Behavioral Parameters of Planarians (Girardia Tigrina) As Fast Screening, Integrative and Cumulative Biomarkers of Environmental Contamination: Preliminary Results

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Article Behavioral Parameters of Planarians (Girardia tigrina) as Fast Screening, Integrative and Cumulative Biomarkers of Environmental Contamination: Preliminary Results

Ana M. Córdova López 1,2 , Althiéris de Souza Saraiva 3, Carlos Gravato 4,* , Amadeu M. V. M. Soares 1,5 and Renato Almeida Sarmento 1

1 Programa de Pós-Graduação em Produção Vegetal, Campus Universitário de Gurupi, Universidade Federal do Tocantins, Gurupi-Tocantins 77402-970, Brazil; [email protected] 2 Estación Experimental Agraria Vista Florida, Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Carretera Chiclayo-Ferreñafe Km. 8, Chiclayo, Lambayeque 14300, Peru; [email protected] 3 Laboratório de Agroecossistemas e Ecotoxicologia, Instituto Federal de Educação, Ciência e Tecnologia Goiano-Campus Campos Belos, Campos Belos-Goiás 73840-000, Brazil; [email protected] 4 Faculdade de Ciências & CESAM, Universidade de Lisboa, 1749-016 Lisboa, Portugal 5 Departamento de Biologia & CESAM, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal; [email protected] * Correspondence: [email protected]

Citation: López, A.M.C.; Saraiva, Abstract: The present study aims to use behavioral responses of the freshwater planarian Girardia A.d.S.; Gravato, C.; Soares, A.M.V.M.; tigrina to assess the impact of anthropogenic activities on the aquatic ecosystem of the watershed Sarmento, R.A. Behavioral Araguaia-Tocantins (Tocantins, Brazil). Behavioral responses are integrative and cumulative tools that Parameters of Planarians (Girardia reflect changes in energy allocation in organisms. Thus, feeding rate and locomotion velocity (pLMV) tigrina) as Fast Screening, Integrative were determined to assess the effects induced by the laboratory exposure of adult planarians to water and Cumulative Biomarkers of samples collected in the region of Tocantins-Araguaia, identifying the sampling points affected by Environmental Contamination: contaminants. Furthermore, physicochemical and microbiological parameters, as well as the presence Preliminary Results. Water 2021, 13, of inorganic compounds (dissolved aluminum, total barium, total chloride, dissolved iron, total 1077. https://doi.org/10.3390/ fluoride, total manganese, nitrates, nitric nitrogen, total sulfate, total zinc) and surfactants, were w13081077 determined on each specific sampling point. The behavioral biomarkers (feeding rate and pLMV)

Academic Editor: Heiko of the freshwater planarians were significantly decreased when organisms were exposed to water L. Schoenfuss samples from four municipalities (Formoso do Araguaia, Lagoa da Confusão, Gurupi and Porto Nacional), sites of the Tocantins-Araguaia hydrographic region—TAHR. Both behavioral biomarkers Received: 20 February 2021 decreased up to ~37–39% compared to organisms in ASTM medium only. Our results showed that Accepted: 11 April 2021 these behavioral biomarkers can be used for fast screening monitoring of environmental samples of Published: 14 April 2021 freshwater ecosystems, since a decrease in feeding rate and locomotor activity was observed in sites impacted by anthropogenic activities. However, the absence of effects observed in some sampling Publisher’s Note: MDPI stays neutral points does not represent the absence of contamination, since several other classes of contaminants with regard to jurisdictional claims in were not determined. In these negative results, the absence of deleterious effects on behavioral published maps and institutional affil- biomarkers might only be indicative that the potential presence of contaminants on such sites does iations. not significantly affect the performance of planarians. This fast screening approach seems to be useful to determine contaminated sites in freshwater ecosystems for biomonitoring purposes. This knowledge will help to develop biomonitoring programs and to decide appropriate sampling sites and analysis. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: biomonitoring; planarians; locomotion; feeding rate This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Water 2021, 13, 1077. https://doi.org/10.3390/w13081077 https://www.mdpi.com/journal/water Water 2021, 13, 1077 2 of 13

1. Introduction Tocantins state (Brazil) is consolidated as the new agricultural frontier of the country, strategically located by environmental conditions and available water resources. In the last few years, Tocantins state has been standing out in the scenario of the national agribusiness of grain production [1], sugar cane [2], melon [3], and watermelon [4]. The increase in agricultural production has facilitated the spread of diseases, pests, and weeds in crops [5,6]. Thus, for achieving high productivity, pesticides and fertilizers have been adopted as the most efficient manner for the short-term control of undesirable organisms [7,8] and have been used in various steps of the production cycle of cultures, whereas such compounds become subject to environmental contamination [9,10]. However, the use of these compounds in agriculture is not unanimous, although its impacts can be considered positive by some segments of society; for others, its impacts are negative, mainly through the environmental point of view [11,12]. In the Tocantins state, agricultural cultivation generally occurs in areas adjacent to aquatic systems (watershed Araguaia-Tocantins). Thus, the aquatic ecosystem can be impacted since the main routes of contamination by xenobiotics, such as metals, in aquatic systems are through runoff, erosion, drift, leaching, and atmospheric deposition [13,14]. Thus, water quality has been influenced by urbanization and modernization, which has brought problems of wastewater disposal and the contamination of surface water, such as lakes [15]. Natural water becomes polluted due to the weathering of rocks, the leaching of soils, and mining processing [16]. The change in land use in agricultural production could increase the use of fertilizers with subsequent leaching to waterways, rivers, and lakes, increasing the risk of eutrophication and the loss of biodiversity [17,18]. Fertilizers have a significant amount of arsenic, chromium, cadmium, lead, zinc, nickel, iron, molybdenum and manganese, which are essential in the healthy growth and maintenance of plants. However, the excess of these metals in sediments of freshwater produces toxic effects to aquatic organisms [19,20]. Heavy metals enter the human body mainly through ingestion, through food, inhalation and dermal contact, such as emissions of waste material in the form of smoke, dust particles, and substance vapors of chemicals from various industrial activities, such as mining and agricultural practices [21]. Exposure to heavy metals can cause their excessive accumulation in body tissues and induce the production of free radicals (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) that lead to an oxidative stress condition. Elevated levels of Zn and Cu can cause various adverse health effects; otherwise, these elements are essential for life and functions of many proteins in the organism. Among the measured elements, arsenic, cadmium, chromium (VI) and nickel are classified as carcinogens [22,23] by the International Agency for Research on Cancer (https://monographs.iarc.who.int/list-of- classifications, accessed on 7 April 2021). Copper is not classified as carcinogenic and there is only one publication on Cu as a tumor promoter [24], cited by Taylor et al. (2019) [25]. Water quality can be assessed by various parameters, such as biochemical oxygen demand (BOD), temperature, conductivity and concentrations of nitrate, phosphorus, potassium and dissolved oxygen. Among many other classes of contaminants, heavy metals are usually of special concern because they might cause poisoning in aquatic animals [15]. At the level of the trophic chain, the most sensitive organisms are those most affected, and in fact, contamination of the aquatic ecosystem, whether due to bioaccumulation or direct exposure, will ultimately lead to the exposure of humans [21,26]. Thus, contamination of aquatic ecosystems has been monitored by analyzing the effects of the presence of contaminants using bioindicator species, which reflects the health state of that specific environment [27,28]. The use of organisms as bioindicators for the evaluation of environmental contamination has been used in several studies, since this strategy offers various advantages concerning the prediction of the flow of contaminants in the populations of the trophic chain [29,30]. Planarians have gained more and more notoriety in ecotoxicology as test organisms with potential for biomonitoring contaminants in freshwater ecosystems, mainly Water 2021, 13, 1077 3 of 13

