Biological Early Warning Systems: the Experience in the Gran Sasso-Sirente Aquifer

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Article Biological Early Warning Systems: The Experience in the Gran Sasso-Sirente Aquifer

Federica Di Giacinto 1,* , Miriam Berti 1, Luigi Carbone 2, Riccardo Caprioli 1,3, Valentina Colaiuda 4,5 , Annalina Lombardi 4, Barbara Tomassetti 4, Paolo Tuccella 5, Gianpaolo De Iuliis 6, Adelina Pietroleonardo 7, Mario Latini 8, Giuseppina Mascilongo 1, Ludovica Di Renzo 1, Nicola D’Alterio 1 and Nicola Ferri 1

1 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZSAM), 64100 Teramo, Italy; [email protected] (M.B.); [email protected] (R.C.); [email protected] (G.M.); [email protected] (L.D.R.); [email protected] (N.D.); [email protected] (N.F.) 2 Officine Inovo, Engineering & Design Studio, 64100 Teramo, Italy; info@officineinovo.it 3 Agenzia Regionale Protezione Ambiente del Lazio—ARPA Lazio, 00187 Rome, Italy 4 CETEMPS, Centre of Excellence—University of L’Aquila, 67100 L’Aquila, Italy; [email protected] (V.C.); [email protected] (A.L.); [email protected] (B.T.) 5 Department of Physical and Chemical Sciences, University of L’Aquila, 67100 L’Aquila, Italy; [email protected] 6 Ruzzo Reti Spa, 64100 Teramo, Italy; [email protected] 7 Consorzio di Bonifica Interno “Bacino Aterno e Sagittario”, 67100 L’Aquila, Italy; [email protected] 8 World Organization for Animal Health—OIE, 75017 Paris, France; [email protected] * Correspondence: [email protected]; Tel.: +39-0861-3321

Citation: Di Giacinto, F.; Berti, M.; Abstract: Biological early warning systems (BEWS) are installed worldwide for the continuous Carbone, L.; Caprioli, R.; Colaiuda, V.; control of water intended for multiple uses. Sentinel aquatic organisms can alert us to contaminant Lombardi, A.; Tomassetti, B.; Tuccella, presence through their physiological or behavioural alterations. The present study is aimed at P.; De Iuliis, G.; Pietroleonardo, sharing the experience acquired with water biomonitoring of the Gran Sasso-Sirente (GS-S) aquifer. A.; et al. Biological Early Warning It represents the major source of the Abruzzi region surface water, also intended as drinkable and Systems: The Experience in the Gran for irrigation use. Besides the biomonitoring of drinkable water of the Teramo Province made by Sasso-Sirente Aquifer. Water 2021, 13, 1529. https://doi.org/10.3390/ Daphnia Toximeter and irrigation water of the L’Aquila Province by Algae Toximeter, a novel sensor w13111529 named “SmartShell” has been developed to register the behaviour of the “pea clam” P. casertanum, an autochthonous small bivalve living in the Nature 2000 site “Tirino River spring”. The valve Academic Editor: movements have been recorded directly on the field. Its behavioural rhythms have been analysed Nikolaos Skoulikidis through spectral analyses, providing the basis for further investigations on their alterations as early warnings and allowing us to propose this autochthonous bivalve species as a novel sentinel organism Received: 30 April 2021 for spring water. Accepted: 27 May 2021 Published: 29 May 2021 Keywords: biological early warning system; Gran Sasso-Sirente aquifer; valvometry; behaviour; Pisidium casertanum Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Contaminants from industrial and urban sources can be accidentally or intentionally discharged at unpredictable times into the water. Common water monitoring programs are inadequate to ensure 24 h control, as they are organized by punctual samplings at pre- Copyright: © 2021 by the authors. established periods of time [1]. Moreover, chemical analysis tools are not able to determine Licensee MDPI, Basel, Switzerland. the concentrations of every compound existing in a water system due to time, cost and This article is an open access article technical limitations [2]. Combined toxic effects, including synergetic and antagonistic ones, distributed under the terms and cannot be identified by chemical analysis tools. These limitations lead to the development conditions of the Creative Commons of biological early warning systems (BEWS), which are based on the response of living Attribution (CC BY) license (https:// sentinels (i.e., molluscs, algae, crustaceans, fish) to a contaminant or mixture of them [3]. creativecommons.org/licenses/by/ BEWSs perform a real-time and continuous (24 h) monitoring of physiological and/or 4.0/).

Water 2021, 13, 1529. https://doi.org/10.3390/w13111529 https://www.mdpi.com/journal/water Water 2021, 13, 1529 2 of 19

behavioural parameters of organism alterations, potentially correlated to water pollution. Early warnings can be sent by SMS, e-mail, etc., to activate response actions [4]. The first research and technological advances on BEWS started in the early 1900s [5,6]. From initial trials based on the simple registration of the organism activities, it passed to the current advanced computational techniques such as video analysis [4–7]. Based on multiple endpoint parameters, numerous instrument models are available on the market [8]. Daphnia Toximeter (DTOX) and Fish Toximeter (®bbe moldaenke) analyse the swimming behaviour of Daphnia magna and fish, respectively. Mosselmonitor® (aquadect company) registers mollusc valve movements [9,10]. Easychem® Tox Early Warning (SYSTEA s.p.a.) and Toxcontrol® (microLAN) register bacteria bioluminescence alterations. Algae Toximeter (ATOX) (®bbe moldaenke) analyses photosynthetic activity inhibition of green microalgae. Moreover, many tools have been developed and applied by research institutes without the commercial scope, i.e., mussel actograph for shell gape monitoring [11], automated Grid Counter device for abnormal activity of D. magna [12], video-based movement analysis system to quantify behavioural stress responses of fish [13]. The Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZSAM) has developed a novel sensor named “SmartShell” for continuous monitoring of valve movements of marine and freshwater bivalves, capable of registering the movements of very small molluscs (height > 2 mm) impossible to allocate in other commercial tools [14]. Preferably, BEWSs are used in multiple panels covering different trophic levels, while the selection has to consider species-sensibility to contaminants according to pollution risk assessment of monitoring locations [15]. Belonging to decomposer trophic level, bacteria are very sensitive to a wide range of chemicals applicable to wastewater, surface water and groundwater [16,17]. Algae, as primary producers, are vulnerable to pesticides and metals [18] and are mainly used for surface water monitoring. Aquatic invertebrates, as primary consumers, are commonly used for ecotoxicological studies. The crustacean Daphnia represents the motile model for behavioural study: its swimming behaviour is recognised as a very sensitive biomarker in toxicity assessment [19,20]. Heavy metals, disinfection by-products, pesticides, etc., have been detected early in groundwater, surface water and wastewater through the water flea swimming alterations [19]. Bivalves, as filter feeder consumers with limited motility, occupy a prominent place in the passive and active biomonitoring [21]. They can accumulate contaminants and their valve movement behaviour is considered an important biomarker in ecotoxicology [22]. Mussels are mainly used for surface water monitoring by alerting the presence of organic and inorganic chemicals [21,22]. As fish are high on the aquatic food chain, they have been studied for decades considering many parameters (swimming, respiratory exchange, etc.) susceptible to low levels of contaminants in surface water and groundwater [8–23]. BEWSs have been installed worldwide, mostly after massive spills or in vulnerable locations. In China, during the Beijing Olympic Games, 40 BEWSs have been installed in water plants and in environmental monitoring stations in Beijing, Tianjin, Ji’nan, Guangzhou and Ningbo [4,24]. After the terrorist attack of 11 September 2001, the United States of America developed the Homeland Security Strategy applying BEWSs such as DTOX, ATOX, and fish monitor in drinking water security [25]. In Europe, biological monitoring was also encouraged by the Water Framework Directive [26] that transformed the con- cept of water quality monitoring, giving more importance to the ecological protection of water [27]. Nowadays, in the Rhine River, several physicochemical probes and BEWSs (DTOX, ATOX, mosselmonitor®) are working to rapidly detect pollution [28]. In Italy, one mosselmonitor® has been installed in the drinking water supply of Turin [29] and another one is present in Ferrara [30]. Currently, DTOXs continuously monitors drinkable water of Rome and Teramo. Tap water of the Teramo province (Abruzzi region) is drained from the Gran Sasso (GS) aquifer, which is recognised as a vulnerable water source subjected to the Water Safety Plans at the national level [31,32], due to the underground infrastructures built inside [33]. In the 1980s, two highway tunnels and the Italian National Institute of Nuclear Physics Water 2021, 13, 1529 3 of 19

