The effect of sampling frequency and spatial and temporal variation in the density of fecal indicator bacteria on the assessment of coastal bathing water quality.

The time and location of sampling as well as the number of samples per season can influence a reliable assessment of bathing water quality. In this study, we investigated the spatio-temporal variation of fecal indicator bacteria (FIB) density and the effects of FIB variability and sampling frequency on the assessment of a single sample and the annual and final assessment of coastal bathing water quality. Increasing the number of samples from 10 to 20 per bathing season had a significant impact on bathing water quality assessment at sites where water quality fluctuations had previously been observed, resulting in a change in water quality to a lower category in 36 % of annual and 54 % of final assessments, suggesting that the minimum number of samples per season should be increased at such sites. Increasing the number of samples at sites assessed as excellent over a longer period had no impact on water quality assessment. Spatial and temporal variations in FIB density were significant at a considerable number of sites both in the single sample assessment and in the annual and final assessment. Bathing water quality was spatially unrepresentative at a quarter of the sites analyzed and temporally unrepresentative at a fifth, as there was at least one additional point with a lower bathing water quality than the official sampling point or the bathing water quality was lower in the afternoon than in the morning. When revising the current recreational water quality regulations, the impact of sampling frequency of and of spatio-temporal variation of FIB density on the relevance of bathing water quality assessment at sites subjected to pollution needs to be seriously considered.

Acetylcholinesterase activity in muscle tissue of Norway lobster Nephrops norvegicus: Importance of body size, season, sex and naturally occurring metals.

The aim of the present study was to determine the levels of acetylcholinesterase (AChE) activity in the tail muscle tissue of wild populations of Nephrops norvegicus from the Northern Adriatic, and correlate it to body size, seasons, sex and the content of mercury, arsenic, cadmium, lead and copper. The animals of both sexes were collected in spring and autumn from two relatively distant fishing grounds. A marked variability of muscle AChE activity was found (0.49 to 11.22 nmol/min/mg prot.), displaying the opposite seasonal trend between two sampling sites. Small, but significant negative correlation has been found between AChE activity and carapace length (rs = - 0.35, p < 0.05). Data reported here provide an essential baseline for future studies of neurotoxicity in crustaceans. The study highlights the necessity for continuous monitoring of potentially toxic metals in edible marine species to avoid possible repercussions of seafood consumption on human health.

Arsenic (As), copper (Cu), zinc (Zn) and selenium (Se) in northwest Croatian seafood: A health risks assessment.

This study focuses on the health risk assessment of arsenic (As), copper (Cu), zinc (Zn), and selenium (Se) concentrations in seafood species commonly consumed in the northwestern region of Croatia. By measuring the concentrations of these elements coupled with data on seafood consumption, the health risks were evaluated using Target Hazard Quotients and Hazard Indexes. The results indicate a slightly increased health risks linked to seafood consumption for As, Cu and Zn in some of the tested seafood species. The findings of this study highlight the critical need for stronger food quality control measures, especially targeting certain types of seafood.

Assessment of health risks associated with heavy metal concentration in seafood from North-Western Croatia.

The following study aims at assessing the health risks associated with the consumption of the most commonly consumed seafood in the north-western part of Croatia due to the presence of heavy metals. Samples of seafood were collected and analysed for lead (Pb), cadmium (Cd), and mercury (Hg) content. Analyses of Cd and Pb were carried out by inductively coupled plasma mass spectrometry (ICP-MS) whereas Hg content was measured using atomic absorption spectrometry (AAS). Metal concentrations were in the following order Hg > Pb > Cd for the gilthead seabream, European hake, sardines, and tuna fish whereas in the Patagonian squid cadmium (Cd) was the heavy metal with the highest concentration, with the order of other metals being Cd > Hg > Pb. The heavy metal concentrations have been used to address the health risks using the Estimated Weekly Intake (EWI), Target Hazard Quotients (THQ), and Hazard Indices (HI). The findings revealed that the concentrations of the tested heavy metals, expressed on a per wet weight basis, did not exceed the Maximum Residue Levels (MRL) for those compounds mandated by national Croatian legislation. However, the HI for Hg was above 1, indicating a risk of adverse health effects due to the presence of this heavy metal in the consumed seafood. We conclude that the consumption of certain type of seafood such as the tuna fish should be limited when sensitive segments of the population such as children, elderly and pregnant women are concerned. Our results strongly advocate for a more stringent seafood quality control in the region.

