Estimation of cellular carbon content based on the cell biovolume of microalgae from a eutrophic estuary (Sea of Marmara, Türkiye).

The cellular carbon content based on the cell biovolume of a total of 61 microalgal species determined in a eutrophic estuary (Golden Horn, Sea of Marmara) was estimated in seawater samples taken during two different sampling periods. Cell biovolume according to geometric dimensions of the cells was then converted to phytoplankton carbon using an appropriate conversion factor. The range of diatom biovolume, in which the majority had small cell sizes (<50 µm), was much wider than that of dinoflagellate biovolume, in which the majority had large cell sizes (>50 µm). The cell biovolume and carbon content ranged from 35 to 4.88 × 105 µm3 and 5 to 1.18 × 104 pgC cell-1 for diatoms and from 3.66 × 102 to 8.68 × 105 µm3 and 55 to 8.14 × 104 pgC cell-1 for dinoflagellates, respectively. The mean carbon density for diatoms and dinoflagellates (excl. Noctiluca scintillans) varied between 0.027 and 0.099 pgC µm-3 0.096 and 0.136 pgC µm-3, respectively. The mean cell carbon content and carbon density of dinoflagellates (6.73 × 103 pgC cell-1 and 0.115 pgC µm-3) were approximately 10 and 2 times greater than those of diatoms, respectively. The carbon content of the other phytoflagellates was lower, whereas their carbon density was higher. As a result, the findings from this study will provide a significant contribution to the assessment and estimation of carbon biomass during algal blooms in this study area.

Imaging and plate counting to quantify the effect of an antimicrobial: A case study of a photo-activated chlorine dioxide treatment.

AIM: To assess removal versus kill efficacies of antimicrobial treatments against thick biofilms with statistical confidence. METHODS AND RESULTS: A photo-activated chlorine dioxide treatment (Photo ClO2 ) was tested in two independent experiments against thick (>100 µm) Pseudomonas aeruginosa biofilms. Kill efficacy was assessed by viable plate counts. Removal efficacy was assessed by 3D confocal scanning laser microscope imaging (CSLM). Biovolumes were calculated using an image analysis approach that models the penetration limitation of the laser into thick biofilms using Beer's Law. Error bars are provided that account for the spatial correlation of the biofilm's surface. The responsiveness of the biovolumes and plate counts to the increasing contact time of Photo ClO2 were quite different, with a massive 7 log reduction in viable cells (95% confidence interval [CI]: 6.2, 7.9) but a more moderate 73% reduction in biovolume (95% CI: [60%, 100%]). Results are leveraged to quantitatively assess candidate CSLM experimental designs of thick biofilms. CONCLUSIONS: Photo ClO2 kills biofilm bacteria but only partially removes the biofilm from the surface. To maximize statistical confidence in assessing removal, imaging experiments should use fewer pixels in each z-slice, and more importantly, at least two independent experiments even if there is only a single field of view in each experiment. SIGNIFICANCE AND IMPACT OF STUDY: There is limited penetration depth when collecting 3D confocal images of thick biofilms. Removal can be assessed by optimally fitting Beer's Law to all of the intensities in a 3D image and by accounting for the spatial correlation of the biofilm's surface. For thick biofilms, other image analysis approaches are biased or do not provide error bars. We generate unbiased estimates of removal and assess candidate CSLM experimental designs of thick biofilms with different pixilations, numbers of fields of view and number of experiments using the included design tool.

Substantial intraspecific genome size variation in golden-brown algae and its phenotypic consequences.

