Fate of Planktothrix-derived toxins in aquatic food webs: A case study in Lake Mindelsee (Germany).

Blooms of the red, filamentous cyanobacterium Planktothrix rubescens occur frequently in pre-alpine lakes in Europe, often with concomitant toxic microcystin (MC) production. Trophic transfer of MCs has been observed in bivalves, fish, and zooplankton species, while uptake of MCs into Diptera species could facilitate distribution of MCs into terrestrial food webs and habitats. In this study, we characterized a Planktothrix bloom in summer 2019 in Lake Mindelsee and tracked possible trophic transfer and/or bioaccumulation of MCs via analysis of phytoplankton, zooplankton (Daphnia) and emergent aquatic insects (Chaoborus, Chironomidae and Trichoptera). Using 16 S rRNA gene amplicon sequencing, we found that five sequence variants of Planktothrix spp. were responsible for bloom formation in September and October of 2019, and these MC-producing variants, provisionally identified as P. isothrix and/or P. serta, occurred exclusively in Lake Mindelsee (Germany), while other variants were also detected in nearby Lake Constance. The remaining cyanobacterial community was dominated by Cyanobiaceae species with high species overlap with Lake Constance, suggesting a well-established exchange of cyanobacteria species between the adjacent lakes. With targeted LC-HRMS/MS we identified two MC-congeners, MC-LR and [Asp3]MC-RR with maximum concentrations of 45 ng [Asp3]MC-RR/L in lake water in September. Both MC congeners displayed different predominance patterns, suggesting that two different MC-producing species occurred in a time-dependent manner, whereby [Asp3]MC-RR was clearly associated with the Planktothrix spp. bloom. We demonstrate an exclusive transfer of MC-LR, but not [Asp3]MC-RR, from phytoplankton into zooplankton reaching a 10-fold bioconcentration, yet complete absence of these MC congeners or their conjugates in aquatic insects. The latter demonstrated a limited trophic transfer of MCs from zooplankton to zooplanktivorous insect larvae (e.g., Chaoborus), or direct transfer into other aquatic insects (e.g. Chironomidae and Trichoptera), whether due to avoidance or limited uptake and/or rapid excretion of MCs by higher trophic emergent aquatic insects.

US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

Functional transepithelial transport measurements to detect nephrotoxicity in vitro using the RPTEC/TERT1 cell line.

The kidney is a frequent target for organ-specific toxicity as a result of its primary function in controlling body fluids, for example, via resorption of amino acids, peptides, nutrients, ions, xenobiotics and water from the primary urine as well as excretion of metabolic waste products and hydrophilic and amphiphilic xenobiotics. Compounds exhibiting dose-limiting nephrotoxicity include drugs from highly diverse classes and chemical structures, e.g., antibiotics (gentamicin), chemotherapeutics (cisplatin), immunosuppressants (cyclosporine A and tacrolimus) or bisphosphonates (zoledronate). All of these compounds elicit nephrotoxicity primarily by injuring renal proximal tubule epithelial cells (RPTECs). However, prediction of a compound's nephrotoxic potential in humans to support early unmasking of risk-bearing drug candidates remains an unmet challenge, mainly due to the complex kidney anatomy as well as pronounced inter- and intraspecies differences and lack of relevant and validated human in vitro models. Accordingly, we used the recently established human RPTEC/TERT1 cell line to carry out toxicity studies with a focus on impairment of functional characteristics, i.e., transepithelial electrical resistance (TEER), vectorial transport of water, cations, and anions. Results were compared to real-time cytotoxicity assessments using cellular impedance (xCELLigence assay) and the routine cell viability readout (MTT). As expected, most toxins caused exposure time- and concentration-dependent cytotoxicity. However, for some compounds (cyclosporine A and tacrolimus), transport processes were strongly impaired in absence of a concomitant decrease in cell viability. In conclusion, these data demonstrate that functional parameters are important, highly sensitive and meaningful additional readouts for nephrotoxicity assessment in human renal proximal tubule epithelial cells.

Total Synthesis of Microcystin-LF and Derivatives Thereof.