Photodynamic inactivation of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents.

There is an increasing need to discover effective methods for treating municipal wastewater and addressing the threat of multidrug-resistant (MDR) strains of bacteria spreading into the environment and drinking water. Photodynamic inactivation (PDI) that combines a photosensitiser and light in the presence of oxygen to generate singlet oxygen and other reactive species, which in turn react with a range of biomolecules, including the oxidation of bacterial genetic material, may be a way to stop the spread of antibiotic-resistant genes. The effect of 5,10,15,20-(pyridinium-3-yl)porphyrin tetrachloride (TMPyP3) without light, and after activation with violet-blue light (VBL) (394 nm; 20 mW/cm2), on MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and K. pneumoniae OXA-48 in tap water and municipal wastewater was investigated. High toxicity (~2 µM) of TMPyP3 was shown in the dark on both strains of K. pneumoniae in tap water, while on P. aeruginosa toxicity in the dark was low (50 µM) and the PDI effect was significant (1.562 µM). However, in wastewater, the toxicity of TMPyP3 without photoactivation was much lower (12.5-100 µM), and the PDI effect was significant for all three bacterial strains, already after 10 min of irradiation with VBL (1.562-6.25 µM). In the same concentrations, or even lower, an anti-adhesion effect was shown, suggesting the possibility of application in biofilm control. By studying the kinetics of photoinactivation, it was found that with 1,562 µM of TMPyP3 it is possible to achieve the complete destruction of all three bacteria after 60 min of irradiation with VBL. This study confirmed the importance of studying the impact of water constituents on the properties and PDI effect of the applied photosensitiser, as well as checking the sensitivity of targeted bacteria to light of a certain wavelength, in conditions as close as possible to those in the intended application, to adjust all parameters and perfect the method.

Surveillance of Legionella pneumophila: Detection in Public Swimming Pool Environment.

The bacterium Legionella pneumophila is a ubiquitous microorganism naturally present in water environments. The actual presence of this opportunistic premise plumbing pathogen in recreational swimming pools and hot tubs in the northwestern part of Croatia has not been investigated. This study aimed to analyze the presence of the opportunistic pathogen L. pneumophila in public swimming pool water in Primorje-Gorski Kotar County (N = 4587) over a four-year period (2018-2021). Additionally, the second aim was to investigate the connection between the presence of L. pneumophila and pool water physicochemical parameters using mathematical predictive models. The presence of L. pneumophila was detected in six pool samples. Five positive samples were found in the water of indoor hot tubs filled with fresh water, and one positive sample in an outdoor recreational saltwater pool. A predictive mathematical model showed the simultaneous influence of chemical parameters dominated by the temperature in saltwater and freshwater pools, as well as the significant influence of free residual chlorine and trihalomethanes. Our results pointed out that keeping all physicochemical parameters in perfect harmony is necessary to reach the best disinfection procedure and to avoid the optimum conditions for L. pneumophila occurrence.

Impacts of Atmospheric and Anthropogenic Factors on Microbiological Pollution of the Recreational Coastal Beaches Neighboring Shipping Ports.

