Study on the diversity and germination of dinoflagellate cysts in the sediments of foreign ships in Shanghai Port.

The dinoflagellate cysts present in the ballast water sediment of foreign ships in Shanghai Port have not been previously studied. Therefore, sediment samples were collected from the ballast water of 16 foreign ships in Shanghai Port, and the types of dinoflagellate cysts were identified and their abundance was calculated, with a specific focus on the analysis of toxic and harmful dinoflagellates. Moreover, simulations of temperature and salinity conditions throughout the year in the Shanghai port waters were conducted to carry out dinoflagellate cyst germination experiments, with analyze and compare the germinated dinoflagellate cysts under different conditions. Dinoflagellate cysts were found in 100 % of the ship sediment samples, including a total of 9 species of toxic and harmful dinoflagellate cysts. In the germination experiment, 15 °C was found to be the optimal temperature for the germination of dinoflagellate cysts in ballast water sediment, and high salinity is more favorable for cyst germination.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Viability assessed with the most probable number dilution culture method after chemical treatment of ballast water reveals the presence of false negatives from an approved vital stain method.

The present study compares the CMFDA/FDA + motility- and the Most Probable Number (MPN) Dilution Culture + Motility methods for testing the viability of ≥10-<50 µm organisms in chlorine treated ballast water. The results of both methods were within the regulatory compliance criterion <10 organisms/mL, but the MPN-method revealed that growth-outs did occur. While the CMFDA/FDA method showed <0.5 organisms/mL, the MPN-method gave approx. 6 organisms/mL. This demonstrated that false negatives, i.e. living but not stained organisms, may occur when using the CMFDA/FDA-method for compliance testing of chemical treated ballast water. Organisms surviving the treatment were primarily the dinoflagellate Scrippsiella sp. and various coccoid chlorophytes present in a brackish- and freshwater test, respectively. It is suggested that their resilience to the chemical treatment is the ability to transform into a temporary cyst (Scrippsiella sp.) or the presence of a chemical resistant cell wall (certain chlorophytes).

Diverse bacterial hosts and potential risk of antibiotic resistomes in ship ballast water revealed by metagenomic binning.

Ship ballast water promoting the long-range migration of antibiotic resistance genes (ARGs) has raised a great concern. This study attempted to reveal ARGs profile in ballast water and decipher their hosts and potential risk using metagenomic approaches. In total, 710 subtypes across 26 ARG types were identified among the ballast water samples from 13 ships of 11 countries and regions, and multidrug resistance genes were the most dominant ARGs. The composition of ARGs were obviously different across samples, and only 5% of the ARG subtypes were shared by all samples. Procrustes analysis showed the bacterial community contributed more than the mobile genetic elements (MGEs) in shaping the antibiotic resistome. Further, 79 metagenome-assembled genomes (46 genera belong to four phyla) were identified as ARG hosts, with predominantly affiliated with the Proteobacteria. Notably, potential human pathogens (Alcaligenes, Mycolicibacterium, Rhodococcus and Pseudomonas) were also recognized as the ARG hosts. Above 30% of the ARGs hosts contained the MGEs simultaneously, supporting a pronounced horizontal gene transfer capability. A total of 43 subtypes (six percent of overall ARGs) of ARGs were assessed with high-risk, of which 23 subtypes belonged to risk Rank I (including rsmA, ugd, etc.) and 20 subtypes to the risk Rank II (including aac(6)-I, sul1, etc.). In addition, antibiotic resistance risk index indicated the risk of ARGs in ballast water from choke points of maritime trade routes was significantly higher than that from other regions. Overall, this study offers insights for risk evaluation and management of antibiotic resistance in ballast water.

Sustainability in maritime transport: Selecting ballast water treatment for a bulk carrier.

