Potentially Poisonous Plastic Particles: Microplastics as a Vector for Cyanobacterial Toxins Microcystin-LR and Microcystin-LF.

The potential of microplastics to act as a vector for micropollutants of natural or anthropogenic origin is of rising concern. Cyanobacterial toxins, including microcystins, are harmful to humans and wildlife. In this study, we demonstrate for the first time the potential of microplastics to act as vectors for two different microcystin analogues. A concentration of up to 28 times from water to plastic was observed for the combination of polystyrene and microcystin-LF achieving toxin concentrations on the plastic of 142 ± 7 µg g-1. Based on the experimental results, and assuming a worst-case scenario, potential toxin doses for daphnids are calculated based on published microplastic ingestion data. Progressing up through trophic levels, theoretically, the concentration of microcystins in organisms is discussed. The experimental results indicate that adsorption of microcystins onto microplastics is a multifactorial process, depending on the particle size, the variable amino acid composition of the microcystins, the type of plastic, and pH. Furthermore, the results of the current study stressed the limitations of exclusively investigating microcystin-LR (the most commonly studied microcystin congener) as a model compound representing a group of around 250 reported microcystin congeners.

Comparison of UV-A photolytic and UV/TiO2 photocatalytic effects on Microcystis aeruginosa PCC7813 and four microcystin analogues: A pilot scale study.

To date, the high cost of supplying UV irradiation has prevented the widespread application of UV photolysis and titanium dioxide based photocatalysis in removing undesirable organics in the water treatment sector. To overcome this problem, the use of UV-LEDs (365 nm) for photolysis and heterogeneous photocatalysis applying TiO2 coated glass beads under UV-LED illumination (365 nm) in a pilot scale reactor for the elimination of Microcystis aeruginosa PCC7813 and four microcystin analogues (MC-LR, -LY, -LW, -LF) with a view to deployment in drinking water reservoirs was investigated. UV-A (365 nm) photolysis was shown to be more effective than the UV/TiO2 photocatalytic system for the removal of Microcystis aeruginosa cells and microcystins. During photolysis, cell density significantly decreased over 5 days from an initial concentration of 5.8 × 106 cells mL-1 until few cells were left. Both intra- and extracellular microcystin concentrations were significantly reduced by 100 and 92 %, respectively, by day 5 of the UV treatment for all microcystin analogues. During UV/TiO2 treatment, there was great variability between replicates, making prediction of the effect on cyanobacterial cell and toxin behavior difficult.

Degradation of microcystin-LR and cylindrospermopsin by continuous flow UV-A photocatalysis over immobilised TiO2.

The increasing presence of freshwater toxins have brought new challenges to preserve water quality due to their potential impact on the environment and human health. Two commonly occurring cyanotoxins, microcystin-LR and cylindrospermopsin, with different physico-chemical properties were used to evaluate the efficiency of photocatalysis using a continuous-flow reactor with immobilized TiO2 on glass tubes and UV-A light. The effect of flow rate and hydrogen peroxide addition on the efficiency of cyanotoxin removal were evaluated. An analysis of the effects on microcystin-LR removal efficiency showed that low flow rates (1 mL/min) and high H2O2 concentrations (120 mg/L) were needed to provide effective degradation. Up to 27.9% and 39.1% removal of MC-LR and CYN, respectively were achieved by UV-A/TiO2 after a single pass through the reactor. A slight increase of the removal of both cyanotoxins was observed when they were in a mixture (35.5% of MC-LR and 51.3% of CYN). The addition of H2O2 to the UV/TiO2 system led to an average removal enhancement of 92.6% of MC-LR and of 29.5% of CYN compared to the UV/TiO2 system. Photolysis assisted by H2O2 degraded MC-LR by up to 77.7%. No significant removal (<10%) was observed by photolysis alone or physical adsorption. This study presents a proof-of-principle that demonstrates the feasibility for this technology to be integrated in large-scale applications.

Aging microplastics enhances the adsorption of pharmaceuticals in freshwater.

