The Impact of Microorganisms Transported in Ships’ Ballast Water on the Fish of the Estuarine Waters and Environmental Sustainability in the Southern Baltic Sea

: Ballast water represents a significant vector for the transfer of aquatic organisms and chemical pollutants. Although various groups of transported microorganisms can have a negative impact on native species of aquatic fauna, the available literature usually focuses on larger organisms. This is important because microorganisms cause changes in the balanced aquatic environment, including a stable trophic pyramid. The objectives of this study were twofold: (i) to determine the seasonal changes in the microbiota of the ballast water of long-and short-range ships entering the southern Baltic port, with a focus on fish pathogenic microorganisms and (ii) to potentially assess the threat to the ichthyofauna caused by the introduction of these microorganisms into the


Introduction
The maritime transport sector serves as a significant conduit for the dissemination of aquatic organisms, exerting notable impacts on port facilities and aquatic ecosystems and causing changes in environmental sustainability [1].Ballast water, in particular, is implicated in the introduction of approximately one-third of the world's alien species [2].Given that ballast water exchange predominantly transpires in coastal areas, its repercussions extend to various water-related industries, such as aquaculture [2,3].
Ballast water constitutes a distinct habitat whose environmental conditions shape the composition of a unique microbiota [4].Firstly, subjecting it to ballast water treatment processes and associated byproduct formation facilitates the acclimatization and survival of microorganisms until the next tank exchange in new areas [5].Microbial communities exhibit varying nutritional preferences, with some adept at utilizing carbon compounds while others rely more on carbohydrates, carboxylic acids, hydrocarbons, or amino acids [5,6].Shipboard ballast water treatment systems modify the chemical composition and availability of organic matter crucial for the proliferation of microorganisms inhabiting ballast tanks.Typically, the elevated concentration of organic matter and metabolites fosters the growth of specialized bacterial groups [7,8], particularly saprophytic, opportunistic, and pathogenic strains [8][9][10].Particles suspended in ballast water and organic debris often settle at the tank bottom, where they amalgamate with older sediments during vessel transit, forming a microbial substrate [7,8].Sediments laden with microbial aggregates undergo partial removal during ballast water exchange, potentially posing ecological hazards upon entry into recipient areas, as indicated by Maglić et al. [11], Shang et al. [12], and Lin et al. [13].
Seasonal variations in the chemical composition of ballast water are also influenced by sunlight exposure, which varies depending on the season and the duration of water retention in the ballast tank [7,8].Although seasonal temperature variations will impact the diversity of microorganisms in ballast waters, the documented ability of microbiota to withstand a wide range of temperatures is well established [6].Despite temperature fluctuations potentially impacting the abundance of transported bacteria, they can transform into spore forms, enabling survival during transport to distant regions [6].The adaptability of microbiota to diverse environments underscores the imperative for global monitoring of microorganisms conveyed in ballast water.
Various ballast water treatment (BWT) systems installed on ships interact with environmental factors, affecting the abundance and diversity of ballast biota [14][15][16].The intensity of this impact largely depends on the specific measures implemented.Different results can be achieved depending on whether a single BWT system (such as filtration, chlorination, or UV radiation) is used, if BWT systems are combined with ballast water exchange (BWE), or if water exchange is conducted only in specific locations [14][15][16].
Briski et al. [14] highlighted that mid-ocean ballast water exchange aims to reduce the risk of introducing organisms to freshwater areas by using osmotic shock associated with different salinity [14,16].Ideally, oceanic species should not survive in freshwater, and freshwater organisms should not survive in saline waters.However, in that study, some euryhaline and marine species were still found alive in freshwater port waters [14].In addition, this type of water exchange is ineffective for ships on short-range, coastal voyages.Finally, its success depends on various factors such as tank design, crew experience, and weather conditions [15].
We should also mention mixed methods of ballast water management, which combine BWE and various BWT technologies [14][15][16].Despite their limitations, these methods are valuable tools for preventing and reducing the introduction of non-native species to new areas [15].Hybrid strategies offer the greatest reduction in ballast biota and, consequently, the highest level of environmental protection [14][15][16].
