1. Introduction
The Marano and Grado Lagoon (MGL) belongs to an extensive network of transitional environments in the northern Adriatic Sea, covering an area of approximately 160 km
2 between the deltas of the Tagliamento and Isonzo rivers [
1]: it is formally divided into the Marano and Grado Basins [
2]. The Marano Basin is the oldest part (5550–4200 years ago), with the current morphology reached around 1600 years ago. The Grado Basin is more recent (4th–6th century AD), and the morphology of its easternmost part was reached after the massive land reclamation that took place since the beginning of the 20th century [
3]. The MGL is classified as a microtidal lagoon of large dimension and is one of the 57 Italian sites under the protection of the Ramsar Convention since 1971 (Valle Cavanata site number 169/1978 and Foci dello Stella number 190/1979) (
https://www.ramsar.org/ accessed on 9 May 2024). As part of the implementation of the Habitats Directive (92/43/EEC), the MGL has been designated as a Site of Community Importance (SCI-IT3320037).
Similar to others Mediterranean coastal areas, the MGL suffers from numerous anthropogenic pressures, such as the presence of industrial sites, marinas, fishing, fish and clam farming and tourism [
4]. However, one of the main concerns is the excess nutrients, particularly nitrate (NO
3−), deriving from upland farming practices [
5], and the MGL has been designated as a Nitrate Vulnerable Zone (NVZ) since 2008 [
6]. Saccon et al. [
5] reported NO
3− concentration ranging from 400 to 31,000 µg L
−1 depending on the water circulation and influencing factors (i.e., mixing between seawater, freshwater and rainwater, tidal oscillation, wave motion, water flow direction, wind speed, atmospheric pressure, tributary discharge, lagoon bottom morphology, and bathymetry). Recently, Acquavita et al. [
1] described a variable range, from oligotrophic to hypertrophic, for the trophic state of the MGL in a 1-year cycle of physicochemical measurements carried out throughout the lagoon using a series of trophic indices (i.e., Carlson Trophic Index, TRIX and ASSETS) [
7,
8,
9]. In addition, more specific efforts have been attempted to study the concentration of nutrients in confined areas, such as dismissed fish farms, and the nutrient recycling at the sediment–water interface throughout benthic chamber deployment [
2,
10].
It is well known that the excess of nutrients (mainly nitrogen and phosphorus) leads to an imbalance of the pristine trophic state, resulting in problems of eutrophication [
11], which is currently defined as “a process driven by the enrichment of water with nutrients, especially compounds of nitrogen and/or phosphorus, leading to: increased growth, primary production and biomass of algae, changes in the balance of organisms and water quality degradation”. The consequences of eutrophication are undesirable if they appreciably degrade ecosystem health and/or the sustainable provision of goods and services [
12]. Fertiliser use, soil leaching, land clearing, livestock farming, wastewater discharge and the burning of fossil fuels are the main sources of nutrients [
13], and, considering the eutrophication problems reported in Europe [
14,
15], policies have been adopted and translated into regional and communitarian programs, to control nutrient inputs and their effects on the environment. These include the Oslo–Paris Convention for the Protection of the Northeast Atlantic [
16], the Helsinki Convention [
17] for the Protection of the Baltic Sea, the Barcelona Convention (MEDPOL) for the Mediterranean, and legislative instruments such as the Urban Wastewater Treatment Directive [
18] and the Nitrates Directive [
19] in the European Union (EU). In recent decades, two more comprehensive pieces of legislation have been introduced: The Water Framework Directive (WFD/2000/60/EC) and the Marine Strategy Framework Directive (MSFD/2008/56/EC) [
20,
21]. Specifically, the WFD/2000/60/EC covers all surface waters and groundwater, while the MSFD/2008/56/EC provides a policy framework up to the 200 nautical miles’ limit of the European Exclusive Economic Zone [
22].
The primary goal of the WFD/2000/60/CE is to achieve conditions of “good ecological status” in selected water bodies by 2015 (or 2027 if certain exemptions are invoked). The Directive takes account of biological, chemical, and morphological elements—which must be supported by physicochemical parameters—and in particular currently sets the limit for DIN (dissolved inorganic nitrogen as the sum of NO
3−, NO
2−, and NH
4+) and phosphorus (as total phosphorus, TP). In Italy, the WFD/2000/60/CE was put into law via Italian Legislative Decree no 152/2006 and by decrees for typing (DM 131/2008), monitoring (DM 56/2009) and classification (DM 260/2010). The analysis of specific descriptors (i.e., geomorphology, tides and salinity) coupled with the application of the DPSIR (driver, pressure, state impact, response) model [
23] is the first tool required by the WFD for the individualisation of water bodies that—based on salinity—corresponds to three types, namely mesohaline, polyhaline, and euhaline.