Plastic pollution is an increasing environmental concern. Pollutants such as microplastics (< 5 mm) and pharmaceuticals often co-exist in the aquatic environment. The current study aimed to elucidate the interaction of pharmaceuticals with microplastics and ascertain how the process of photo-oxidation of microplastics affected the adsorption of the pharmaceuticals. To this end, a mixture containing ibuprofen, carbamazepine, fluoxetine, venlafaxine and ofloxacin (16 µmol L-1 each) was placed in contact with one of six either virgin or aged microplastic types. The virgin microplastics were acquired commercially and artificially aged in the laboratory. Polypropylene, polyethylene, polyethylene terephthalate, polyamide, polystyrene, and polyvinyl chloride microparticles at two sizes described as small (D50 < 35 µm) and large (D50 95-157 µm) were evaluated. Results demonstrated that the study of virgin particles may underestimate the adsorption of micropollutants onto microplastics. For virgin particles, only small microparticles of polypropylene, polyethylene, polyvinyl chloride, and both sizes of polyamide adsorbed pharmaceuticals. Aging the microplastics increased significantly the adsorption of pharmaceuticals by microplastics. Fluoxetine adsorbed onto all aged microplastics, from 18 % (large polyethylene terephthalate) to 99 % (small polypropylene). The current investigation highlights the potential of microplastics to act as a vector for pharmaceuticals in freshwater, especially after aging.

Solar-driven semi-conductor photocatalytic water treatment (TiO2, g-C3N4, and TiO2+g-C3N4) of cyanotoxins: Proof-of-concept study with microcystin-LR.

Cyanobacteria and their toxins are a threat to drinking water safety as increasingly cyanobacterial blooms (mass occurrences) occur in lakes and reservoirs all over the world. Photocatalytic removal of cyanotoxins by solar light active catalysts is a promising way to purify water at relatively low cost compared to modifying existing infrastructure. We have established a facile and low-cost method to obtain TiO2 and g-C3N4 coated floating photocatalysts using recycled glass beads. g-C3N4 coated and TiO2+g-C3N4 co-coated beads were able to completely remove microcystin-LR in artificial fresh water under both natural and simulated solar light irradiation without agitation in less than 2 h. TiO2 coated beads achieved complete removal within 8 h of irradiation. TiO2+g-C3N4 beads were more effective than g-C3N4 beads as demonstrated by the increase reaction rate with reaction constants, 0.0485 min-1 compared to 0.0264 min-1 respectively, with TiO2 alone found to be considerably slower 0.0072 min-1. g-C3N4 based photocatalysts showed a similar degradation pathway to TiO2 based photocatalysts by attacking the C6-C7 double bond on the Adda side chain.

Characterisation of microplastics is key for reliable data interpretation.

Microplastic research has gained attention due to the increased detection of microplastics (<5 mm size) in the aquatic environment. Most laboratory-based research of microplastics is performed using microparticles from specific suppliers with either superficial or no characterisation performed to confirm the physico-chemical information detailed by the supplier. The current study has selected 21 published adsorption studies to evaluate how the microplastics were characterised by the authors prior experimentation. Additionally, six microplastic types described as 'small' (10-25 µm) and 'large' (100 µm) were commercially acquired from a single supplier. A detailed characterisation was performed using Fourier transform infrared spectroscopy (FT-IR), x-ray diffraction, differential scanning calorimetry, scanning electron microscopy, particle size analysis, and N2-Brunauer, Emmett and Teller adsorption-desorption surface area analysis. The size and the polymer composition of some of the material provided by the supplier was inconsistent with the analytical data obtained. FT-IR spectra of small polypropylene particles indicated either oxidation of the particles or the presence of a grafting agent which was absent in the large particles. A wide range of sizes for the small particles was observed: polyethylene (0.2-549 µm), polyethylene terephthalate (7-91 µm) and polystyrene (1-79 µm). Small polyamide (D50 75 µm) showed a greater median particle size and similar size distribution when compared to large polyamide (D50 65 µm). Moreover, small polyamide was found to be semi-crystalline, while the large polyamide displayed an amorphous form. The type of microplastic and the size of the particles are a key factor in determining the adsorption of pollutants and subsequent ingestion by aquatic organisms. Acquiring uniform particle sizes is challenging, however based on this study, characterisation of any materials used in microplastic-related experiments is critical to ensure reliable interpretation of results, thereby providing a better understanding of the potential environmental consequences of the presence of microplastics in aquatic ecosystems.

Adsorption of cyanotoxins on polypropylene and polyethylene terephthalate: Microplastics as vector of eight microcystin analogues.