BACKGROUND AND AIMS: While nuclear DNA content variation and its phenotypic consequences have been well described for animals, vascular plants and macroalgae, much less about this topic is known regarding unicellular algae and protists in general. The dearth of data is especially pronounced when it comes to intraspecific genome size variation. This study attempts to investigate the extent of intraspecific variability in genome size and its adaptive consequences in a microalgal species. METHODS: Propidium iodide flow cytometry was used to estimate the absolute genome size of 131 strains (isolates) of the golden-brown alga Synura petersenii (Chrysophyceae, Stramenopiles), identified by identical internal transcribed spacer (ITS) rDNA barcodes. Cell size, growth rate and genomic GC content were further assessed on a sub-set of strains. Geographic location of 67 sampling sites across the Northern hemisphere was used to extract climatic database data and to evaluate the ecogeographical distribution of genome size diversity. KEY RESULTS: Genome size ranged continuously from 0.97 to 2.02 pg of DNA across the investigated strains. The genome size was positively associated with cell size and negatively associated with growth rate. Bioclim variables were not correlated with genome size variation. No clear trends in the geographical distribution of strains of a particular genome size were detected, and strains of different genome size occasionally coexisted at the same locality. Genomic GC content was significantly associated only with genome size via a quadratic relationship. CONCLUSIONS: Genome size variability in S. petersenii was probably triggered by an evolutionary mechanism operating via gradual changes in genome size accompanied by changes in genomic GC content, such as, for example, proliferation of transposable elements. The variation was reflected in cell size and relative growth rate, possibly with adaptive consequences.

Protist Diversity and Seasonal Dynamics in Skagerrak Plankton Communities as Revealed by Metabarcoding and Microscopy.

Protist community composition and seasonal dynamics are of major importance for the production of higher trophic levels, such as zooplankton and fish. Our aim was to reveal how the protist community in the Skagerrak changes through the seasons by combining high-throughput sequencing and microscopy of plankton collected monthly over two years. The V4 region of the 18S rRNA gene was amplified by eukaryote universal primers from the total RNA/cDNA. We found a strong seasonal variation in protist composition and proportional abundances, and a difference between two depths within the euphotic zone. Highest protist richness was found in late summer-early autumn, and lowest in winter. Temperature was the abiotic factor explaining most of the variation in diversity. Dinoflagellates was the most abundant and diverse group followed by ciliates and diatoms. We found about 70 new taxa recorded for the first time in the Skagerrak. The seasonal pattern in relative read abundance of major phytoplankton groups was well in accordance with microscopical biovolumes. This is the first metabarcoding study of the protist plankton community of all taxonomic groups and through seasons in the Skagerrak, which may serve as a baseline for future surveys to reveal effects of climate and environmental changes.

Effects of eutrophication on diatom abundance, biovolume and diversity in tropical coastal waters.

Diatom abundance, biovolume and diversity were measured over a 2-year period along the Straits of Malacca at two stations with upper (Klang) and lower (Port Dickson) states of eutrophication. Diatom abundance, which ranged from 0.2 × 104 to 21.7 × 104 cells L-1 at Klang and 0.9 × 103- 41.3 × 103 cells L-1 at Port Dickson, was influenced partly by nutrient concentrations. At Klang, the diatoms were generally smaller and less diverse (H' = 0.77 ± 0.48) and predominated by Skeletonema spp. (60 ± 32% of total diatom biomass). In contrast, diatoms were larger and more diverse (H' = 1.40 ± 0.67) at Port Dickson. Chaetoceros spp. were the most abundant diatoms at Port Dickson but attributed only 48 ± 30% of total diatom biomass. Comparison of both Klang and Port Dickson showed that their diatom community structure differed and that eutrophication reduced diatom diversity at Klang. We also observed how Si(OH)4 affected the abundance of Skeletonema spp. which in turn influenced the temporal variation of diatom community at Klang. Our results highlighted how eutrophication affects diatom diversity and community structure.

An evaluation of a handheld spectroradiometer for the near real-time measurement of cyanobacteria for bloom management purposes.

A commercially available handheld spectroradiometer, the WISP-3, was assessed as a tool for monitoring freshwater cyanobacterial blooms for management purposes. Three small eutrophic urban ponds which displayed considerable within-pond and between-pond variability in water quality and cyanobacterial community composition were used as trial sites. On-board algorithms provide field measurements of phycocyanin (CPC) and chlorophyll-a (Chl-a) from surface reflectance spectra measured by the instrument. These were compared with laboratory measurements. Although significant but weak relationships were found between WISP-3 measured CPC and cyanobacterial biovolume measurements and WISP-3 and laboratory Chl-a measurements, there was considerable scatter in the data due likely to error in both WISP-3 and laboratory measurements. The relationships generally differed only slightly between ponds, indicating that different cyanobacterial communities had little effect on the pigment retrievals of the WISP-3. The on-board algorithms need appropriate modification for local conditions, posing a problem if it is to be used extensively across water bodies with differing optical properties. Although suffering a range of other limitations, the WISP-3 has a potential as a rapid screening tool for preliminary risk assessment of cyanobacterial blooms. However, such field assessment would still require adequate support by sampling and laboratory-based analysis.