due to: (i) a useful potential to screen contaminant toxicity and assess the quality of freshwater ecosystems; (ii) several interesting biological characteristics (for example, behavior, regeneration, and reproduction) that can be used to evaluate the sub-lethal and chronic toxicity of environmental contaminants; (iii) some physiological systems that share resem- blances to those in mammals (e.g., the nervous system); (iv) being a representative group of invertebrates with wide geographical distribution, functioning as prey and predators; (v) ease of capture and low laboratory maintenance costs [31–34]. Among the freshwater planarians used as test organisms in ecotoxicological bioassays, Girardia tigrina (Paludi- cola, Dugesiidae; Girard, 1850) has been used to evaluate lethal and sub-lethal toxicity of xenobiotics and potential biomonitoring [35–37]. Behavioral biomarkers, such as feeding rate and locomotor activity, have been de- veloped as low-cost tools, but with high sensitivity and acceptance as integrative and cumulative responses induced by xenobiotics that are indicative of deleterious cellular alterations and effects at higher levels of biological organization [37]. Moreover, although scarce, the effects of metals on freshwater planarians have been reported in the literature, such as copper’s effects on antioxidant defenses in Dugesia japonica [38], the genotoxic effects of copper in Girardia schubarti [39], and aluminum’s neurotoxic effects in Dugesia estrusca [40]. Additionally, although not a metal, the effects of chlorine have been reported on the feeding, locomotion, regeneration and reproduction of Girardia tigrina [28]. Thus, our present study aimed to determine the usefulness of such biomarkers to assess the environmental contamination of water samples coming from the watershed of Araguaia-Tocantins through the laboratory exposure of adult planarians. These water samples were collected in areas of intense agricultural production and exploitation of mineral resources. In addition, this study also aims to gather information about the behavioral biomarkers and its potential use as fast screening tools that can be adopted to determine important decisions concerning further biomonitoring studies complemented with chemical analysis of different freshwater compartments.

2. Material and Methods 2.1. Study Area Water samples were collected in four municipalities, Formoso do Araguaia (11◦48002.800 S and 49◦37026.300 W), Lagoa da Confusão (10◦50064.300 S and 49◦42051.000 W), Gurupi (11◦51033.9600 S and 48◦53015.1400 W) and Porto Nacional (10◦47041.8300 S and 49◦37022.85400 W), of Tocantins-Araguaia Hydrographic region (TAHR). Those sampling sites represent areas of intense agricultural production and exploitation of mineral resources (Figure1). More- over, a local reference site for water sampling was chosen considering a location far from nearby sources of human impact and therefore less contaminated (Figure1). The sampling period was conducted during the wet season, i.e., January to February.

2.2. Water Analysis The sampling, storage and transport of the water samples to the laboratory were carried out following the specifications of the sampling of surface waters according to the National Water Agency [41]. Then, samples were subsequently sent to the Conagua Ambiental laboratory for analysis (Goiás State, Brazil—https://conaguaambiental.com.br/ wp/, accessed on 15 July 2020) and also used in the laboratory for exposure of planarians. For the analysis of the water samples, they were collected in borosilicate glass bottles and PET containers for the first use, containing the necessary preservatives (Conagua Ambiental protocols—https://conaguaambiental.com.br/wp/, accessed on 15 July 2020) to avoid the deterioration of the sample. The collection was carried out at a depth no greater than 30 cm. Later, the flasks were sealed and transported to the laboratory at a temperature of 4 ◦C. The water samples for the exposures were collected and transferred at 4 ◦C and expected until the sample reached the temperature for conducting the experimental tests, 22 ± 1 ◦C, without the addition of additives. Water 2021, 13, 1077 4 of 13

Water 2021, 13, x FOR PEER REVIEW 4 of 14

Water 2021, 13, x FOR PEER REVIEW Analyses were carried out on the water samples to determine chemical, physical4 of 14 and biological parameters following the methods of the Environmental Protection Agency and Standard Methods for the Examination of Water and Wastewater [42].

Figure 1. Map of Tocantins-Araguaia hydrographic region (TAHR), Brazil. (A) Formoso do Araguaia, 4 (Taboca), 5 (small Figure 1. Map of Tocantins-Araguaia hydrographic region (TAHR), Brazil. (A) Formoso do Araguaia, 4 (Taboca), 5 (small irrigationFigure channel), 1. Map 6 (large of Tocantins-Araguaia volumes of water in hydrographic the channel); region (B) Lagoa (TAHR), da Confusão, Brazil. (A 1 )(lake), Formoso 2 (water do Araguaia, channel), 4 3(Taboca), (Urubu 5 (small river); (Cirrigation) Gurupi,irrigation channel),10 channel), (Dam 6Brejo (large 6 (large Pantanal), volumes volumes 11of ofwater(Santo water in Antônio inthe the channel); channel); river), ( B12) ( BLagoa(Reference,) Lagoa da daConfusão, Confusdam withã 1o, (lake),crystal 1 (lake), 2 clean(water 2 (water water); channel), channel), (D) 3 (Urubu 3 (Urubu Porto Nacional,river);river); (7C ((Tocantins)C Gurupi,) Gurupi, 10 river), 10 (Dam (Dam 8 Brejo(large Brejo Pantanal), Pantanal),volumes of 11 11 wate (Santor in AntAntônio theô niochannel), river),river), 9 12 12(small (Reference, (Reference, irrigation dam dam channel); with with crystal crystal adapted clean clean water);from water); (D) ( PortoD) Google MapsPortoNacional, and Nacional, Google 7 (Tocantins 7 Earth(Tocantins (2020). river), river), 8= (largeWater 8 (large volumes collection volumes of watersites. of wate Water in ther in channel),collected the channel), 9in (small each 9 (small site irrigation was irrigation used channel); to channel); expose adapted adultadapted from from Google