Microcystins (MCs) are highly toxic natural products which are produced by cyanobacteria. They can be released to the water during harmful algal blooms and are a serious threat to animals and humans. Described is the total synthesis of the cyanotoxin microcystin-LF (MC-LF, 1a) and two derivatives thereof. Deuterated derivative 1b is of interest as an internal standard during MC quantification in biological samples by mass spectrometry and alkyne-labeled 1c can be employed for toxin derivatization by click chemistry with an azide-containing reporter molecule or as an activity-based probe to identify interaction partners. Application of tert-butyl ester protecting groups for erythro-ß-d-methylaspartic acid and γ-d-glutamic acid were key for an isomerization-free synthesis. The analytical data of synthetic MC-LF were identical to those of an authentic sample of the natural product. All derivatives 1a-c were determined to be potent inhibitors of protein phosphatase-1 with similar activity.

Human cost burden of exposure to endocrine disrupting chemicals. A critical review.

Recently published papers have alleged that exposures to endocrine disrupting chemicals (EDCs) are causing substantial disease burdens in the EU and US and are consequently costing society hundreds of billions of dollars annually. To date, these cost estimates have not undergone adequate scientific scrutiny, but nevertheless are being used aggressively in advocacy campaigns in an attempt to fundamentally change how chemicals are tested, evaluated and regulated. Consequently, we critically evaluated the underlying methodology and assumptions employed by the chief architects of the disease burden cost estimates. Since the vast majority of their assigned disease burden costs are driven by the assumption that "loss of IQ" and "increased prevalence of intellectual disability" are caused by exposures to organophosphate pesticides (OPPs) and brominated flame retardants (PBDEs), we have taken special care in describing and evaluating the underlying toxicology and epidemiology evidence that was relied upon. Unfortunately, our review uncovered substantial flaws in the approach taken and the conclusions that were drawn. Indeed, the authors of these papers assumed causal relationships between putative exposures to EDCs and selected diseases, i.e., "loss of IQ" and "increased prevalence of intellectual disability", despite not having established them via a thorough evaluation of the strengths and weaknesses of the underlying animal toxicology and human epidemiology evidence. Consequently, the assigned disease burden costs are highly speculative and should not be considered in the weight of evidence approach underlying any serious policy discussions serving to protect the public and regulate chemicals considered as EDCs.

Novel insights into renal D-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo.

Chronic exposure to propiverine, a frequently prescribed pharmaceutical for treatment of overactive bladder and incontinence, provokes massive protein accumulation in the cytosol and nucleus of renal proximal tubule epithelial cells in rats. Previously, the accumulating protein was identified as D-amino acid oxidase (DAAO), a peroxisomal flavoenzyme expressed in kidney, liver and brain. The cellular mechanism of propiverine-induced DAAO accumulation, however, remains unexplained and poorly characterized. Therefore, to further increase the understanding of DAAO accumulation in rat kidney, this study aimed to characterize DAAO accumulations using differential immunofluorescent staining of rat kidney sections as well as in vitro binding analyses and proteasomal activity studies. We demonstrated that propiverine is neither a ligand of DAAO nor an inhibitor of the proteasome in vitro. However, propiverine treatment resulted in a significant decrease of peroxisomal size in rat proximal tubule epithelial cells. Moreover, peroxisomal catalase also accumulated in the cytosol and nuclei of propiverine-treated rats concurrently with DAAO. Taken together, our study indicates that propiverine treatment affects the trafficking and/or degradation of peroxisomal proteins such as DAAO and catalase by a so far unique and unknown mechanism.

Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon.

Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats.

Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O6-methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6 months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis.

Molecular cloning and functional characterization of a rainbow trout liver Oatp.

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9µM and 13.4µM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103µM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications.

PODO/TERT256 - A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels.

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 µM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 µM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.

Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro: an initial characterization.

BACKGROUND: A good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney. RESULTS: We carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics. CONCLUSIONS: On the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells.

The cyanobacterial neurotoxin ß-N-methylamino-l-alanine (BMAA) induces neuronal and behavioral changes in honeybees.

