysis in fish tissue using sodium hydroxide as a digestion agent and Lemieux oxidation to obtain the 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) moiety, common to all microcystin congeners. The use of laser diode thermal desorption-atmospheric pressure chemical ionization coupled with Q-Exactive mass spectrometry (LDTD-APCI-HRMS) led to an analysis time of approximately 10 s per sample and high-resolution detection. Digestion/oxidation and solid phase extraction recoveries ranged from 70 to 75% and from 86 to 103%, respectively. Method detection and quantification limits values were 2.7 and 8.2 µg kg(-1), respectively. Fish samples from cyanobacteria-contaminated lakes were analyzed, and concentrations ranging from 2.9 to 13.2 µg kg(-1) were reported.

High resolution/accurate mass (HRMS) detection of anatoxin-a in lake water using LDTD-APCI coupled to a Q-Exactive mass spectrometer.

A new innovative analytical method combining ultra-fast analysis time with high resolution/accurate mass detection was developed to eliminate the misidentification of anatoxin-a (ANA-a), a cyanobacterial toxin, from the natural amino acid phenylalanine (PHE). This was achieved by using the laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) coupled to the Q-Exactive, a high resolution/accurate mass spectrometer (HRMS). This novel combination, the LDTD-APCI-HRMS, allowed for an ultra-fast analysis time (<15 s/sample). A comparison of two different acquisition modes (full scan and targeted ion fragmentation) was made to determine the most rigorous analytical method using the LDTD-APCI interface. Method development focused toward selectivity and sensitivity improvement to reduce the possibility of false positives and to lower detection limits. The Q-Exactive mass spectrometer operates with resolving powers between 17500 and 140000 FWHM (m/z 200). Nevertheless, a resolution of 17500FWHM is enough to dissociate ANA-a and PHE signals. Mass accuracy was satisfactory with values below 1 ppm reaching precision to the fourth decimal. Internal calibration with standard addition was achieved with the isotopically-labeled (D5) phenylalanine with good linearity (R(2)>0.999). Enhancement of signal to noise ratios relative to a standard triple-quadrupole method was demonstrated with lower detection and quantification limit values of 0.2 and 0.6 µg/L using the Q-Exactive. Accuracy and interday/intraday relative standard deviations were below 15%. The new method was applied to 8 different lake water samples with signs of cyanobacterial blooms. This work demonstrates the possibility of using an ultra-fast LDTD-APCI sample introduction system with an HRMS hybrid instrument for quantitative purposes with high selectivity in complex environmental matrices.

Total microcystins analysis in water using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

A new approach for the analysis of the cyanobacterial microcystins (MCs) in environmental water matrices has been developed. It offers a cost efficient alternative method for the fast quantification of total MCs using mass spectrometry. This approach permits the quantification of total MCs concentrations without requiring any derivatization or the use of a suite of MCs standards. The oxidation product 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) was formed through a Lemieux oxidation and represented the total concentration of free and bound MCs in water samples. MMPB was analyzed using laser diode thermal desorption-atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS). LDTD is a robust and reliable sample introduction method with ultra-fast analysis time (<15 s sample(-1)). Several oxidation and LDTD parameters were optimized to improve recoveries and signal intensity. MCs oxidation recovery yield was 103%, showing a complete reaction. Internal calibration with standard addition was achieved with the use of 4-phenylbutyric acid (4-PB) as internal standard and showed good linearity (R(2)>0.999). Limits of detection and quantification were 0.2 and 0.9 µg L(-1), respectively. These values are comparable with the WHO (World Health Organization) guideline of 1 µg L(-1) for total microcystin-LR congener in drinking water. Accuracy and interday/intraday variation coefficients were below 15%. Matrix effect was determined with a recovery of 91%, showing no significant signal suppression. This work demonstrates the use of the LDTD-APCI-MS/MS interface for the screening, detection and quantification of total MCs in complex environmental matrices.

Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments.

In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee's AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.

Spatial and temporal occurrence of N-nitrosamines in seven drinking water supply systems.

