Field and laboratory studies of fluorescence-based technologies for real-time tracking of cyanobacterial cell lysis and potential microcystins release.

Elevated levels of dissolved microcystins (MCs) in source water due to rapid cell lysis of harmful cyanobacterial blooms may pose serious challenges for drinking water treatment. Catastrophic cell lysis can result from outbreaks of naturally-occurring cyanophages - as documented in Lake Erie during the Toledo water crisis of 2014 and in 2019, or through the application of algaecides or water treatment chemicals. Real-time detection of cyanobacterial cell lysis in source water would provide a valuable tool for drinking water plant and reservoir managers. In this study we explored two real-time fluorescence-based devices, PhycoSens and PhycoLA, that can detect unbound phycocyanin (uPC) as a potential indication of cell lysis and MCs release. The PhycoSens was deployed at the Low Service pump station of the City of Toledo Lake Erie drinking water treatment plant from July 15 to October 19, 2022 during the annual cyanobacteria bloom season. It measured major algal groups and uPC in incoming lake water at 15-min intervals during cyanobacteria dominant and senescence periods. Intermittent uPC detections from the PhycoSens over a three-month period coincided with periods of increasing proportions of extracellular MCs relative to total (intracellular and extracellular) MCs, indicating potential for uPC use as an indicator of cyanobacterial cell integrity. Following exposures of laboratory-cultured MCs-producing Microcystis aeruginosa NIES-298 (120 µg chlorophyll/L) to cyanophage Ma-LMM01, copper sulfate (0.5 and 1 mg Cu/L), sodium carbonate peroxyhydrate (PAK® 27, 6.7 and 10 mg H2O2/L), and potassium permanganate (2.5 and 4 mg/L), appearance of uPC coincided with elevated fractions of extracellular MCs. The PhycoLA was used to monitor batch samples collected daily from Lake Erie water exposed to algaecides in the laboratory. Concurrence of uPC signal and surge of dissolved MCs was observed following 24-h exposures to copper sulfate and PAK 27. Overall results indicate the appearance of uPC is a useful indicator of the onset of cyanobacterial cell lysis and the release of MCs when MCs are present.

Toxic effects of thallium (Tl+) on prokaryotic alga Microcystis aeruginosa: Short and long-term influences by potassium and humic acid.

Thallium (Tl) is a priority pollutant regulated by the US EPA. It is also a critical element commonly used in high technology industries; with an increasing demand for semiconductors nowadays, wastewater discharges from manufacturing plants or metal mining activities may result in elevated levels of thallium in receiving water harming aquatic organisms. Regarding the impact of thallium on freshwater algae, little attention has been paid to prokaryotic physiology through various exposure periods. In this bench-scale study, prokaryotic alga Microcystis aeruginosa PCC 7806 was cultured in modified BG11 medium and exposed to Tl+ (TlNO3) ranging from 250 to 1250 µg/L for 4 and 14 days. Throughout the experiment using flow cytometry assays, algal population, cell membrane integrity, oxidation stress level, and chlorophyll fluorescence were exacerbated following the exposure to 750 µg Tl/L (approximately 4-day effective concentration of Tl+ for reducing 50% of algal population). Potassium and humic acid (HA) (1-5 mg/L) were added to study their influences on the thallium toxicity. With the additions of potassium, thallium toxicities to algal population and physiology were not significantly changed within 4 days, while they were alleviated within 14 days. With the addition of HA at 1 mg/L, cell membrane integrity was significantly attenuated within 4 days; ameliorating effects on algal population and oxidative stress were not observed until day 14. Thallium toxicities on oxidative stress level and photosynthesis activity were exacerbated in the presence of HA at 3-5 mg/L. The study provides useful information for further studies on the mode of toxic action of Tl+ in prokaryotic algae; it also demonstrates the necessity of considering short and long-term exposure durations while incorporating water chemistry into assessment of thallium toxicity to algae.

Characterization of Dissolved Organic Matter from Wildfire-induced Microcystis aeruginosa Blooms controlled by Copper Sulfate as Disinfection Byproduct Precursors Using APPI(-) and ESI(-) FT-ICR MS.

Copper-based algaecides are usually used for controlling algae bloom triggered by the elevated levels of nutrients after wildfires, resulting in the promoted reactivity of dissolved organic matter (DOM) in forming disinfectant byproducts (DBPs). To identify the best strategy for handling this source water, we employed Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to characterize the DBPs precursors after 4-d Microcystis aeruginosa bloom cultured with black (BE) and white (WE) ash water extracts under 0, 0.5, and 1.0 mg-Cu/L. The disappeared DOM during disinfections, primarily composed of O1-14, N1O1-14 and N2O1-14, had a higher average molecular weight (MW) and double-bond equivalent (DBE), relative to DOM after incubation, regardless of disinfects and Cu2+. This result suggests assigned features with larger MW and more double bonds/rings as preferable DBP precursors. We observed a larger number of disappeared assigned features with low DBE of 1-10 in control without Cu2+ addition, possibly explaining lower DOM chlorine reactivity in forming carbonaceous and oxygenated DBPs, relative to the treatments with Cu2+ addition. We found a larger number of O1-14 and N1O1-14 with DBE=5-16 in the treatments, potentially explaining higher DOM chloramine reactivity in forming N-nitrosodimethylamine (NDMA), compared to the control. Our study suggests removing oxygen- and nitrogen-containing organic compounds with more double bonds/aromatic rings as a preferable strategy for handling source water after controlling post-fire algae blooms with copper sulfate.