The effectiveness of BWT systems varies depending on the number of processes they involve [14].Two-step processes, such as filtration followed by chlorination, UV radiation, or ozonation, yield better results and significantly reduce the concentration of ballast biota compared to single-step treatments (e.g., using only radiation, ozonation, or chlorination) [14][15][16].Additionally, the efficiency of these systems depends on environmental factors (ionic composition, turbidity, and temperature), which influence the ability of ballast biota to tolerate the environmental conditions and their assimilation in ballast tanks [14].
It is noteworthy that pathogenic bacteria such as Vibrio cholerae and indicator bacteria like Escherichia coli are also transported within ballast water [8,10,17,18].The unregulated movement of microorganisms, including pathogens, raises concerns not only due to their significant impact on marine ecosystems but also due to the exacerbation of antibiotic resistance among such microbiota, posing substantial threats to human and animal health [19][20][21][22].
The International Maritime Organization (IMO) introduced the International Convention for the Control and Management of Ballast Water and Sediments from Ships (BWM Convention), advocating for the monitoring of pathogen presence in ballast water using three bacteria-E.coli, Enterococcus faecalis, and V. cholerae-as indicator species [23].However, numerous scientific studies have indicated the colonization of ballast water by other pathogenic species, including Pseudomonas pseudoalcaligenes, Enterococcus hirae, Stenomorphomonas maltophilia, Shigella sonnei, and Bacillus anthracis, which are pathogenic to fish and humans [21,24,25].Several studies failed to demonstrate a strong correlation between the IMO-proposed indicator bacteria and potential pathogens introduced into aquatic environments [4,26,27].The indicator bacteria suggested by the IMO for ballast water monitoring lack reliability compared to the species (pathogenic or opportunistic) that are frequently reported in scientific literature.This underscores the necessity for further examination, not only of potential pathogen presence in ballast water and sediments but also of the associated ecological risks upon entry into recipient environments.
It is also important to note that the diversity and complexity of pathogenic microbiota in both sediment and ballast water are crucial aspects of monitoring the presence of pathogens in ballast water [8,28], especially as ballast water and its sediments represent two distinct types of microbiota habitats [4].
This research is important because obtained results will contribute to obtaining information on the possibility of disturbing the balanced, stable aquatic ecosystem of the Oder estuary.Therefore, the objectives of this study were twofold: (i) to determine the seasonal changes in the microbiota of the ballast water of long-and short-range ships entering the southern Baltic port, with a focus on fish pathogenic microorganisms and (ii) to potentially assess the threat to the ichthyofauna caused by the introduction of these microorganisms into the aquatic environment.

Sampling
The study utilized material in the form of ship ballast water samples obtained from twelve general cargo, bulk carrier, and cargo ships (the latter denoting merchant ships of an unknown type), with sizes between 89.95 m and 199.90 m, berthed at the Police Sea Port (Poland, Odra River estuary).The study was conducted at baseline for the local waters in the southern Baltic Sea (53 • 33 ′ 44.8 ′′ N 14 • 35 ′ 15.2 ′′ E).The vessels were divided into two groups based on their cruising regions: short-range (European routes) and long-range (transatlantic routes).Samples were collected in spring, summer, autumn, and winter (Table 1).
In order to ensure that the samples were representative, the sampling process was performed in accordance with the International Maritime Organization (IMO) International Guidelines for Ballast Water Sampling (IMO 2013).The samples were taken approximately 15 min after the ballast tanks were opened while the ship was docked.The process itself consisted of pumping water through the ballast water pump until suction was lost.Each sample was then transferred into 20 L tanks.Subsequently, in order to obtain a homogeneous mixture, each sample tank was secured and mixed further before being separated into smaller samples.The volume of each collected bulk sample was divided equally (into 2 L tanks) for each analysis that would be performed.Each sample was taken from ballast tanks (AFT Peek) with an average capacity of 500 tons.Each ship was equipped with a type-approved BWT system utilizing either filtration and electrochlorination (EC) or filtration and ultraviolet radiation (UV) (Table 1).None of the ballast water treatment systems were operational at the time of sampling.In preparation for loading, the ballast tanks were partially emptied.The ships were specifically selected because they had type-approved BWT systems installed.