The objective of this study was to assess the spatial distribution and seasonal variability of physicochemical parameters and nutrients in order to characterise the water quality of the lagoon and to classify it in accordance with the implementation of the WFD/2000/60/CE. Given that the primary pressure on the system is the input of riverine freshwater, an analysis was conducted to assess the degree of precipitation at two selected inland sites. The occurrence of a correlation between precipitation and physicochemical parameters and nutrients was also tested. To assess the trophic status, numerous multimetric indices are available, as reported in Bonometto et al. [
24]. The metrics related to macrophytes—macroalgae cover, seagrass cover, and benthic macrophytes—were not fully available. Therefore, the simpler and widely applied TSI and TRIX [
7,
8] were used. Based on evidence from previous studies conducted in an abnormally rainy year and considering that temporal variability is a key issue in monitoring and explaining the dynamics of ecological systems [
25], especially in transitional environments [
26], the time-series was also discussed to detect the occurrence of significant positive or negative trends of the considered parameters. This is of paramount importance considering that the prediction that emissions of N, a major contributor, will increase significantly between now and 2050 [
27] and that the concomitant effect of climate change [
28,
29] calls for special attention in the monitoring and control of trophy in marine–coastal systems.
4. Discussion and Conclusions
As a member state of the European Union, Italy has implemented the WFD 2000/60/CE. Since 2010, the Friuli Venezia Giulia region, which includes the Marano and Grado lagoon, has been conducting a monitoring programme. The programme focuses on collecting surface water samples seasonally to define the physicochemical conditions that support the biological quality elements. This allows for a better understanding of the trophic state of the lagoon. Furthermore, long-term studies have not been conducted in the MGL, and it is well known that physicochemical parameters that describe the water column are susceptible to significant changes, both positive and negative, in response to climate change and anthropogenic pressures [
45,
46,
47,
48,
49,
50].
The Mediterranean Sea is renowned for its significant spatiotemporal variability of physicochemical parameters and nutrients in coastal marine areas. Fluctuations in lagoons are typically substantial due to their location between land and sea, shallow depth, tidal effects, wind action, and runoff from inland. This has been observed in numerous studies [
26,
51,
52,
53]. Similar observations were made in the MGL, where steep spatial distribution gradients and fluctuations in the form of outliers were found for all parameters except water temperature and dissolved oxygen concentration (see
Figures S3 and S4). Additionally, clear seasonal variability was detected, except for total phosphorus. The observed variabilities were statistically significant, as confirmed by the application of the Kruskal–Wallis test for equal medians (see
Table 6).
The spatial variability in the MGL is mainly influenced by freshwater input and morphology. The impact of river discharge is supported by the significant inverse correlation between salinity and all nutrient species (except for N-NH
4+ and TP) as well as the positive correlation with accumulated rainfall. However, the pattern of rainfall with nutrient level could be limited by the active exchange with the more diluted seawater through the tidal channel (Grado (390 m wide/10 m deep) 22% of the total water exchange, Porto Buso (430 m, 30%), and Lignano (310 m/11 m, 35%) [
54]. The Marano basin is characterised by the flow of the Stella and Cormor rivers, which have average discharges of 36.1 and 10.7 m
3 s
−1 respectively. This basin has the lowest salinity values and the highest nutrient concentration. The morphology of the basin affects the water residence time, which can be up to 20 days under certain conditions [
32]. As a result, the surface water temperature is higher on average within the confined water bodies.
The primary cause of nutrient loading in the upper Friulian Plain is runoff resulting from intensive agricultural activity [
6]. This is evidenced by the high levels of the main dissolved nitrogen form, N-NO
3−, which derives from lixiviate enriched in plant fertilisers [
55,
56,
57,
58]. Additionally, there are direct and inverse significant correlations between N-NO
3− and accumulated rainfall and salinity. The reduced form, N-NH
4+, displays the highest values in summer and is inversely correlated with the O
2 content. This is likely due to intense biogeochemical processes, such as uptake by phytoplankton, excretion by zooplankton, bacterial remineralisation, and denitrification processes, which are favoured in low oxygen conditions and stimulated by high temperatures [
59,
60,
61] and have already been observed in different lagoon environments [
62,
63,
64]. The concentration of PO
43− was significantly lower (three orders of magnitude) than that of N-NO
3−. However, its spatial distribution was similar to that of nitrogen species, thus supporting the common origin of nutrients. The anthropogenic impact in the MGL is well evident if the level of nutrients is compared to that observed outside of the lagoon area in the adjacent Gulf of Trieste and in the northern Adriatic Sea. Cozzi et al. [
65] analysed a long-term series of data (1992–2018) at the station C1, which belongs to the Long-term Ecosystem Research Network (
http://www.lteritalia.it/ accessed on 9 May 2024). On average, DIN was mostly lower than 4 µM (value integrated over water column), which is one order of magnitude lower than that observed in the MGL (surface waters). In addition, Giani et al. [
66] reported a typical range from <lod to 16 µM of N-NO
3− for all the northern Adriatic. On the contrary, P-PO
43− was comparable, with a minimum of 0.18 and a maximum of 1.5 µM. This suggests that the main concern in the MGL is the anthropogenic input of N species. It is noteworthy that due to the exchange of water masses, there is an active export of nutrients from the MGL to the Gulf of Trieste. In fact, two external water bodies recently considered by Bellese [
67] showed values of N-NO
3− up to 130 µM (mean 15.92 ± 17.73 μM), which is a dilution of about one third respect to the value found in the lagoon basin.
