Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Assessment of the eutrophication status at Mediterranean sub-basin scale, within the European Marine Strategy Framework Directive.

The aim of this work is to define harmonized reference conditions and assessment thresholds for selected criteria elements of the Marine Strategy Framework Directive (MSFD) Descriptor 5 (Eutrophication) in the Eastern Mediterranean Sea and to test if a tool for integrated assessment of the status of marine systems can be used as a common methodological approach. In this frame, we tested two statistical approaches in order to set threshold values for four criteria of Descriptor 5: nutrients, chlorophyll a, transparency and dissolved oxygen in the bottom waters. It is noteworthy that this work revealed the need to apply common procedures in data treatment and assessment evaluation. This is the first attempt to set common methods for the assessment of eutrophication in the Eastern Mediterranean, which is essential in marine environments, especially those shared by several countries. To this end, we have applied common criteria and metrics and established thresholds "Good" and "Moderate" for nutrients, chlorophyll a, transparency and dissolved oxygen in the bottom waters for the different Water Types of the Adriatic and Aegean Seas (I, II, IIIW, IIIE), based on datasets provided by Italy, Slovenia, Croatia and Greece. The selected criteria elements were common for all countries, providing a unified approach to GES assessment of two case study areas: the Adriatic Sea and the Saronikos Gulf. Dissolved Inorganic Nitrogen (DIN) threshold values of 15.6, 6.85, 1.61 and 2.11 µmol L-1 were set for the Water Types I, II, IIIW and IIIE, respectively. We also tested if an aggregation tool for GES assessment, such as Nested Environmental status Assessment Tool (NEAT), could be used as a common methodological approach. The comparison of NEAT with TRIX showed good comparability. In this end, NEAT can be used as a useful and much needed assessment tool for assessing eutrophication status of the marine.

Exploring the mesoscale connectivity of phytoplankton periodic assemblages' succession in northern Adriatic pelagic habitats.

An appropriate model for phytoplankton distribution patterns is critical for understanding biogeochemical cycles and trophic interactions in the oceans and seas. Because phytoplankton dynamics in coastal waters are more complex due to shallow depth and proximity to land, more accurate models applied to the correct spatial and temporal scales are needed. Our study investigates the role of the atmosphere and hydrosphere in pelagic habitat by modelling phytoplankton assemblages at two Long Term Ecological Research sites in the northern Adriatic Sea using niche-forming environmental variables (wind, temperature, salinity, river discharge, rain, and water column stratification). To study the synchronization between the phytoplankton community and these environmental variables at the two LTER sites, we applied current linear and nonlinear numerical methods for ecological modelling. The aim was to use periodic and/or non-periodic properties of the environmental variables to classify the phytoplankton assemblages at one LTER site (Gulf of Trieste - Slovenia) and then predict them at another LTER site 100 km away (Gulf of Venice - Italy). We found that periodicity played a role in the explanatory and predictive power of the environmental variables and that it was more important than non-periodic events in defining the common structure of the two pelagic habitats. The non-linear classification functions of the neural networks further increased the predictive power of these variables. We observed partial synchronization of communities at the mesoscale and differences between the original and predicted assemblages under similar environmental conditions. We conclude that mesoscale connectivity plays an important role in phytoplankton communities in the northern Adriatic. However, the loss of periodicity of niche-forming variables due to more frequent extreme meteorological and hydrological events could loosen these connections and affect the temporal succession of phytoplankton assemblages.

Corrigendum: Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera.

[This corrects the article DOI: 10.3389/fmicb.2023.1071379.].

Evaluation of the rbcL marker for metabarcoding of marine diatoms and inference of population structure of selected genera.