laboratory (INFN) were excavated in the core of the aquifer, posing a potential risk on its quality. In 2003, the presence of anomalous organic substances was found in its spring water [34]. Furthermore, on 9 May 2017, Teramo citizens were not authorized to drink potable water [35]. At the moment, an official multidisciplinary team is implementing the action plan according to the Water Safety Plans principles to ensure safe drinking water in Teramo city. The continuous monitoring of tap water is one of the planned key actions. Within this framework, DTOX was installed at the collection of potable water of Teramo, close to the INFN and motorway tunnel, as a vulnerable site to be controlled 24 h. Water flowing from the Gran Sasso-Sirente (GS-S) aquifer is also exploited for irrigation use, since it constitutes the major part of regional surface water [36]. Pesticide pollution in aquatic ecosystems and in groundwater is a worldwide problem to be solved. A recent study assessed the ecological risk of pesticide mixtures in the alluvial aquifers in the Abruzzi region [37]. Continuous monitoring of irrigation water quality would avoid contamination and illegal use of banned substances that are yet persistent. Hence, IZSAM has started the monitoring of regional irrigation water through the BEWS “ATOX”, highly sensitive to pesticides. The GS-S aquifer feeds the Tirino River, which is considered one of the most important Italian “chalk-streams” [38]. Mainly constituted by groundwater, it exhibits less seasonal variation than other rivers, and it constitutes a very precious habitat. The first part of the Tirino River has been recognized as a Nature 2000 site (IT7110209). The surrounding area is subjected to agricultural and industrial activities, mostly in the urbanized area of Bussi sul Tirino and Popoli. Some water channellings have changed the natural assets of the river [39]. Considering the high environmental value, this water body should be under continuous control to avoid the loss of biodiversity. For this purpose, the novel sensor “SmartShell” presented in this study has been used for the first time to assess the behaviour of the pea clam (Pisidium casertanum), living in this area. It has been installed directly in the field, without any environmental modifications, which are necessary in the case of allochthonous indicator species use. The main aim of this work is to report the experience on water biomonitoring of the GS-S aquifer, focusing on the development of a novel bio-sensor. A never-tested autochthonous bivalve has been proposed as a new sentinel organism for spring water. For further information, an overview of the monitoring results of the GS-S aquifer as a whole is briefly reported in the results section. Innovative technologies and methods internationally recognised as efficient tools have been used for water quality control, safeguarding human and ecosystem health.

2. Materials and Methods 2.1. Study Area: The Gran Sasso-Sirente Aquifer (GS-S) The GS-S is situated in Central Italy and belongs to the Apennines ridge area, corresponding to part of the Gran Sasso–Laga National Park and part of the Sirente-Velino regional Park (Abruzzi region, Figure1). The aquifer system has an extent of about 700 km2 [40], and the total drained discharge estimated on its springs is more than 18 m3/s [41]. The hydrogeological system can be divided into two sub-units, corresponding to the GS aquifer, the wider the northern part, and the Sirente aquifer, in the south-western side of the Aterno River. From a structural-stratigraphic point of view, both units consist of fractured and karstified carbonate platforms [42]. According to Amoruso et al. [43], the GS is ~1000 m thick and retains 1010 m3 of water. It is surrounded by well-defined aquitards, made by flysch deposits laying in the northern and western boundaries down to the Bussi town: no subterranean water exchange is present in those areas due to the tectonical overlapping with clayish layers alternated to sandstone. Fluvial and lacustrine deposits and debris layers with low permeability are mainly located on the south-western border of the GS aquifer and inside the massifs. With its very high permeability, the endoeric basin of Campo Imperatore, in the middle of the Water 2021, 13, x FOR PEER REVIEW 5 of 19

The chemical/physical characteristics of the monitored water are listed in Table 1. The parameters have been registered with chemical/physical probe (AP-2000 model, aquaread®, Kent, UK).

Table 1. Characteristics of the monitored water.

BEWS婉Monitoring Dissolved Oxy- Conductivity婉 Monitoring Site Water Temp.婉(°C) pH Period gen (%) µS/cm 30/03–12/04/18婉–婉 Capestrano Tirino River spring 12.30–13.50 6.64–7.14 89.7–87.4 668–726 05/07–27/07/18

Other water biomonitoring experiences of GS-S have been placed at (Figure 1): • Casale San Nicola monitoring station (Isola del Gran Sasso, Teramo, Italy), where Water 2021, 13, 1529 groundwater collected close to highway tunnels and INFN laboratory (technically4 of 19 named “Ruzzo sbarramento destro + sinistro”) is continuously controlled by DTOX in the aqueduct network of the Teramo province (managed by Ruzzo Reti S.p.a.); • main Corfinio, ridge, is theMarane most and important Raiano rechargechannel areaand tanks for the (Sulmona, springs systems, L’Aquila, mainly Italy) located in the ir- in the northernrigation network and eastern managed boundary by Consorzio and characterized di Bonifica by de quitell’Aterno constant Sagittario discharge are mon- rates betweenitored 0.5 by and ATOX. 7 m3 /s.

Figure 1. Map of the monitoring sitesite inin thethe GS-SGS-S aquifer aquifer where where SmartShell SmartShell has has been been installed installed and and other other water water biomonitoring biomonitor- experienceing experience sites sites using using BEWSs. BEWSs. Boni et al. [44] have estimated a mean recharge rate in Campo Imperatore of about 2.3. SmartShell in the Tirino River Springs 700 mm/y, corresponding to the occurrence of an average precipitation of 1200 mm/y overSmartShell the same basin has been [45]. installed The hydrogeological in March 2018 structure at the isCapestrano NW-SE oriented, crayfish organized hatchery (Capestrano,in a sequence L’Aquila, of complex Italy). interconnected It is a novel sensor karstic developed reservoirs, by frequently IZSAM, capable interrupted to meas- by urelow-permeable the valve movements layers and faultsof bivalves, that influence remotely the and underground continuously, circulation with high [46 ],resolution resulting (>0.1in progressively mm). Differently decreasing from piezometricother systems, levels it does with not a hydrostructural require any attachments peak in the on upper the shells,part [47 thus]. Most reducing of the disturbance subterranean to the drainage mollusc flows and allowing south-westward, the use of feedingsmall size the bivalve Tavo, L’Aquila and Tirino springs, while a residual fraction is collected in the northern part, flowing into the Ruzzo springs. A hydrological subterranean exchange has been described between the GS carbonate massif and the Sulmona plain in the southern part [48]. The Gran Sasso mountain hosts the A24 motorway tunnel connecting the cities of L’Aquila and Teramo, as well as the INFN laboratories. The impressive infrastructure was built between 1969 and 1987 and consists of two 10 km-long tunnels placed at 970 m a.s.l, crossing the mountain core, and three 36,000 m3 rooms, serving as INFN laboratories, that are connected by minor passages. The artificial structure intercepts carbonate rocks and the argillic-marly deposit and had a great impact on the massif hydrostructure during its building, causing the piezometric surface to vertically decrease about 600 m [47–49]. As a consequence, springs discharge significantly decreased, especially in Ruzzo springs. Well points have been installed along the tunnel path to drain water coming from the upper reservoir: a system of two 5 km-long channels subdivides the drainage water between L’Aquila and Teramo provinces, where it is exploited for drinking uses, for a total amount of about 1.5 m3/s [42]. Water 2021, 13, 1529 5 of 19

