Snapshot of cyanobacterial toxins in Pakistani freshwater bodies.

Cyanobacteria are known to produce diverse secondary metabolites that are toxic to aquatic ecosystems and human health. However, data about the cyanotoxins occurrence and cyanobacterial diversity in Pakistan's drinking water reservoirs is scarce. In this study, we first investigated the presence of microcystin, saxitoxin, and anatoxin in 12 water bodies using an enzyme-linked immunosorbent assay (ELISA). The observed cyanotoxin values for the risk quotient (RQ) determined by ELISA indicated a potential risk for aquatic life and human health. Based on this result, we made a more in-depth investigation with a subset of water bodies (served as major public water sources) to analyze the cyanotoxins dynamics and identify potential producers. We therefore quantified the distribution of 17 cyanotoxins, including 12 microcystin congeners using a high-performance liquid chromatography-high-resolution tandem mass spectrometry/mass spectrometry (HPLC-HRMS/MS). Our results revealed for the first time the co-occurrence of multiple cyanotoxins and the presence of cylindrospermopsin in an artificial reservoir (Rawal Lake) and a semi-saline lake (Kallar Kahar). We also quantified several microcystin congeners in a river (Panjnad) with MC-LR and MC-RR being the most prevalent and abundant. To identify potential cyanotoxin producers, the composition of the cyanobacterial community was characterized by shotgun metagenomics sequencing. Despite the noticeable presence of cyanotoxins, Cyanobacteria were not abundant. Synechococcus was the most abundant cyanobacterial genus found followed by a small amount of Anabaena, Cyanobium, Microcystis, and Dolichospermum. Moreover, when we looked at the cyanotoxins genes coverage, we never found a complete microcystin mcy operon. To our knowledge, this is the first snapshot sampling of water bodies in Pakistan. Our results would not only help to understand the geographical spread of cyanotoxin in Pakistan but would also help to improve cyanotoxin risk assessment strategies by screening a variety of cyanobacterial toxins and confirming that cyanotoxin quantification is not necessarily related to producer abundance.

Early and late cyanobacterial bloomers in a shallow, eutrophic lake.

Cyanobacterial blooms present challenges for water treatment, especially in regions like the Canadian prairies where poor water quality intensifies water treatment issues. Buoyant cyanobacteria that resist sedimentation present a challenge as water treatment operators attempt to balance pre-treatment and toxic disinfection by-products. Here, we used microscopy to identify and describe the succession of cyanobacterial species in Buffalo Pound Lake, a key drinking water supply. We used indicator species analysis to identify temporal grouping structures throughout two sampling seasons from May to October 2018 and 2019. Our findings highlight two key cyanobacterial bloom phases - a mid-summer diazotrophic bloom of Dolichospermum spp. and an autumn Planktothrix agardhii bloom. Dolichospermum crassa and Woronichinia compacta served as indicators of the mid-summer and autumn bloom phases, respectively. Different cyanobacterial metabolites were associated with the distinct bloom phases in both years: toxic microcystins were associated with the mid-summer Dolichospermum bloom and some newly monitored cyanopeptides (anabaenopeptin A and B) with the autumn Planktothrix bloom. Despite forming a significant proportion of the autumn phytoplankton biomass (>60%), the Planktothrix bloom had previously not been detected by sensor or laboratory-derived chlorophyll-a. Our results demonstrate the power of targeted taxonomic identification of key species as a tool for managers of bloom-prone systems. Moreover, we describe an autumn Planktothrix agardhii bloom that has the potential to disrupt water treatment due to its evasion of detection. Our findings highlight the importance of identifying this autumn bloom given the expectation that warmer temperatures and a longer ice-free season will become the norm.

Occurrence of BMAA Isomers in Bloom-Impacted Lakes and Reservoirs of Brazil, Canada, France, Mexico, and the United Kingdom.