The spatiotemporal presence of eight N-nitrosamines in the water of seven supply systems in Quebec considered to be susceptible to these emerging disinfection by-products was evaluated. This is the first study on the presence of N-nitrosamines in drinking water utilities in Quebec. Seven sampling campaigns were carried out at several sampling points in each of the systems over a period of 1 year. The results show that N-nitrosamines, primarily N-nitrosodimethylamine (NDMA), were not commonly detected in the water of the facilities under study (10 % of samples). The concentrations measured were lower than those reported in recent North American studies. None of the 195 samples taken exceeded the Ontario standard of 9 ng/L for NDMA (maximum value observed of 3.3 ng/L). N-nitrosomethylethylamine and N-nitrosopiperidine were detected once, with concentrations of 3.7 and 6.0 ng/L, respectively. Chloramination was identified as being the main risk factor regarding the presence of N-nitrosamines, but water quality and some operating parameters, in particular disinfectant residual, also seem to be related to their presence. NDMA concentrations at the end of the distribution systems were generally higher than water leaving the plant. No seasonal trends were observed for the formation of N-nitrosamines in the investigated supply systems. Finally, an association between the presence of N-nitrosamines and the levels of trihalomethanes and haloacetic acids was observed in some facilities.

Performance evaluation of phycocyanin probes for the monitoring of cyanobacteria.

The performance of two field probes (YSI 6600 and TriOS), used for the measurement of in vivo phycocyanin fluorescence, was compared and validated in the laboratory in 2008 and 2009 with cultures of Microcystis aeruginosa and field samples. The background noise of the two probes was low and the detection limits were estimated at 1500 cells mL(-1) for the YSI and 0.69 µg PC L(-1) for the TriOS. The linearity and repeatability of both probes have been excellent. Strong relationships were observed between the in vivo fluorescence and the total cyanobacterial biovolume (R(2) = 0.82 YSI; 0.83 TriOS) or the abundance (R(2) = 0.71 YSI; 0.75 TriOS) of cyanobacteria. However, the difference between cell densities determined by microscopy and measured by the YSI can be very large and has been associated to the variability of cell volume among cyanobacteria. This last observation makes the YSI a qualitative tool if a post-calibration is not done. The analysis of filtrated samples showed that dissolved phycocyanin (extracellular) may represent a significant fluorescence signal. No relationship could be established between the abundance, the total cyanobacterial biovolume or the in vivo fluorescence of phycocyanin and the concentrations of cyanotoxins (R(2) ≤ 0.22).

Study design, water quality, morphometrics and age of the bullfrog, Rana catesbeiana, in sub-watersheds of the Yamaska River drainage basin, Québec, Canada.

The Yamaska River basin is renowned for its poor water quality, which has been attributed to intensive agriculture (corn, soya, high-density pork and poultry production). Six locations within the Yamaska watershed were selected to evaluate the impact of agriculture on water habitats and study the bullfrog as a sentinel species of potential exposure and effects. The selected sub-watersheds were chosen according to the percentage of surface area under cultivation and classified as low (0-19%), moderate (20-59%) or high (>60%). In 2004 and 2005, analysis of surface water samples demonstrated that pesticide concentrations and most water quality parameters increased with increasing agricultural activity. Sixteen adult bullfrogs were sampled from each site. Animals were weighed, measured for length (total, snout-vent, tibia, tympanum), sexed, and evaluated for the colour of the throat. Skeletochronology was used to estimate the growth and age of the frogs. Cross-sections of decalcified phalanges and femurs were treated in order to count LAGs (lines of arrest growth) and for the observations of other parameters related to bone growth. The bullfrogs from highly contaminated sites had the lowest mean age and the smallest snout-vent length compared to sites of low contamination.

Pesticides in fluvial wetlands catchments under intensive agricultural activities.

