Anthropogenic litter in a Mediterranean coastal wetland: A heterogeneous spatial pattern of historical deposition.

Coastal wetlands represent areas that can testify historical accumulation of litter. We analyzed the anthropogenic litter deposited on the channel bottom of a coastal wetland area that experienced water stress due to extreme summer dryness after about 20 years. We hypothesize that the litter accumulated in the different areas over the years reflects the different social user categories (i.e., fishermen, beach users, hunters) and exposure to meteo-marine events. Our findings highlight that historically accumulated litter is composed of plastics (78.8 %), clothes (8.9 %), and glass (4.9 %). Moreover, litter concentration averages 53.6 items/ha in the 8 sectors. The most found categories were common household items (25.4 %), diverse (professional and consumer) items (24.2 %), and food and beverages packaging (21.4 %). Finally, litter diversity indices and the Detrended Correspondence Analysis showed sector and litter type similarities. We reported for the first time the presence of litter accumulated for 20 years testifying non-more occurring recreational activities.

The under-investigated plastic threat on seagrasses worldwide: a comprehensive review.

Marine plastic pollution is a well-recognised and debated issue affecting most marine ecosystems. Despite this, the threat of plastic pollution on seagrasses has not received significant scientific attention compared to other marine species and habitats. The present review aims to summarise the scientific data published in the last decade (January 2012-2023), concerning the evaluation of plastic pollution, of all sizes and types, including bio-based polymers, on several seagrass species worldwide. To achieve this goal, a comprehensive and critical review of 26 scientific papers has been carried out, taking into consideration the investigated areas, the seagrass species and the plant parts considered, the experimental design and the type of polymers analysed, both in field monitoring and in laboratory-controlled experiments. The outcomes of the present review clearly showed that the dynamics and effects of plastic pollution in seagrass are still under-explored. Most data emerged from Europe, with little or no data on plastic pollution in North and South America, Australia, Africa and Antarctica. Most of the studies were devoted to microplastics, with limited studies dedicated to macroplastics and only one to nanoplastics. The methodological approach (in terms of experimental design and polymer physico-chemical characterisation) should be carefully standardised, beside the use of a model species, such as Zostera marina, and further laboratory experiments. All these knowledge gaps must be urgently fulfilled, since valuable and reliable scientific knowledge is necessary to improve seagrass habitat protection measures against the current plastic pollution crisis.

Diatom and macroinvertebrate communities dynamic: A co-occurrence pattern analysis on plastic substrates.

Wetlands are habitats that provide numerous ecosystem services, but are often understudied and threatened by anthropogenic pollution, particularly plastic pollution. Macroplastics are a significant component of plastic litter that have high biological impacts but are often understudied. Previous studies have highlighted negative impacts on biota, but there is a lack of information about the communities of micro and macro organisms that settle on macroplastic litter. In this context, we investigated the colonization patterns and community structures of diatoms and macroinvertebrates on virgin substrates composed of two different plastic polymers, polystyrene and polyethylene terephthalate, located at two different depths in a protected wetland in Central Italy over a period of 10 months. The results show that diatom community is not highly structured by competitive forces and aggregation patterns emerges. In contrast, macroinvertebrate community appears to be randomly structured, without the presence of patterns following specific assembly rules. Randomness in macroinvertebrates assemblages could highlight the presence of different niches available for settlement of different taxa. Combined matrix analyses show that diatoms and macroinvertebrates co-occur, and their community assemblages are sometimes structured, while they appeared to be randomly assembled at other times. Whenever non-randomness of diatoms and macroinvertebrates co-occurrences was detected, it suggested aggregation. Moreover, the possible predatory relationship between different macroinvertebrates taxa should be investigated, as it could reveal important scenarios in the establishment of macroinvertebrate structured communities on plastic litter, including taxa that exploit different ecological niches. This could lead to an enrichment of the biological community within areas impacted by plastics.

Dune plants as a sink for beach litter: The species-specific role and edge effect on litter entrapment by plants.