The GS aquifer consists of 7 groundwater bodies, whose characteristics are summa- rized in Supplementary Table S1. The Sirente sub-system includes the southern part of the GS-S aquifer, respect to the Aterno river valley. Eastward, the Sirente unit is conﬁned by the Sulmona plain, where relevant groundwater exchange with the ﬂuvial and lacustrine deposits is present. The southern side of the aquifer is delimited by other overlapping carbonate deposits from the Marsicano massif, with no relevant groundwater exchanges, while the south-western limit is represented by the carbonate massif of Monte Pianeccia, where groundwater exchanges have been observed in the Celano plain. Finally, the northern boundary is characterized by water exchanges with the L’Aquila plain. The groundwater circulation scheme is described in Supplementary Table S2.

2.2. Monitoring Site SmartShell has been mounted directly on the field, at the monitoring site (Figure1 ) of GS-S water at Capestrano crayﬁsh hatchery (Capestrano, L’Aquila, Itay). Here, the Tirino spring water ﬂows directly in the hydraulic system of the hatchery. The Capestrano site is within Tirino springs which belong to the Colle Madonna groundwater catchment in the GS aquifer, but receive water also from other northern groundwater bodies up to the hydrostructural ridge in Gran Sasso. It is a group of springs that originates the namesake river, in three points: the Capo d’Acqua Lake (373 m a.s.l.), the Capestrano Lake (337 m a.l.m.) and Presciano (329 m a.l.m.). The left branch of the Tirino River arises from Capo d’Acqua, while the right branch results from the merging between Capestrano and Presciano. The chemical/physical characteristics of the monitored water are listed in Table1 . The parameters have been registered with chemical/physical probe (AP-2000 model, aquaread®, Kent, UK).

Table 1. Characteristics of the monitored water.

BEWS Temp. Dissolved Conductivity Monitoring Site Water pH Monitoring Period (◦C) Oxygen (%) µS/cm 30/03–12/04/18 Tirino River Capestrano – 12.30–13.50 6.64–7.14 89.7–87.4 668–726 spring 05/07–27/07/18

Other water biomonitoring experiences of GS-S have been placed at (Figure1): • Casale San Nicola monitoring station (Isola del Gran Sasso, Teramo, Italy), where groundwater collected close to highway tunnels and INFN laboratory (technically named “Ruzzo sbarramento destro + sinistro”) is continuously controlled by DTOX in the aqueduct network of the Teramo province (managed by Ruzzo Reti S.p.a.); • Corﬁnio, Marane and Raiano channel and tanks (Sulmona, L’Aquila, Italy) in the irrigation network managed by Consorzio di Boniﬁca dell’Aterno Sagittario are monitored by ATOX.

2.3. SmartShell in the Tirino River Springs SmartShell has been installed in March 2018 at the Capestrano crayﬁsh hatchery (Capestrano, L’Aquila, Italy). It is a novel sensor developed by IZSAM, capable to measure the valve movements of bivalves, remotely and continuously, with high resolution (>0.1 mm). Differently from other systems, it does not require any attachments on the shells, thus reducing disturbance to the mollusc and allowing the use of small size bivalve species (height > 2 mm). The measurement principle is based on the detection of the distance between the proximity infrared (IR) sensor and mollusc shell. The principal components of the SmartShell are shown in Figure2. Water 2021, 13, x FOR PEER REVIEW 6 of 19

species (height > 2 mm). The measurement principle is based on the detection of the dis- Water 2021, 13, 1529 tance between the proximity infrared (IR) sensor and mollusc shell. The6 of principal 19 components of the SmartShell are shown in Figure 2.

FigureFigure 2. 2.SmartShell SmartShell components: components: 1—sensor 1—sensor tank; 2—sensor tank; 2—sen tank cap;sor 3—IR tank sensor cap; 3—IR containment sensor containment chamber;chamber; 4—positioning 4—positioning system system of sensor; of 5—externalsensor; 5—external case containing case hub; containing 6—mollusc hub; support. 6—mollusc support.

It is based on the IR proximity sensor (model GP2Y0A41SK0F, sharp corporation, Japan)It and is anbased open-source on the hardwareIR proximity and software sensor platform(model calledGP2Y0A41SK0F, “Arduino” (https: sharp corporation, //www.arduino.cc/Japan) and an (accessedopen-source on 27 Novemberhardware 2020)) and [50 software]. Each mollusc platform is glued called on “Arduino” sensor(https://www.arduino.cc/ support and IR light direction (accessed can beon regulated27 November on the 2020)) shell by [50] the. positioningEach mollusc is glued on systemsensor of support the sensor. and The IR sensorlight direction detects the can distance be regulated of the object on and the convertsshell by it the into positioning sys- an analogue signal that is acquired by the hub acting as a data distribution node. The hubtem converts of the thesensor. signal The into asensor digital onedetects with the a resolution distance of 10of bitsthe and object sends and it to converts the it into an excelanalogue software signal on a scale that ranging is acquired from 0 toby 1023 the values. hub acting The IR sensoras a data has adistribution light emitting node. The hub diodeconverts (emitter), the asignal position-sensible into a digital photo one detector—PSD with a re (receiver)solution andof 10 a signal bits processingand sends it to the excel circuit.software After on reflecting a scale onranging the mollusc from shell, 0 to the1023 emitted values. light The beam IR hitssensor the PSD,has a whose light emitting diode conductivity(emitter), a depends position-sensible on beam fall position. photo Thedetector—P analogue distanceSD (receiver) value is proportionatedand a signal processing cir- to the outputted conductivity. The optical path of the reflected light beam is influenced by thecuit. different After refractivereflecting indices on the (n) mollusc of the media shell, it passesthe emitted through. light Emitted beam and hits reflected the PSD, whose con- lightductivity beam of depends SmartShell on sensor beam passes fall throughposition. air The (n = 1),analogue polycarbonate distance (n = 1.492)value and is proportionated waterto the (n outputted= 1.333). This conductivity. generates a difference The optical between path the measuredof the reflected distance light and actual beam is influenced distanceby the different in water according refractive to the indices description (n) of of the Figure media3. it passes through. Emitted and reflected All plastic components of SmartShell are designed ad hoc and manufactured in polyamidelight beam using of SubtractiveSmartShell Rapid sensor Prototyping passes technology.through air Except (n = for 1), the polycarbonate cap, the sensor (n = 1.492) and remainswater ( whollyn = 1.333). immersed This in generates the water during a difference the test. between the measured distance and actual distanceValve movementsin water according are registered to the every description 30 s, and a of graph Figure per mollusc3. is elaborated by theAll customized plastic components instrument software of SmartShell reporting theare opening designed of thead valveshoc and during manufactured the in pol- experimental time. An example has been reported in Figure4. yamide using Subtractive Rapid Prototyping technology. Except for the cap, the sensor remains wholly immersed in the water during the test.