The study provides a comprehensive assessment of ballast water treatment systems (BWTS) selection regarding crucial parameters such as energy efficiency, fuel consumption, and CO2 emissions. The focus of the study is investigating the environmental impacts of BWTS and how these impacts can be considered in decision-making processes. In this context, it comprehensively analyzes the importance of decision-making parameters and the environmental consequences of BWTS applications. The potential impacts of the system on the sustainability of the maritime industry are highlighted. In this context, seven different BWTSs for a bulk carrier of 83,000 deadweight tonnes are evaluated using Analytical Hierarchy Process (AHP) and Preference Ranking Technique by Similarity to Ideal Solution (TOPSIS). According to the analysis, Operational Expenditure (0.314508), Capital Expenditure (0.249515), and Capacity (0.159952) are the most critical factors. Among the seven systems analysed, product G (0.8561137) emerges as the most suitable option. The analysis represents 3% of total emissions (456.5 tonnes CO2) and highlights the impact of BWTS on CO2 emissions. The results underline the need for innovative approaches aimed at ensuring the long-term sustainability of the maritime industry.

Exploitation of environmental DNA (eDNA) for ecotoxicological research: A critical review on eDNA metabarcoding in assessing marine pollution.

The rise in worldwide population has led to a noticeable spike in the production, consumption, and transportation of energy and food, contributing to elevated environmental pollution. Marine pollution is a significant global environmental issue with ongoing challenges, including plastic waste, oil spills, chemical pollutants, and nutrient runoff, threatening marine ecosystems, biodiversity, and human health. Pollution detection and assessment are crucial to understanding the state of marine ecosystems. Conventional approaches to pollution evaluation usually represent laborious and prolonged physical and chemical assessments, constraining their efficacy and expansion. The latest advances in environmental DNA (eDNA) are valuable methods for the detection and surveillance of pollution in the environment, offering enhanced sensibility, efficacy, and involvement. Molecular approaches allow genetic information extraction from natural resources like water, soil, or air. The application of eDNA enables an expanded evaluation of the environmental condition by detecting both identified and unidentified organisms and contaminants. eDNA methods are valuable for assessing community compositions, providing indirect insights into the intensity and quality of marine pollution through their effects on ecological communities. While eDNA itself is not direct evidence of pollution, its analysis offers a sensitive tool for monitoring changes in biodiversity, serving as an indicator of environmental health and allowing for the indirect estimation of the impact and extent of marine pollution on ecosystems. This review explores the potential of eDNA metabarcoding techniques for detecting and identifying marine pollutants. This review also provides evidence for the efficacy of eDNA assessment in identifying a diverse array of marine pollution caused by oil spills, harmful algal blooms, heavy metals, ballast water, and microplastics. In this report, scientists can expand their knowledge and incorporate eDNA methodologies into ecotoxicological research.

Validation of an adenosine triphosphate (ATP) model for 10-50 µm plankton.

A recent model demonstrated that the adenosine triphosphate (ATP) content of spherical aquatic organisms with a 10 to 50 µm diameter is between 0.16 and 19.9 pg cell-1. Here, the model is validated by comparing microscopy-based counts with ATP concentrations from a commercial ATP kit. The measured ATP content of both freshwater and marine organisms 10 to 50 µm size range falls in the 0.16 to 19.9 pg cell-1 model range. On average, freshwater organisms contain 0.33 pg ATP cell-1, have a spherical equivalent diameter (SED) of 13 µm, while marine organisms have 0.89 pg ATP cell-1 and a SED of 18 µm. In addition, their 13 to 18 µm size is within the 10 to 50 µm ballast water size range and in agreement with the 15 µm mean SED of a coastal plankton size-distribution model. This study concludes that the ATP-model is reliable, emphasizing the need for caution when converting three-dimensional biomass proxies into linear cell concentrations.

Estimation of the introduction risk of non-indigenous species through ship ballast water in the Port of Douala (Cameroon).

The transport of non-indigenous species in ship's ballast water represents a threat to marine biodiversity. This study is the first on marine bioinvasion in Sub-Saharan Africa. The Port of Douala (PoD), located in the Gulf of Guinea, is experiencing increasing maritime traffic, hence the importance of preventing biological invasions. PoD received ballast water from 41 ports and 20 ecoregions during the study period (2018-2021). We used a biological invasion model and showed that ships from the ports of Antwerp, Durban, Dar es Salaam, Pointe-Noire (Southern Gulf of Guinea) and Dakar (Sahelian Upwelling), with their associated ecoregions present a major invasion risk. Treating ballast water from these ships to IMO D-2 standards could reduce their probability of biological invasion by 97.18, 98.43, 98.80, 98.77 and 98.84 %, respectively. Climate change may also mitigate the risk of biological invasion, particularly for ships in the North Sea ecoregion from the port of Antwerp.