The neurotoxic alkaloid ß-N-methyl-amino-l-alanine (BMAA) and related isomers, including N-(2-aminoethyl glycine) (AEG), ß-amino-N-methyl alanine (BAMA), and 2,4-diaminobutyric acid (DAB), have been reported previously in cyanobacterial samples. However, there are conflicting reports regarding their occurrence in surface waters. In this study, we evaluated the impact of amending lake water samples with trichloroacetic acid (0.1 M TCA) on the detection of BMAA isomers, compared with pre-existing protocols. A sensitive instrumental method was enlisted for the survey, with limits of detection in the range of 5−10 ng L−1. Higher detection rates and significantly greater levels (paired Wilcoxon's signed-rank tests, p < 0.001) of BMAA isomers were observed in TCA-amended samples (method B) compared to samples without TCA (method A). The overall range of B/A ratios was 0.67−8.25 for AEG (up to +725%) and 0.69−15.5 for DAB (up to +1450%), with absolute concentration increases in TCA-amended samples of up to +15,000 ng L−1 for AEG and +650 ng L−1 for DAB. We also documented the trends in the occurrence of BMAA isomers for a large breadth of field-collected lakes from Brazil, Canada, France, Mexico, and the United Kingdom. Data gathered during this overarching campaign (overall, n = 390 within 45 lake sampling sites) indicated frequent detections of AEG and DAB isomers, with detection rates of 30% and 43% and maximum levels of 19,000 ng L−1 and 1100 ng L−1, respectively. In contrast, BAMA was found in less than 8% of the water samples, and BMAA was not found in any sample. These results support the analyses of free-living cyanobacteria, wherein BMAA was often reported at concentrations of 2−4 orders of magnitude lower than AEG and DAB. Seasonal measurements conducted at two bloom-impacted lakes indicated limited correlations of BMAA isomers with total microcystins or chlorophyll-a, which deserves further investigation.

Fast screening of saxitoxin, neosaxitoxin, and decarbamoyl analogues in fresh and brackish surface waters by on-line enrichment coupled to HILIC-HRMS.

The proliferation of harmful cyanobacterial algal blooms is of concern due to the associated release of toxins affecting ecosystems and human health. The paralytic shellfish poison saxitoxin (STX) is a small polar alkaloid that can occur in inland and marine aquatic environments. Here, we optimized a fast and sensitive analytical method for the determination of STX, neosaxitoxin (NeoSTX), and their decarbamoyl analogues in surface waters. The method involves a simple filtration, addition of isotope-labelled internal standard (ILIS), and analysis by on-line solid-phase extraction coupled to hydrophilic interaction liquid chromatography high-resolution mass spectrometry (on-line SPE-HILIC-HRMS). Except glass fiber filters, other tested materials (e.g., nylon, nitrocellulose) provided suitable filtration performance. Time-dependent adsorptive losses occurred during the LC-MS batch sequence if glass autosampler vials were used, while no such effect was observed for polypropylene autosampler vials. Matrix effects were evaluated for 4 different quantification scenarios, including external vs. internal curves and neat reagent water vs. matrix-matched curves. Matrix-matched calibration with ILIS correction (NeoSTX-15N7) provided the best performance overall. The analytical method was validated in freshwater lake water and estuarine brackish water (30‰ salinity), with suitable determination coefficients (R2 > 0.9975), matrix spike accuracy (90-107%), and intraday/interday precision (RSD of 0.61-16%). Method limits of detection (LOD in lake water: 0.72-3.9 ng/L) are also improved over most of the recent literature. The method was applied to a set of 302 surface water samples collected in Canada, France, and the United Kingdom, and positive detections were reported for STX (max: 98 ng/L), decarbamoyl-STX (max: 15 ng/L), and NeoSTX (max: 87 ng/L).

Occurrence of microcystins, anabaenopeptins and other cyanotoxins in fish from a freshwater wildlife reserve impacted by harmful cyanobacterial blooms.

Harmful algal blooms of cyanobacteria (CyanoHABs) can lead to the release of potent toxins that can seriously affect ecosystem integrity. Some freshwater watersheds are particularly at risk considering the threats to already imperiled wildlife. The consumption of tainted drinking water and contaminated food also raises concerns for human health. In the present study, a pilot survey was conducted in the riverine ecosystem of the Pike River Ecological Reserve (QC, Canada) near Missisquoi Bay, Lake Champlain. We examined the occurrence of multiclass cyanotoxins including 12 microcystins, anatoxins, cylindrospermopsin (CYN), anabaenopeptins (AP-A, AP-B), and cyanopeptolin-A in surface waters and wild-caught fish during the summer 2018. Out of the 18 targeted cyanotoxins, 14 were detected in bloom-impacted surface water samples; toxins peaked during early-mid September with the highest concentrations for MC-LR (3.8 µg L-1) and MC-RR (2.9 µg L-1). Among the 71 field-collected fish from 10 species, 30% had positive detections to at least one cyanotoxin. In positive samples, concentration ranges in fish muscle were as follows for summed microcystins (∑MCs: 0.16-9.2 µg kg-1), CYN (46-75 µg kg-1), AP-A (1.1-5.4 µg kg-1), and AP-B (0.12-5.0 µg kg-1). To the best of our knowledge, this is one the first reports of anabaenopeptins occurrence in wildlife. The maximum ∑MCs in fish was 1.15-fold higher than the World Health Organization (WHO) daily intake recommendation for adults and nearly equated the derived value for young children. The concentration of CYN was also about 3-fold higher than the limit derived from the human health guideline values.