Water samples from the ballast tanks in specific seasons were matched with port water samples taken from the Police Seaport area (southern Baltic estuary), which served as a baseline for local waters.Port water samples were collected from an area adjacent to the port quay, approximately 3 m from the quay.A polyethylene container mounted on metal holders, which allowed for water sampling at depths up to 1.5 m, was used to collect the samples.All port water samples were taken from three locations.These samples were combined into a single composite sample and transferred to a 20 L canister, identical to the one used for the ballast water samples.To ensure the quality and accuracy of the samples, all analyses were conducted within one hour of collection, shortly after safely transporting them at room temperature to the university laboratory.

Sequencing
As a result of the conducted macroscopic analysis, dominant colonies were selected for phylogenetic analysis of the microbiota using sequencing.Representatives of the most abundant colonies underwent a genotypic differentiation analysis.For this purpose, DNA was isolated according to the Genomic Mini AX protocol (A&A Biotechnology, Gdansk, Poland) using lysozyme, lysostaphin, and mutanolysin (Sigma-Aldrich, Saint Louis, MO, USA).Universal primers 338G (5 ′ -CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAGT-3 ′ ) and RP534 (5 ′ -ATT ACC GCG GCT GCT GG-3 ′ ) were used for DNA amplification [30].The PCR reaction was carried out in reaction mixture with a final volume of 25 µL consisting of 1.0 µL of MgCl 2 , 2.5 µL of dNTP, 2.0 µL of each primer, 1U Taq DNA polymerase (Eppendorf, Sigma-Aldrich, Saint Louis, MO, USA), 2.5 µL of polymerase buffer, and 3.0 µL of DNA template.The PCR reaction was performed in a thermocycler (Eppendorf, Hamburg, Germany) with the following thermal profile conditions: denaturation for 180 s at 94 • C; 35 cycles of 60 s at 94 • C, 30 s at 55 • C, and 60 s at 72 • C; and final elongation for 10 min at 72 • C [30].The amplified products were separated by electrophoresis, according to the generally accepted principles of the DGGE method, in an 8% polyacrylamide gel stained with ethidium bromide [31].The results of the electrophoretic separation were visualized under UV light using a GelDoc apparatus (Bio-Rad, Hercules, CA, USA).To conduct a similarity analysis for the obtained amplification profiles, the UPGMA method was used.

Statistical Analysis
In order to ascertain whether the distribution of the obtained results is normal and exhibits a constant variance, the test of homogeneity of variance and Levene's test were employed.Subsequent to this, an analysis of variance (ANOVA) was conducted, with the Tukey test serving as a post hoc test.The levels of similarity between the microbiota profiles of each sample were determined using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analysis with a Pearson distribution.The statistical analysis was conducted using the Statistica 13.3 software.

Results
The analysis of microbiota abundance identified statistically significant differences (p < 0.05) depending on the sampling period (Table 2).Generally, the highest microbial counts (1.970-2.55log cfu/mL) were observed in samples collected in autumn, while the lowest counts were found in samples from spring (1.15-1.29 log cfu/mL) and summer (1.16-1.29 log cfu/mL).In spring, the highest count was observed in a sample from a long-range vessel (L1; 1.29 log cfu/mL), whereas in the sample from a short-range vessel (S1), the count was lower (1.15 log cfu/mL) than in the control sample, which was port water (P1; 1.20 log cfu/mL).In summer, the highest overall microbial colonization was observed in a sample from a near-range vessel (S2; 1.29 log cfu/mL), while the least colonized sample was from a long-range vessel (L2; 1.16 log cfu/mL).The autumn sampling period was characterized by the highest overall microbial counts in port water (P3; 2.55 log cfu/mL), with slightly lower counts in some samples from near-range vessels (S3; 2.49 log cfu/mL) and long-range vessels (L3; 2.40 log cfu/mL).Conversely, the lowest counts were recorded in the second sample from long-range vessel L4 (1.97 log cfu/mL).In winter, the lowest overall microbial counts were recorded in samples from long-range vessel L6 (1.49 log cfu/mL) and near-range vessel S6 (1.49 log cfu/mL), which were lower than in port water (P4; 1.77 log cfu/mL).The highest counts were noted in samples from long-range vessel L5 (2.27 log cfu/mL) and near-range vessel S5 (2.25 log cfu/mL).The statistical significance of the differences observed in individual sampling periods had a p value < 0.05.a,b,c,d,e -statistical significance of individual groups of microorganisms between samples with p < 0.05; TAMC-total bacteria count; TYMC-total yeast count; TMMC-total mold count; TCPs-total Pseudomonas count; TCPsf-total Pseudomonas fluorescens count; THC-total halophilic bacteria count; TLC-total lipolytic bacteria count; TAC-total amylolytic bacteria count; TPC-total proteolytic bacteria count.