Plastics are utilised globally but are of environmental concern due to their persistence. The global presence of microplastics (particles <5 mm in all dimensions) in freshwater environments is increasingly reported, as has the presence of cyanobacterial toxins, including the microcystins. We elucidated the potential role of microplastics as a vector for eight microcystin analogues. Two sizes of polypropylene (PP) and polyethylene terephthalate (PET) microparticles were evaluated. The median particle size distribution (D50) was 8-28 µm for small particles, and 81-124 µm for large particles. Additionally, microcystin-LR and -LF were evaluated individually using small PP and PET to elucidate the adsorption behaviour in the absence of competition. Microcystin hydrophobicity, polymer material, and particle size were key factors influencing adsorption to the plastic microparticles. The small size PP microparticles demonstrated a high affinity for the 8 microcystin analogues. The proportion of microcystin adsorbed onto the small particles of PP after 48 h contact was between 83 and 100%, depending on the analogue. Of all analogues investigated, only microcystin-LW and -LF adsorbed onto the larger sized PP and PET microparticles. Individually, greater amounts of MC-LF adsorbed onto the small PET (19%) compared to when it was present in the mixture of microcystins (11%). While MC-LR did not adsorb onto small PET microparticles in the mixture, 5% adsorption was observed when individually in contact with small PET microparticles. The results demonstrated that microplastics can adsorb eight different microcystin analogues and that more hydrophobic analogues are more likely to adsorb than less hydrophobic analogues.

Suppressing cyanobacterial dominance by UV-LED TiO2-photocatalysis in a drinking water reservoir: A mesocosm study.

Cyanobacteria and their toxic secondary metabolites present challenges for water treatment globally. In this study we have assessed TiO2 immobilized onto recycled foamed glass beads by a facile calcination method, combined in treatment units with 365 nm UV-LEDs. The treatment system was deployed in mesocosms within a eutrophic Brazilian drinking water reservoir. The treatment units were deployed for 7 days and suppressed cyanobacterial abundance by 85% while at the same time enhancing other water quality parameters; turbidity and transparency improved by 40 and 81% respectively. Genomic analysis of the microbiota in the treated mesocosms revealed that the composition of the cyanobacterial community was affected and the abundance of Bacteroidetes and Proteobacteria increased during cyanobacterial suppression. The effect of the treatment on zooplankton and other eukaryotes was also monitored. The abundance of zooplankton decreased while Chrysophyte and Alveolata loadings increased. The results of this proof-of-concept study demonstrate the potential for full-scale, in-reservoir application of advanced oxidation processes as complementary water treatment processes.

Detection of morphological changes caused by chemical stress in the cyanobacterium Planktothrix agardhii using convolutional neural networks.

The presence of harmful algal bloom in many reservoirs around the world, alongside the lack of sanitation law/ordinance regarding cyanotoxin monitoring (particularly in developing countries), create a scenario in which the local population could potentially chronically consume cyanotoxin-contaminated waters. Therefore, it is crucial to develop low cost tools to detect possible systems failures and consequent toxin release inferred by morphological changes of cyanobacteria in the raw water. This paper aimed to look for the best combination of convolutional neural network (CNN), optimizer and image segmentation technique to differentiate P. agardhii trichomes before and after chemical stress caused by the addition of hydrogen peroxide. This method takes a step towards accurate monitoring of cyanobacteria in the field without the need for a mobile lab. After testing three different network architectures (AlexNet, 3ConvLayer and 2ConvLayer), four different optimizers (Adam, Adagrad, RMSProp and SDG) and five different image segmentations methods (Canny Edge Detection, Morphological Filter, HP filter, GrabCut and Watershed), the combination 2ConvLayer with Adam optimizer and GrabCut segmentation, provided the highest median accuracy (93.33%) for identifying H2O2-induced morphological changes in P. agardhii. Our results emphasize the fact that the trichome classification problem can be adequately tackled with a limited number of learned features due to the lack of complexity in micrographs from before and after chemical stress. To the authors' knowledge, this is the first time that CNNs were applied to detect morphological changes in cyanobacteria caused by chemical stress. Thus, it is a significant step forward in developing low cost tools based on image recognition, to shield water consumers, especially in the poorest regions, against cyanotoxin-contaminated water.

Oxidative stress in the cyanobacterium Microcystis aeruginosa PCC 7813: Comparison of different analytical cell stress detection assays.