Stability issues of microcystins, anabaenopeptins, anatoxins, and cylindrospermopsin during short-term and long-term storage of surface water and drinking water samples.

Reproducible analytical procedures and rigorous quality control are imperative for an accurate monitoring of cyanobacterial toxins in environmental water samples. In this study, the short-term and long-term storage stability of diverse cyanotoxins (anatoxins, cylindrospermopsin, anabaenopeptins, and 12 microcystins) was evaluated in water samples, under different scenarios. Transport controls were performed at three monitoring sites in spiked ultrapure water and lake water to investigate short-term stability issues. Medium-term storage stability was evaluated for up to 14-28 days in ultrapure water, chlorine-treated drinking water (amended with reductant), and surface water (filtered and unfiltered) stored at different temperatures (20 °C, 4 °C, and -20 °C). Substantial decreases of cylindrospermopsin and anabaenopeptins were observed in tap water (20 °C) and unfiltered surface water (20 °C or 4 °C). Regardless of matrix type, cyanotoxin recoveries generally remained within an 80-120% range when the water samples were kept frozen. After a prolonged storage duration of 365 days at -20 °C, most cyanotoxins experienced decreases in the range of 10-20%. The notable exception was for the tryptophan-containing MC-LW and MC-WR, with more substantial variations (30% to 50% decrease) and conversion to N-formylkynurenine analogs. Reanalysis of field-collected surface waters after long-term storage at -20 °C also indicated significantly decreasing trends of cyanotoxins (between 6% and 23% decrease). In view of the above, short sample hold times should be favored as recommended in EPA methods.

Analysis of the neurotoxin ß-N-methylamino-L-alanine (BMAA) and isomers in surface water by FMOC derivatization liquid chromatography high resolution mass spectrometry.

The neurotoxin ß-N-methylamino-L-alanine (BMAA), suspected to trigger neurodegenerative diseases, can be produced during cyanobacterial bloom events and subsequently affect ecosystems and water sources. Some of its isomers including ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl) glycine (AEG), and 2,4-diaminobutyric acid (DAB) may show different toxicities than BMAA. Here, we set out to provide a fast and sensitive method for the monitoring of AEG, BAMA, DAB and BMAA in surface waters. A procedure based on aqueous derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was investigated for this purpose. Under optimized conditions, a small aqueous sample aliquot (5 mL) was spiked with BMAA-d3 internal standard, subjected to FMOC-Cl derivatization, centrifuged, and analyzed. The high-throughput instrumental method (10 min per sample) involved on-line pre-concentration and desalting coupled to ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). Chromatographic gradient and mobile phases were adjusted to obtain suitable separation of the 4 isomers. The method limits of detection were in the range of 2-5 ng L-1. In-matrix validation parameters including linearity range, accuracy, precision, and matrix effects were assessed. The method was applied to surface water samples (n = 82) collected at a large spatial scale in lakes and rivers in Canada. DAB was found in >70% of samples at variable concentrations (<3-1,900 ng L-1), the highest concentrations corresponding to lake samples in cyanobacterial bloom periods. BMAA was only reported (110 ng L-1) at one HAB-impacted location. This is one of the first studies to report on the profiles of AEG, BAMA, DAB, and BMAA in background and impacted surface waters.

Transcriptome sequencing (RNA-seq) analysis of the effects of metal nanoparticle exposure on the transcriptome of Chlamydomonas reinhardtii.

The widespread use of nanoparticles (NPs) raises concern over their potential toxicological effects in humans and ecosystems. Here we used transcriptome sequencing (RNA-seq) to evaluate the effects of exposure to four different metal-based NPs, nano-Ag (nAg), nano-TiO2 (nTiO2), nano-ZnO (nZnO), and CdTe/CdS quantum dots (QDs), in the eukaryotic green alga Chlamydomonas reinhardtii. The transcriptome was characterized before and after exposure to each NP type. Specific toxicological effects were inferred from the functions of genes whose transcripts either increased or decreased. Data analysis resulted in important differences and also similarities among the NPs. Elevated levels of transcripts of several marker genes for stress were observed, suggesting that only nZnO caused nonspecific global stress to the cells under environmentally relevant conditions. Genes with photosynthesis-related functions were decreased drastically during exposure to nTiO2 and slightly during exposures to the other NP types. This pattern suggests either toxicological effects in the chloroplast or effects that mimic a transition from low to high light. nAg exposure dramatically elevated the levels of transcripts encoding known or predicted components of the cell wall and the flagella, suggesting that it damages structures exposed to the external milieu. Exposures to nTiO2, nZnO, and QDs elevated the levels of transcripts encoding subunits of the proteasome, suggesting proteasome inhibition, a phenomenon believed to underlie the development and progression of several major diseases, including Alzheimer's disease, and used in chemotherapy against multiple myeloma.