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Figure 3. SmartShell: IR optical path and measurement of object distance.

Valve movements are registered every 30 s, and a graph per mollusc is elaborated by the customized instrument software reporting the opening of the valves during the experimental time. An example has been reported in Figure 4. Further data analysis has established the maximum percentage of opening per specimen, elaborating the valve gape (VG). Five ranges of valve gapes (VG) have been established for the frequency calculation of results: VG ≤ 20%, 21–40%, 41–60%, 61–80% and ≥81% [51]. Time spent in each VG has been calculated by multiplying the number of observations in each VG type per registration time (30 s). Figure 3.3. SmartShell: IR optical path and measurement of object distance.

FigureFigure 4. 4. GraphGraph continuously continuously calculated calculated by by SmartShell SmartShell software software with with the the XX-axis-axis showing showing “time” “time” and and the the YY-axis-axis showing showing “valve“valve opening”. opening”. Each mollusc (named(named withwith code code “HUBxx-xx) “HUBxx-xx) has has a distincta distinct pattern pattern highlighted highlighted with with speciﬁc specific colours, colours, dark darkblue blue and and light light blue. blue.

TheFurther sentinel data organism analysis chosen has established for the study the maximum was the “pea percentage clam” P. of casertanum opening per, a small speci- freshwatermen, elaborating bivalve thespecies valve autochthonous gape (VG). Five in rangesthe Tirino of valve River. gapes Study (VG) animals have beenwereestab- col- lectedlished near for thethe frequencymonitoring calculation site, measuring of results: an average VG ≤ 20%, shell 21–40%,length equal 41–60%, to 361–80% mm. They and were≥81% immediately [51]. Time observed spent in eachby stereomicroscope VG has been calculated (wild M3, by Heerbrugg, multiplying Switzerland) the number to of verifyobservations their vitality. in each The VG live type specimens per registration were first time acclimatized (30 s). in a tank with an open river

water Theflow sentinel rate of almost organism 20 chosenL/h, in IZSAM’s for the study crayfish was hatc the “peahery clam”at the P.Tirino casertanum River., a small Figure 4. Graph continuouslyfreshwater calculated bivalve by SmartShell species software autochthonous with the inX-axis the Tirinoshowing River. “time” Study and animalsthe Y-axis were showing collected “valve opening”. Each molluscnear (named the monitoring with code site,“HUBxx-xx) measuring has a an distinct average pattern shell highlighted length equal with to specific 3 mm. colours, They were dark blue and light blue. immediately observed by stereomicroscope (wild M3, Heerbrugg, Switzerland) to verify their vitality. The live specimens were first acclimatized in a tank with an open river water flowThe rate sentinel of almost organism 20 L/h, inchosen IZSAM’s for the crayfish study hatcherywas the “pea at the clam” Tirino P. River.casertanum, a small freshwaterThe taxonomic bivalve species investigations autochthonous were carried in the outTirino in theRiver. laboratory Study animals with the were use col- of morphometriclected near the analytical monitoring keys site [52, measuring] using a scanning an average electron shell microscope—SEM length equal to 3 (Zeiss mm. DSMThey 940were A—Carl immediately Zeiss Oberkochen, observed by Germany) stereomicroscope (Figure5)[ (wild53] and M3, through Heerbrugg, the biomolecular Switzerland) test. to verify their vitality. The live specimens were first acclimatized in a tank with an open river water flow rate of almost 20 L/h, in IZSAM’s crayfish hatchery at the Tirino River.

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The taxonomic investigations were carried out in the laboratory with the use of mor- phometric analytical keys [52] using a scanning electron microscope—SEM (Zeiss DSM 940 A—Carl Zeiss Oberkochen, Germany) (Figure 5) [53] and through the biomolecular test. The taxonomic investigations were carried out in the laboratory with the use of mor- phometricTotal DNA analytical was extracted keys from [52] soft using tissue a samples scanning by Maxwell electron® 16 microscope—SEM Tissue DNA Pu- (Zeiss DSM rification940 A—Carl Kit (Promega, Zeiss Oberkochen,Madison, WI, USA) Germany) and tested (Figure by conventional 5) [53] andPCR withthrough specific the biomolecular primerstest. [54] amplifying a portion of 700 bp of the Cytochrome Oxidase I (COI) gene. All PCR products were sequenced using the Sanger method. The consensus sequences were Total DNA was extracted from soft tissue samples by Maxwell® 16 Tissue DNA Pu- obtained using Unipro UGENE v.32 (Okonechnikov K, Golosova O, Fursov M, the UGENErification team. Kit Unipro (Promega, UGENE: Madison, a unified WI, bioinfor USA)matics and toolkit. tested Bioi bynformatics conventional 2012 28:PCR with specific 1166–1167.primers [54]doi:10.1093/bioinformatics/bts091) amplifying a portion of 700 and bp BLASTn of the analysisCytochrome was performed Oxidase toI (COI) gene. All

Water 2021, 13, 1529 identifyPCR products the closest were publicly sequenced available sequen usingce the in the Sanger Genbank method. database. The The consensus highest8 ofnu- 19 sequences were cleotideobtained identity using (100%) Unipro was evidencedUGENE withv.32 Pisidium (Okonechnikov casertanum, alreadyK, Golosova registered O, as Fursov M, the autochthonous species in the Tirino River [55]. UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 2012 28: 1166–1167. doi:10.1093/bioinformatics/bts091) and BLASTn analysis was performed to identify the closest publicly available sequence in the Genbank database. The highest nucleotide identity (100%) was evidenced with Pisidium casertanum, already registered as autochthonous species in the Tirino River [55].

(a) (b)

Figure 5. Morphometrics analyses of valves of P. casertanum.(. (a) Insight view of right valve with cardinal tooth c3, anterior lateral teeth a1 and a3, posterior lateral teeth p1 and p3. (b) Insight view of left valve with cardinal teeth c2 and c4, anterior lateral teeth a1 and a3, posterior lateral teeth p1 and p3. (b) Insight view of left valve with cardinal teeth c2 and c4, anterior lateral tooth a2, posterior lateral tooth p2. lateral tooth a2, posterior lateral tooth p2.