Diatoms are one of the most important phytoplankton groups in the world's oceans. There are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Traditionally, these have been assessed using microscopy, which in some cases is not reliable or reproducible. Next-generation sequencing enabled us to study diversity in a high-throughput manner and uncover new distribution patterns and diversity. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often insufficient because they cannot distinguish between some taxa. In this work, we demonstrate the performance of the chloroplast-encoded rbcL marker for metabarcoding marine diatoms compared to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT), northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 metabarcoding or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. We were able to spot misidentification of genera obtained with microscopy such as Pseudo-nitzschia galaxiae, which was mistaken for Cylindrotheca closterium, as well as genera that were completely overlooked, such as Minidiscus and several genera from the Cymatosiraceae family. Furthermore, on the example of two well-studied genera in the region, namely Chaetoceros and particularly Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Despite a very thorough community analysis obtained by rbcL the incompleteness of reference databases was still evident, and we shed light on possible improvements. Our work has further implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.

Ecological time series and integrative taxonomy unveil seasonality and diversity of the toxic diatom Pseudo-nitzschia H. Peragallo in the northern Adriatic Sea.

Pseudo-nitzschia H. Peragallo (1900) is a globally distributed genus of pennate diatoms that are important components of phytoplankton communities worldwide. Some members of the genus produce the neurotoxin domoic acid, so regular monitoring is in place. However, the identification of toxic members in routine samplings remains problematic. In this study, the diversity and seasonal occurrence of Pseudo-nitzschia species were investigated in the Gulf of Trieste, a shallow gulf in the northern Adriatic Sea. We used time series data from 2005 to 2018 to describe the seasonal and inter-annual occurrence of the genus in the area and its contribution to the phytoplankton community. On average, the genus accounted for about 15 % of total diatom abundance and peaked in spring and autumn, with occasional outbreaks during summer and large inter-annual fluctuations. Increased water temperature and decreased salinity positively affected the presence of some members of the genus, while strong effects could be masked by an unsuitable definition of the species complexes used for monitoring purposes. Therefore, combining morphological (TEM) and molecular analyses by sequencing the ITS, 28S and rbcL markers, eight species were identified from 83 isolated monoclonal strains: P. calliantha, P. fraudulenta, P. delicatissima, P. galaxiae, P. mannii, P. multistriata, P. pungens and P. subfraudulenta. A genetic comparison between the isolated strains and other strains in the Mediterranean was carried out and rbcL was inspected as a potential barcode marker in respect to our results. This is the first study in the Gulf of Trieste on Pseudo-nitzschia time series from a long-term ecological research (LTER) site coupled with molecular data. We show that meaningful ecological conclusions can be drawn by applying integrative methodology, as opposed to the approach that only considers species complexes. The results of this work will provide guidance for further monitoring efforts as well as research activities, including population genetics and genomics, associated with seasonal distribution and toxicity profiles.

Phytoplankton diversity in Adriatic ports: Lessons from the port baseline survey for the management of harmful algal species.

An inventory of phytoplankton diversity in 12 Adriatic ports was performed with the port baseline survey. Particular emphasis was put on the detection of harmful aquatic organisms and pathogens (HAOP) because of their negative impact on ecosystem, human health, and the economy. Phytoplanktonic HAOP are identified as species, either native or non-indigenous (NIS), which can trigger harmful algal blooms (HAB). A list of 691 taxa was prepared, and among them 52 were classified as HAB and five as NIS. Records of toxigenic NIS (Pseudo-nitzschia multistriata, Ostreopsis species including O. cf. ovata) indicate that the intrusion of non-native invasive phytoplankton species has already occurred in some Adriatic ports. The seasonal occurrence and abundance of HAOP offers a solid baseline for a monitoring design in ports in order to prevent ballast water uptake and possible expansion of HAOP outside their native region.

Oceanographic characteristics of the Adriatic Sea - Support to secondary HAOP spread through natural dispersal.