A comparative study of the two northeastern ports of the Adriatic Sea indicated that the port of Rijeka is microbiologically more loaded than the port of Pula and posing a greater threat to other ports through a potential transfer of pathogens by ballast water. Fecal indicator bacteria, Escherichia coli and intestinal enterococci, were investigated seasonally in 2014-2015 in the ports and during the bathing season monitoring in the two bays where ports are located in 2009-2020. In addition, the indicators and pathogens related to human health were determined in the ports' seawater and sediment. The determined factors contributing to microbiological pollution were higher number of tourists and locals, potential wastewater and ballast water discharge and enclosed port configuration, with high solar radiation and low precipitation reducing the negative effects. Our research points to the necessity of including Clostridium perfringens in monitoring beach sand during the bathing seasons and a wider list of pathogens in port monitoring due to a potential transfer by shipping ballast water.

Decreasing Pasteurization Treatment Efficiency against Amoeba-Grown Legionella pneumophila-Recognized Public Health Risk Factor.

Legionellae are gram-negative bacteria most commonly found in freshwater ecosystems and purpose-built water systems. In humans, the bacterium causes Legionnaires' disease (LD) or a Pontiac fever. In this study, the different waters (drinking water, pool water, cooling towers) in which Legionella pneumophila has been isolated were studied to assess the possible risk of bacterial spreading in the population. The influence of physical and chemical parameters, and interactions with Acanthamoeba castellanii on L. pneumophila, were analyzed by Heterotrophic Plate Count, the Colony-forming units (CFU) methods, transmission electron microscopy (TEM), and Sequence-Based Typing (SBT) analysis. During the study period (2013-2019), a total of 1932 water samples were analyzed, with the average annual rate of Legionella-positive water samples of 8.9%, showing an increasing trend. The largest proportion of Legionella-positive samples was found in cooling towers and rehabilitation centers (33.9% and 33.3%, respectively). Among the isolates, L. pneumophila SGs 2-14 was the most commonly identified strain (76%). The survival of Legionella was enhanced in the samples with higher pH values, while higher electrical conductivity, nitrate, and free residual chlorine concentration significantly reduced the survival of Legionella. Our results show that growth in amoeba does not affect the allelic profile, phenotype, and morphology of the bacterium but environmental L. pneumophila becomes more resistant to pasteurization treatment.

Is a Proactive Approach to Controlling Legionella in the Environment Justified?

RESEARCH BACKGROUND: Legionella are Gram-negative bacteria that are ubiquitous in the natural environment. Contaminated water in man-made water systems is a potential source of transmission of legionnaires' disease. The aim of this study is to explore the prevalence of Legionella pneumophila in the drinking water distribution system (DWDS) of Primorje-Gorski Kotar (PGK) County, Croatia, for the period 2013-2019, coupled with the incidence of legionnaires' disease. A number of L. pneumophila-positive samples (>100 CFU/L), serogroup distribution and the degree of contamination of specific facilities (health and aged care, tourism, and sports) were assessed. Based on the obtained results, the reasoning for the implementation of a mandatory Legionella environmental surveillance program was assessed. EXPERIMENTAL APPROACH: Sample testing for Legionella was carried out according to the standard method for enumeration of this bacterium. A heterotrophic plate count (HPC) and Pseudomonas aeruginosa number were analysed along with the basic physicochemical indicators of drinking water quality. The research period was divided into two parts, namely, the 2013-2018 period (before implementation of the prevention program, after the outbreak of legionnaires' disease), and the year 2019 (proactive approach, no disease cases recorded). RESULTS AND CONCLUSION: During the 7-year observation period in PGK County, an increase in the number of samples tested for Legionella was found. An increase in Legionella-positive samples (particularly pronounced during the warmer part of the year) was recorded, along with a growing trend in the number of reported legionnaires' disease cases. In addition to hot water systems, the risk of Legionella colonisation also applies to cold water systems. Health and aged care facilities appear to be at highest risk. In addition to the higher proportion of positive samples and a higher degree of microbiological load in these facilities, the highest proportion of L. pneumophila SGs 2-14 was identified. Due to the diagnostic limitations of the applied tests, the number of legionnaires' disease cases is underdiagnosed. NOVELTY AND SCIENTIFIC CONTRIBUTION: The introduction of a mandatory preventive approach to monitoring Legionella in DWDS water samples, along with the definition of national criteria for the interpretation of the results will create the preconditions for diagnosis and adequate treatment of larger numbers of legionnaires' disease cases.