Cyanobacterial blooms are observed when high cell densities occur and are often dangerous to human and animal health due to the presence of cyanotoxins. Conventional drinking water treatment technology struggles to efficiently remove cyanobacterial cells and their metabolites during blooms, increasing costs and decreasing water quality. Although field applications of hydrogen peroxide have been shown to successfully suppress cyanobacterial growth, a rapid and accurate measure of the effect of oxidative stress on cyanobacterial cells is required. In the current study, H2O2 (5 and 20 mg L-1) was used to induce oxidative stress in Microcystis aeruginosa PCC 7813. Cell density, quantum yield of photosystem II, minimal fluorescence and microcystin (MC-LR, -LY, -LW, -LF) concentrations were compared when evaluating M. aeruginosa cellular stress. Chlorophyll content (determined by minimal fluorescence) decreased by 10% after 48 h while cell density was reduced by 97% after 24 h in samples treated with 20 mg L-1 H2O2. Photosystem II quantum yield (photosynthetic activity) indicated cyanobacteria cell stress within 6 h, which was considerably faster than the other methods. Intracellular microcystins (MC-LR, -LY, -LW and -LF) were reduced by at least 96% after 24 h of H2O2 treatment. No increase in extracellular microcystin concentration was detected, which suggests that the intracellular microcystins released into the surrounding water were completely removed by the hydrogen peroxide. Thus, photosynthetic activity was deemed the most suitable and rapid method for oxidative cell stress detection in cyanobacteria, however, an approach using combined methods is recomended for efficient water treatment management.

Effect of hydrogen peroxide on natural phytoplankton and bacterioplankton in a drinking water reservoir: Mesocosm-scale study.

Cyanobacterial blooms are increasingly reported worldwide, presenting a challenge to water treatment plants and concerning risks to human health and aquatic ecosystems. Advanced oxidative processes comprise efficient and safe methods for water treatment. Hydrogen peroxide (H2O2) has been proposed as a sustainable solution to mitigate bloom-forming cyanobacteria since this group presents a higher sensitivity compared to other phytoplankton, with no major risks to the environment at low concentrations. Here, we evaluated the effects of a single H2O2 addition (10 mg L-1) over 120 h in mesocosms introduced in a reservoir located in a semi-arid region presenting a Planktothrix-dominated cyanobacterial bloom. We followed changes in physical and chemical parameters and in the bacterioplankton composition. H2O2 efficiently suppressed cyanobacteria, green algae, and diatoms over 72 h, leading to an increase in transparency and dissolved organic carbon, and a decrease in dissolved oxygen and pH, while nutrient concentrations were not affected. After 120 h, cyanobacterial abundance remained low and green algae became dominant. 16S rRNA sequencing revealed that the original cyanobacterial bloom was composed by Planktothrix, Cyanobium and Microcystis. Only Cyanobium increased in relative abundance at 120 h, suggesting regrowth. A prominent change in the composition of heterotrophic bacteria was observed with Exiguobacterium, Paracoccus and Deinococcus becoming the most abundant genera after the H2O2 treatment. Our results indicate that this approach is efficient in suppressing cyanobacterial blooms and improving water quality in tropical environments. Monitoring changes in abiotic parameters and the relative abundance of specific bacterial taxa could be used to anticipate the regrowth of cyanobacteria after H2O2 degradation and to indicate where in the reservoir H2O2 should be applied so the effects are still felt in the water treatment plant intake.

Removal of microcystins from a waste stabilisation lagoon: Evaluation of a packed-bed continuous flow TiO2 reactor.

Photocatalysis has been shown to successfully remove microcystins (MC) in laboratory experiments. Most research to date has been performed under ideal conditions in pure or ultrapure water. In this investigation the efficiency of photocatalysis using titanium dioxide was examined in a complex matrix (waste stabilisation lagoon water). A flow-through photocatalytic reactor was used for the photocatalytic removal of four commonly occurring microcystin analogues (MC-YR, MC-RR, MC-LR, and MC-LA). Up to 51% removal for single MC analogues in waste lagoon water was observed. Similar removal rates were observed when a mixture of all four MC analogues was treated. Although treatment of MC-containing cyanobacterial cells of Microcystis aeruginosa resulted in no decline in cell numbers or viability with the current reactor design and treatment regime, the photocatalytic treatment did improve the overall quality of waste lagoon water. This study demonstrates that despite the presence of natural organic matter the microcystins could be successfully degraded in a complex environmental matrix.

Modeling phosphorus exchange between bottom sediment and water in tropical semiarid reservoirs.