Recommendations for representative sampling methodologies in ballast water: A case study from the land-based test.

Accurate assessment of ballast water's community composition and organism concentrations is crucial for effective management. Yet, the lack of standardized global sampling methods presents challenges to achieving this objective. Inconsistencies hinder data comparison across regions and vessels, impeding efficient ballast water treatment and discharge regulation. This study conducted land-based tests to establish suitable methodologies. For organisms ≥50 µm and ≥10 µm to <50 µm, the recommended max flow rate is 50 L/min; for <10 µm organisms, 25 L/min is advised to prevent cell damage. Sampling should cover substantial discharge durations. To ensure representation, ≥50 µm organisms require ≥1m3, while ≥10 µm to <50 µm and <10 µm organisms need 20 L. The ultimate aim is standardized methods for assessing ballast water across regions and vessel types, facilitating effective management to curb invasive species and protect aquatic ecosystems.

Exploring the bacterial diversity and composition with special emphasis on pathogens in ship ballast water and sediments using full-length 16S rRNA gene sequencing.

Accurate detecting bacterial communities in ballast water and sediments supports risk management. This study uses full-length 16S rRNA gene sequencing to investigate the bacterial communities in ballast water and sediments, focusing on detecting pathogens. The results indicate that full-length sequencing more accurately reveals the species diversity. There is a significant difference (P < 0.05) in bacterial communities between ballast water and sediments, despite both being dominated by the Proteobacteria phylum. Thirty human and fish pathogens were identified by full-length sequencing, yet only five pathogens were detected from V3-V4 sequencing. Notably, emerging pathogens such as Citrobacter freundii and Nocardia nova are detected in samples, which are harmful to aquaculture and human health. Several opportunistic pathogens were also identified. In summary, this study provides important insights into the bacterial communities in ballast water and sediments, highlighting the need for strict management.

Evaluate the compliance of ballast water management system on various types of operational vessels based on the D-2 standard.

The transfer of ship ballast water poses significant risks to the aquatic ecosystem and human health. To mitigate the influences of non-native species, ballast water management systems (BWMS) have been installed on international ships to ensure proper treatment of ballast water before discharge. This study investigates whether ballast water discharges managed by BWMS meet the requirements of the D-2 standard for organisms in different size classes. Representative ballast water samples were collected from 28 ships (a total of 20 different BWMS) arriving in Shanghai during the period 2020-2022. Results have shown that two samples (7.1 %) exceeded the D-2 Standard. The compliance rates varied among different vessel types, with cargo vessels achieving a compliance rate of 81.8 %, while LNG vessels and container vessels achieved 100 % compliance. The potential to achieve higher levels of ballast water management will increase as crews improve their skills in operating BWMS and BWMS is further developed.

Responses of compositions, functions, and assembly processes of bacterial and microeukaryotic communities to long-range voyages in simulated ballast water.

Ballast water is one of the main vectors for the spread of harmful organisms among geologically isolated waters. However, the successional processes of microbial functions and assembly processes in ballast water during the long-term shipping voyage remain unclear. In this study, the compositions, ecological functions, community assembly, and potential environmental drivers of bacteria and microeukaryotes were investigated in simulated ballast water microcosms for 120 days. The results showed that the diversity and compositions of the bacterial and microeukaryotic communities varied significantly in the initial 40 days (T0∼T40 samples) and then gradually converged. The relative abundance of Proteobacteria showed a distinct tendency to decrease (87.90%-41.44%), while that of Ascomycota exhibited an increasing trend (6.35%-62.12%). The functional groups also varied significantly over time and could be related to the variations of the microbial community. The chemoheterotrophy and aerobic chemoheterotrophy functional groups for bacteria decreased from 44.80% to 28.02% and from 43.77% to 25.39%, respectively. Additionally, co-occurrence network analysis showed that the structures of the bacterial community in T60∼T120 samples were more stable than those in T0∼T40 samples. Stochastic processes also significantly affected the community assembly of bacteria and microeukaryotes. pH played the most significant role in driving the structures and assembly processes of the bacterial and microeukaryotic communities. The results of this study could aid in the understanding of variations in the functions and ecological processes of bacterial and microeukaryotic communities in ballast water over time and provide a theoretical basis for its management.