planariansGoogle (MapsG. tigrina Maps and) Google inand the Google laboratory. Earth Earth (2020). (2020).= Water collection= Water collection sites. Water sites. collected Water collected in each site in each was usedsite was toexpose used to adult expose planarians adult planarians(G. tigrina (G.) in tigrina the laboratory.) in the laboratory. 2.2. Water Analysis The2.2. sampling,2.3. Water Planarians Analysis storage and transport of the water samples to the laboratory were carried out followingTheThe sampling, the specimens specifications storage of G. and tigrina of transporttheused sampling in of the the of bioassays water surface samples werewaters obtainedto according the laboratory from to thethe were University car- Nationalried Waterof out Sao Agency following Paulo and [41]. the maintained Then, specifications samples in laboratory we ofre the subsequently sampling cultures inof sentthe surface Federalto the waters Conagua University according Am- of Tocantins, to the biental NationallaboratoryTocantins Water State-Brazil,for Agency analysis [41]. since Then, 2014.(Goiás samples American State, we re StandardBrazil—https://conaguaambien- subsequently Test and sent Materials to the Conagua (ASTM) Am- hard tal.com.br/wp/,bientalwater accessed waslaboratory used on as 15 a forJuly culturing 2020)analysis and medium also(Goiás used [43 ]. in OrganismsState, the laboratoryBrazil—https://conaguaambien- were for maintained exposure inof controlled 3 planarians.tal.com.br/wp/,conditions in accessed plastic boxes on 15 (30July× 2020)18 × and10 also cm ),used containing in the laboratory 1 L of ASTM for exposure medium, of at ◦ Forplanarians. the22 analysis± 1 C, of in the the water dark andsamples, constant they aeration.were collected The planarians in borosilicate were glass fed withbottles bovine liver and PET containers(adFor libitum the for analysis) twice the first a of week, use,the watercontaini followed samples,ng by the medium necessarythey were renewal preservatives collected two in hours borosilicate (Conagua after the Am-glass beginning bottles of biental protocols—https://conagandfeeding PET containers activity, except for theuaambiental.com.br/wp/, the first 96 use, h prior containi to exposureng the necessaryaccessed and the entirepreservativeson 15 exposureJuly 2020) (Conagua period. to Am- avoid thebiental deterioration protocols—https://conag of the sample. Theuaambiental.com.br/wp/, collection was carried out accessed at a depth on no 15 greater July 2020) to Development and Design Test of Behavioral Responses than 30 cm.avoid Later, the deterioration the flasks were of thesealed sample. and transportedThe collection to wasthe laboratorycarried out at at a a tempera-depth no greater ture of 4than °C. The30The cm. water planariansLater, samples the flasks have for theirthe were exposu eating sealedres behavior an wered transported collected altered according andto the transferred laboratory to the chemical at at 4 a°C tempera- composi- and expectedturetion of until of4 °C. the the The water sample water in thereached samples natural the for environment temperature the exposures [ 44for ].were conducting Despite collected this, the weand experimental did transferred not determine at 4 °C the tests, 22 and± 1hard °C, expected without water inuntil the the theaddition different sample of water reachedadditives. samples the temperature studied. In this for way,conducting we used the ASTM experimental hard water Analysestests,as a22 were control ± 1 °C,carried treatment, without out on the since the addition thewater planarian samplesof additives. culture to determine is maintained chemical, in this physical culture and medium and we were able to determine the number of salts (sodium hydrogen carbonate, magnesium biological parametersAnalyses following were carried the outmethods on the of water the Environmental samples to determine Protection chemical, Agency physical and and sulfate, potassium chloride, and calcium sulfate) available to the organisms. For example, Standardbiological Methods parameters for the Examination following of the Water methods and ofWastewater the Environmental [42]. Protection Agency and Standardcalcium Methods ions in the for aquatic the Examination environment of Water are considered and Wastewater to be indispensable [42]. for the feeding behavior of planarians, and exposure to higher levels of calcium ions enhanced the feeding 2.3. Planarians 2.3.behavior, Planarians showing that there was a good correlation between the concentration of calcium The specimensions and theof G. responsiveness tigrina used in of the planarians bioassays to were foods obtained [44]. In addition, from the the University amount of calcium of Sao Paulosalt andThe can maintainedspecimens be up to ﬁfty of in G. laboratory times tigrina the used amountcultur in thees of in bioassays potassiumthe Federal were salt University obtained without of unfavorable from Tocantins, the University results for Tocantinsof State-Brazil,Sao Paulo and since maintained 2014. American in laboratory Standard cultur Testes in and the FederalMaterials University (ASTM) ofhard Tocantins, water wasTocantins used as State-Brazil, a culturing sincemedium 2014. [43]. American Organisms Standard were Testmaintained and Materials in controlled (ASTM) hard conditionswater in plastic was used boxes as (30a culturing × 18 × 10 mediumcm3), containing [43]. Organisms 1 L of ASTM were medium, maintained at 22 in ±controlled 1 conditions in plastic boxes (30 × 18 × 10 cm3), containing 1 L of ASTM medium, at 22 ± 1

Water 2021, 13, 1077 5 of 13

planarians [45]. On the other hand, in the collection of water samples in the ﬁeld, it was not possible for us to determine an uncontaminated reference point to adopt as a control (in addition to the control with ASTM medium). Individual G. tigrina (0.83 ± 0.11 cm in total length) were exposed (n = 10 per condition) during 96 h to 20 mL of 12 water samples, on Petri dishes (Ø = 4.6 cm), including the reference site (Figure1) and a laboratory control with ASTM water only. The exposed organisms were maintained at 22 ◦C ± 1 under dark conditions and no food was provided. After four days exposure, planarians from each condition were transferred to clean medium and locomotor velocity and feeding rate were determined as post-exposure analysis. Brieﬂy, planarians were individually transferred (n = 10 per condition) to new crystal- lizing dishes containing 20 mL of ASTM medium, and twenty live larvae of Chironomus xanthus (six days old) were released on each plate. Then, the feeding rate per hour was calculated, analyzing the number of larvae consumed per planarian at the end of 12 h. To determine the locomotor velocity (pLMV), planarians were placed in a plate (Ø 35 cm) with 200 mL of ASTM hard water and an adhered millimeter paper (grid lines spaced 0.1 cm apart) below. The pLMV was measured individually, after scoring the number of crossed grid lines for each planarian for 2 min (taking as reference the crossing of the planarian head) according to the methodology already adopted by our research group [28,35,36]. There was no mortality of planarians during the experimental assay.

2.4. Statistical Analysis For pLMV and feeding rate data, we used one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons in order to determine differences between sampling and reference sites and the laboratory control. Prior to ANOVA, the homogeneity of variances of data was assessed using the Brown–Forsythe test and the normality of data by using the D’Agostino and Pearson test. Locomotion data were transformed (Y = rank(Y)) for the correction of homogeneity of variance. Statistical analysis was performed using the software GraphPad Prism version 7.0 for Windows (GraphPad Software, La Jolla, San Diego, CA, USA).

3. Results 3.1. Sampling Sites Disturbing concentrations of aluminum were found in the surface water samples at points 2, 3, 7, 10 and 11—at these points, concentrations of 0.10, 0.13, 1.02, 0.27 and 0.38 mg L−1 of aluminum were detected, respectively (Table1—concentrations higher up to 10 times the limit acceptable). For these points already indicated, the pH was variable, obtaining values of 5.44, 7.1, 7.25, and 6.97, respectively. The water sample collected at site 1 (Lagoa da Confusão lake) affected the planarian locomotion, decreasing to 36.18%; in this sample, concentrations below the acceptable limit of inorganic compounds (total ﬂuoride, total manganese, nitrates, nitric nitrogen) were found (Table1). The water sample collected at site 2 (water channel, Lagoa da Confus ão) altered the pLMV of the planarian, decreasing when compared to the control. In this sample, concentrations above the permissible limit of inorganic compounds (dissolved aluminum, dissolved iron) were found (Table1), as well as an acid pH of 5.44, affected the feeding rate and pLMV of the planarian.