New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

Parasites of fish larvae: do they follow metabolic energetic laws?

Eumetazoan parasites in fish larvae normally exhibit large body sizes relative to their hosts. This observation raises a question about the potential effects that parasites might have on small fish. We indirectly evaluated this question using energetic metabolic laws based on body volume and the parasite densities. We compared the biovolume as well as the numeric and volumetric densities of parasites over the host body volume of larval and juvenile-adult fish and the average of these parasitological descriptors for castrator parasites and the parasites found in the fish studied here. We collected 5266 fish larvae using nearshore zooplankton sampling and 1556 juveniles and adult fish from intertidal rocky pools in central Chile. We considered only the parasitized hosts: 482 fish larvae and 629 juvenile-adult fish. We obtained 31 fish species; 14 species were in both plankton and intertidal zones. Fish larvae exhibited a significantly smaller biovolume but larger numeric and volumetric densities of parasites than juvenile-adult fish. Therefore, fish larvae showed a large proportion of parasite biovolume per unit of body host (cm(3)). However, the general scaling of parasitological descriptors and host body volume were similar between larvae and juvenile-adult fish. The ratio between the biovolume of parasites and the host body volume in fish larvae was similar to the proportion observed in castrator parasites. Furthermore, the ratios were different from those of juvenile-adult fish, which suggests that the presence of parasites implies a high energetic cost for fish larvae that would diminish the fitness of these small hosts.

Nuclear genome of dinoflagellates: Size variation and insights into evolutionary mechanisms.

Recent progress in high-throughput sequencing technologies has dramatically increased availability of genome data for prokaryotes and eukaryotes. Dinoflagellates have distinct chromosomes and a huge genome size, which make their genomic analysis complicated. Here, we reviewed the nuclear genomes of core dinoflagellates, focusing on the genome and cell size. Till now, the genome sizes of several dinoflagellates (more than 25) have been measured by certain methods (e.g., flow cytometry), showing a range of 3-250 pg of genomic DNA per cell. In contrast to their relatively small cell size, their genomes are huge (about 1-80 times the human haploid genome). In the present study, we collected the genome and cell size data of dinoflagellates and compared their relationships. We found that dinoflagellate genome size exhibits a positive correlation with cell size. On the other hand, we recognized that the genome size is not correlated with phylogenetic relatedness. These may be caused by genome duplication, increased gene copy number, repetitive non-coding DNA, transposon expansion, horizontal gene transfer, organelle-to-nucleus gene transfer, and/or mRNA reintegration into the genome. Ultimate verification of these factors as potential causative mechanisms would require sequencing of more dinoflagellate genomes in the future.

Assessment of in-situ monitoring and tracking the vertical migration of cyanobacterial blooms using LISST-HAB.

Cyanobacterial harmful algal blooms (cyanoHABs) are becoming increasingly common in aquatic ecosystems worldwide. However, their heterogeneous distributions make it difficult to accurately estimate the total algae biomass and forecast the occurrence of surface cyanoHABs by using traditional monitoring methods. Although various optical instruments and remote sensing methods have been employed to monitor the dynamics of cyanoHABs at the water surface (i.e., bloom area, chlorophyll a), there is no effective in-situ methodology to monitor the dynamic change of cell density and integrated biovolume of algae throughout the water column. In this study, we propose a quantitative protocol for simultaneously measurements of multiple indicators (i.e., biovolume concentration, size distribution, cell density, and column-integrated biovolume) of cyanoHABs in water bodies by using the laser in-situ scattering and transmissometry (LISST) instrument. The accuracy of measurements of the biovolume and colony size of algae was evaluated and exceeded 95% when the water bloom was dominated by cyanobacteria. Furthermore, the cell density of cyanobacteria was well estimated based on total biovolume and mean cell volume measured by the instrument. Therefore, this methodology has the potential to be used for broader applications, not only to monitor the spatial and temporal distribution of algal biovolume concentration but also monitor the vertical distribution of cell density, biomass and their relationship with size distribution patterns. This provides new technical means for the monitoring and analysis of algae migration and early warning of the formation of cyanoHABs in lakes and reservoirs.