This study investigated phosphorus (P) dynamics in the sediment-water interface of three distinct reservoirs located in a tropical semiarid region. Sequential chemical fractioning of the P content in the sediment and controlled experiments of the sediment-water interface were performed to understand and model the effect of the different P fractions on the exchange dynamics under anoxic and oxic scenarios. The results revealed that the older the reservoir, the higher the amount of iron and aluminum-bound P in the sediment, and that this fraction was responsible for a 10-fold increase in P concentration in the water during anoxic conditions. After aeration, P in water decreased but did not return to its initial concentration. The most recently constructed reservoir showed the lowest P concentration in the sediment and dominance of the unavailable P fraction, resulting in no potential impact on water quality. Phosphorus release and precipitation rates were well described by zero- and first-order models, respectively. Reservoirs with high P availability in the sediment, not only released more phosphorus but also presented a lower precipitation rate, resulting in higher potential damage to water quality and making some in-lake treatment techniques potentially ineffective.

Photocatalytic removal of the cyanobacterium Microcystis aeruginosa PCC7813 and four microcystins by TiO2 coated porous glass beads with UV-LED irradiation.

Cyanobacteria and their toxic secondary metabolites are a challenge in water treatment due to increased biomass and dissolved metabolites in the raw water. Retrofitting existing water treatment infrastructure is prohibitively expensive or unfeasible, hence 'in-reservoir' treatment options are being explored. In the current study, a treatment system was able to photocatalytically inhibit the growth of Microcystis aeruginosa and remove released microcystins by photocatalysis using titanium dioxide coated, porous foamed glass beads and UV-LEDs (365 nm). A 35% reduction of M. aeruginosa PCC7813 cell density compared to control samples was achieved in seven days. As a function of cell removal, intracellular microcystins (microcystin-LR, -LY, -LW, and -LF) were removed by 49% from 0.69 to 0.35 µg mL-1 in seven days. Microcystins that leaked into the surrounding water from compromised cells were completely removed by photocatalysis. The findings of the current study demonstrate the feasibility of an in-reservoir treatment unit applying low cost UV-LEDs and porous foamed beads made from recycled glass coated with titanium dioxide as a means to control cyanobacteria and their toxins before they can reach the water treatment plant.

The effect of water treatment unit processes on cyanobacterial trichome integrity.

Many toxic and/or noxious cyanobacteria appear in nature with a filamentous, stacked cell arrangement called trichomes. Although water treatment can be optimized to keep cyanobacterial cells intact and avoid the release of toxic and/or noxious compounds, many physical and chemical stresses encountered during the treatment process may result in trichome truncation, decreasing treatment efficiency by allowing single cells or short trichomes to reach the product water. This makes it possible for harmful/noxious compounds as well as organic matter to enter the distribution system. Investigations in a pilot and three full-scale water treatment plants were carried out in order to elucidate the degree of trichome truncation across different unit processes. It was found that genera (Pseudanabaena, Planktolyngbya) with short trichomes (<10-12 cells per trichome), are hardly affected by the unit processes (loss of one to four cells respectively), while genera (Planktothrix, Geitlerinema, Dolichospermum) with longer trichomes (30+ cells per trichome) suffer from high degrees of truncation (up to 63, 30, and 56 cells per trichome respectively). The presence of a rigid sheath and/or mucilaginous layer appears to offer some protection from truncation. It was observed that certain unit processes alter the sensitivity or resilience of trichomes to disruption by physical stress. Some genera (Planktothrix, Geitlerinema) were sensitive to pre-oxidation making them more susceptible to shear stress, while Dolichospermum sp. appears more robust after pre-oxidation. While the potential of toxicogenic genera breaking through into the product water is a real danger, in the current study no toxicogenic cyanobacteria were observed. This work stresses the need for plant operators to study the incoming cyanobacterial composition in the raw water in order to adjust treatment parameters and thus limit the potential of toxic/noxious compound breakthrough.

Treatment challenge of a cyanobacterium Romeria elegans bloom in a South Australian wastewater treatment plant - a case study.