3.2. Feeding Rate Planarians exposed in the laboratory to water samples from the Tocantins Araguaia hydrographic region caused the alteration of their post-exposure feeding rate. Signiﬁcant differences were found in sampling sites when compared with the ASTM condition in the laboratory (p < 0.0001, Figure2). Planarians, when exposed to water samples from site 3 (Urubu river—Lagoa da Confusão), site 8 (large volumes of water in the channel—Porto Nacional), site 2 (water channel—Lagoa da Confusão), site 6 (large volumes of water in the channel—Formoso do Araguaia), and site 9 (small irrigation channel—Porto Nacional), Water 2021, 13, x FOR PEER REVIEW 6 of 14 Water 2021Water, 13 2021, xWater FOR, 13 ,PEER2021 xWater FOR, 13REVIEW 2021PEER, xWater FOR, 13REVIEW , PEER2021x FOR, REVIEW13 PEER, x FOR REVIEW PEER REVIEW 6 of 14 6 of 14 6 of 146 of 14 6 of 14

The water sample collected at site 1 (Lagoa da Confusão lake) affected the planarian The waterThe watersampleThe samplewater collectedThe watersample collectedThe at sample sitewater collected at1 (Lagoa site samplecollected 1at (Lagoa siteda collected Confusão at1 (Lagoa siteda Confusão1 at (Lagoa sitedalake) Confusão 1 affected(Lagoada lake) Confusão affected dalake) the Confusão planarian affectedlake) the planarianaffected lake) the planarian affected the planarian the planarian locomotion, decreasing to 36.18%; in this sample, concentrations below the acceptable locomotion,locomotion, decreasinglocomotion, locomotion,decreasing to decreasinglocomotion, 36.18%; to decreasing 36.18%; in to thisdecreasing 36.18%; in sample,to this 36.18%; insample, to thisconcentrations 36.18%; in sample, thisconcentrations insample, thisconcentrations below sample, concentrations belowthe concentrationsacceptable belowthe acceptable below the acceptable thebelow acceptable the acceptable limit of inorganic compounds (total fluoride, total manganese, nitrates, nitric nitrogen) limit oflimit inorganic oflimit inorganic compoundsoflimit inorganic ofcompoundslimit inorganic (tcompoundsofotal inorganic fluoride,(tcompoundsotal fluoride, (tcompoundsotal total (tfluoride, otalmanganese, total fluoride, (t manganese,otal total fluoride, nitrates, manganese,total nitrates,manganese, totalnitric nitrates,manganese, nitrogen)nitric nitrates, nitrogen)nitric nitrates, nitricnitrogen) nitrogen) nitric nitrogen) were found (Table 1). The water sample collected at site 2 (water channel, Lagoa da Con- were foundwere found(Tablewere (Tablefoundwere1). The found (Table1). werewater The (Table found1). watersample The 1).(Table samplewater collectThe 1). watersample collected The at sample sitewater edcollect at2 (water site samplecollected 2at (water channel,siteed collect at2 (watersitechannel,ed Lagoa 2 at (water channel,site Lagoada 2 Con-channel,(water daLagoa Con- channel, Lagoa da Con- daLagoa Con- da Con- fusão) altered the pLMV of the planarian, decreasing when compared to the control. In fusão) fusão)altered fusão)altered the pfusão) LMValtered the p ofalteredLMVfusão) thethe pofplanarian, LMV alteredthe the p planarian,ofLMV thethe decreasing ofpplanarian,LMV the decreasing planarian,of thewhen decreasing planarian, whencompared decreasing comparedwhen decreasing to when comparedthe tocontrol. compared whenthe control.to In comparedthe tocontrol. Inthe control.to In the control. In In this sample, concentrations above the permissible limit of inorganic compounds (dis- this sample,this sample, thisconcentrations sample, thisconcentrations sample, thisconcentrations above sample, concentrations abovethe concentrationsperm abovetheissible perm above the issible limitperm theabove ofissiblelimitperm inorganic the issibleof limitperm inorganic oflimitissiblecompounds inorganic of compoundslimit inorganic of(dis-compounds inorganic compounds(dis- (dis-compounds (dis- (dissolved aluminum, dissolved iron) were found (Table 1), as well as an acid pH of 5.44, solvedsolved aluminum, solvedaluminum, solveddissolved aluminum, dissolved solvedaluminum, iron) dissolved aluminum, wereiron) dissolved foundwere iron) dissolved found (Twereiron)able found (Twere1), iron)able as found (Twell1),wereable as as (T foundwell1), ablean as acidas 1),(Twell anable aspH acidas well 1), ofan pH5.44, as asacid wellofan pH5.44, acid as of an pH 5.44, acid of 5.44,pH of 5.44, affected the feeding rate and pLMV of the planarian. affectedaffected the feedingaffected the feedingaffected rate the and feeding affectedrate the pLMV andfeeding rate the pofLMV and feedingthe rate pplanarian.of LMVand the rate p planarian.ofLMV and the ofplanarian.pLMV the planarian. of the planarian. Table 1. Analysis of surface water samples collected at eleven sampling sites (1–11) and the reference (12) site in Tocantins. Table 1.Table Analysis 1.Table Analysis of surface 1.Table Analysis of surface 1.waterTable Analysis of samples surfacewater 1. Analysis of samplessurface watercollected of samples water surfacecollected at eleven samples watercollected at sampeleven samples collected atling samp eleven sites collected atling eleven samp(1–11) sites atling sampand (1–11) eleven sites theling and referencesamp(1–11) sites theling and(1–11)reference (12)sites the and site reference(1–11) (12) thein Tocantins. sitereferenceand (12) in the Tocantins. site reference (12) in Tocantins.site (12)in Tocantins. site in Tocantins. Sites of Hydrographic Region Tocantins Water 2021, 13, 1077 Sites ofSites Hydrographic ofSites Hydrographic ofSites Hydrographic Region ofSites Hydrographic Region Tocantinsof Hydrographic Region Tocantins Region Tocantins Region Tocantins Tocantins 6 of 13 Test AL LQ Unit Araguaia Test Test TestAL TestAL LQ Test AL LQ UnitALLQ Unit ALLQ UnitLQ Unit AraguaiaUnit Araguaia Araguaia Araguaia Araguaia 1 2 3 4 5 6 7 8 9 10 11 12 1 21 32 1 43 21 54 32 1 65 43 2 76 54 3 87 65 4 98 76 5 109 87 6 1110 98 7 121110 9 8 121110 9 121110 1211 12 Biochemical oxygen BiochemicalBiochemical oxygenBiochemical oxygenBiochemical oxygenBiochemical 5 oxygen 0.2 oxygen mg L−1 demand 5 5 0.2 5 0.2mg 5 L0.2−1mg 5 L0.2 showed− 1 mg L0.2 − 1mga signiﬁcantly L − 1 mg L − 1 decreased feeding activity of 38, 34, 28, 26 and 25%, respectively, demanddemand demand demand demand Dissolved oxygen >5 0.1 mg L−1 when compared to the4.7 ASTM3.9 control condition (Figure 2 ). 2.8 DissolvedDissolved oxygenDissolved oxygen Dissolved >5 oxygen Dissolved >5oxygen 0.1 >5 oxygen 0.1 mg >5 L 0.1−1 mg >5 L 0.1 − 1 mg L 0.1 − 1mg L − 1 mg 4.7 L − 1 3.94.7 3.9 4.7 3.9 4.7 3.9 4.7 3.9 2.8 2.8 2.8 2.8 2.8 Turbidity 100 0.21 NTU TurbidityTurbidity Turbidity 100Turbidity Table 100Turbidity0.21 1. Analysis 1000.21 NTU 100 of0.21 surface NTU 1000.21 water NTU samples0.21 NTU collected NTU at eleven sampling sites (1–11) and the reference (12) site in Tocantins. Acidity/alkalinity 6.0–9.0 0.1 pH 5.44 Acidity/alkalinityAcidity/alkalinityAcidity/alkalinity Acidity/alkalinity6.0–9.0 Acidity/alkalinity6.0–9.0 0.16.0–9.0 0.16.0–9.0 pH 0.1 6.0–9.0 pH 0.1 pH5.44 0.1 pH5.44 pH 5.44 5.44 5.44 Total dissolved Sites of Hydrographic Region Tocantins Araguaia Total dissolvedTotal dissolvedTotal dissolvedTotal 500 dissolvedTotal Test dissolved0.05 mgAL L−1 LQ Unit solids 500 5000.05 5000.05 mg500 0.05L −1mg 500 0.05L − 1 mg 0.05L − 1mg L − 1 mg L − 1 1 2 3 4 5 6 7 8 9 10 11 12 solids solids solids solids solids Cyanobacterial Biochemical oxygen −1 CyanobacterialCyanobacterialCyanobacterial Cyanobacterial 50,000Cyanobacterial 1 Cel 5mL−1 0.2 mg L demand −1 −1 −1 −1 −1 density 50,000 50,0001 50,0001 Cel 50,000 mL1 Cel 50,000 mL1 Cel mL Cel1 mL Cel− mL densitydensity density densityDissolved density oxygen >5 0.1 mg L 1 4.7 3.9 2.8 Dissolved aluminum 0.1Turbidity 0.004 mg 100 L−1 0.21 0.11 NTU 0.13 1.02 0.27 0.38 DissolvedDissolved aluminumDissolved aluminumDissolved aluminum 0.1Dissolved aluminum 0.1 0.004 aluminum 0.1 0.004 mg 0.1 0.004L − 1 mg 0.1 0.004 L − 1 mg 0.11 0.004L− 1 mg 0.13 0.11 L−1 mg0.13 0.11 L − 1 0.130.11 0.13 0.11 1.02 0.13 1.02 1.02 0.27 1.02 0.38 0.27 1.02 0.380.27 0.380.27 0.380.27 0.38 Total barium Acidity/alkalinity 0.7 0.005 mg 6.0–9.0 L−1 0.1 pH 5.44 Total bariumTotal bariumTotal Total barium dissolved Total 0.7 barium 0.7solids 0.005 0.7 0.005 500−1 mg 0.7 0.005 L −10.05 mg 0.005L− 1 mg mg L L−1 − mg 1 L − 1 Total barium 0.7 0.005 mg L −1 Total chloride 250Cyanobacterial 0.5 mg L Total chlorideTotal chlorideTotal chlorideTotal 250 chlorideTotal 250 0.5 chloride 250 0.5 mg 250 50,000L 0.5−1mg 250 L 0.5 − 1 mg 1 L 0.5 − 1 mg Cel L mL − 1 − mg1 L − 1 Total chlorine 0.01density 0.01 mg L−1 0.04 0.07 Total chlorineTotal chlorineTotal chlorineTotal 0.01 chlorineTotal 0.01 0.01 chlorine 0.01 0.01 mg 0.01 0.01L −1 mg 0.01 0.01L− 1 mg 0.01L − 1mg L − 1 − mg 10.04 L − 1 0.04 0.04 0.04 0.04 0.07 0.07 0.07 0.07 0.07 Dissolved aluminum 0.1−1 0.004 mg L 0.11 0.13 1.02 0.27 0.38 Dissolved iron 0.3 0.04 mg L 0.36 0.34− 0.62 0.88 DissolvedDissolved ironDissolved ironDissolved 0.3 iron TotalDissolved 0.3 0.04bariumiron 0.3 0.04 iron mg 0.3 0.04L 0.7−1 mg 0.3 0.04L − 10.005 mg 0.36 0.04L− 1 mg mg0.340.36 L L−1 mg1 0.34 0.36 L − 1 0.340.36 0.34 0.36 0.62 0.34 0.62 0.62 0.88 0.62 0.88 0.62 0.88 0.88 0.88 Total fluoride 1.4 0.04 mg L−1 − 1 Total chloride 250−1 0.5 −1 mg L−1 −1 Total fluorideTotal fluorideTotal fluorideTotal 1.4 fluorideTotal 1.4 0.04 fluoride 1.4 0.04 mg 1.4 0.04L −1 mg 1.4 0.04L mg 0.04L mg L − mg L Total manganese 0.1Total chlorine 0.007 mg 0.01 L−1 0.01 mg L 1 0.04 0.07 Total manganeseTotal manganeseTotal manganeseTotal 0.1 manganese DissolvedTotal 0.1 0.007 manganese iron 0.1 0.007 mg 0.1 0.007L 0.3−1 mg 0.1 0.007 L − 10.04 mg 0.007L − 1mg mg L L− 1 − mg 1 L − 1 0.36 0.34 0.62 0.88 −1 Nitrates 10 0.1 mg L − 1 NitratesNitrates Nitrates 10Nitrates Total10 ﬂuorideNitrates 0.1 100.1 mg 10 L0.1 1.4−1mg 10 L0.1 − 10.04 mg L0.1 − 1mg mg L L− 1 mg L − 1 Total manganese 0.1−1 0.007 mg L−1 Nitric nitrogen 1 0.001 mg L − 1 Nitric Nitricnitrogen nitrogenNitric Nitricnitrogen 1 nitrogenNitric 1 0.001 nitrogen 1 0.001 mg 1 0.001L−1 mg 0.0011 L − 1 mg 0.001L − 1mg L − 1 − mg1 L Total sulfate 250Nitrates 0.11 mg 10 L−1 0.1 mg L Total sulfateTotal sulfate Total sulfateTotal 250 Nitric sulfate 250Total nitrogen0.11 sulfate 250 0.11 mg 250 0.11L − 1 mg 250 0.11L − 10.001 mg 0.11L − 1mg mg L L− 1 − mg 1 L − 1 Total zinc 0.18 0.007 mg L−1 − 1 0.44 Total zincTotal zincTotal 0.18 zincTotal Total 0.18zincTotal 0.007 sulfate 0.18 zinc 0.007 mg 0.18 0.007L 250−1 mg 0.18 0.007 L− 10.11 mg 0.007L − 1mg mg L L− 1 mg L − 1 0.44 0.44 0.44 0.44 0.44 SurfactantsWater 2021NR, 13Total , x FOR zinc0.001 PEER REVIEWmg 0.18 L−1 0.0070.74 0.61mg L0.68−1 0.76 0.64 0.63 0.64 0.620.44 0.7 0.61 0.59