The analysis of the overall microbial count in the tested ballast water samples revealed that Pseudomonas fluorescens bacteria (TPCPsf) and halophilic bacteria (THC) were significantly less abundant (p < 0.05) than the other analyzed microorganisms.The highest mold counts (TMMC) were observed in autumn samples S4 and L4, while no presence of mold was noted in samples P1, P2, P4, S1, S2, S6, L1, L2, and L6.The highest yeast presence (TYMC) was observed in spring sample S1 and summer sample S2, while no presence was noted in autumn sample S4 and winter samples S6, L5, and L6.Lipolytic bacteria (TLC) were found in the highest numbers in samples P3, S1, S5, and L4, and the lowest in sample L6.Proteolytic bacteria were most abundant in samples S1 and L1, while they were least abundant in samples P3 and L4.The presence of proteolytic bacteria was not noted in the winter sample S5.It was also determined that the quantitatively and qualitatively expressed proportions of isolated microorganisms from the samples depended on the origin of the tested samples and the time of their collection (Figure 1).These relationships were also confirmed by the cluster analysis results.All analyzed clusters exhibited high intra-group similarity while maintaining a high species diversity between samples (Figure 2).
The conducted phylogenetic analysis revealed significant genetic diversity across all the examined samples.Beyond the microorganisms quantitatively assessed in the samples, additional species were identified, and species diversity was found to be influenced by both the sampling period and origin.Port waters during the spring-summer period primarily harbored Bacillus bacteria, accompanied by Intestinimonas, Oceanobacillus, and Virgibacillus bacteria.Spring samples of ballast water also exhibited distinctive species compositions.The samples from near-range vessels were predominantly inhabited by Bordetella bacteria, alongside Oligella, Brackiella, Basilea, and Derxia.In contrast, the spring sample from a long-range vessel displayed the highest microbiota biodiversity, predominantly featuring Acholeplasma bacteria, alongside Clostridium, Bacillus, Peptosteptococcus, Intestinibacter, Terrisporobacter, Anaerobacillis, and Listeria bacteria.also confirmed by the cluster analysis results.All analyzed clusters exhibited high intragroup similarity while maintaining a high species diversity between samples (Figure 2).The conducted phylogenetic analysis revealed significant genetic diversity across all the examined samples.Beyond the microorganisms quantitatively assessed in the samples, additional species were identified, and species diversity was found to be influenced by both the sampling period and origin.Port waters during the spring-summer period primarily harbored Bacillus bacteria, accompanied by Intestinimonas, Oceanobacillus, and Virgibacillus bacteria.Spring samples of ballast water also exhibited distinctive species compositions.The samples from near-range vessels were predominantly inhabited by Bordetella bacteria, alongside Oligella, Brackiella, Basilea, and Derxia.In contrast, the spring sample from a long-range vessel displayed the highest microbiota biodiversity, predominantly featuring Acholeplasma bacteria, alongside Clostridium, Bacillus, Peptosteptococcus, Intestinibacter, Terrisporobacter, Anaerobacillis, and Listeria bacteria.