A bloom of the non-toxic cyanobacterium Romeria elegans in waste stabilisation ponds (WSPs) within Angaston waste water treatment plant (WWTP) has posed an unprecedented treatment challenge for the local water utility. The water from the WSPs is chlorinated for safety prior to reuse on nearby farmland. Cyanobacteria concentrations of approximately 1.2 × 106 cells mL-1 increased the chlorine demand dramatically. Operators continuously increased the disinfectant dose up to 50 mg L-1 to achieve operational guideline values for combined chlorine (0.5-1.0 mg L-1) prior to reuse. Despite this, attempts to achieve targeted combined chlorine residual (CCR) failed. In this study, samples from the waste stabilisation pond at Angaston WWTP were chlorinated over a range of doses. Combined chlorine, disinfection by-product formation, cyanobacteria cell concentration, Escherichia coli inactivation, as well as dissolved organic carbon and free ammonia were monitored. This study shows that, in the occurrence of cyanobacterial blooms, CCR does not directly suggest pathogen removal efficiency and is therefore not an ideal parameter to evaluate the effectiveness of disinfection process in WWTP. Instead, E. coli removal is a more direct and practical parameter for the determination of the efficiency of the disinfection process.

Fate of cyanobacteria in drinking water treatment plant lagoon supernatant and sludge.

In conventional water treatment processes, where the coagulation and flocculation steps are designed to remove particles from drinking water, cyanobacteria are also concentrated into the resultant sludge. As a consequence, cyanobacteria-laden sludge can act as a reservoir for metabolites such as taste and odour compounds and cyanotoxins. This can pose a significant risk to water quality where supernatant from the sludge treatment facility is returned to the inlet to the plant. In this study the complex processes that can take place in a sludge treatment lagoon were investigated. It was shown that cyanobacteria can proliferate in the conditions manifest in a sludge treatment lagoon, and that cyanobacteria can survive and produce metabolites for at least 10days in sludge. The major processes of metabolite release and degradation are very dependent on the physical, chemical and biological environment in the sludge treatment facility and it was not possible to accurately model the net effect. For the first time evidence is provided to suggest that there is a greater risk associated with recycling sludge supernatant than can be estimated from the raw water quality, as metabolite concentrations increased by up to 500% over several days after coagulation, attributed to increased metabolite production and/or cell proliferation in the sludge.

Photocatalytic degradation of eleven microcystin variants and nodularin by TiO2 coated glass microspheres.

Microcystins and nodularin are toxic cyanobacterial secondary metabolites produced by cyanobacteria that pose a threat to human health in drinking water. Conventional water treatment methods often fail to remove these toxins. Advanced oxidation processes such as TiO2 photocatalysis have been shown to effectively degrade these compounds. A particular issue that has limited the widespread application of TiO2 photocatalysis for water treatment has been the separation of the nanoparticulate powder from the treated water. A novel catalyst format, TiO2 coated hollow glass spheres (Photospheres™), is far more easily separated from treated water due to its buoyancy. This paper reports the photocatalytic degradation of eleven microcystin variants and nodularin in water using Photospheres™. It was found that the Photospheres™ successfully decomposed all compounds in 5 min or less. This was found to be comparable to the rate of degradation observed using a Degussa P25 material, which has been previously reported to be the most efficient TiO2 for photocatalytic degradation of microcystins in water. Furthermore, it was observed that the degree of initial catalyst adsorption of the cyanotoxins depended on the amino acid in the variable positions of the microcystin molecule. The fastest degradation (2 min) was observed for the hydrophobic variants (microcystin-LY, -LW, -LF). Suitability of UV-LEDs as an alternative low energy light source was also evaluated.

Extraction method for total microcystins in cyanobacteria-laden sludge.

Cyanobacteria in water treatment sludge pose a health risk as they continue to be viable, multiply, and produce potentially harmful secondary metabolites. To date, little research has focused on accurately determining cell bound microcystin (MC) concentrations of cyanobacterial cells in water treatment sludge. Three extraction methods (freeze-thaw, lyophilisation, direct methanolic extraction) with three different pre-treatments (homogenisation, (ultra)sonication, combination of both, and controls) were investigated for their MC extraction recovery. It was found that lyophilisation with prior sonication achieved the highest toxin recovery across the two MC analogues (MC-LR, MC-LA) tested. The method was able to extract 69 and 56% of MC-LR and MC-LA, respectively with good reproducibility. Comparable results were also obtained with direct methanolic extraction, with poor reproducibility. The least efficient method was freeze-thawing which achieved poor recoveries and was less reproducible. This study highlights a rapid, efficient, low-cost extraction method for determining total microcystins in cyanobacterial-laden sludge.