0.00.0 rate Feeding (n° consumed/hour) larvae Feeding rate Feeding (n° consumed/hour) larvae Feeding rate Feeding (n° consumed/hour) larvae rate Feeding (n° consumed/hour) larvae ASTM 1 2 3 4 5 6 7 8 9 10 11 12 ASTMASTMASTM11 221 332 443 554 665 776 887 998 10109 111110 121211 12 Sites hydrographic region:Tocantins Araguaia SitesSites hydrographic hydrographicSites hydrographicSites region:Tocantinshydrographic region:Tocantins region:Tocantins region:Tocantins Araguaia Araguaia Araguaia Araguaia Figure 2. Feeding rate (number of C. xanthus larvae consumed per hour) of G. tigrina after 96 h Figure 2. Feeding rate (number of C. xanthus larvae consumed per hour) of G. tigrina after 96 h expo- FigureFigure Figure2. 2. Feeding Feeding 2. Feeding rate rate (number (number rate (number of of C. C. xanthus xanthusof C. xanthus larvae larvae consumedlarvae consumed consumed per per hour) hour) per of hour)of G. G. tigrina tigrinaof G. tigrinaafter after 96 96after h h 96 h exposure with water samples from Tocantins-Araguaia Sites Hydrographic Region: Lagoa da Con- Figuresure with 2. Feeding water samples rate (number from Tocantins-Araguaia of C. xanthus larvae Sites consumed Hydrographic per Region:hour) of Lagoa G. tigrina da Confus after ã96o h exposureexposureexposure with with water waterwith sampleswater samples samples from from Tocantins-Arag Tocantins-Aragfrom Tocantins-Araguaiauaia Sites Sitesuaia Hydrographic HydrographicSites Hydrographic Region: Region: Region: Lagoa Lagoa daLagoa da Con- Con- da Con-fusão (1–3); Formoso do Araguaia (4–6); Porto Nacional (7–9); Gurupi (10–12); Reference (12). A exposure(1–3); Formoso with water do Araguaia samples (4–6); from Porto Tocantins-Arag Nacional (7–9);uaia Gurupi Sites (10–12); Hydrographic Reference Region: (12). A laboratory Lagoa da Con- fusãofusão (1–3); fusão(1–3); fusãoFormoso (1–3);Formoso (1–3); Formoso do do AraguaiaFormoso Araguaia do Araguaia (4–6); do(4–6); Araguaia Porto (4–6);Porto Na PortoNa cional(4–6);cional Na (7–9);Portocional (7–9); Gurupi Na(7–9);Gurupicional Gurupi (10–12); (10–12); (7–9); (10–12); Reference ReferenceGurupi Reference (10–12);(12). (12). A A (12). Reference Alaboratory (12). control A with ASTM only was also used to expose planarians. Values represent the mean laboratorycontrol control with with ASTM ASTM only only was was also also used used to exposeto expose planarians. planarians. Values Values represent represent the the mean mean(±( SEM),± SEM), n = 10. Different letters indicate significant differences between study sites when p < 0.05. laboratorylaboratory controllaboratory control with with controlASTM ASTM only onlywith was was ASTM also also usedonly used to wasto expose expose also planarians. usedplanarians. to expose Values Values planarians. represent represent the theValues mean mean represent the mean (±(± SEM), SEM),(± nSEM), n = =10. n10. =nDifferent Different= 10. 10. Different Different letters letters letters indicateletters indicate indicate indicate si significantgnificant signiﬁcant significant differences differences differencesdifferences between between betweenbetween study study sitesstudystudy sites when when sitessites p when p< <0.05. 0.05. p < 0.05. 0.05.Colors Colors used in graph bars are associated with analysis of water performed: very high concen- (± SEM), n = 10. Different letters indicate significant differences between study sites when p < 0.05. ColorsColors Colorsused used in inused graph graph in bars graph bars are are bars associated associated are associated with with an anwith alysisalysis an analysis ofalysis of water water of performed:water performed: performed: performed: very very high veryhigh very concen- highconcen-high concentrations,concen-trations, high concentrations, above acceptable limits, acceptable limit. trations,trations,trations, Colors high highhigh concentrations, concentrations, highconcentrations,used concentrations, in graph bars above above above are acceptable above acceptableacceptable associated acceptable limits, limits,limits, with limits, anacceptable acceptable acceptablealysis acceptable of limit. limit.water limit. limit.performed: very high concentrations, high concentrations, above acceptable limits, acceptable limit. 3.3. Planarian Locomotor Velocity (pLMV) 3.3.3.3. Planarian Planarian3.3. Planarian Locomotor Locomotor Locomotor Velocity Velocity Velocity (pLMV) (pLMV) (pLMV) The post-exposure locomotion velocity (pLMV) of G. tigrina was significantly affected TheThe post-exposure post-exposure3.3.The Planarianpost-exposure locomotion locomotionLocomotor locomotion velocity velocityVelocity velocity ( p ((pLMV)pLMV)LMV) (p LMV)of of G. G. tigrina oftigrina G. tigrina was was significantly significantly was significantly affected affected affected (p < 0.0001) in planarians after 96 h exposure to water samples from site 10 (Dam Brejo (p( p< < 0.0001) 0.0001)(p < 0.0001) in in planarians Theplanarians in post-exposureplanarians after after 96 after96 h h locomotionexposure 96exposure h exposure to to watervelocity water to watersamples samples (pLMV) samples from from of siteG. fromsite tigrina 10 10 site (Dam (Dam 10was (DamBrejo Brejo significantly Brejo Pantanal-Gurupi), affected site 1 (lake—Lagoa da Confusão), site 4 (Taboca-Formoso do Ara- Pantanal-Gurupi),Pantanal-Gurupi),Pantanal-Gurupi),(p < 0.0001) site site 1 in 1 site(lake—Lagoa planarians(lake—Lagoa 1 (lake—Lagoa afterda da Confusão), Confusão),96 da h Confusão), exposure site site 4 4 tosite(Taboca-Formoso (Taboca-Formoso water 4 (Taboca-Formoso samples dofromdo Ara- Ara- do site Ara-guaia), 10 (Dam site Brejo5 (small irrigation channel—Formoso do Araguaia), site 3 (Urubu river), site guaia),guaia),guaia), site sitePantanal-Gurupi), 5 5 (smallsite (small 5 (small irrigation irrigation irrigation channel—Formoso channel—Formososite 1channel—Formoso (lake—Lagoa do do Araguaia), Araguaia),da do Confusão), Araguaia), site site 3 3 (Urubusite site(Urubu 3 4(Urubu river),(Taboca-Formoso river), river),site site 11site (Santo do Antônio Ara- river), site 2 (water channel), site 7 (Tocantins river) and site 8 (large 1111 (Santo (Santo11 (Santo Antônioguaia), Antônio Antônio siteriver), river), 5 river),(smallsite site 2 2(watersite irrigation(water 2 (water channel), channel), channel—Formoso channel), site site 7 7 (Tocantinssite (Tocantins 7 (Tocantins do river) river) Araguaia), river)and and site siteand 8site 8(largesite (large 3 8(Urubu (large volumes river), of water site in the channel), showing decreases of 39, 36, 35, 34, 33, 30, 28, 26 and volumes of water in the channel), showing decreases of 39, 36, 35, 34, 33, 30, 28, 26 and volumesvolumes of of11 water water(Santo in in theAntônio the channel), channel), river), showing showing site 2de de(watercreasescreases channel), of of 39, 39, 36, 36, 35,site 35, 34, 734, (Tocantins33, 33, 30, 30, 28, 28, 26 26river) and and and24%, site respectively, 8 (large when compared to the control condition using ASTM only (Figure 3). 24%, 24%,respectively, respectively, when when compared compared to the to control the control condition condition using using ASTM ASTM only only(Figure (Figure 3). 3). 24%, respectively,volumes when of water compared in the to thechannel), control showingcondition deusingcreases ASTM of only 39, 36,(Figure 35, 34,3). 33, 30, 28, 26 and 24%, respectively, when compared to the control condition using ASTM only (Figure25 3). 2525 25 a ab a a a abab ab 20 abc 20 25 abcd 2020 abcabc abc bcd bcd bcd cd bcd abcdabcd abcd cd bcdbcda bcd bcdbcd bcd abbcd 15 cd cd cd bcd cd d cdcd cd cdcd 1515 15 cd20cd cd cdcd cdcd cd d d d abc abcd bcd bcd bcd 10 cd cd 1010 10 15 cd cd cd d 5