AfterTotal DNAacclimatization was extracted of 15–20 from days, soft tissue molluscs samples were by inserted Maxwell on® 16the Tissue support DNA of SmartShellPurification (Figure Kit (Promega, 6). Two groups Madison, of 2 WI, indivi USA)duals and were tested observed by conventional directly on PCR the field, with respectively,specific primers for [5412] and amplifying 23 days, a by portion putting of 700 them bp ofin thethe Cytochromesame tank of Oxidase the acclimatization I (COI) gene. (withAlla) PCR a continuous products river were flow sequenced rate. using the Sanger method. The consensus(b) sequences were obtained using Unipro UGENE v.32 (Okonechnikov K, Golosova O, Fursov M, the Figure 5. Morphometrics analysesUGENE of team. valves Unipro of P. UGENE:casertanum a unified. (a) Insight bioinformatics view of right toolkit. valve Bioinformatics with cardinal 2012 tooth 28: c3, anterior lateral teeth a1 and a3, posterior1166–1167. lateral doi:10.1093/bioinformatics/bts091) teeth p1 and p3. (b) Insight view andof left BLASTn valve with analysis cardinal was performed teeth c2 and to c4, anterior lateral tooth a2, posterior lateralidentify tooth the closestp2. publicly available sequence in the Genbank database. The highest nucleotide identity (100%) was evidenced with Pisidium casertanum, already registered as autochthonousAfter acclimatization species in the Tirino of River 15–20 [55 ].days, molluscs were inserted on the support of After acclimatization of 15–20 days, molluscs were inserted on the support of SmartShell SmartShell (Figure 6). Two groups of 2 individuals were observed directly on the field, (Figure6). Two groups of 2 individuals were observed directly on the field, respectively, for 12respectively, and 23 days, by for putting 12 and them 23 in days, the same by tankputting of the them acclimatization in the same with tank a continuous of the acclimatization riverwith flow a continuous rate. river flow rate.

Figure 6. One specimen of P. casertanum installed on the support of SmartShell.

FigureFigure 6. One6. One specimen specimen of P. casertanum of P. casertanuminstalled oninstalled the support on the of SmartShell. support of SmartShell.

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2.4. Spectral Analysis of Valve Movements In order to investigate dominant frequencies of molluscs’ valve behaviour, the Fourier transform has been applied to opening time series of molluscs HUB03-01 of the ﬁrst test and HUB03-02 of the second test [4,56]. The transform decomposes a signal function represented in a time domain (i.e., the opening time-series) into its characteristic sinusoidal components in the complex ﬁeld, characterized by their own amplitudes, phases and frequencies. The absolute value of the transform is the original frequency value of the original time- series, while the complex argument represents the phase offset of predominant sinusoidal functions that compose the signal. The discrete Fourier transform Y(k) for a given time- series of n samplings is derived by the following formula:

j=n (j−1)(k−1) Y(k) = ∑ X(j)Wn , (1) j=1

where k = 0, . . . ,n − 1 and function W is given by:

(−2πi)/n Wn = e . (2)

Before carrying out the spectral analysis, the raw signal of the opening of both molluscs has been filtered, in order to remove spike values able to alter results. Primarily, the first two days of sampling were removed from the two time-series, as they correspond to an acclimatization period for the P. casertanum due to the instrument installation that inevitably perturbed surrounding environmental conditions. Subsequently, spike opening values lower than 701 and 652 were removed from mollusc HUB03-01 and mollusc HUB03-02 measurements, respectively. Those values can be considered unreliable opening data because they are under the minimum value appointed during the positioning closed mollusc. Finally, the resulting signal has been smoothed using a moving average filter with a span of 240 samplings. Before the application of the Fourier transform, the presence of a trend in both molluscs opening time-series has been verified through a regression analysis, consequently, data have been detrended using the ordinary last squares method as described in Warner [57].

2.5. Other Biomonitoring Experiences in GS-S: DTOX in Drinkable Water of Teramo Province and ATOX for Control of Irrigation Water of L’Aquila DTOX has been installed in April 2019 at Casale San Nicola monitoring station (Isola del Gran Sasso, Teramo, Italy). It is a BEWS able to detect toxic substances in the water through the registration of water flea behaviour anomalies. The measurement principle is based on a statistical analysis of swimming trajectories of 20 crustaceans (Daphnia magna) in a flow of water sample of 2–3 L/h, passing through the two measurement chambers [58,59]. The scheme of the principal components and the analytical procedures are available at https://www.bbe-moldaenke.de (accessed on 27 November 2020). The results of the monitoring programme (24 h) with DTOX, the weekly Toxic Index, are presented in this article from 4 January 2020 until 31 March. ATOX has been installed in April 2019 at IZSAM laboratory of Teramo city. It is a BEWS enable to detect toxic substances in the water through the registration of green microalgae photosynthetic activity inhibition. The measurement principle is based on the determination of the fluorescence spectrum and fluorescence kinetics of algae [60]. The scheme of the principal components and the analytical procedures are available at https://www.bbe-moldaenke.de (accessed on 27 November 2020). Surface water has been sampled at the irrigation net managed by the Consorzio di Bonifica del Bacino dell’Aterno—Sagittario. Three hundred litres of water were collected from each sampling site and immediately transported in a tank to the lab. The duration of each analysis was almost 7 days. Data on % inhibition have been analysed. Water 2021, 13, x FOR PEER REVIEW 10 of 19

Water 2021, 13, x FOR PEER REVIEW 10 of 19 3. Results Water 2021, 13, 1529 3.1. SmartShell in Tirino River Spring 10 of 19 3. ResultsValve movements of four specimens belonging to P. casertanum have been observed 3.1.3.directly SmartShell Results on in Tirinothe field River at Spring the crayfish hatchery of Capestrano [AQ]. Two individuals have 3.1.beenValve SmartShell simultaneously movements in Tirino of River four analysed Spring specimens from belonging 30 March to P. casertanum 2018 to 12have April been 2018observed (first study group). directlyTheValve onother the movements twofield wereat the of four crayfishexamined specimens hatche from belongingry of 5 CapestranoJuly to P. to casertanum 28 July[AQ]. have2018 Two been (secondindividuals observed study have group). The first beendirectlygroup simultaneously on showed the field atanalyseddissimilar the crayfish from behaviours: hatchery 30 March of Capestrano2018 one to individual12 [AQ].April Two 2018 individuals(HUB03-01) (first study have group). registered a flapping been simultaneously analysed from 30 March 2018 to 12 April 2018 (first study group). The Thegraph other two with were the examined alternation from of 5 activityJuly to 28 and July inactivity2018 (second cycles study ofgroup). valve The movements. first The other groupother showed two were dissimilar examined frombehaviours: 5 July to 28one July individual 2018 (second (HUB03-01) study group). registered The first groupa flapping showedone (HUB03-02) dissimilar behaviours: had the onevalves individual almost (HUB03-01) always opened, registered awithout flapping graphany flapping activity (Fig- graph with the alternation of activity and inactivity cycles of valve movements. The other withure the7). alternation At the end of activity of the and test, inactivity the mollusc cycles of valvewas movements.alive and Thewas other incubating one seven juveniles one(HUB03-02) (HUB03-02) had had the the valves valves almost almost always always opened, opened, without without any flapping any flapping activity (Figure activity7). (Fig- ureAt ready7). the At end theto of beend the released test,of the the test, mollusc (Figure the wasmollusc 8). alive was and wasalive incubating and was sevenincubating juveniles seven ready juveniles to readybe released to be released (Figure8 (Figure). 8).

FigureFigureFigure 7. RawRaw 7. Raw patterns patterns patterns of valveof valvevalve openings openings openings of of two two of individuals individuals two individuals of P.P. casertanumcasertanum of P.included casertanumincluded in thein the ﬁrstincluded first study study group, in group,the from first from 30 study 30 group, from 30 March 2018 to 12 April 2018: HUB03-01 in blue colour, HUB03-02 in heavenly colour. MarchMarch 2018 2018 to 12 to 12April April 2018: HUB03-01 HUB03-01 in blue in colour,blue colour, HUB03-02 HUB03-02 in heavenly colour.in heavenly colour.