Anthropogenic litter accumulates along coasts worldwide. In addition to the flowing litter load, wind, sea currents, geomorphology and vegetation determine the distribution of litter trapped on the sandy coasts. Although some studies highlighted the role of dune plants in trapping marine litter, little is known about their efficiency as sinks and about the small-scale spatial distribution of litter across the dune area. Here, we explore these gaps by analysing six plant species widespread in Mediterranean coastal habitats, namely Echinophora spinosa, Limbarda crithmoides, Anthemis maritima, Pancratium maritimum, Thinopyrum junceum, and Salsola kali. The present study analyses for the first time the capture of litter by dune vegetation at a multi-species level, considering their morphological structure. Data on plastic accumulation on dune plants were compared with unvegetated control plots located at embryo-dune and foredune belts. We found that dunal plants mainly entrapped macrolitter (> 0.5 cm). Particularly, E. spinosa, L. crithmoides, A. maritima and P. maritimum mostly accumulated litter in the embryo dune while T. junceum and S. kali entrapped more in the foredune area. Moreover, beach litter was mainly blocked at the edge of the plant patches rather than in the core, highlighting the 'Plant-edge litter effect'. As A. maritima and S. kali entrapped respectively more litter in embryo and foredune habitats, these species could be used to monitor and recollect litter. In this light, our findings provide further insight into the role of dune plants in the beach litter dynamics, suppling useful information for beach clean-up actions.

Teratogenic effects of environmental concentration of plastic particles on freshwater organisms.

Given the widespread presence of plastics, especially in micro- and nanoscale sizes, in freshwater systems, it is crucial to identify a suitable model organism for assessing the potential toxic and teratogenic effects of exposure to plastic particles. Until now, the early life stage of freshwater organisms and the regeneration capacity in relation to plastic particles exposure is a still poorly investigated topic. In this study, we examine the teratogenic effect on diatom Cocconeis placentula and cnidarian Hydra vulgaris under controlled exposure conditions of poly(styrene-co-methyl methacrylate) (P(S-co-MMA)) particles. Significant effects were observed at the lowest concentrations (0.1 µg/L). A significant increase in the teratological frequency in C. placentula and a significant decrease in the regeneration rate in H. vulgaris were found at the lowest concentration. The delay in hydra regeneration impaired the feeding capacity and tentacles reactivity at 96 h of exposure. No effects on diatom growth were observed upon exposure to P(S-co-MMA) particles (0.1, 1, 100, 10,000 µg/L) for 28 days and these findings agree with other studies investigating algal growth. The application of the Teratogenic Risk Index, modified for diatoms, highlighted a moderate risk for the lowest concentration evaluating C. placentula and low risk at the lowest and the highest concentrations considering H. vulgaris. This study suggests the importance of testing organisms belonging to different trophic levels as diverse teratogenic effects can be found and the need to evaluate environmentally relevant concentrations of plastic particles.

Nanoplastic exposure inhibits feeding and delays regeneration in a freshwater planarian.

The concentration of nanoplastics (NPs) is expected to increase in aquatic environments thus potentially threatening freshwater organisms through interactions with plastic particles that variously float, circulate in the water column or sink into the benthos. Studies into the mechanisms of any NP effects are still scarce, particularly with respect to the regenerative ability of biota for which there is no recognised model organism. The present study therefore aimed to investigate behavioural and regeneration responses of the freshwater planarian Girardia tigrina after 10 days exposed to along a gradient 0.01-10 mg/L of poly (styrene-co-methyl methacrylate) NPs (∼426 ± 175 nm). Exposure to NPs induced a significant reduction in planarian feeding rate even at low concentrations (LOEC of 0.01 mg/L), while head regeneration was delayed in a clear dose response way (LOEC of 0.1 mg/L for blastema length). Planaria locomotion assessed was not affected. Our results highlight the potential adverse effects of exposure to poly (styrene-co-methyl methacrylate) NPs and show that feeding behaviour and regeneration of a freshwater benthic organism can be indicators of the resulting toxicity. Planarians are becoming widely used model organisms in ecotoxicology and can help to address potential effects of plastic polymers on regeneration.

Dunal plants as sink for anthropogenic marine litter: The entrapping role of Salsola kali L. (1753) in a Mediterranean remote beach (Sardinia, Italy).

The ability to retain anthropogenic marine litter by a halo-psammophilous plant formation dominated by a single prostrate species (Salsola kali) on a Sardinian beach was measured. We hypothesized that the anthropogenic litter (i) is trapped by plants to a greater extent than in control areas, and (ii) has more elongated size, mimicking the organic Posidonia wrack, largely occurring locally as 'banquettes'. Salsola kali patches show an apparently higher anthropogenic litter density than control sites without vegetation. Salsola kali plants trap litter items significantly longer and a larger number of size length categories than control plots. These effects may be due to the prostrate structure of the plant with small thorns at the apex. Also, litter entrapped by plants can interfere with the mechanisms of dune deposition and structuration, in turn affecting food chains by decreasing the availability of organic material for pedofauna.