Staphylococcus aureus-An Additional Parameter of Bathing Water Quality for Crowded Urban Beaches.

During the last years, the report of the occurrence of waterborne disease symptoms related to non-enteric pathogens has increased, without any record of higher levels of indicator bacteria (Escherichia coli and intestinal enterococci). Therefore, the use of current indicators is not always adequate when assessing the overall potential health risk and the inclusion of additional parameters needs to be examined. This paper reports on the incidence and levels of Staphylococcus aureus at 258 locations in Primorje-Gorski Kotar County (Croatia) recorded by official bathing water quality monitoring, as well as supplemental monitoring carried out at the two most frequented beaches in the City of Rijeka. The number of bathers was found to be the main factor affecting S. aureus levels (r = 0.321, p < 0.05). The share of S. aureus positive samples from the official monitoring was significantly lower, when compared to the share of samples from supplemental monitoring (2.2% and 36.3%, respectively; p < 0.01). Besides the number of bathers, one of the main reasons is likely the higher sampling frequency. No correlation was found between S. aureus levels and the indicator bacteria. The results indicate that the determination of S. aureus and increased sampling frequency is recommended for overcrowded beaches.

Study of nitrogen pollution in the Republic of North Macedonia by moss biomonitoring and Kjeldahl method.

In the summer of 2005 and 2010, moss samples were collected from 72 sampling sites evenly distributed all over the territory of Republic of North Macedonia. Kjeldahl method was used to determine the nitrogen content in the samples. Descriptive statistics and distribution maps were prepared. Data obtained from these two surveys were compared, and additional comparison was done with data obtained from similar studies in the South-Eastern European countries and Finland as a clean area. The median value of N content in the samples collected in 2005 is 1.21%, varies from 0.70% to 1.54%, while the content of N in samples collected in 2010 ranges between 0.68% and 1.75% with the median value of 1.06%. High contents of N were found in the Northern and Central parts of the country mainly as a result of agricultural activities, industry and traffic.

Status of faecal pollution in ports: A basin-wide investigation in the Adriatic Sea.

Ports are subject to a variety of anthropogenic impacts, and there is mounting evidence of faecal contamination through several routes. Yet, little is known about pollution in ports by faecal indicator bacteria (FIB). FIB spatio-temporal dynamics were assessed in 12 ports of the Adriatic Sea, a semi-enclosed basin under strong anthropogenic pressure, and their relationships with environmental variables were explored to gain insight into pollution sources. FIB were abundant in ports, often more so than in adjacent areas; their abundance patterns were related to salinity, oxygen, and nutrient levels. In addition, a molecular method, quantitative (q)PCR, was used to quantify FIB. qPCR enabled faster FIB determination and water quality monitoring that culture-based methods. These data provide robust baseline evidence of faecal contamination in ports and can be used to improve the management of routine port activities (dredging and ballast water exchange), having potential to spread pathogens in the sea.

A baseline study of the metallothioneins content in digestive gland of the Norway lobster Nephrops norvegicus from Northern Adriatic Sea: Body size, season, gender and metal specific variability.

Metallothioneins content was investigated in digestive gland of two wild-caught Norway lobster Nephrops norvegicus populations from the Northern Adriatic Sea, in relation to body size, season and gender. Concomitant accumulation of cadmium, mercury, arsenic, lead, chromium and manganese, reactive oxygen species concentration and energy reserves in digestive gland were also assessed. While differences between genders were not recorded, metallothioneins content seasonal trends were affected by body size. Most of parameters displayed inconsistent trends across sampling sites. Significant correlation between metallothioneins content and cadmium, arsenic and mercury concentrations was recorded only for larger lobsters. A negative correlation of reactive oxygen species concentration and metallothioneins content was observed for small, but not large lobsters. Energy reserves, in particular lipids, could considerably influence biochemical and chemical parameters variations. The present results constitute the essential baseline for future studies aimed at evaluating the N. norvegicus health in relation to metal contamination of coastal sediments.