Control wildfire-induced Microcystis aeruginosa blooms by copper sulfate: Trade-offs between reducing algal organic matter and promoting disinfection byproduct formation.

Elevated levels of nutrients due to wildfire ash input into stream waters will likely cause algal blooms. When source water is impeded by algae and requires immediate restoration, copper algaecides are usually applied. Previous studies indicate that Cu2+ can promote reactivity of dissolved organic matter in forming disinfection byproducts (DBPs). However, it is unclear that how DBP formation is changed after the treatment of post-fire algal bloom by copper algaecide. In this study Microcystis aeruginosa was cultured in the medium containing black and white ash water extracts (BE and WE) to study DBP concentrations before and after 4-days exposures to low and high copper sulfate (0.5 and 1.0 mg-Cu/L). Dissolved organic matter (DOM) was characterized by UV-VIS absorption and fluorescence spectroscopy and chlorination/chloramination-based DBP formation potential (FP) experiments. DOM concentrations and algal population in the treatments were lower than that in control, regardless of types of water extract. N-nitrosodimethylamine FP in the treatments were 4-6 times higher than the control (0.23-0.34 vs. 0.05-0.06 µg/L), while haloacetonitrile FP revealed no significant difference (132-191 vs. 167-185 µg/L). Trade-offs between reducing algal population and promoting DBP-FP were more pronounced for the solutions containing BE than WE. Low copper concentration was as effective as high concentration in inhibiting algal growth while minimizing promotion of DBP formation. The results can serve to support risk evaluations of algal population and DBP concentration when wildfire-induced algal bloom is left untreated and treated by copper algaecides.

Dynamic Changes of Disinfection Byproduct Precursors following Exposures of Microcystis aeruginosa to Wildfire Ash Solutions.

Wildfires can elevate dissolved organic matter (DOM) levels due to ash input and algal growth in source waters, and consequently impacting disinfection byproduct (DBP) formation in finished water; however, it remains unclear how quality and quantity of overall allochthonous and autochthonous DOM as well as associated DBP formation are changed during an entire algal life cycle. Microcystis aeruginosa was cultured in the medium containing low and high concentrations [10% and 65% (v/v)] of black and white ash water extracts (BE and WE) to study dynamic changes of carbonaceous, nitrogenous, and oxygenated DBP precursors during algal growth. DOM was characterized by absorption and fluorescence spectroscopy and chlorination/chloramination-based DBP formation experiments. Throughout the entire experiment, C-DBP precursors in the control ranged from 2.41 to 3.09 mmol/mol-C. In the treatment with 10% BE, the amount of C-DBP precursors decreased from 6.8 to 3.0 mmol/mol-C at initial-exponential phase then increased to 4.2 mmol/mol-C at death phase. The same trend was observed for O-DBP precursors. However, these dynamic changes of C- and O-DBP precursors exhibited opposite patterns in 65% extracts. Similar patterns were also observed in the WE treatments. On the other hand, N-DBP precursors continuously declined in all treatments. These results indicate that postfire ash loading and algal bloom stage may significantly affect DBP formation in source water.

Growing Algae Alter Spectroscopic Characteristics and Chlorine Reactivity of Dissolved Organic Matter from Thermally-Altered Forest Litters.

Previous studies demonstrated that wildfires alter spectroscopic characteristics of terrestrial dissolved organic matter (DOM) and increase specific disinfection byproduct formation potential (SDBP-FP). However, it is unclear whether characteristics of thermally altered DOM (TA-DOM) are altered by biogeochemical processes (e.g., transformed by growing algae) before entering water treatment facilities. The freshwater green algae Pseudokirchneriella subcapitata and blue-green algae Microcystis aeruginosa were separately incubated in the mixture of cultural medium and pine (Pinus palustris) litter-derived TA-DOMs (50 °C, 250 °C, and 400 °C) over 7 days to demonstrate the effects of algal growth on alterations in SDBP-FP. TA-DOM optical characteristics and SDBP-FP were quantified by absorption and fluorescence spectroscopy and chlorination-based DBP-FP experiments. After the inoculation with P. subcapitata, TA-DOM aromaticity (indicated by SUVA254) increased from 1.19 to 1.90 L/mg/m for 50 °C-extract but decreased from 4.95 to 3.75 L/mg/m for 400 °C-extract. The fraction of tyrosine-like components decreased from 25.9 to 9.3% for 50 °C-extract but increased from 0.9 to 1.3% for 400 °C-extract. Same patterns were also observed for M. aeruginosa. Growing algae generally increased chlorine reactivities and formations of trihalomethanes, haloacetonitriles, chloral hydrate, and haloketones. Our data suggest that the biodegradable dissolved organic carbon in TA-DOM decreases as fire intensity (i.e., temperature) increases. Postfire algal blooms can increase chlorine reactivity of fire-affected terrestrial DOM for DBP formation.