New ecological frontiers in the plastisphere: Diatoms and macroinvertebrates turnover assessment by a traits-based approach.

To date, there are very few studies regarding the colonization of artificial substrates in wetlands by macroinvertebrates and diatoms and even fewer are the studies in Italy that take into consideration the diatomic guilds and the biological and ecological traits proposed in literature. Wetlands are at the forefront through the most delicate and threatened freshwater ecosystems. In this study, we want to evaluate the colonization capacity of plastics of diatoms and macroinvertebrates and characterize the diatomic and macroinvertebrate communities using a "traits-based" approach focusing on the colonization of virgin substrates made of polystyrene and polyethylene terephthalate. The study was conducted within the 'Torre Flavia wetland Special Protection Area' a protected wetland area in Central Italy. The study was conducted from November 2019 to August 2020. The results obtained in this study show a tendency of diatom species to colonize artificial plastic supports placed in lentic environments without differences related to the plastic type and water depth. There is also a greater number of species belonging to the "Motile" guild, endowed with a high motility that they exploit to search for more ecologically suitable habitats for settlement. Macroinvertebrates, prefer settlement on polystyrene supports, those on the surface, probably due to the anoxic conditions present on the bottom and the physical structure of the polystyrene that provides shelter to many animal taxa. The analysis on traits highlighted the establishment of an ecologically diverse community mainly formed by univoltine organisms, with dimensions between 5 and 20 mm, predators, choppers and scrapers feeding on plant organisms and animals, but without the formation of a clear ecological system, that is, without evidence of ecological relationships established between two or more taxa. Our research can contribute to underline the ecological complexity of biota inhabiting plastic litter in freshwaters and the implications for plastic-impacted ecosystems biodiversity enrichment.

Dunal plants intercepting macrolitter: Implications for beach clean-ups.

Coastal vegetation intercepts macroplastics and, consequently, it may represent a reservoir of anthropogenic litter and organic wrack. We aimed at investigating (i) the abundance variation of macrolitter from the beach to foredune and backdune (three cross-shore plots over 20 long-shore sectors) and (ii) the role of the halo-psammophilous plants and Phragmites australis reedbed in intercepting the macrolitter, respectively, in the foredunes and backdunes. The vegetation in the foredunes (mainly halo-psammophilous species) acted as a first interception belt for macrolitter, while the bigger litter reached the backdunes. Our results might be of great concern with implications for beach clean-ups - which must also be mainly focused in foredunes and backdunes, however warning operators in advance that they could damage the vegetation by trampling on.

Epiplastic microhabitats for epibenthic organisms: a new inland water frontier for diatoms.

Plastic pollution is widespread in each type of ecosystems. However, the colonization events of microorganisms on plastics seem to be neglected in inland waters. Therefore, in this study we analyze the possible colonization on the surface (hereafter epiplastic microhabitats) of two typology of plastic supports by diatom community. Specifically, we located 20 supports in expanded polystyrene and 20 in polyethylene terephthalate both floating and dipped (~ 1 m) in a central Italian shallow water pond, in order to evaluate the diachronic colonization of diatoms from November 2019 to August 2020. Our result showed the tendency in colonizing both epiplastic microhabitats without significant differences in number of species; additionally, depth does not appear to affect the number of species. As regard the temporal colonization, the number of species tends to increase over time from autumn-winter to spring-summer in both types of epiplastic microhabitats and depth. Instead, increase in dominance of some species over time has been demonstrated: only a few species keep a high number of individuals compared to the others; therefore, the number of individuals within the species is not uniformly distributed. These results suggest the tendency of diatom community to colonize plastic supports in lentic waters, and this evidence can be very important because artificial supports can increase the surface available for the settlement of the algae community with an increase of productivity and the colonization of new communities of different taxa. Further studies are mandatory to investigate the possible effects on the epiplastic community and the ecological implications in freshwater environments.

From city to sea: Spatiotemporal dynamics of floating macrolitter in the Tiber River.