The Redfield N:P ratio (RR) is commonly used to describe the relative importance of N and P as factors capable of limiting the growth of phytoplankton [
68]. It is important to note that the ratio of 16:1, originally calculated, can be significantly deviated due to the uncontrolled supply of nutrients, as observed in our lagoon. However, it is worth mentioning that the RR is currently being continuously revised, as reported in [
69]. In this work the RR showed a wide range (3–55,967) and was, on average, 1130 ± 3033. These data are comparable to those observed in the nearby Gulf of Trieste and Isonzo River basin, thus confirming that the northern Adriatic Sea is experiencing P-limitation that is extreme in some cases [
65,
67,
70,
71]. This is a common occurrence in coastal environments with anthropogenic inputs that are enriched in N compared to P [
72,
73] and is a well-recognised characteristic of the Mediterranean Sea [
74,
75] and of other Mediterranean coastal lagoons [
56,
75,
76,
77]. The RR significantly decrease in summer due to the reduced inputs of dissolved nitrogen form and to the hypoxic conditions that cause the sediment to be an internal source of nutrients (e.g., ammonium and phosphates) [
78].
Since the early 1970s, the European Council has introduced several environmental directives to establish threshold values for the concentration of dissolved inorganic nitrogen (DIN) species in various aspects of water quality. These include drinking water, protection from dangerous substances, support of fish life, and regulation of urban wastewater (74/440/EEC; 76/464/EC; 78/659/E; 80/68/EC; 98/15/EC). Despite the high DIN concentrations, the recommended values have never been exceeded. The WFD/2000/60/CE is currently in force and sets limits for DIN and P-PO43− as physicochemical elements that support ecological status. Proposed thresholds differentiate between euhaline water bodies and poli- and mesohaline ones. In euhaline water bodies, the limits for DIN and P-PO43− are 18 and 0.48 µM, respectively. For poli- and mesohaline ones, the limit for DIN is 30 µM. For P-PO43−, the limit value was never exceeded. However, all water bodies in the Marano basin were affected by DIN, including TEU2, TEU3, FM2, and FM4 in the Grado Basin.
The detection of eutrophication-related issues commonly involves measuring the concentration of chlorophyll
a. In our transitional system, both the average and maximum values are lower than those reported for other Mediterranean lagoons, which in some cases reached extreme values close to 50 µg L
−1 [
57,
61,
76,
79,
80] and are indication of oligotrophic/mesotrophic conditions [
81,
82]. Phytoplankton growth rate is commonly sustained by the absolute concentration of nitrogen and phosphorus [
83] and limited by light availability [
84,
85]. Despite P-limitation, chlorophyll
a concentration in our lagoon is correlated with TP but not with dissolved and total nitrogen species, as the signal of the latter is superimposed with respect to that of phosphorus [
61]. In shallow-water systems like the MGL, turbidity caused by suspended solid material may limit the production of chlorophyll
a. This is due to reduced availability of light for photosynthesis and soluble phosphorus, which adheres to solid particles [
86]. It is possible that this is the reason for the observed correlation with TP, but unfortunately no direct measurements of total suspended matter are available in this study to confirm the hypothesis. Overall, the traditional models that assume a direct response of phytoplankton to nutrient load (Phase I
sensu Cloern) [
11] is not applicable in this lagoon environment and at the same time indicates that phytoplankton do not efficiently control nutrient concentrations [
87].