55 5 /min) crossed (gridlines LMV 10 p LMV (gridlines crossed /min) crossed (gridlines LMV LMV (gridlines crossed /min) crossed (gridlines LMV LMV (gridlines crossed /min) crossed (gridlines LMV p p p 0 0 0 ASTM 1 2 3 4 5 6 7 8 9 10 11 12 0 5 ASTMASTMASTM11 212 323 434 545 656 767 878 989 10109 111011 121112 12 Sites of Tocantins Araguaia hydrographic region LMV (gridlines crossed /min) crossed (gridlines LMV

p SitesSites of Sitesof Tocantins Tocantins of Tocantins Araguaia Araguaia Araguaia hydrographic hydrographic hydrographic region region region 0 ASTM 1 2 3 4 5 6 7 8 9 10 11 12 Sites of Tocantins Araguaia hydrographic region

Water 2021, 13, x FOR PEER REVIEW 7 of 14

Nacional), showed a significantly decreased feeding activity of 38, 34, 28, 26 and 25%, respectively, when compared to the ASTM control condition (Figure 2).

2.0

a 1.5 ab ab abc abc abc abc abc bc bc bc bc c 1.0

0.5

0.0 Feeding rate Feeding (n° consumed/hour) larvae ASTM 1 2 3 4 5 6 7 8 9 10 11 12 Sites hydrographic region:Tocantins Araguaia

Figure 2. Feeding rate (number of C. xanthus larvae consumed per hour) of G. tigrina after 96 h exposure with water samples from Tocantins-Araguaia Sites Hydrographic Region: Lagoa da Con- fusão (1–3); Formoso do Araguaia (4–6); Porto Nacional (7–9); Gurupi (10–12); Reference (12). A laboratory control with ASTM only was also used to expose planarians. Values represent the mean (± SEM), n = 10. Different letters indicate significant differences between study sites when p < 0.05. Colors used in graph bars are associated with analysis of water performed: very high concentrations, high concentrations, above acceptable limits, acceptable limit. Water 2021, 13, 1077 7 of 13 3.3. Planarian Locomotor Velocity (pLMV) The post-exposure locomotion velocity (pLMV) of G. tigrina was significantly affected 3.3. Planarian Locomotor Velocity (pLMV) (p < 0.0001) in planarians after 96 h exposure to water samples from site 10 (Dam Brejo Pantanal-Gurupi),The post-exposure site locomotion1 (lake—Lagoa velocity da (p LMV)Confusão), of G. tigrina site was4 (Taboca-Formoso signiﬁcantly affected do Ara- (p < 0.0001) in planarians after 96 h exposure to waterWater 2021 samples, 13, x FOR from PEER site REVIEW 10 (Dam Brejo 7 of 14 guaia), site 5 (small irrigation channel—Formoso do Araguaia), site 3 (Urubu river), site WaterWater 2021 2021Water, 13, 13 2021, x, xFOR FOR, 13 ,PEER x PEER FOR REVIEW REVIEWPEER REVIEW Pantanal-Gurupi), site 1 (lake—Lagoa da Confusão), site 4 (Taboca-Formoso7 7of of 14 do14 7 of Araguaia), 14

11site (Santo 5 (small Antônio irrigation river), channel—Formoso site 2 (water channel), do Araguaia), site site 7 (Tocantins 3 (Urubu river), river) site and 11 (Santosite 8 (large volumes of water in the channel), showing decreases of 39, 36, 35, 34, 33, 30, 28, 26 and Antônio river), site 2 (water channel), site 7 (Tocantins river) and site 8 (large volumesNacional), of showed a significantly decreased feeding activity of 38, 34, 28, 26 and 25%, 24%,water respectively, in the channel), when showing compared decreases to ofthe 39, control 36, 35, 34,condition 33, 30, 28, using 26 and ASTM 24%, respectively, only (Figure 3). Nacional),Nacional),Nacional), showed showed showed a a significantly significantly a significantly decreased decreased decreased fe feedingeding fe edingactivity activity activity of of 38, 38, of34, 34, 38, 28, 28, 34, 26 26 28,and and 26 25%, 25%,and 25%,respectively, when compared to the ASTM control condition (Figure 2). when compared to the control condition using ASTM only (Figure3). respectively,respectively,respectively, when when whencompared compared compared to to the the toASTM ASTM the ASTM control control control condition condition condition (Figure (Figure (Figure 2). 2). 2).