Figure 8. Mollusc HUB03-02: adult incubating 7 juveniles ready to be released (stereomicroscope 40× magnification).

In the second group of molluscs, the HUB03-01 mollusc (dark blue in Figure 9) was almostFigureFigure always 8. Mollusc8. Mollusc open HUB03-02: with HUB03-02: frenetic adult incubating and adult cont incuba 7inuous juvenilesting valve ready 7 juveniles tomovements be released ready (stereomicroscopefor to the be first released 10 days, (stereomicroscope then4040× ×themagniﬁcation). magnification). specimen died (Figure 9). The other bivalve HUB03-02, highlighted with the light blue colourIn the had second clearly group distinguishable of molluscs, the periods HUB03-01 of activity mollusc and (dark inactivity, blue in Figure repeated9) was within 24 almosth. SuchIn always periods the opensecond did with not group freneticseem to andof be molluscs, continuousrelated to valvethe photoperiod. HUB03-01 movements forThismollusc the mollusc ﬁrst 10(dark days,was blue alive in Figure 9) was at thethenalmost end the of specimen always the test. died open (Figure with9). Thefrenetic other bivalve and cont HUB03-02,inuous highlighted valve movements with the light for the first 10 days, blue colour had clearly distinguishable periods of activity and inactivity, repeated within 24then h. Such the periods specimen did not died seem (Figure to be related 9). toThe the photoperiod.other bivalve This HUB03-02, mollusc was alive highlighted at with the light theblue end colour of the test. had clearly distinguishable periods of activity and inactivity, repeated within 24 h. Such periods did not seem to be related to the photoperiod. This mollusc was alive at the end of the test.

Figure 9. Raw patterns of valve openings of the two individuals of P. casertanum included in the second study group, from 5 to 28 July 2018: HUB03-01 in blue colour, HUB03-02 in heavenly colour.

Water 2021, 13, x FOR PEER REVIEW 10 of 19

3. Results 3.1. SmartShell in Tirino River Spring Valve movements of four specimens belonging to P. casertanum have been observed directly on the field at the crayfish hatchery of Capestrano [AQ]. Two individuals have been simultaneously analysed from 30 March 2018 to 12 April 2018 (first study group). The other two were examined from 5 July to 28 July 2018 (second study group). The first group showed dissimilar behaviours: one individual (HUB03-01) registered a flapping graph with the alternation of activity and inactivity cycles of valve movements. The other one (HUB03-02) had the valves almost always opened, without any flapping activity (Fig- ure 7). At the end of the test, the mollusc was alive and was incubating seven juveniles ready to be released (Figure 8).

Figure 7. Raw patterns of valve openings of two individuals of P. casertanum included in the first study group, from 30 March 2018 to 12 April 2018: HUB03-01 in blue colour, HUB03-02 in heavenly colour.

Figure 8. Mollusc HUB03-02: adult incubating 7 juveniles ready to be released (stereomicroscope 40× magnification).

In the second group of molluscs, the HUB03-01 mollusc (dark blue in Figure 9) was almost always open with frenetic and continuous valve movements for the first 10 days, then the specimen died (Figure 9). The other bivalve HUB03-02, highlighted with the light blue colour had clearly distinguishable periods of activity and inactivity, repeated within Water 2021, 13, 1529 24 h. Such periods did not seem to be related to the photoperiod. This mollusc11 was of 19 alive at the end of the test.

WaterWater 2021 2021, 13, 13, x, FORx FOR PEER PEER REVIEW REVIEW 1111 of of 19 19 Figure Figure 9. Raw 9. patternsRaw patterns of valve of valve openings openings of ofthe the two two individuals individuals ofofP. P. casertanum casertanumincluded included in the in secondthe second study study group, group, from 5 from 5 to 28to July 28 July 2018: 2018: HUB03-01 HUB03-01 in inblue blue colour, colour, HUB03-02 HUB03-02 inin heavenly heavenly colour. colour.

TheTheThe patterns patterns patterns of of ofthe the the two two two molluscs molluscs molluscs included included included in in inthe the the second second second study study study group group group are are are better better better detailed detailed detailed inin inFigures Figures Figures 10 10 10and and and 11. 11. 11 The The. The HUB03-01 HUB03-01 HUB03-01 was was was always always always open open open with with with a acontinuous continuous a continuous flapping flapping ﬂapping from from from10 10 toto 1012 12 toJuly July 12 July(Figure (Figure (Figure 10) 10) until10 until) until its its death itsdeath death (Figur (Figur (Figuree e11). 11). 11 The ).The The HUB03-02 HUB03-02 HUB03-02 (light (light (light blue blue blue in in inthe the the graph), graph), graph), presentedpresentedpresented an an aninitial initial initial trend trend trend more more more irregular irregular irregular in in init sitits srhythms rhythms rhythms (Figure (Figure (Figure 10), 10), 10 with), with with long long long intervals intervals intervals of of of activityactivityactivity interspersed interspersed interspersed with with with short short short periods periods periods of of ofin inactivity. inactivity.activity. This This This behaviour behaviour behaviour became became became more more more regular regular regular duringduringduring the the the following following following days, days, days, with with with three three three or or orfour four four activity/inactivity activity/inactivity activity/inactivity cycles cycles cycles per per perday day day (Figure (Figure (Figure 11). 11). 11 ).

FigureFigureFigure 10. 10. 10. Zoom ZoomZoom of of ofthe the the raw raw raw patterns patterns patterns of of ofvalve valve valve openings openings openings of of oftwo two two individuals individuals individuals of of ofP. P. P.casertanum casertanum casertanum included includedincluded in in inthe the the second second second study study study group,group,group, from from from 10 10 10to to to12 12 12July July July 2018: 2018: 2018: HUB03-01 HUB03-01 HUB03-01 in in inblue blue blue colour, colour, colour, HUB03-02 HUB03-02 HUB03-02 in in inheavenly heavenly heavenly colour. colour. colour.

FigureFigureFigure 11. 11. 11. Zoom ZoomZoom of of ofthe the the raw raw raw patterns patterns patterns of of ofvalve valve valve openings openings openings of of oftwo two two individuals individuals individuals of of ofP. P. P.casertanum casertanum casertanum included includedincluded in in inthe the the second second second study study study group,group,group, from from from 19 19 19to to to21 21 21July July July 2018: 2018: 2018: HUB03-01 HUB03-01 HUB03-01 in in inblue blue blue colour, colour, colour, HUB03-02 HUB03-02 HUB03-02 in in inheavenly heavenly heavenly colour. colour. colour.

TheTheThe mollusc mollusc mollusc behaviour behaviour behaviour was was was further further further investig investig investigatedatedated by by by evaluating evaluating evaluating the the the extent extent extent of of ofvalve valve valve openingopeningopening during during during the the the study study study period. period. period. Figures Figures Figures 12 12 12 an an andd d13 13 13 show show show the the the average average average percentage percentage percentage of of oftime time time spentspentspent by by by molluscs molluscs molluscs with with with given given given VG VG VG ranges. ranges. ranges. InIn the the first first study study group group (Figure (Figure 12), 12), mollus mollusc cHUB03-01 HUB03-01 showed showed a aflapping flapping behaviour, behaviour, withwith about about 50% 50% of of the the time time spent spent in in the the VG VG category category of of 21–40%. 21–40%. Conv Conversely,ersely, mollusc mollusc HUB03- HUB03- 0202 was was always always fixed fixed at at VG VG equal equal to to 61–80% 61–80% without without any any valve valve movements movements (Figure (Figure 12). 12). InIn the the second second study study group, group, the the mollusc mollusc HUB0 HUB03-013-01 spent spent 90% 90% of of the the time time at at VG VG of of 61– 61– 80%,80%, the the other other HUB03-02 HUB03-02 ranged ranged equally equally between between the the two two VG VG categories categories 21–40% 21–40% and and 41– 41– 60%60% (Figure (Figure 13). 13).