Bloom forming species transported by ballast water under the management of D-1 and D-2 standards-Implications for current ballast water regulations.

Ballast water (BW) is a well-known transporter for introducing non-indigenous aquatic organisms. To reduce such risks associated with BW discharge, the International Maritime Organization (IMO) adopted the International Convention for the Control and Management of Ships' Ballast Water and Sediments (BWM Convention). We examined the abundance and diversity of bloom forming species in BW under the management of Regulation D-1 Ballast Water Exchange Standard and D-2 Ballast Water Performance Standard. The abundance and richness of bloom forming species were also examined in relation to ballast water age. Our findings indicate the abundance and diversity of bloom forming species were significantly lower in BW under the management of D-2 standard than that under D-1 standard. The abundance and richness represent no statistically significant correlation with BW age (p = 0.76 and p = 0.43, respectively). Some resistant species persist in ballast water. Thereby, we further provide some advice to overcome the existing challenges for the implementation of the Regulation D-2.

Electrokinetic motion and viability assessment of algae with a polyethylene glycol-dextran interface.

At present, there is still limited report on the electrokinetic (EK) behavior of bioparticles at the interface of an aqueous two-phase system. In this paper, the EK motion and viability assessment of live algae mixed with the NaClO treated dead algae were carried out at the interface formed by polyethylene glycol (PEG)-rich phase and dextran (DEX)-rich phase in a straight microchannel. The experimental results show that both the live and dead algae at the PEG-DEX interface migrate from the negative electrode to the positive electrode, and the EK velocity of live algae at the interface is slightly larger than that of the dead ones with similar diameters. For either live or dead algae, the EK velocity at the interface decreases with the increase in diameter. A size-velocity curve was used to evaluate the viability of the algae. As most of the microorganisms in ballast water are algae, the method in this paper provides a promising way to detect and evaluate the live microorganism in treated ballast water of a ship.

The dispersal of dinoflagellate cyst caused by international ships under repair conditions: a potential invasion risk to the Yangtze River Estuary, China.

The foreign dinoflagellate cysts in ships' ballast water tank sediments (BWTS) can be viable for a long time under hostile storage conditions. It is crucial to understand the detailed mechanisms of harmful biological invasions in estuary ecosystems. To study the relationship between the abundance of dinoflagellate cysts and environmental factors, cyst assemblages were analyzed in 7 sediment samples, collected from one international commercial ship that arrived in Shanghai in August 2020. Twenty-three dinoflagellate cyst taxa were identified in 5 groups, including autotrophic (9) and heterotrophic (14) species. Distribution of dinoflagellate cysts in the different ballast water tanks is heterogeneous. Dinoflagellate cysts in BWTS of the repaired ship were dominated by Scrippsiella acuminata, Protoperidinium leonis, Protoperidinium oblongum, Lingulodinium polyedra, Alexandrium tamarense/A. catenella, Protoperidinium pentagonum, and Protoperidinium subinerme. The abundance of the dinoflagellate cysts in each tank ranged from 80.69 to 330.85 cysts g-1 DS (dry sediment). Multivariate statistical analyses revealed that the variation in cysts from different tanks had positive correlations with total nitrogen (TN), total phosphorus (TP), and pH and negative correlations with total organic carbon (TOC) except for sample TK5. For germination of dinoflagellate cysts in BWTS, 12 species were germinated in 40 days and cysts of potentially toxic dinoflagellate species were more abundant than those of non-toxic species. Results show that potentially viable and harmful/toxic dinoflagellate cysts are present in BWTS of ships arriving in Shanghai, China. Consequently, knowledge obtained in this study can be valuable for further managing of potential biological invasion of the Yangtze River Estuary.

Examining the performance of three ballast water compliance monitoring devices for quantifying live organisms in both regulated size classes (≥50 µm and ≥10-<50 µm).