Rivers are undoubtedly the main pathway of waste dispersed in the environment that from land reaches oceans and seas increasing the amount of marine litter. Major cities are a great source of riverine litter as large urbanization can originate pressure on the integrated waste management resulting in litter entering the rivers. Within this study, we aim to investigate the dynamic of floating riverine macrolitter (items >2.5 cm) in the city of Rome before it reaches the sea by assessing the composition, amount, and seasonal trends of litter transported from the urban centre to the main river mouth of Tiber River. Visual surveys for a whole year (March 2021-February 2022) were conducted from two bridges, Scienza Bridge (in the city) and Scafa Bridge (at the main river mouth) and followed JRC/RIMMEL protocol for riverine litter monitoring. Overall, similar litter composition was observed from the city centre to the mouth with a prevalence of plastic material, mainly related to fragmentation process (i.e. plastic pieces) and single use items, mainly in food and beverage sectors. An extrapolated annual loading of 4 × 105 items/year was estimated at the main mouth of Tiber River. The litter flux seems to be influenced by the seasonal variability and hydrometeorological parameters. The frequency of size classes decreases with increasing size in both sites, and more than half of the recorded items were below 10 cm. Specific categories belonging to "other plastics" have been reported related to anti-Covid-19 behaviour such as face masks and beverage sector, e.g. bottle lids and rings. The main colour of plastics was white, suggesting weathering process of floating riverine litter. This study contributes to increasing knowledge of the origin, composition and spatiotemporal dynamics of riverine floating litter from the city and entering the sea.

General macro-litter as a proxy for fishing lines, hooks and nets entrapping beach-nesting birds: Implications for clean-ups.

Fishing lines, hooks and nets represent a sub-category of macro-litter potentially entrapping plover birds nesting on sandy beaches. Here, during a winter period, the accumulation pattern of both general beach litter and fishing lines, hooks and nets was analysed on four central Italy beaches. Despite the active monthly litter removal by clean-ups, there was not a decrease in its density during the winter period, due to the continuous accumulation by frequent winter storms. However, the entrapping litter was very low (<2.5 % of the general litter) and appeared directly correlated to the general litter density. Following a DPSIR approach, the general litter can act as an indirect pressure indicator (proxy) of the amount of entrapping litter. Therefore, an increase in general macro-litter should alarm those involved in the conservation of entanglement-sensitive bird species, such as plovers, suggesting that they should implement high-frequency clean-up activities aimed at removing it.

Another one bites the plastics.

Old-growth forests host a rich diversity of invertebrate assemblages. Among them, saproxylic insects play a fundamental role in the nutrient cycle and ecosystem functioning. In these environments, coevolution between insect and plants have reached a stable equilibrium over millions of years. These delicate ecosystems are threatened mainly by habitat loss and fragmentation, and to date, they have to face the new "plastic threat." Plastics are widespread in all biomes and ecosystems accumulating throughout the years due to their low degradation rate. Once accumulated, large pieces of plastics can be degraded into smaller particles, the latter representing a great threat to biodiversity and ecosystem health, producing detrimental effects on biota. Since the effects of plastics on terrestrial systems remain largely unexplored, this study aimed at contributing to increasing the knowledge on the interaction between plastics and terrestrial biota. We put our emphasis on the novel and broad topic of plastic degradation by saproxylic beetle larvae, describing how they fragmented macroplastics into microplastics. To investigate whether saproxylic cetonid larvae could degrade expanded polystyrene, we performed an experiment. Thus, we put larvae collected in the field in an expanded polystyrene box. We observed that larvae dug in the thickness of the box fragmenting macroplastics into microplastics and producing a total of 3441 particles. Then, we removed the larvae from the EPS box and isolated them in glass jars filled with natural substrate. The substrate was checked for EPS microplastics previously ingested and now egested by larvae. Additionally, we pointed out that plastics remained attached to cetonid larvae setae, with a mean number of 30.7 ± 12.5 items. Although preliminary, our results highlighted that microplastics attached to saproxylic cetonid larvae might be transported into habitats and transferred along the food web. In conclusion, plastic pollution might affect vulnerable species and ecosystem services representing a risk also for human health.

Temporal changes of plastic litter and associated encrusting biota: Evidence from Central Italy (Mediterranean Sea).

We investigated the temporal changes from spring to summer of the stranded litter and the composition of plastic encrusting biota along an Italian beach. Our findings highlight a higher quantity of litter (average value 1510.67 ± 581.27 items) in spring, particularly plastic material with a composition driven by currents, winds and waves transported from rivers to sea. During summer the source was caused by anti-social behaviours (e.g. cigarettes). Regarding the plastic size, the most is macroplastic (85.96 %), followed by mesoplastic (13.74 %) and megaplastic (0.30 %) overall, and no seasonal trend was observed. Concerning the encrusting biota, Mollusca was the most frequent phylum found on plastic beach litter, whereas Porifera the most abundant overall. During spring a greater abundance of individuals was recorded compared to summer. The trend of taxa richness was decreasing from spring to summer. Arthropoda, Porifera and Mollusca phyla were significantly more abundant in spring, while Algae in summer.