Comparison between resident and caged mussels: Polycyclic aromatic hydrocarbon accumulation and biological response.

The aim of this study was to compare the capability of "passive" and "active" biomonitoring to determine the environmental pressure. For this purpose, PAHs content and several biological responses in resident and caged mussels (Mytilus galloprovincialis) at five sampling sites (Rijeka Bay, Adriatic Sea) were analsed. Resident mussels were found better in reflecting the level of PAH loads at particular sites while only caged mussels could detect input of HMW PAHs. When data of each investigated parameter were compared separately, the majority of differences between resident and caged mussels' results were site-specific. Integration of biological response patterns expressed as Index of Biological Response (IBR) resulted with different sampling sites ranking for resident and caged mussels. Multiple Factor Analysis (MFA) based on integration of tissue PAH concentration and biological response revealed resident mussels as more powerful for detection of environmental pressure. The use of resident mussels is recommended as appropriate and less costly approach for monitoring the effect of pollution.

Escherichia coli in marine water: Comparison of methods for the assessment of recreational bathing water samples.

Bathing Water Directive (2006/7/EC) specifies two reference methods for Escherichia coli detection: ISO 9308-1 and 9308-3. The revised ISO 9308-1 is recommended only for waters with a low bacterial background flora. Considering the extended time needed for analysis and, generally, the lack of experience in using ISO 9308-3 in the Mediterranean, the suitability of ISO 9308-1 for the examination of E. coli in bathing water was evaluated. The present study was aimed at a comparison of data obtained by the reference method in seawater samples (110 beaches, N=477) with data received from six alternative methods. Results show that recently used TSA/TBA method may overestimate E. coli numbers in marine waters. The temperature modified ISO 9308-1 (44°C) did not significantly alter the results, but outperformed the antibiotic supplemented agar at reducing non-E. coli bacteria on the plates, allowing the use of the respective method for monitoring coastal water.

Study of nitrogen pollution in Croatia by moss biomonitoring and Kjeldahl method.

During the summer and autumn of 2006 moss samples were collected from 98 sampling sites evenly distributed all over the territory of Croatia. Moss sampling was repeated in 2010 when additional sampling sites were added and a total of 121 samples were collected. Kjeldahl method was used to determine the nitrogen content in the samples. Descriptive statistics and distribution maps were prepared. Data obtained from these two surveys were compared, and additional comparison was done with data obtained from similar studies in neighbouring countries and Finland as a clean area. The median value of N content in the samples collected in 2006 is 1.60% and varies from 0.79% to 3.16%. The content of N in samples collected in 2010 ranges between 0.71% and 2.93% with the median value of 1.49%. High contents of N (2.32% - 3.17%) were found in the regions of Slavonia, Podravina, Posavina and cities Zagreb and Sisak as a result of agricultural activities, industry and traffic.

Influence of cadmium on metallothionein expression and products of lipid peroxidation in the organs of hares (Lepus europaeus Pallas).