Summer monsoon promotes the energy transfer efficiency of the zooplankton community in northern South China sea.

The summer monsoon shows a fundamental influence on the pelagic ecosystem of the South China Sea. Zooplankton are a major link for energy transfer between primary producers and upper trophic levels. Therefore, evaluating the energy transfer efficiency (ETE) of zooplankton is crucial to understand the function of pelagic ecosystem under the influence of monsoon. In this study, field surveys were conducted during May (intermonsoon) and August 2021 (summer monsoon) focusing on the variation of zooplankton size and trophic structures across the shelf and slope. The result showed that the summer monsoon reinforced the gradient of abundance, biovolume, and biomass from slope to shelf, and greatly intensified the role of environmental factors in driving spatial variation in most taxa. Both the results of size and trophic structures indicated that the ETE of zooplankton decreased from slope to shelf. The size structure also indicated that the ETE of zooplankton significantly increased under the influence of summer monsoon. These results were consistent with previous studies by different methods, suggesting that these approaches of size and trophic structures had important potential value in assessing changes in the function of marine pelagic ecosystem, especially when compared with sufficient historical data or reanalyzing historical samples.

Dataset on meteorological forcing mechanisms impacting marine circulation and oceanographic variables in the northern part of the Veracruz reef system.

The dataset is divided in two main groups. The first group, referred to as "Meteorological data", consists of air temperature, sea level pressure and U and V components of wind direction and intensity: The second group, referred to as "Oceanographic data", includes biovolume, sea level and water temperature measurements. The meteorological data is derived from model data obtained from the NAAR-NCEP reanalysis for North America, calculated over the area of the Veracruz reef system, Mexico. On the other hand, the oceanographic data was collected in situ using four ADCPs (Acoustic Doppler Current Profilers) anchored at a depth of 20 m at four different reefs within the Veracruz reef system. Both datasets cover a period of 10 days in November 2008, during which successive low-and high-pressure systems occurred over the southwestern Gulf of Mexico. These datasets can be used to evaluate the effect of the pressure changes on marine circulation, residual current and oceanographic variables.

Uncertainties of cell number estimation in cyanobacterial colonies and the potential use of sphere packing.

Cyanobacteria are notorious bloom formers causing various water quality concerns, such as toxin production, extreme diurnal variation of oxygen, pH, etc., therefore, their monitoring is essential to protect the ecological status of aquatic systems. Cyanobacterial cell counts and biovolumes are currently being used in water management and water quality alert systems. In this study, we investigated the accuracy of traditional colonial biovolume and cell count estimation approaches used in everyday practice. Using shape realistic 3D images of cyanobacterial colonies, we demonstrated that their shape cannot be approximated by ellipsoids. We also showed that despite the significant relationship between overall colony volume and cell biovolumes, because of the considerable scatter of cell count data the regressions give biased estimates for cyanobacterial cell counts. We proposed a novel approach to estimate cell counts in colonies that was based on the random close sphere packing method. This method provided good results only in those cases when overall colony volumes could be accurately measured. The visual investigation of colonies done by skilled experts has given precise but lower estimates for cell counts. The estimation results of several experts were surprisingly good, which suggests that this capability can be improved and estimation bias can be reduced to the level acceptable for water quality estimations.

Effects of space and environment on phytoplankton distribution in subtropical reservoirs depend on functional features of the species.