Water 2021, 13, 1529 12 of 19 WaterWater 2021 2021, ,13 13, ,x x FOR FOR PEER PEER REVIEW REVIEW 1212 of of 19 19

Figure 12. Average percentage of time spent in established valve gape ranges by molluscs in the Figure 12. AverageFigure 12. percentage Average percentage of time spent of time in established spent in estab valvelished gape valve ranges gape by ranges molluscs byin molluscs the ﬁrst in study the group, from firstfirst study study group, group, from from 30 30 March March 2018 2018 to to 12 12 April April 2018. 2018. Valve Valve gapes gapes is is reported reported as as % % of of valve valve 30 March 2018 to 12 April 2018. Valve gapes is reported as % of valve openings. openings.openings.

Figure 13. AverageFigureFigure 13. 13. percentage Average Average ofpercentagepercentage time spent of of intime time established spent spent in in valveestabestablishedlished gape rangesvalve valve gape bygape molluscs ranges ranges by inby themolluscs molluscs second in in study the the group, from second study group, from 5 to 28 July 2018. Valve gapes is reported as % of valve openings. 5 to 28 Julysecond 2018. Valve study gapes group, is reportedfrom 5 toas 28 % July of valve2018. openings.Valve gapes is reported as % of valve openings.

FourierFourier transformtransformIn the analysisanalysis first study ofof valve groupvalve movemove (Figurementsments 12), molluschashas beenbeen HUB03-01 appliedapplied to showedto thethe molluscmollusc a flapping behaviour, showingshowing aa flappingflappingwith about behaviour:behaviour: 50% of HUB03-01HUB03-01 the time spent ofof thth inee firstfirst the VG studystudy category group,group, of fromfrom 21–40%. 3030 MarchMarch Conversely, 20182018 toto mollusc HUB03- 1212 AprilApril 2018,2018, 02 andand was toto alwaysHUB03-02HUB03-02 fixed ofof at thethe VG secondsecond equal to studystudy 61–80% group,group, without fromfromany 55 toto valve 2828 JulyJuly movements 2018.2018. AsAs (Figure 12). shownshown inin FigureFigure 14,14, thethe graphgraph ofof thethe dominantdominant periodperiod resultingresulting fromfrom spectralspectral analysisanalysis ofof

Water 2021, 13, 1529 13 of 19

In the second study group, the mollusc HUB03-01 spent 90% of the time at VG of 61–80%, the other HUB03-02 ranged equally between the two VG categories 21–40% and 41–60% (Figure 13). Fourier transform analysis of valve movements has been applied to the mollusc Water 2021, 13, x FOR PEER REVIEW 13 of 19 showing a flapping behaviour: HUB03-01 of the first study group, from 30 March 2018 to 12 April 2018, and to HUB03-02 of the second study group, from 5 to 28 July 2018. As shown in Figure 14, the graph of the dominant period resulting from spectral analysis of thethe mollusc mollusc HUB03-01 HUB03-01 detected detected ~4 ~4 daily daily domi dominantnant periods periods and and 2 2 periods periods slightly slightly exceed- exceeding ing1 1 day. day.The The secondsecond mollusc,mollusc, HUB03-02, showed showed dominant dominant periods periods in in ~5–6 ~5–6 days, days, and and ~4 ~4 activityactivity periods periods per per day day (Figure (Figure 15). 15 In). Table In Table 2, 2further, further detail detail on on the the first first six six dominant dominant periodsperiods for for both both molluscs molluscs is given. is given.

FigureFigure 14. 14.FourierFourier transform transform analysis analysis of ofvalve valve openin openingsgs of of mollusc mollusc HUB03-01 HUB03-01 in in the the first ﬁrst study study group, group,from from 30 March 30 March 2018 2018 to 12 to April 12 April 2018 2018 (x-axis (x-axis in semi-logarithmic in semi-logarithmic scale). scale).

Figure 15. Fourier transform analysis of valve openings of mollusc HUB03-02 in the second study FigureFigure 15. 15.FourierFourier transform transform analysis analysis of valve of valve openin openingsgs of mollusc of mollusc HUB03-02 HUB03-02 in the in second the second study study group, from 5 to 28 July 2018 (x-axis in semi-logarithmic scale). group,group, from from 5 to 5 28 to 28July July 2018 2018 (x-a (x-axisxis in semi-logarithmic in semi-logarithmic scale). scale).

st th Table 2. Dominant periods (hours/cycles) in descending order from the 1st to the 6th for HUB03-01 and HUB03-02.

Dominant Period HUB03-01 HUB03-02 1st 25.70 124.00 2nd 40.04 166.03 3rd 34.33 26.20

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Table 2. Dominant periods (hours/cycles) in descending order from the 1st to the 6th for HUB03-01 and HUB03-02.

Water 2021, 13, x FOR PEER REVIEW Dominant Period HUB03-01 HUB03-02 14 of 19

1st 25.70 124.00 2nd 40.04 166.03 3rd 34.33 26.20 4th4th 14.14 14.14 29.29 29.29 5th5th 16.02 16.02 13.46 13.46 6th6th 10.92 10.92 14.65 14.65

3.2.3.2. DTOXDTOX inin DrinkableDrinkable Water Water of of Teramo Teramo Province Province and and ATOX ATOX for for Control Control of of Irrigation Irrigation Water Water of ofL’Aquila L’Aquila BiomonitoringBiomonitoring ofof drinkable water of of Teramo Teramo province province from from 4 4January January 2020 2020 until until 31 31March March 2020 2020 waswas performed performed continuously continuously pe performedrformed by by DTOX (24(24 h)h) withoutwithout anyany launchedlaunched alarms. alarms. Examples Examples of of the the recorded recorded weekly weekly toxic toxic index index are are reported reported in in Figure Figure 16 16..

FigureFigure 16. 16.Toxic Toxic index index combined combined of of analysis analysis from from 20 20 to to 27 27 February February 2020. 2020.

AllAll surface surface samples samples collected collected from from the the irrigation irrigation net net showed showed almost almost 0% 0% of of inhibition inhibition inin ATOX ATOX (Table (Table3). 3). Accordingly, Accordingly, no no alarms alarms were were launched. launched.

TableTable 3. 3.ATOX ATOX monitoring monitoring data. data.