A survey on pesticides (73 compounds) in the Bay St. François wetland and its catchment (part of the wetlands of Lake St. Pierre area [St. Lawrence River, Québec]) was achieved in 2006. The metabolites as well as the active ingredients of pesticides (11 compounds) were detected in the wetland and its catchment. This wetland ecosystem was active in the degradation of agricultural pesticides (e.g., atrazine). The measured pesticides were individually below the criteria for aquatic species in natural water, except chlorpyrifos. Overall, the pesticides lost from agricultural field towards the streams were <1% of the quantity applied. The environmental fates of the pesticides were found to vary according to the size of the watershed. Over large catchments, half-life times were important in terms of global loss from the agricultural lands to wetlands whereas over small catchments, soil organic carbon/water distribution coefficient (Koc) was an important term for pesticides losses to water system since half-life times were not limiting factors.

Pesticides measured in air and precipitation in the Yamaska Basin (Québec): occurrence and concentrations in 2004.

Air and precipitation samples were collected and analyzed for 91 pesticides or metabolites from May to September 2004 at St. Damase, an agricultural site located in Yamaska basin in Québec, Canada. A broad range of pesticides was detected during this experiment where 40 different compounds were measured. Most of the samples showed more than 10 simultaneous pesticide detections and sometimes reaching 19 simultaneous detections. The most detected pesticides in air were trifluralin, metolachlor and captan, which were found in all the samples during the 5 months of measurements. For the detected compounds, the average concentrations ranged from 4 pg/m3 to 8 ng/m3. Some of the pesticides detected in air were found in precipitation samples as well showing that wet deposition can occur and have an impact on aquatic ecosystems. The most important pesticides detected were related to corn and soya cultivations, the two main cultures in this region highlighting that the major sources come from local applications. On the other hand, the detection of some pesticides in precipitation which were undetectable in air implies that some compounds may have a long range transport origin.

Hepatic retinoids of bullfrogs in relation to agricultural pesticides.

Agricultural pesticides often have been cited as a factor affecting indigenous amphibian populations, but possible effects of pesticides and other factors associated with agricultural practices are understood poorly. Adult bullfrogs (Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) in subwatersheds representing low, medium, and high agricultural activities and 53 pesticides were analyzed in surface water. More pesticides were detected in subwatersheds associated with high agricultural activities like Rivière Noire and Rivière à la Barbue and pesticide concentrations were higher compared to the other study sites. Female and male body weights differed between sites. In the case of males, body weight was significantly less at Rivière à la Barbue. Liver retinol stores were decreased significantly in male bullfrogs from Rivière Noire, although total retinyl esters concentrations varied between sites having the highest concentration at Yamaska-Nord where the agricultural activity was considered low. The ratio of hepatic retinyl palmitate to retinol tended to be higher for male bullfrogs from Rivière Noire and Rivière à la Barbue. These results suggest that factors associated with intensive agricultural practices may affect the body weight and retinoid stores in male bullfrogs living in these agroecosystems.

Oxidative stress and endocrine endpoints in white sucker (Catostomus commersoni) from a river impacted by agricultural chemicals.

The effects of agricultural chemicals on cortisol secretion, antioxidants, and lipid peroxidation were investigated in hepatic and adrenal tissue of white sucker (Catostomus commersoni) from a river (Yamaska) that drains an agricultural region in Québec (Canada). Plasma cholinesterase (ChE) activity, used as a biomarker of exposure to pesticides, was elevated in fish from the reference site compared to fish from the contaminated sites. Plasma concentrations of cortisol and thyroid hormones (T3 and T4) were higher in fish from the reference site compared to contaminated sites; reduced glutathione (GSH) levels, catalase (CAT), and glutathione peroxidase (GPx) activities were higher and lipid peroxidation (LPO) was lower. Levels of antioxidants (CAT, Gpx, and GSH) were higher (10-90%) and LPO levels were lower (50%) in the liver than in the adrenal tissue. The present in situ study provided evidence that antioxidants, lipid peroxidation, and plasma hormones were altered in fish sampled in areas impacted by agricultural chemicals. Endocrine-disrupting effects were associated with oxidative stress. The results suggest that antioxidants and lipid peroxidation could be used as markers of contaminant exposure in fish.