Anthropogenic particles in coypu (Myocastor coypus; Mammalia, Rodentia)' faeces: first evidence and considerations about their use as track for detecting microplastic pollution.

Anthropogenic plastic litter is widespread in all environments, with particular emphasis on aquatic habitats. Specifically, although freshwater mammals are important as they are at the top of food web, research mainly focus on marine animals, while only few studies have been carried out on freshwater mammals. The main gap is that microplastics (MP) are completely understudied in freshwater mammals. Here, we reported the first evidence of the presence of anthropogenic particles (including MP) in coypu (Myocastor coypus)' faeces. Coypu is a rodent mammal inhabiting rivers and wetland areas, and we discussed our preliminary data suggesting the use of these tracks as possible future bioindicator of MP pollution in wetlands and freshwaters. We collected 30 coypu's faeces in "Torre Flavia wetland" nature reserve. Then, in laboratory, faeces were digested in 30 ml hydrogen peroxide (30%) for a week a 20 °C and analysed under stereoscope. All the suspected found MP were isolated in a petri dish, using FT-IR analysis to confirm the polymers. Overall, we recorded 444 natural and anthropogenic particles with most of items being fibres. FT-IR analysis of the 10% of the particles recovered revealed that 72% of them was not MP (mainly, polyethylene, polyethylene terephthalate, and polyamide). Also, the number of anthropogenic particles is not correlated with the faecal weight. Given that alien species, such as coypu, are widespread species, our results might have a great importance as these species and MP in faecal tracks may be used as undirect proxy of environmental bioavailability of MP pollution.

Microplastics distribution and possible ingestion by fish in lacustrine waters (Lake Bracciano, Italy).

Understanding the spatial distribution patterns of microplastics (plastics < 5 mm) contributes to the assessment of sources and sinks of pollution thus providing information for the management of biota safety and overall ecosystem functionality. We chose a semi-closed study area, Lake Bracciano (Italy), to assess the environmental variability of contamination, focusing on the water compartment and the exposure of biota, specifically fish, by analysing the ingestion of microplastics. The focus of this study is to evaluate the concentration of microplastics in water (surface and column) across the lake and the ingestion of microplastics by two fish species of economic interest: Atherina boyeri and Coregonus lavaretus, inhabiting demersal and pelagic habitats respectively. Results show a surface contamination of 392,000 ± 417,000 items km-2 and a column one of 0.76 ± 1.00 items m-3. Fragments were the most abundant in surface while fibres in the column. Microplastics were found in C. lavaretus specimens, corresponding to contamination frequency of 5% and concentration of 0.15 items/fish. The main polymer found in water was polyethylene (81%); of minor percentages, there were various other polymers, including polystyrene and acrylic, which were also found in fish. As scientific literature provides few research where water and fish are simultaneously sampled, this investigation wants to contribute filling this knowledge gap by investigating for the first time a volcanic lake.

Spatial distribution of microplastics in volcanic lake water and sediments: Relationships with depth and sediment grain size.

Microplastics (plastics <5 mm) are globally widespread pollutants of aquatic ecosystems. As microplastics contaminate both water and sediments, research on their spatial distribution in these different environmental matrices has increased. However, fresh waters are poorly studied and even less so are lentic ecosystems. To contribute filling this knowledge gap, this study analyses the distribution of microplastics in the water column and surface sediments of a volcanic lake, namely Lake Bracciano. Furthermore, it analyses in more detail the relationship between the concentration of microplastics in sediments, its grain size and the sampling depth (i.e. nearshore or deep). Water and sediment sampling was carried out in different sectors of the lake (northern, eastern, southern, western) using a plankton net and a van Veen grab sampler, respectively. Two sediment samples were collected at each station in order to analyse the abundance of microplastic and to perform grain size analysis. Results show a mean concentration of 2.4 items m-3 in water and 42 items kg-1 in sediments. The distribution of microplastics is uneven between the different sampling stations, with the northern sector being the most contaminated in both matrices. The chemical composition and shape of microplastics vary between water and sediment. In particular, polyethylene terephthalate and polyvinyl chloride are the most abundant polymers in water and sediments, respectively. Fibres are the main shape of microplastics in water while fragments are more abundant in sediments. In-depth analysis of sediment shows that sediments from deep stations are more contaminated than nearshore samples and have more fragment-shaped microplastics than fibre-shaped ones. Furthermore, there is a significant positive correlation between the concentration of microplastics and the abundance of silt, confirming data emerging from the scientific literature on marine and lotic ecosystems.