Vessels, specifically ballast water and hull fouling, are a major vector for the introduction of non-indigenous species (NIS) in European seas. The Mediterranean is one of the world's marine regions where their invasion is heaviest. The shallow Adriatic basin is a highly sensitive area that is already experiencing its consequences. The secondary spread of NIS over a wider area through natural dispersion is a complex process that depends on a wide range of oceanographic factors. This work analysed the dataset of the BALMAS project, in whose framework twelve ports in the Adriatic Sea were subjected to a Port Baseline Survey (PBS), to estimate the natural spread of NIS organisms from their port of arrival to the wider Adriatic basin. Its findings indicate that the prevailing water circulation patterns facilitate the natural dispersal of harmful aquatic organisms and pathogens (HAOP).

Quantifying indicatively living phytoplankton cells in ballast water samples--recommendations for Port State Control.

Different phytoplankton analysis methods (pulse-amplitude modulated fluorometry (PAM) and microscopy) were compared in preparation for compliance monitoring and enforcement with ballast water discharge standards. The key objective was to practically evaluate the performance of different new methods and tools to identify indicatively living phytoplankton cells of the size <50 µm in minimum dimension and ≥ 10 µm in minimum dimension as addressed by the Ballast Water Performance Standard (Regulation D-2, International Convention for the Control and Management of Ships' Ballast Water and Sediments, 2004). Four different PAM instruments were selected for the tests based upon knowledge and experience gained in different ballast water sample processing studies. The measurements of the PAM instruments were compared with epifluorescence microscope algae cell counts using fluorescein diacetate as viability stain. It was concluded that PAM fluorometry is a suitable method for indicative phytoplankton analysis of ballast water and the most accurate PAM instruments were identified.

A new index (MediSkew) for the assessment of the Cymodocea nodosa (Ucria) Ascherson meadow's status.

In the Mediterranean region Cymodocea nodosa is widely distributed throughout shallow sites. Therefore, a correct assessment of the status of its meadows is of great importance for the implementation of the Water Framework Directive (WFD), the Marine Strategy Framework Directive (MSFD), and the Habitat Directive (HD), especially for areas where Posidonia oceanica meadows (the most frequently used indicator in the Mediterranean Sea) are rare or not present. The previously proposed index for the evaluation of the status of C. nodosa meadows (CymoSkew) is based on ln-transformed relative frequencies of photosynthetic leaf lengths, which we believe is statistically questionable. Therefore, we further improved the methodology and developed a new index named MediSkew, where ln-transformation is applied to raw data of leaf lengths. More specifically, the index is a combination of two metrics, both based on C. nodosa leaf length: deviation from the reference median length (Medi-) and skewness of the length frequency distribution (-Skew), though greater importance was assigned to the first. To develop the classification criteria for the assessment of the status, also a Pressure Index for Seagrass Meadows (PISM), for the evaluation of pressure-impact relationships, was developed. The MediSkew is meant to be a rapid screening method for wide areas, therefore the index should be tested for the assessment of the status of C. nodosa meadows throughout Mediterranean coastal waters.

Ecological characterization of toxic phytoplankton species (Dinophysis spp., Dinophyceae) in Slovenian mariculture areas (Gulf of Trieste, Adriatic Sea) and the implications for monitoring.

Diarrhetic shellfish poisoning (DSP) events are often registered in Slovenian mariculture areas (Gulf of Trieste, Adriatic Sea) and are related to the occurrence of Dinophysis spp. The annual dynamic of this genus and succession of the most important species were studied at two shellfish farms during monitoring fieldwork in the period 1995-2003. Results indicate that the Dinophysis genus maintains a relatively stable inter-annual dynamic at both sites. The Dinophysis community is characterized by two surface maxima in June and September, while in the middle layer only the autumn peak is pronounced (peak median 92 cells l(-1)). Occasional abundance maxima of around 2000 cells l(-1) in the surface layer indicate that potential outbursts of toxic species are less predictable than their seasonal dynamic. On the basis of multivariate analysis, Dinophysis sacculus was characterized as a typical late spring-early summer species, and Dinophysis caudata and Dinophysis fortii as autumn species. Correlation analysis revealed the influence of stratified conditions only on the most abundant species, D. sacculus. Ecological characteristics of the species were combined with shellfish safety requirements towards a more effective monitoring.