Environmental factors and dispersal can influence the structure of biological communities. Their effects can depend on the functional features of the species in the community. Since species belonging to the same trophic level, such as phytoplankton, may show functional differences, we investigated whether the effects of environment and dispersal differ among phytoplankton species from different functional groups. We analyzed data from a rainy and a dry period in 30 reservoirs in a subtropical region. In both periods, the environment as well as high and limited dispersal influenced the metacommunity structure. The functional groups had a low correspondence in their response to both dispersal and environment. Our results showed that the influence of the processes underlying the structure of the metacommunities, such as species sorting (environment influence), mass effect (high dispersal), and neutral dynamics (limited dispersal), depended on the functional characteristics of the organisms and could vary even among species of the same trophic level. These findings suggested that species at the same trophic level could not be considered ecological equivalents. This paper includes a Portuguese and Spanish version of the abstract in the online resources. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00027-021-00837-0.

The impact of micropollutants on native algae and cyanobacteria communities in ecological filters during drinking water treatment.

An attractive alternative for drinking water production is ecological filtration. Previous studies have reported high removal levels of pharmaceutical and personal care products (PPCPs) by this technology. Algae and cyanobacteria play an important role in the biological activity of ecological filters. The aim of this study was to characterize and identify the community of algae and cyanobacteria in relation to its composition, density and biovolume from 22 ecological filters that received spikings of 2 µg L-1 PPCPs. For algae and cyanobacteria species, triplicate samples were collected before and 96 h after each spiking from the interface between the top sand layer of the ecological filters and the supernatant water. Results show that Chlorophyceae and Cyanobacteria were present in high numbers of taxa and abundance. The specie Lepocinclis cf. ovum (Euglenophyceae) had the highest percentage occurrence/abundance and frequency into the filters, indicating a possible tolerance by Lepocinclis cf. ovum to the concentration of selected PPCPs. Although the concentration of PPCPs did not affect the treated water quality, they did affect the algae and cyanobacteria community. No differences were detected between filters that received a single PPCP and filters that received a mixture of the six compounds. Also, changes in the composition of algae and cyanobacteria communities were observed before and 96 h after the spikings.

Changes in Phytoplankton Community Composition and Phytoplankton Cell Size in Response to Nitrogen Availability Depend on Temperature.

The climate-driven changes in temperature, in combination with high inputs of nutrients through anthropogenic activities, significantly affect phytoplankton communities in shallow lakes. This study aimed to assess the effect of nutrients on the community composition, size distribution, and diversity of phytoplankton at three contrasting temperature regimes in phosphorus (P)-enriched mesocosms and with different nitrogen (N) availability imitating eutrophic environments. We applied imaging flow cytometry (IFC) to evaluate complex phytoplankton communities changes, particularly size of planktonic cells, biomass, and phytoplankton composition. We found that N enrichment led to the shift in the dominance from the bloom-forming cyanobacteria to the mixed-type blooming by cyanobacteria and green algae. Moreover, the N enrichment stimulated phytoplankton size increase in the high-temperature regime and led to phytoplankton size decrease in lower temperatures. A combination of high temperature and N enrichment resulted in the lowest phytoplankton diversity. Together these findings demonstrate that the net effect of N and P pollution on phytoplankton communities depends on the temperature conditions. These implications are important for forecasting future climate change impacts on the world's shallow lake ecosystems.

Activity density at a continental scale: What drives invertebrate biomass moving across the soil surface?

Activity density (AD), the rate that an individual taxon or its biomass moves through the environment, is used both to monitor communities and quantify the potential for ecosystem work. The Abundance Velocity Hypothesis posited that AD increases with aboveground net primary productivity (ANPP) and is a unimodal function of temperature. Here we show that, at continental extents, increasing ANPP may have nonlinear effects on AD: increasing abundance, but decreasing velocity as accumulating vegetation interferes with movement. We use 5 yr of data from the NEON invertebrate pitfall trap arrays including 43 locations and four habitat types for a total of 77 habitat-site combinations to evaluate continental drivers of invertebrate AD. ANPP and temperature accounted for one-third to 92% of variation in AD. As predicted, AD was a unimodal function of temperature in forests and grasslands but increased linearly in open scrublands. ANPP yielded further nonlinear effects, generating unimodal AD curves in wetlands, and bimodal curves in forests. While all four habitats showed no AD trends over 5 yr of sampling, these nonlinearities suggest that trends in AD, often used to infer changes in insect abundance, will vary qualitatively across ecoregions.