MaximumMaximum 婉% Inhibi- MinimumMinimum婉% In- AnalysingAnalysing Date Date Location Location % Inhibitiontion % Inhibitionhibition 23–2623–26 June June 20 20 Marane Marane 0.34 0.34 −0.94−0.94 − 20–2720–27 July July 20 20 Corfinio Corﬁnio 1.40 1.40 0.96−0.96 27–31 July 20 Raiano 0.12 −0.96 27–31 July 20 Raiano 0.12 −0.96

4.4. DiscussionDiscussion InIn bivalves, bivalves, the the behaviour behaviour ofof valve valve movementmovement isis correlatedcorrelated toto several several activities,activities, i.e.,i.e., ﬁltration,filtration, feeding, feeding, locomotion, locomotion, reproduction, reproduction, etc. etc. For For this this reason, reason, it it has has been been investigated investigated forfor a a long long time, time, also also through through the development the developmen of differentt of different kinds of kinds registering of registering “valvometers” “val- usefulvometers” for ethological useful for and ethological ecotoxicological and ecotoxicological studies [51]. Behavioural studies [51]. sublethal Behavioural effects sublethal reﬂect theeffects complex reflect system the complex of the system organism’s of the responseorganism’s to response disturbances. to disturbances. The combination The combi- of physiological and environmental factors causes a reaction that could represent a set of nation of physiological and environmental factors causes a reaction that could represent acute cumulative effects, not detectable by classical methods. They occur precociously and a set of acute cumulative effects, not detectable by classical methods. They occur preco- at very low contaminant concentrations. Hence, the behavioural endpoint is very useful to ciously and at very low contaminant concentrations. Hence, the behavioural endpoint is evaluate how toxicants exert their effects and for early warning signalling in BEWS. very useful to evaluate how toxicants exert their effects and for early warning signalling in BEWS. To date, some bivalve species such as P. casertanum have never been allocated in a “valvometer” due to their small dimensions. Indeed, the available commercial tools are based on sensors too big to be glued on the valves of this small mollusc. Moreover, such sensors do not have the sensibility needed to detect their little movements.

Water 2021, 13, 1529 15 of 19

To date, some bivalve species such as P. casertanum have never been allocated in a “valvometer” due to their small dimensions. Indeed, the available commercial tools are based on sensors too big to be glued on the valves of this small mollusc. Moreover, such sensors do not have the sensibility needed to detect their little movements. The SmartShell tool developed by IZSAM is able to register at distance the behaviour of molluscs with shell height >2 mm. This system is still unpublished in the scientific literature, as only preliminary results concerning laboratory testing with eight species of marine and freshwater mollusc species and reference toxicant have been described in a poster during EUROTOX 2019 [14]. This tool can be used both as a “valvometer” and as an “early warning system” with the appropriate algorithm. The basic idea in SmartShell is that each bivalve species has a very characteristic rhythm of valve opening. Any deviation from the basic pattern should be considered as an early signal of water disturbances. Starting from this hypothesis, the initial step is to analyse in detail the normal behaviour of each bivalve species in order to elaborate a specific early warning algorithm for each one. In this study, the behaviour of P. casertanum has been recorded for the first time, showing very particular characteristics. From the spectral analysis of valve movements data, the normal rhythms of activity and inactivity period were repeated about four times per day, and another one is registered on the fifth day. A circadian rhythm has been registered one time a day in Mytilus galloprovincialis [56] and in Anodonta anatina [61]. Conversely, multiple rhythms have been observed in Anodonta cygnea [62], Unio pictorum [63], in Corbicula fluminea [64]. Sometimes these rhythms are correlated to diurnal/nocturnal cycles, in other cases to feeding behaviour or tidal frequency [65]. Moreover, examining valve movements for a long period of time is useful to comprehend the effect of natural environmental changes and natural conditions (temperature, food, etc.) [66,67]. In this study, the behaviour of an adult mollusc (HUB 03-02 of the first study group) ready to release seven juveniles was also recorded. Pisidium species, in fact, are hermaphrodites incubating their progeny until release [53]. This mollusc was almost fixed at VG ranging from 61 to 80%. It did not show any rhythmical pattern. This demonstrates that behaviour of viviparous bivalves is greatly influenced by the reproductive period. Before juveniles release, the mollusc did not move valves for 12 days at least. Two P. casertanum individuals showed valve gapes ranging from 21 to 40% for most of the observation period. This behaviour is quite unusual since studies conducted using “valvometers” on other bivalve species showed that their valves were are almost completely opened for most of the time [9,68]. Instead, it was almost confirmed in the study on the normoxic and anoxic heat output of the freshwater bivalves Pisidium amnicum and Sphaerium corneum [69]. They registered regular periods of behavioural and metabolic quiescence in normoxic conditions through the use of heat-flow microcalorimeter. A normoxic rhythm of heat output was registered with short bursts of high heat output of almost 1.5 h alternated with longer low heat flux suggesting a period of shell closure of 9 h. Therefore, the observed behaviour detected a long quiescence period surely almost three times per day. Metabolic quiescence in aquatic invertebrates is usual in the unfavourable environmental conditions such as lack of food, extreme temperature and anoxia. For P. casertanum, this first study should be extended by investigating the behaviour pattern of this species during different seasonal periods, including the reproductive ones. Further studies are also needed to confirm the observations on the extent and the rhythms of valve gapes, and to identify potential interfering causes, with the aim of developing a reliable algorithm for early warning signalling. Being a cosmopolitan species, P. casertanum is easy to find in the environment, and, being an autochthonous species, its use is allowed in Natura 2000 sites like in the Tirino springs according to Habitat Directive [70]. Living in oligotrophic cold waters, this bivalve represents the best sentinel for this kind of water. Using the combination SmartShell with P. casertanum, the continuous monitoring of river springs could be arranged without any environmental modifications. Water 2021, 13, 1529 16 of 19

Concerning the other biomonitoring experiences carried out in the GS-S aquifer, DTOX has been chosen for continuous control of tap water, since D. magna is one of the most sensitive species for a wide range of compounds. ATOX has been selected for irrigation water, being the most reliable for pesticide detection. They did not launch any kind of early warnings by representing a good practical experience for safeguarding water bodies intended for multiple uses.

5. Conclusions This study reported the experience gained through the use of different BEWS in different sensitive points of the waters of the GS-S aquifer, choosing the most appropriate tool and sentinel for each site. A never tested autochthonous bivalve, P. casertanum, has been studied trough SmartShell. Being immotile and ﬁlter-feeding, this autochthonous mollusc represents a good sentinel candidate to estimate natural environments as spring water through BEWS. Our results may represent the basis to establish stable multiple trophic BEWSs for the control of the GS-S aquifer in speciﬁc sites, to be appointed according to the European and Italian Legislations concerning the Water Security Plan [71] and the protection zones (Italian National Decree 152/2006) [72]. BEWSs exploiting innovative technology are essential to ensure water protection, which also means to guarantee human and ecosystem health.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/w13111529/s1, Table S1: Classification of the Gran Sasso aquifer groundwater bodies, main hydrological characteristics and groundwater circulation, Table S2: Classification of the Sirente aquifer groundwater bodies, main hydrological characteristics and groundwater circulations. Author Contributions: Conceptualization, F.D.G.; methodology, F.D.G. and M.B.; software, L.C.; formal analysis, R.C., G.M., L.D.R.; data curation, V.C. and P.T.; writing—original draft preparation, F.D.G. and V.C.; writing—review and editing, R.C., M.B., A.L., B.T.; visualization, G.D.I., A.P., M.L.; supervision, N.F., M.B., N.D.; project administration, N.F.; funding acquisition, N.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by REGIONE ABRUZZO—Legge Regionale 27 agosto 1982 n. 59 “Controllo della salubrità delle carni ittiche”—year 2017. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article and supplementary materials. More detailed data may be requested from authors. Acknowledgments: We would like to express special thanks to Sandro Pelini and Paola Di Giuseppe for map and graphics, Annarita D’Angelo for SEM analysis, Maurilia Marcacci and Valentina Curini for PCR, Gianfranco Diletti and Roberta Ceci for their technical supervision. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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