A number of ballast water compliance monitoring devices (CMDs) have been made commercially available to verify the efficacy of ballast water management systems by quantifying the living organisms for both plankton size classes (≥50 µm and ≥10-<50 µm). This study aimed to examine whether new CMDs can provide a reliable indication of compliance regarding Regulation D-2 and to evaluate their performance for indicative analysis of organisms by assessing their accuracy (comparison to microscopy) and precision (comparison within measurement). Challenge fresh water samples were collected in four locations of Lake Ontario, Canada, whereas marine challenge water samples were collected around the Bay of Fundy, New Brunswick, Canada. Ballast water samples were collected from ships visiting several ports across Canada. Overall, accuracy was higher (>80%) in estimating organisms from prepared-challenge water (Ballast Eye and BallastWISE) than from ballast water samples (>70%) (B-QUA only). The sensitivity ranged from 50 to 100% for the ≥50 µm organism size class, whereas for the ≥10-<50 µm organism size class, it was higher for freshwater samples (>75%) than for marine samples (>50%). The performance of CMDs should be assessed under real-world conditions for a better understanding and to improve their use.

A port statistics-based generic ballast water estimation and risk assessment approach and its application to Chinese ports.

The Ballast Water Management Convention now enters the experience-building stage, but developing countries have no adequate ballast water information yet. Against this background, considering the richness and generality of common port statistics, we develop a new generic approach to estimate discharge volumes and assess associated risks. This is one of the few efficient and feasible ways for port authorities to manage real discharged ballast water. Discharge volumes during 2017-2020 and risks in 2017 are analyzed for bulker and tanker. Results show that: (1) Ports in Yangtze River Delta and Bohai Rim received most ballast water, especially Ningbo-Zhoushan port as high-risk region receiving about 65 million tons per year; (2) With a higher ratio of discharge volumes to cargo throughputs, the tanker tends to release more ballast water than the bulker; (3) Chinese ports suffer more than 0.9 of the probability of non-indigenous species introduction. All these findings help implement convention globally.

A temporal assessment of risk of non-indigenous species introduction by ballast water to Canadian coastal waters based on environmental similarity.

The environmental similarity scores between source and recipient locations are essential in ballast water risk assessment (BWRA) models used to estimate the potential for non-indigenous species (NIS) introduction, survival, and establishment, and to guide management strategies aiming to minimize biodiversity loss and economic impacts. Previous BWRA models incorporate annual-scale environmental data, which may overlook seasonal variability. In this study, temporal variation in sea surface temperature and salinity data were examined at global ports, and the influence of this variation on environmental distance calculations (and corresponding risk of NIS) was examined for ballast water discharges in Canada by comparing outputs from monthly and annual scale assessments in a BWRA model. Except for some outliers in the Pacific region, the environmental distances based on monthly scale data generally become smaller in all regions, demonstrating that the model using annual decadal average environmental data to inform environmental matching can underestimate risk of NIS survival and establishment in comparison to monthly data. The results of this study suggest future evaluations incorporating the date of ballast water uptake and discharge can provide a more sensitive assessment of risk reflecting seasonal variability compared to an annual average risk model.

The hidden diversity of microbes in ballast water and sediments revealed by metagenomic sequencing.

With the rapid globalization of trade, the worldwide spread of pathogens through ballast water is becoming a major concern. Although the international maritime organization (IMO) convention has been adopted to prevent the spread of harmful pathogens, the limited species resolution of the current microbe-monitoring methods challenged the ballast water and sediments management (BWSM). In this study, we explored metagenomic sequencing to investigate the species composition of microbial communities in four international vessels for BWSM. Our results showed the largest species diversity (14,403) in ballast water and sediments, including bacteria (11,710), eukaryotes (1007), archaea (829), and viruses (790). A total of 129 phyla were detected, among which the Proteobacteria, followed by Bacteroidetes, and Actinobacteria were the most abundant. Notably, 422 pathogens that are potentially harmful to marine environments and aquaculture were identified. The co-occurrence network analysis showed that most of these pathogens were positively correlated with the commonly used indicator bacteria Vibrio cholerae, Escherichia coli, and intestinal Enterococci species, validating the D-2 standard in BWSM. The functional profile showed prominent pathways of methane and sulfur metabolism, indicating that the microbial community in the severe tank environment still utilizes the energy to sustain such a high level of microbe diversity. In conclusion, metagenomic sequencing provides novel information for BWSM.