Riparian vegetation as a trap for plastic litter.

Plastic pollution represents the most widespread threaten throughout the world and, amongst aquatic habitats, freshwaters and in particular riparian zones seems to be highly disturbed. Since the plastic storage and accumulation on the riparian vegetation have not yet been deeply investigated, here, we focussed on the riparian zone's function in trapping plastic litter. To do so, we assessed the occurrence and density of plastics in different vegetated (arboreal, shrubby, herbaceous, reed, bush) and unvegetated types in 8 central Italian rivers, running in different land use contexts. Our results showed that plastic pieces, bags, bottles and food containers were the most abundant specific categories on the vegetated types, demonstrating the riparian vegetation role in trapping plastic litter. Specifically, the highest plastic density was found on the shrubby type suggesting that a tree shape retains plastics more easily than all other vegetated and unvegetated types. Shape and size classification of plastics are not significantly different between vegetated and unvegetated types. These findings allow to collect important information on how the riparian vegetation can be exploited in management activities for removing plastic litters from both freshwater and sea, being the former considered the main plastic source for the latter. This study highlights a further ecosystem service as mechanical filter provided by the riparian zone, even if further studies ought to be performed to understand the role of vegetation as plastic trap and the possible detrimental effects of plastics on the plant health status.

Field evidence for microplastic interactions in marine benthic invertebrates.

Microplastics represent an important issue of concern for marine ecosystems worldwide, and closed seas, such as the Mediterranean, are among the most affected by this increasing threat. These pollutants accumulate in large quantities in benthic environments causing detrimental effects on diverse biocenoses. The main focus of this study is on the 'polychaetes-microplastics' interactions, particularly on two species of benthic polychaetes with different ecology and feeding strategies: the sessile and filter feeder Sabella spallanzanii (Gmelin, 1791) and the vagile carnivorous Hermodice carunculata (Pallas, 1766). Since not standardized protocols are proposed in literature to date, we compared efficiencies of diverse common procedures suitable for digesting organic matter of polychaetes. After the definition of an efficient digestion protocol for microplastics extraction for both polychaetes, our results showed high microplastics ingestion in both species. Microplastics were found in 42% of individuals of S. spallanzanii, with a mean of 1 (± 1.62) microplastics per individual, in almost all individuals of H. carunculata (93%), with a mean of 3.35 (± 2.60). These significant differences emerged between S. spallanzanii and H. carunculata, is probably due to the diverse feeding strategies. The susceptibility to this pollutant makes these species good bioindicators of the impact of microplastics on biota.

An integrated approach for chemical water quality assessment of an urban river stretch through Effect-Based Methods and emerging pollutants analysis with a focus on genotoxicity.

The impact of emerging chemical pollutants, on both status and functionality of aquatic ecosystems is worldwide recognized as a relevant issue of concern that should be assessed and managed by researchers, policymakers, and all relevant stakeholders. In Europe, the Reach Regulation has registered more than 100.000 chemical substances daily released in the environment. Furthermore, the effects related to the mixture of substances present in aquatic ecosystems may not be predictable on the basis of chemical analyses alone. This evidence, coupled with the dramatic effects of climate changes on water resources through water scarcity and flooding, makes urgent the application of innovative, fast and reliable monitoring methods. In this context, Effect-Based Methods (EBMs) have been applied in the urban stretch of the Tiber River (Central Italy) with the aim of understanding if detrimental pressures affect aquatic environmental health. In particular, different eco-genotoxicological assays have been used in order to detect genotoxic activity of chemicals present in the river, concurrently characterized by chemical analysis. Teratogenicity and embryo-toxicity have been studied in order to cover additional endpoints. The EBMs have highlighted the presence of diffuse chemical pollution and ecotoxicological effects in the three sampling stations, genotoxicological effects have been also detected through the use of different tests and organisms. The chemical analyses confirmed that in the aquatic ecosystems there is a diffuse presence, even at low concentrations, of emerging contaminants such as pharmaceuticals, not routinely monitored pesticides, personal care products, PFAS. The results of this study can help to identify an appropriate battery of EBMs for future studies and the application of more appropriate measures in order to monitor, mitigate or eliminate chemical contamination and remediate its adverse/detrimental effects on the ecosystem health.