The cyanobacterially produced neurotoxin beta-N-methylamino-l-alanine (BMAA) is thought to induce amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC)-like symptoms. However, its mechanism of action and its pathway of intoxication are yet unknown. In vivo animal models suitable for investigating the neurotoxic effect of BMAA with applicability to the human are scarce. Hence, we used the honeybee (Apis mellifera) since its nervous system is relatively simple, yet having cognitive capabilities. Bees fed with BMAA-spiked sugar water had an increased mortality rate and a reduced ability to learn odors in a classical conditioning paradigm. Using (14)C-BMAA we demonstrated that BMAA is biologically available to the bee, and is found in the head, thorax and abdomen with little to no excretion. BMAA is also transferred from one bee to the next via trophallaxis resulting in an exposure of the whole beehive. BMAA bath application directly onto the brain leads to an altered Ca(2+) homeostasis and to generation of reactive oxygen species. These behavioral and physiological observations suggest that BMAA may have effects on bee brains similar to those assumed to occur in humans. Therefore the bee could serve as a surrogate model system for investigating the neurological effects of BMAA.

Intracellular conformations of human telomeric quadruplexes studied by electron paramagnetic resonance spectroscopy.

In-cell DEER (in-cell double electron electron resonance) is applied to investigate quadruplex formation of the human telomeric repeat d[AGGG(TTAGGG)(3)]. The initially unfolded DNA sequence forms a mixture of different quadruplex topologies upon injection into living cells. In addition, time-dependent distance measurements are carried out to monitor quadruplex folding.

Physiological oxygen and co-culture with human fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells.

Reliable prediction of compound mediated nephrotoxicity in humans is still unsatisfactory irrespective of the recent advancements in in silico, in vitro and in vivo models. Therefore, current in vitro approaches need refinement to better match the human in vivo situation, specifically with regard to the potential influence of other cell types (e.g. fibroblasts) and to the potential biases introduced by the excessive 21% O2 (AtmOx) as employed in routine cell culturing. We used a transwell co-culture model combining human renal proximal tubule epithelial cells (RPTEC/TERT1) and human fibroblasts (fHDF/TERT166) to compare the functional properties and expression of selected marker proteins at 21% O2 and at the physiologically normal 10% O2 tension (PhysOx) commensurate with in vivo conditions. Culturing at PhysOx and co-culturing with fibroblasts significantly improved epithelial barrier integrity, expression of transporters (e.g. aquaporin 2; OCT-MATE; MRP-OAT) and metabolism. Moreover, beyond culturing these human cells in co-culture for up to 41 days, we were able to demonstrate increased functionality of cation transport, as shown via ASP+ (OCT-MATE axis), and anion transport, as shown via LY (MRP-OAT axis). Thus, adjusting the in vitro system to near physiological conditions had a major impact on functionality and provides the basis for the future development of true flow-through microfluidic renal testing systems with better predictability of human renal proximal toxicity.

Investigation of microcystin conformation and binding towards PPP1 by molecular dynamics simulation.

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [ß-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

Label-free identification and differentiation of different microplastics using phasor analysis of fluorescence lifetime imaging microscopy (FLIM)-generated data.

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.

Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC-MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake.

Variability in microcystin quotas during a Microcystis bloom in a eutrophic lake.

Microcystis is a bloom-forming genus of cyanobacteria with some genotypes that produce highly toxic microcystin hepatotoxins. In waterbodies where biological and physical factors are relatively homogenous, toxin quotas (the average amount of toxin per cell), at a single point in time, are expected to be relatively constant. In this study we challenged this assumption by investigating the spatial distribution of microcystin quotas at a single point in time on two separate occasions in a lake with a major Microcystis bloom. Microcystis cell concentrations varied widely across the lake on both sampling occasions (730- and 137-fold) together with microcystin quotas (148- and 362-fold). Cell concentrations and microcystin quotas were strongly positively correlated (R2 = 0.89, P < 0.001, n = 28; R2 = 0.67, P < 0.001, n = 25). Analysis of Microcystis strains using high-throughput sequencing of the 16S-23S rRNA intergenic spacer region showed no relationship between microcystin quota and the relative abundance of specific sequences. Collectively, the results of this study indicate an association between microcystin production and cell density that magnifies the potential for bloom toxicity at elevated cell concentrations.