Bioaccumulation and effects of CdTe/CdS quantum dots on Chlamydomonas reinhardtii - nanoparticles or the free ions?

In order to properly assess the environmental risk of engineered nanoparticles (ENP), it is necessary to determine their fate (including dissolution, aggregation, and bioaccumulation) under representative environmental conditions. CdTe/CdS quantum dots (QD), such as those used in medical imaging, are known to release Cd(2+) due (mainly) to the dissolution of their outer shell. In this study, Chlamydomonas reinhardtii was exposed to either a soluble Cd salt or QD at similar concentrations of total Cd. Free Cd concentrations were measured using the Absence of Gradients and Nernstian Equilibrium Stripping technique. QD dissolution increased with decreasing pH and with increasing QD concentration. When exposed to QD, bioaccumulation was largely accounted for by dissolved Cd. Nonetheless, QD were shown to be taken up by the cells and to provoke unique biological effects. Whole transcriptome screening using RNA-Seq analysis showed that the free Cd and the QD had distinctly different biological effects.

In situ evaluation of cadmium biomarkers in green algae.

In situ measurements provide data that are the highly representative of the natural environment. In this paper, laboratory-determined biomarkers of Cd stress that were previously identified for the green alga Chlamydomonas reinhardtii, were tested in two French rivers: a contaminated site on the Riou Mort River and an "uncontaminated" reference site on the Lot River. Transcript abundance levels were determined by real time qPCR for biomarkers thought to be Cd sensitive. Transcript levels were significantly higher (>5 fold) for organisms exposed to the contaminated site as compared to those exposed at the uncontaminated site. Biomarker mRNA levels were best correlated to free Cd (Cd(2+)) rather than intracellular Cd, suggesting that they may be useful indicators of in situ stress. The paper shows that biomarker expression levels increased with time, were sensitive to metal levels and metal speciation and were higher in the "contaminated" as opposed to the "reference" site.

A novel in situ tool for the exposure and analysis of microorganisms in natural aquatic systems.

To evaluate the effects of contaminants or nutrient limitation in natural waters, it is often desirable to perform controlled exposures of organisms. While in situ exposures are routine for caged organisms or macrophytes, they are extremely difficult to perform for microorganisms, mainly due to difficulties in designing an exposure device that isolates the cells while allowing rapid equilibration with the external media. In this paper, a stirred underwater biouptake system (SUBS) based on the diffusion of chemicals across a semipermeable membrane housing a controlled population of microorganisms is reported. Cd diffusion through the semipermeable membrane was evaluated by voltammetry using a microelectrode. Comparison of stirred and unstirred solutions demonstrated a significantly increased diffusive flux in the presence of stirring. Lab tests using Chlamydomonas reinhardtii showed that diffusion across the semipermeable membrane was not limiting with respect to the biouptake of Cd. The SUBS device was field tested and the results of viability studies and trace metal biouptake by C. reinhardtii are reported. No diffusion limitation due to the SUBS was observed for Cd under the tested field conditions. The SUBS device was also shown to be useful for field exposures and subsequent measurements of trace metal uptake and viability. The results support the future use of the SUBS for the in situ measurement of phytochelatin/metallothionein production, photosynthetic efficiency, or reporter gene induction of controlled organisms.

Global expression profiling of Chlamydomonas reinhardtii exposed to trace levels of free cadmium.

In the natural environment, cadmium is often found as a trace contaminant. Due to the complexity of Cd speciation and the heterogeneity of natural systems and processes, it is often difficult to determine clear relationships between analytical measurements of Cd and its induced biological response. Measurements of gene induction can be used to identify molecular mechanisms underlying toxicity and to quantify sublethal responses to trace contaminants. In the present paper, genes that could be involved in the tolerance of Cd to green algae were examined using two global transcriptome profiling strategies. Microarray and differential display techniques were used for a global transcriptome analysis of Chlamydomonas reinhardtii exposed to micromolar and lower Cd(2+) concentrations for a short period (2 h). Real-time quantitative polymerase chain reaction analysis confirmed that a small set of 10 genes was differentially expressed in response to trace Cd(2+) exposures ranging from 7.8 nM to 9.0 microM. Since induction was only observed for a few genes, none of which are known to function in a general stress response, it was likely the result of relevant responses to Cd exposure. The identified genes are discussed with respect to their possible involvement in Cd tolerance and to their future use as biomarkers for monitoring Cd bioavailability in natural soils and waters.