The use of indices can assess eutrophication based on a few diagnostic physical and biogeochemical variables. To account for the variability of physicochemical parameters in the lagoon environment, such as salinity, this work applies criteria suitable for fresh, marine–coastal, and transitional waters [
7,
8,
38]. When more variables that act as drivers (i.e., different species of nutrients) and well-known responses (i.e., chlorophyll
a, dissolved oxygen) are considered for the calculation, the application of Carlson’s indices can result in a wide range of classifications. This is a common finding, as reported by Acquavita et al. and Coelho et al. [
1,
57], which is also supported by the present study. The MGL exhibited greater eutrophication when the TSI (TP) was applied, as opposed to the TSI (CHla). This finding is consistent with the results reported in [
57,
76,
88]. Specifically, 8.5% of the samples were in a eutrophic condition in the former case, while only 1.34% were in a eutrophic condition in the latter. The multimetric TRIX considers both drivers and responses in the same formula, making it more suitable for assessing the trophic state of coastal environments. However, TRIX was calibrated on the coastline of the Adriatic and Tyrrhenian Seas [
8,
32,
38]. As previously suggested [
57], specific classification criteria should be proposed to assess the correspondence between TRIX and water quality in lagoons. The trophic condition of the MGL varied, but the system is mostly oligo-mesotrophic. This suggests that several factors may contribute to buffering the input of nutrients that limit primary production (i.e., P-limitation, mineralization processes, water transparency, active exchange of water with the open sea, presence of microphytobenthos and macroalgae) [
11,
61,
89,
90]. It is important to note that the assessment of trophic conditions in transitional environments is critical. It is challenging to distinguish between the effects of anthropogenic pressures and the trophic status that is supported by the natural background of the system (EC, 2024) [
24]. The indices TSI (TP and Chl-a) and TRIX have been a significant influence in the past literature [
24] due to the ease of collection of the parameters that compose the indices. In this context, it is evident that the simplicity of these indices—particularly that of the TSI, which functions as a linear cause-and-effect mode (as defined by Phase-1 Cloern [
11]) and which was calibrated for freshwater ecosystems—provides only a preliminary indication of the trophic state. This is primarily applicable to mesohaline water types. It is evident that the state of the art of the previous multimetric indices assessed for transitional waters [
9,
91,
92,
93,
94] must be surpassed in order to reach the more recent TWEAM (transitional water eutrophication assessment method) [
24]. It is recommended that further parameters, particularly biological ones, be considered in order to provide a more accurate assessment of the water quality of the Marano and Grado Lagoon. However, this is complicated by the difficulty of collecting samples at an appropriate frequency.
Temporal patterns observed for physicochemical parameters and nutrients suggests that significant changes can also be detected, if a long-term series of data is available, without human intervention (i.e., land reclamation, restoration). The latter are in fact causes of significant improvement of the trophic state, or better reoligotrophication, in such sensitive environments [
95,
96,
97]. For natural systems that have not been disturbed by human intervention, there is a limited body of scientific literature on the remediation of environmentally degraded conditions, but two works similar to ours have been carried out in the nearby Lagoon of Venice [
98,
99]. The authors described a general increase in DIN and a decrease in PO
43− because of the reduced amounts of phosphorous compounds, from 8% to 1%, in domestic and industrial Italian detergents, following Italian laws enforced in 1983. There is no overlap between the time periods under consideration and the time period of our study, and therefore, no direct comparison can be made. In fact, it is surprising that in our case P-PO
43− significantly increase, but the improvement effects observed in water quality of marine coastal environment [
100] was followed by a slight counter-trend [
67,
101]. Positive and significant increases were also found for the indices TSI (Chla) and TRIX. This evidence reflects the positive trend observed for Chl
a, TP, and N-NO
3− (see
Supplementary Materials). On the other hand, N-NH
4+ showed a significant negative trend, which could be related to improvement in the efficiency of the local sewage treatment plants. A positive trend was observed for the temperature which is in accordance with the evidence reported for the whole Mediterranean area [
102] and in the adjacent Gulf of Trieste [
103,
104], whereas salinity showed a decrease. This latter parameter was found to generally increase in the Mediterranean Sea [
105], the Gulf of Trieste [
103], the Venice Lagoon [
106], and—considering the presence of macrozoobenthos in relation with salinity—the MGL [
107]. Thus, the negative pattern found in our study is probably influenced by the limited time series and by the recent anomaly of rainfall that occurred in 2019.
This study concludes that the MGL shows clear patterns in the distribution and seasonal variation of environmental parameters, with high nutrient levels, especially nitrogen, in the western part due to anthropogenic (effluent) sources and, as a consequence, high variability of trophic state using both TSI and TRIX indices depending on where and when the indices are applied. The current state of the lagoon requires careful management given its importance as an ecosystem service and should consider the maintenance of long-term series to better define future strategies, especially in the light of actual climate change. Future works and research programmes dedicated to the biogeochemical budget, biological processes, and integration with a modelling approach could provide further understanding, and this will be part of our future study.