Ballast waters collected in the summer were predominantly colonized by Clostridium bacteria, with a near-range vessel sample also showing the presence of Desnuesuella and Oxobacter bacteria.Conversely, the analysis of species diversity in the summer sample from a long-range vessel also indicated the presence of Anaerofustis and Oxobacter bacteria.Representatives of the genera Aspergillus, Cladosporium, Mucor, and Penicillium were identified consistently across all analyzed autumn-winter samples.Based on the conducted microbiological and phylogenetic analysis, it was established that each sample exhibited species diversity.This diversity depended on the sampling period (spring, summer, autumn, or winter) and the range of vessel navigation.In port water samples from the spring-summer period, while maintaining a 74-76% quantitativequalitative diversity similarity profile (Figure 2a), the prevalence of Bacillus bacteria was noted.The spring sample of ballast water from a short-range vessel (Figure 2b) was pre- Based on the conducted microbiological and phylogenetic analysis, it was established that each sample exhibited species diversity.This diversity depended on the sampling period (spring, summer, autumn, or winter) and the range of vessel navigation.In port water samples from the spring-summer period, while maintaining a 74-76% quantitativequalitative diversity similarity profile (Figure 2a), the prevalence of Bacillus bacteria was noted.The spring sample of ballast water from a short-range vessel (Figure 2b) was predominantly characterized by the presence of Bordetella bacteria (84-89% diversity according to UPGMA cluster analysis).The highest microbial biodiversity in ballast water was exhibited by the spring sample from a long-range vessel, mainly represented by Acholeplasma bacteria, with a quantitative-qualitative diversity similarity ranging from 75 to 77% (Figure 2d).Summer water samples were predominantly colonized by Clostridium bacteria, with a quantitative-qualitative diversity similarity ranging 78-84% for the short-range vessel sample (Figure 2c) and 80-90% for the long-range vessel sample (Figure 2e).Conversely, all analyzed autumn-winter samples were found to contain representatives of the genera Aspergillus, Cladosporium, Mucor, and Penicillium.Based on the quantitative-qualitative diversity similarity profiles (Figure 2f) of these samples, it was observed that all analyzed clusters exhibited high intra-group similarity while maintaining high species diversity between samples.

Discussion
The increase in global maritime transport and the potential for invasive species to be carried in ballast water have led to ballast water being recognized as one of the greatest threats to the health of aquatic ecosystems [1,10,18,19].Compared to higher organisms, the abundance of microbiota transported in ballast water is significantly greater [10].Although our results indicate a much lower number of microorganisms isolated from ballast water than those reported in the literature [10,18,19,24,28], their adaptive capabilities in new environments still make them particularly dangerous for local hydrobionts and entire ecosystems [32,33].
The aforementioned facts show the significance of the proper management of ballast water and its sediments.Despite its limitations, this management remains a valuable tool for reducing and preventing the transfer of alien species to new environments [16].
Our results do not show a significant difference in microbiota abundance between port and ballast waters, but extensive research has emphasized that the rational combined use of ballast water exchange (BWE) and ballast water treatment systems (BWTSs) significantly reduces the microbiota transported by ships [14][15][16].Briski et al. [14] indicated that midocean ballast water exchange, when conducted according to regulations, should protect aquatic ecosystems through osmotic shock, especially for transoceanic ships docking at ports that connect marine and freshwater ecosystems [14,15].Unfortunately, this does not apply to ships operating over short distances in waters with a similar salinity.
Using BWTSs alone, where ballast tanks are filled with freshwater port waters, does not yield significant results [14].A different situation arises when BWE is combined with a shipboard BWTS [14][15][16].This is particularly noticeable in studies on changes in the abundance and species diversity of zooplankton and phytoplankton in ballast waters [14,15].Bailey [15] in her analyses pointed out that physical water exchange significantly reduces the risk of freshwater invertebrate invasions.However, she considers it a temporary and short-term solution whose effectiveness depends on tank construction, crew experience, skills, and weather conditions [15].
To sum up, using BWT systems alone do not fully clean or treat ballast water, and the combination of BWE and various BWTS technologies is much more effective [14][15][16].Hybrid strategies provide the greatest reduction in ballast biota and, consequently, the highest level of environmental protection [14][15][16].
The effectiveness of BWT systems varies depending on the number of processes involved [14].Using two-step processes for ballast water management, such as filtration followed by chlorination, UV radiation, or ozonation, yields better results and significantly reduces the concentration of plankton and microorganisms in ballast tanks compared to single-step treatment processes (e.g., using only radiation, ozonation, or chlorination) [14][15][16].