Biovolume and surface area calculations for microalgae, using realistic 3D models.

Morphology and spatial dimensions of microalgal units (cells or colonies) are among the most relevant traits of planktic algae, which have a pronounced impact on their basic functional properties, like access to nutrients or light, the velocity of sinking or tolerance to grazing. Although the shape of algae can be approximated by geometric forms and thus, their volume and surface area can be calculated, this approach cannot be validated and might have uncertainties especially in the case of complicated forms. In this study, we report on a novel approach that uses real-like 3D mesh objects to visualize microalgae and calculates their volume and surface area. Knowing these dimensions and their intraspecific variabilities, we calculated specific shape and surface area constants for more than 300 forms, covering more than two thousand taxa. Using these constants, the accurate volume and surface area can be quickly computed for each taxon and having these values, morphology-related metrics like surface area/volume ratio, the diameter of spherical equivalent can also be given quickly and accurately. Besides their practical importance, the volume and surface area constants can be considered as size-independent morphological traits that are characteristic for the microalgal shapes, and provide new possibilities of data analyses in the field of phytoplankton ecology.

Acoustic scattering by gas-bearing cyanobacterium Microcystis: Modeling and in situ biomass assessment.

Cyanobacterial harmful algal blooms (HABs) are increasing in a growing number of aquatic ecosystems around the world due to eutrophication and climatic change over the past few decades. Quantitative monitoring of HABs remains a challenge because their distributions are spatially heterogeneous and temporally variable. Most of the standard biological sampling methods are labor intensive and time consuming. In this paper, we present an efficient acoustic method to assess the biomass (biovolume) concentration of the cyanobacterium Microcystis in aquatic ecosystems. Acoustic backscattering vertical profiles from a gas-bearing Microcystis population were measured with echosounders at three frequencies (70, 120, and 333 kHz) in Lake Kinneret (case study). Concurrently, the volume concentration of Microcystis colonies and cyanobacteria-related Chlorophyll a were evaluated. We developed a partially coherent acoustic scattering model to quantify the cyanobacterium biomass based on depth-dependent acoustic backscattering signals. We also evaluated empirical regression models to obtain the Microcystis biomass from acoustically measured volume backscattering strength, Sv. It is demonstrated that both methods can convert the Sv to Microcystis biovolume concentrations reasonably well. Pro and cons of these methods are discussed. The results suggest that the presented methods may have a potential to be used for broader applications to monitor and quantify the gas-containing plankton in large aquatic ecosystems.

Reproductive cycle progression arrest and modification of cell morphology (shape and biovolume) in the alga Pseudokirchneriella subcapitata exposed to metolachlor.

Metolachlor (MET) is an herbicide widely used and frequently found (at µg L-1) in aquatic systems. This work aimed to study the modes of action of MET on the green microalga Pseudokirchneriella subcapitata. Algae exposed to 115 or 235 µg L-1 MET, for 48 or 72 h, presented a reduction of metabolic activity, chlorophyll a and b content and photosynthetic efficiency. The exposure to 115 or 235 µg L-1 MET also induced growth yield reduction, mean cell biovolume increase and alteration of the typical algae shape (cells lunate or helically twisted) to "French croissant"-type; at these MET concentrations, algal population was mainly composed by multinucleated cells (≥ 4 nuclei), which suggest that MET impairs the normal progression of the reproductive cycle but did not hinder nuclear division. The accumulation of multinucleated cells seems to be the consequence of the incapacity of the parent cell to release the autospores. In conclusion, MET disrupts the physiology of P. subcapitata cells; the disturbance of the progression of the reproductive cycle should be in the origin of growth slowdown (or even its arrest), increase of mean cell biovolume and modification of algal shape. This work contributed to elucidate, in a systematically and integrated way, the toxic mechanism of MET on the non-target organism, the alga P. subcapitata.