Effects of two copper compounds on Microcystis aeruginosa cell density, membrane integrity, and microcystin release.

Microcystin release following Microcystis aeruginosa cell lysis after copper-based algaecide treatment is often cited as a concern leading to restricted use of algaecide in restoration of natural water resources. To examine this concern, bench-scale experiments were conducted to study responses of M. aeruginosa to 8-day copper exposures as copper sulfate and copper-ethanolamine (Cu-EA). M. aeruginosa UTEX 2385 was cultured in BG11 medium to cell density of 10(6)cells/mL with total and extracellular microcystin of 93 and 53µg/L, respectively. Exposures of copper concentration ranged from 40 to 1000µgCu/L. Cell membrane integrity was indicated by erythrosine B. In the end of experiment, total microcystin and cell density in untreated control (313µg/L and 10(7)cells/mL) was 3.3 and 10 times greater than pretreatment value, respectively. Minimum amount of copper required to reduce M. aeruginosa population within 8 days was 160µgCu/L as copper sulfate and 80µgCu/L as Cu-EA, where total and extracellular microcystin concentrations (47 and 44µg/L for copper sulfate; 56 and 44µg/L for Cu-EA) were degraded with degradation rate coefficient 0.1 day(-1) and were less than pretreatment values. Given a copper concentration at 80µgCu/L as Cu-EA, M. aeruginosa cells were intact and less microcystin were released compared to treatments at 160-1000µgCu/L, where lysed cells and relatively greater microcystin release were observed. Based on the laboratory results, a minimum amount of copper required for reducing M. aeruginosa population could decrease total microcystin concentration and not compromise cells and minimize microcystin release.

An algal toxicity database of organic toxicants derived by a closed-system technique.

The current study presents the toxicity data of 90 organic compounds with various modes of actions to Pseudokirchneriella subcapitata. The assessment was conducted using a closed-system technique, and a biomass-type end point based on the cell density was employed. The above toxicity data were compared with test results from ciliate (Tetrahymena pyriformis), water flea (Daphnia magna), fish (Pimephales promelas), and luminescent bacteria (Photobacterium phosphoreum). Satisfactory correlation relationships between toxicity data from algae and other aquatic organisms were found (r2 = 0.66-0.82). Pseudokirchneriella subcapitata revealed considerably higher sensitivity to organic toxicants compared with other organisms. Benzenes, aldehydes, and alkanes also were highly selective to the test alga. In addition, the results show that conventional algal batch tests tend to underestimate the toxicity of organic compounds, except in the case of 4-chlorophenol. Toxicity observed from the closed-system test is approximately 2- to 380-fold higher than that estimated by conventional batch tests. Such a phenomenon can be found in nearly all organic compounds, regardless of the chemical's Henry's law constant. In the risk assessment of chemicals, following the European Union's practice, approximately 30% (7 of 23) of the cases may result in a more strict classification when the batch test is replaced by the closed-system test. More effort therefore is needed to revise the algal toxicity database using the closed-system test method.

The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata.

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).

A novel algal toxicity testing technique for assessing the toxicity of both metallic and organic toxicants.

This study presents a closed-system algal toxicity test technique that is capable of detecting the effects of both organic and metallic toxicants. Toxicity testing was conducted by transferring adequate amounts of algal suspension, dilution water (with culture growth medium), and toxicants into 300-mL BOD bottles. The BOD bottles were completely filled up with no head-space left. The initial cell density and the exposure time were 15,000 cells/mL and 48 h, respectively. The performance of the above test method was evaluated using three heavy metals and six organic toxicants based on three different test endpoints, i.e., dissolved oxygen production, algal growth rate, and cell density. The proposed test revealed excellent test sensitivity and reproducibility. Currently, none of the existing algal toxicity test protocols is adequate for assessing the toxicity of organic chemicals. The closed-system algal toxicity tests developed by previous researchers also may not be ideal because the enlarged headspace and/or enriched bicarbonate buffer may result in either underestimations of the exposure concentrations or insensitive responses to both heavy metals and organic toxicants. Compared to the aforementioned algal toxicity test methods, the proposed technique in the present study has a more general applicability under conditions such as effluent samples containing both metals and organic toxicants or samples with unknown compositions.