Nonylphenolic compounds in drinking and surface waters downstream of treated textile and pulp and paper effluents: a survey and preliminary assessment of their potential effects on public health and aquatic life.

Eleven drinking water treatment plants, located downstream of textile plants or pulp and paper mills, have been sampled monthly during a year for the analysis of 17 nonylphenol ethoxylates (NP1-17EO) and two nonylphenoxycarboxylic acids (NP1-2EC). At all but one plant, results in the drinking water, for the sum of these 19 substances, range between below detection levels and 6.7 microg/l. Annual means are between 0.02 and 2.8 microg/l. At the other plant, the yearly average concentration is 10.4 microg/l and the monthly maximum is 43.3 microg/l. In the surface (pre-treatment) water, the annual mean concentrations of the 11 plants range between 0.14 and 17.8 microg/l and the recorded instantaneous maximum is 55.3 microg/l. According to Canadian health authorities, drinking water is a negligible route of human exposure to nonylphenolic compounds, even at the highest concentrations found in this study. After transformation of the data into nonylphenol equivalents, about 20% of the surface water samples exceed the Canadian 1 microg/l nonylphenol water quality guideline for the protection of aquatic life. Some results also exceed Québec's 6 microg/l nonylphenol guideline. The efficiency of the plants in removing nonylphenolic compounds from drinking water is highly variable, ranging from 11% to 99%.

Vitellogenin in tilapia male fishes exposed to organochlorine pesticides in Ouémé River in Republic of Benin.

In many African countries, the economy largely depends on agriculture. Pesticides are therefore likely to represent an important source of xenoestrogens in contaminated rivers and lagoons. The largely uncontrolled use of diverse pesticides led us to hypothesize that these agents, and particularly organochlorine compounds, may pose a serious problem in the Republic of Benin. To verify our hypothesis, tilapia (Sarotherodon melanotheron) from five sites in the southern part of the main Ouémé River were analyzed. Ouémé River drains the southern region of the country. Vitellogenin (Vtg) was used as an indicator of contaminated sites. This approach has its limitations, because there are a wide variety of man-made chemicals present in the aquatic environment likely to induce Vtg in male fish. Therefore, in this study this approach allows us to define potential contaminated target sites. In order to determine whether the presence of Vtg could be attributable to pesticides, organochlorine pesticides in the flesh of tilapia were also analyzed. Significant amounts of Vtg in fish from contaminated sites were detected, and were correlated with organochlorine pesticide levels in tissue. These results indicate that organochlorine pesticides are present in the Ouémé River and that these compounds can act as endocrine modulators in this ecosystem. Eating fish from contaminated rivers, such as the Ouémé River, may contribute to the accumulation of high concentrations of these pesticides in the body, leading to exposure to their negative effects.

Liquid chromatographic-tandem mass spectrometric determination of nonylphenol polyethoxylates and nonylphenol carboxylic acids in surface water.

This paper presents a new LC-MS-MS method for the determination of the concentration of nonylphenol ethoxylates (NPEOs) and nonylphenol carboxylic acids (NPECs) in surface and drinking water using a reversed-phase column, which is fast and specific by nature. This method allows the simultaneous analysis of the two families of compounds in the same extract. Liquid-solid extraction of 100 ml of sample is performed on graphitized carbon black (GCB) cartridges. Reversed-phase chromatography is performed on a C8 column with isocratic elution. The electrospray interface is used to monitor the [M+NH4]+ ion for NPEOs and the [M-H]- ion for NPECs. Detection limits range from 0.01 to 0.05 microg/l for NP(1-17)EOs and are 0.01 microg/l for NP(1-2)ECs. Mean recoveries range from 78 to 107% with relative standard deviations ranging from 6 to 16%. Applicability of the method is demonstrated by results from a monthly sampling of river water at 11 sampling points located downstream of suspected polluting industries in Quebec (Canada).