Our study also found that the season of sample collection and the route of the ships impact the variability of ballast microbiota.Microorganism abundance changes mainly with the season, possibly due to biological factors such as temperature, general weather conditions, and the microbiota's adaptive capabilities for the prevailing conditions.Regardless of the fact that ballast water treatment systems were installed on the ships from which the samples came, the autumn and winter samples were characterized by a significantly higher microbiota count.Comparing the obtained results, it was noticeable that the differences between the number of microorganisms in port waters and samples from ships were not large.Significant differences were visible in the case of individual groups of microbiotas, such as yeasts and molds.Our results showed that ballast water samples collected in spring and summer had the highest species richness for the microbiota, while autumn and winter samples had the lowest.
Additionally, the region where ships operate significantly affects the abundance and diversity of ballast microbiota.Although our results showed only minor differences, with comparable overall microbiota numbers, there was generally a lower abundance of microbiota in samples from long-range ships compared to short-range ships.Bailey's study also indicated this same trend, suggesting that it might be due to weaker adaptive capabilities and high mortality among the studied plankton [15].She particularly noted the presence of smaller soft-bodied plankton species, such as Copepoda, Rotifera, and mollusks, which selectively survived the ballast water treatment process, slowing the settlement rate of new species in the tanks.Similar phenomena were noted for phytoplankton [15].
The presence of diverse microbiota in the analyzed samples of ballast and port waters, particularly species pathogenic for fish, raises concerns that it may not only antagonistically affect the native environmental microbiota but also pose a threat to the health of both humans and fish [8,19,25].This threat becomes more significant as the likelihood of disrupting the biological balance increases, even at the lowest levels of the aquatic food chain [9,10].The introduced microbial species occupy areas previously inhabited by native microbiota, potentially leading to the disruption of symbiotic interactions through the displacement of symbiotic microbiota [12,13].This scenario is particularly worrisome when opportunistic bacteria may take the place of symbionts [9][10][11][12][13].
The microbiota identified in this study's ballast waters primarily consisted of pathogenic species, such as Pseudomonas, Bordetella, Bacillus, and Clostridium, as confirmed by the phylogenetic analysis.Under favorable conditions, these organisms can lead to various fish diseases, presenting a significant challenge for fisheries and aquaculture [34,35].This highlights the intricate relationship between environmental conditions, pathogens, and hosts, stressing the necessity for multidisciplinary research on potential fish pathogens and a deeper understanding of global environmental factors influencing fish health [36].Notably, the observed microorganisms belonging to the Proteobacteria group were predomi-nantly represented by Pseudomonas bacteria.These bacteria, commonly detected in ballast waters, are pathogenic factors for fish, resulting in chronic or acute symptoms [37,38].Pseudomonas bacteria were present in nearly all the analyzed samples, which bears epidemiological significance.Particularly noteworthy is P. fluorescens, identified in 75% of all the analyzed samples, which can cause infections in carp, salmon, trout, pike, perch, and catfish, manifesting as erythema, septicemia, and even mortality [37].Diseases caused by P. fluorescens often manifest as changes in gills, such as edema, increased mucus secretion, pale discoloration of thickened gill filaments, and necrotic changes [37,38].Higher temperatures lead to the rapid replacement of pathogenic Pseudomonas bacteria by competitive Aeromonas bacteria, which are recognized as the most common pathogens in both freshwater and marine fish [39].Infections caused by Aeromonas bacteria are associated with skin ulcers, hemorrhages, hemorrhagic enteritis, red blood cell disease, septicemia, and epizootic ulcerative syndrome.When introduced into freshwater, these bacteria pose a threat to aquaculture in the breeding of catfish, tilapia, carp, and eels [40][41][42][43].