Cadmium occurs naturally in the environment and as an anthropogenic pollutant. Exposure to low concentrations of cadmium is inevitable and may produce toxic effects. Another important aspect of cadmium toxicity is its interaction, often antagonistic, with essential elements such as selenium. The aim of this study was to highlight the risks of long-term exposure to low cadmium concentrations, using a scientific and chemical approach and hares (Lepus europaeus Pallas) as model organisms in a field study. Two study areas were monitored. Levels of cadmium and selenium were quantified in the organs of hares, the expression of metallothioneins I + II and the products of lipid peroxidation were determined. The median cadmium concentrations (wet weight) in the muscle, liver, kidney and brain of hares from an exposed group ranged from 0.033 to 0.037, 0.763 to 1.054, 3.090 to 16.594 and 0.016 to 0.087 µg g(-1), respectively; whereas, the median selenium concentrations (wet weight) ranged from 0.100 to 0.108, 0.153 to 0.332, 0.677 to 0.701 and 0.078 to 0.116 µg g(-1), respectively. Expression of the metallothioneins I + II proteins was observed in tissues. Lipid peroxidation (LPO) levels, measured as malondialdehyde (MDA) equivalents, increased with the cadmium concentration. Further research on long-term exposure to low concentrations of cadmium in the environment is needed.

Evaluation of equivalence between different methods for enumeration of fecal indicator bacteria before and after adoption of the new Bathing Water Directive and risk assessment of pollution.

The quality of bathing water is of considerable public importance due to the possibility of fecal contamination. In 2009, Croatia implemented the new European Bathing Water Directive (BWD, 2006/7/EC) establishing stricter microbiological standards for new parameters with new reference methods. This study aims to evaluate the equivalence of different methods according to the old and revised BWD and to provide the possibility of data comparison. Furthermore, the directive requires the establishment of the bathing water profile (BWP) for pollution risk assessment. The estimation of consistency of pollution risk assessment with obtained microbiological results was also performed. Six marine beaches of the Municipality of Rijeka (Croatia) were examined during the 2009 season. Statistical analysis showed equivalence between determination methods for fecal contamination indicators. Based on the current water classification results, the need for correction of estimated pollution risks and recommendations for inclusion of historical microbiological data during BWP enactment was noticed.

Effects of mercury on glutathione and glutathione-dependent enzymes in hares (Lepus europaeus Pallas).

The aim of this study was to analyze and evaluate risks of long-term exposure to mercury in hares (Lepus europaeus Pallas), with a chemical-analytical approach evaluating median mass fraction of toxic mercury in the hares organs (liver, kidney, muscle and brain). To obtain better insight into possible effects of mercury, the study included screening of the oxidative status after long term exposure to low concentrations of mercury. Hares organs were analyzed for total mercury concentration by AAS. Glutathione and glutathione-dependent enzymes status was also investigated. The median mercury concentrations (wet weight) in the liver, kidney, muscle and brain of the hares ranged from 0.058-0.189, 0.138-0.406, 0.013-0.046 and 0.022-0.102 µg/g respectively. Concentration of the glutathione (GSH), glutathione-peroxidase (GPx), and glutathione-reductase (GR) activity increased with the mercury concentration. However, glutathione S-transferase (GST) and superoxide-dismutase (SOD) activity decreased with the mercury concentration. The results of this study show the impact of environmentally absorbed mercury on the antioxidant status of the examined hares. Further research on long-term exposure to low concentrations of mercury is needed.

Ecotoxicological characterization of marine sediment in Kostrena coastal area.

Samples of marine sediment were taken on 4 selected sites close to the shipyard industry in Kostrena coastal area. Concentration of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and heavy metals (Cu, Pb, Zn, Hg, Fe) were analyzed from chemical-analytical and toxicological aspect. For toxicity detection, the bacterial bioluminescence test (Vibrio fisheri) was used. Concentration of total PAHs varied in the range from 697 to 7807 microg/kg dry weight in marine sediments. The concentration of PCBs in sediment was 1.1 mg/kg dry weight. The highest concentrations of heavy metals were found at the station within the shipyard. PAH toxicity was not correlated with the toxic potential of sediments. The obtained results indicate a high degree of environmental risk, especially at stations within the shipyard, with the 54% possibility of toxic effects. Chemical determination of the concentration of conventional pollutants is not sufficient for assessing the quality of the marine environment and it is necessary to use other approaches in order to evaluate the biological impact.