In recent years, significant changes have been observed in bacterial fish diseases, where some Gram-negative pathogens are being replaced by species that are potentially neutral or with unknown pathogenic potential for ichthyofauna [38].Among these are bacteria from the genus Bordetella (also detected in our study), which often accompany other opportunistic microorganisms contributing to fish diseases [44].One representative of this genus, B. petrii, isolated from sediments of the analyzed ballast waters, is frequently found in various ecosystems, including aquatic ones, with the ability to degrade many organic substances, including aromatic compounds [45].Moreover, it occasionally expands its metabolic capabilities, contributing to respiratory infections in humans and aquatic organisms [46].B. petrii has been isolated from patients with cystic fibrosis, indicating its potential pathogenicity [46].Its presence in marine organisms such as sponges and seagrass roots further underscores its ecological impact [47,48].The metabolic versatility of B. petrii suggests its involvement in various environmental processes, necessitating a deeper understanding of its versatility at the genomic level.In particular, Bordetella bacteria are phylogenetically linked to other opportunistic pathogens such as Achromobacter and Alcaligenes [44].It is worth noting that in some fish, B. petrii may contribute to the development of the intestinal inflammation caused by the widely distributed waterborne Acetinobacter sp., ultimately leading to mortality.Infections caused by opportunistic bacteria, often accompanying Bordetella spp., are characterized by petechiae on the body surface, scale loss, fin fraying, gill congestion, and eye protrusion in trout and carp [37,38].
Mollicutes, represented by Acholeplasma sp.bacteria isolated from our ballast water samples, are frequently isolated from diseased fish.These mycoplasma-like microorganisms commonly inhabit fish gastrointestinal tracts, but their presence in other organs can lead to pathomorphological changes [48].Moreover, these opportunistic bacteria are considered fish parasites [49].The symptoms associated with their presence include inflammatory reddening and necrotic changes in external layers, and petechiae in the peritoneum, muscles, and sometimes in internal organs [50].Infections caused by this group of microorganisms can result in multisystemic infections, leading to necrotic changes preceded by extensive lymphocyte loss and hemorrhage.Fish infected by Mollecutes spp.show damage to the spleen, liver, pancreas, swim bladder, and gills with hyperplasia in subepithelial layers [50].Moreover, under favorable environmental conditions and stress factors, the presence of mycoplasmas with other opportunistic bacteria may facilitate the development of fish diseases [50].
Due to the significant interdependencies between the impact of microorganisms, particularly pathogenic and opportunistic ones, on the environment and fish health, monitoring of the structure of microorganisms inhabiting ballast waters is of utmost importance.Monitoring the colonization of ballast waters using microorganisms will facilitate the assessment of the impact of ballast microbiota on specific areas and the human activities associated with them (such as fishing and aquaculture).Additionally, microbial monitoring of ballast water is a vital component of assessing the state of waters and their dependent ecosystems, as well as creating maps of potential routes for the spread of fish diseases facilitated by the appearance of opportunistic microorganisms.

Conclusions
Despite the widespread implementation of ballast water treatment systems on ships, the risk of transferring microorganisms to new aquatic environments persists.These microorganisms frequently demonstrate extensive adaptive capabilities and can outcompete indigenous microbiota in the colonized areas.This may cause changes in the trophic pyramid and disruptions in environmental sustainability.This phenomenon is influenced by interactions between fish and saprophytic bacteria that inhabit their digestive tracts, gills, and skin.The introduction of alien microbiota often disrupts fish immune systems, contributing to various diseases.However, our current understanding of the intricate mechanisms by which certain bacteria become pathogenic in ballast waters and their role in inducing immune disturbances in fish remains limited.Hence, it seems that research on the transmission of fish pathogenic microorganisms is crucial for comprehending the changes currently observed in aquatic ecosystems and stopping changes in environmental sustainability.

Figure 2 .
Figure 2. Dendrogram of the cluster analysis of the similarity of the microbiological profiles of the analyzed samples based on phylogenetic analysis of microbiota [%]; (a) port waters of the springsummer period; ballast water of a short-range ship (b) in spring and (c) in summer; ballast water of a long-range ship (d) in spring, (e) in summer, and (f) in autumn and winter.

Figure 2 .
Figure 2. Dendrogram of the cluster analysis of the similarity of the microbiological profiles of the analyzed samples based on phylogenetic analysis of microbiota [%]; (a) port waters of the springsummer period; ballast water of a short-range ship (b) in spring and (c) in summer; ballast water of a long-range ship (d) in spring, (e) in summer, and (f) in autumn and winter.

Table 1 .
Sampling periods and terminology of the ships with installed BWT systems using ultraviolet radiation (UV) and electrochlorination (EC) used in the work.

Table 2 .
Quantitative diversity of microorganisms isolated from water samples.