25 2.0 2.0 2.0 2.0 a ab 20 a abc abc a a a abcd 1.5 ab ab abc bcd bcd abc abc abc 1.5 ab ab bcd abcabc abc 1.51.5 abc abab abab abcabc abc cd bc bc bc abcabc cd abcabc abc abcabc 15 cd cd bc cd bc bc bcbc bc bcbc bcbc d c bc c bcbc 1.0 1.0 c c 1.01.0 10 0.5 0.50.5 0.5 5 LMV (gridlines crossed /min) crossed (gridlines LMV p 0.0 0.00.0 0.0 0 rate Feeding (n° consumed/hour) larvae ASTM 1 2 3 4 5 6 7 8 9 10 11 12 Feeding rate Feeding (n° consumed/hour) larvae Feeding rate Feeding (n° consumed/hour) larvae Feeding rate Feeding (n° consumed/hour) larvae ASTM 1 2 3 4 5 6 7 8 9 10 11 12 ASTMASTMASTM11 221 332 443 554 665 776 887 998 10109 111110 121211 12 Sites hydrographic region:Tocantins Araguaia SitesSites hydrographic hydrographicSites hydrographicSites ofregion:Tocantins region:Tocantins Tocantins region:Tocantins Araguaia Araguaia Araguaia Araguaia hydrographic region Figure 2. Feeding rate (number of C. xanthus larvae consumed per hour) of G. tigrina after 96 h Figure 3. Planarian locomotor velocity (pLMV) of G. tigrina (gridlines crossed/min), after 96 h of FigureFigure Figure2. 2. Feeding Feeding 2. Feeding rate rate (number (number rate (number of of C. C. xanthus xanthusof C. xanthus larvae larvae consumedlarvae consumed consumed per per hour) hour) per of hour)of G. G. tigrina tigrinaof G. tigrinaafter after 96 96after h h 96 h exposure with water samples from Tocantins-Araguaia Sites Hydrographic Region: Lagoa da Con- exposure to water samples of Sites of Tocantins-Araguaia Hydrographic region: Lagoa da Confusão exposureexposureexposure with with water waterwith sampleswater samples samples from from Tocantins-Arag Tocantins-Aragfrom Tocantins-Araguaiauaia Sites Sitesuaia Hydrographic HydrographicSites Hydrographic Region: Region: Region: Lagoa Lagoa daLagoa da Con- Con- da Con-fusão (1–3); Formoso do Araguaia (4–6); Porto Nacional (7–9); Gurupi (10–12); Reference (12). A (1–3); Formoso do Araguaia (4–6); Porto Nacional (7–9); Gurupi (10–12); Reference (12). A laboratory fusãofusão (1–3); fusão(1–3); Formoso (1–3);Formoso Formoso do do Araguaia Araguaia do Araguaia (4–6); (4–6); Porto (4–6);Porto Na PortoNacionalcional Na (7–9);cional (7–9); Gurupi (7–9);Gurupi Gurupi (10–12); (10–12); (10–12); Reference Reference Reference (12). (12). A A (12). Alaboratory control with ASTM only was also used to expose planarians. Values represent the mean ± laboratorylaboratorylaboratory control controlcontrol control with with with ASTM ASTMwith ASTM ASTMonly only only was wasonly was also also was also used used also used to toused expose toexpose exposeto expose planarians. planarians. planarians. planarians. Values Values Values Values represent represent represent represent the the mean themean the mean mean(± (SEM), SEM), n = 10. Different letters indicate significant differences between study sites when p < 0.05. (± SEM),n =n = 10. 10. Different Different letters letters indicate indicate signiﬁcant significant differencesdifferences betweenbetween studystudy sitessites when p < 0.05. 0.05. Colors (±(± SEM), SEM), n n = =10. 10. Different Different letters letters indicate indicate si significantgnificant differences differences between between study study sites sites when when p p< <0.05. 0.05. Colors used in graph bars are associated with analysis of water performed: very high concen- ColorsColors Colorsused used in inused graph graph in bars graph bars are are bars associated associated are associated with with an anwith alysisalysis an analysis ofalysis of water water of performed:water performed: performed: performed: very very high veryhigh very concen- highconcen-high concentrations,concen-trations, high concentrations, above acceptable limits, acceptable limit. trations,trations,trations, high highhigh concentrations, concentrations, highconcentrations, concentrations, above above above acceptable above acceptableacceptable acceptable limits, limits,limits, limits, acceptable acceptable acceptable acceptable limit.limit. limit. limit. 3.3. Planarian Locomotor Velocity (pLMV) 4. Discussion 3.3.3.3. Planarian Planarian3.3. Planarian Locomotor Locomotor Locomotor Velocity Velocity Velocity (pLMV) (pLMV) (pLMV) The post-exposure locomotion velocity (pLMV) of G. tigrina was significantly affected Alternative methodologies involving invertebrates, including freshwater planarians, TheThe post-exposure post-exposureThe post-exposure locomotion locomotion locomotion velocity velocity velocity ( p(pLMV)LMV) (p LMV)of of G. G. tigrina oftigrina G. tigrina was was significantly significantly was significantly affected affected affected (p < 0.0001) in planarians after 96 h exposure to water samples from site 10 (Dam Brejo have been awakened worldwide. In this context, the effects observed on the feeding rate (p( p< < 0.0001) 0.0001)(p < 0.0001) in in planarians planarians in planarians after after 96 after96 h h exposure 96exposure h exposure to to water water to watersamples samples samples from from site fromsite 10 10 site (Dam (Dam 10 (DamBrejo Brejo Brejo Pantanal-Gurupi), site 1 (lake—Lagoa da Confusão), site 4 (Taboca-Formoso do Ara- and pLMV of G. tigrina are considered sensitive and relevant parameters from the point of Pantanal-Gurupi),Pantanal-Gurupi),Pantanal-Gurupi), site site 1 1 site(lake—Lagoa (lake—Lagoa 1 (lake—Lagoa da da Confusão), Confusão), da Confusão), site site 4 4 site(Taboca-Formoso (Taboca-Formoso 4 (Taboca-Formoso do do Ara- Ara- do Ara-guaia), site 5 (small irrigation channel—Formoso do Araguaia), site 3 (Urubu river), site view of environmental monitoring of different contaminants. Additionally, these endpoints guaia),guaia),guaia), site site 5 5 (smallsite (small 5 (small irrigation irrigation irrigation channel—Formoso channel—Formoso channel—Formoso do do Araguaia), Araguaia), do Araguaia), site site 3 3 (Urubusite (Urubu 3 (Urubu river), river), river),site site 11site (Santo Antônio river), site 2 (water channel), site 7 (Tocantins river) and site 8 (large may be linked to changes in higher levels of biological organization, as they are the result of 1111 (Santo (Santo11 (Santo Antônio Antônio Antônio river), river), river),site site 2 2(watersite (water 2 (water channel), channel), channel), site site 7 7 (Tocantinssite (Tocantins 7 (Tocantins river) river) river)and and site siteand 8 8(largesite (large 8 (large volumes of water in the channel), showing decreases of 39, 36, 35, 34, 33, 30, 28, 26 and a series of biochemical and physiological processes caused by environmental stress [47–49]. volumesvolumesvolumes of of water water of waterin in the the inchannel), channel), the channel), showing showing showing de decreasescreases decreases of of 39, 39, of36, 36, 39, 35, 35, 36, 34, 34, 35, 33, 33, 34, 30, 30, 33, 28, 28, 30, 26 26 28,and and 26 and24%, respectively, when compared to the control condition using ASTM only (Figure 3). The results of our study showed the alteration of various physical and chemical 24%, respectively, when compared to the control condition using ASTM only (Figure 3). 24%,24%, respectively, respectively,parameters when when andcompared compared the presence to to the the control ofcontrol various condition condition heavy using metalsusing ASTM ASTM in water only only samples (Figure (Figure 3). that 3). altered the behavior of the planarian (Table1). We observed that the behavioral results of G. tigrina 25 were shown to be sensitive to the TAHR water samples, as a possible result of synergistic or 2525 25 antagonistic interactions of the physical-chemical parameters of each sample, illustrating a ab a athata these are important integrativeabab ab and cumulative ecotoxicological parameters with20 abc 20 abcd 2020 ecological relevance. The biomarkers of environmental contaminationabcabc abc evaluated showed bcd bcd bcd cd bcd abcdabcd abcd cd differencesbcdbcd in sensitivitybcd dependingbcdbcd onbcd thebcd waterbcd sample location in TAHR. For pLMV,15 cd cd cd cd cdcd cd d 15signiﬁcantcdcd cd differencescdcd werecd observedcd when planarians were exposed to water samples from 1515 cdcd cdcd d most of the sampling points of TAHR (sampling pointsd d 1, 2, 3, 4, 5, 7, 8, 10, 11—Figure3 ). 10 On the other hand, the feeding rate of planarians was affected when organisms were 1010 10 exposed to less contaminated sites (2, 3, 6, 8, 9—Figure2). Despite the fact that planarians’ locomotion is achieved mainly by gliding (ciliary5

55 5 /min) crossed (gridlines LMV beating), the locomotor behavior is related to the proper functioning of the nervous system,p LMV (gridlines crossed /min) crossed (gridlines LMV LMV (gridlines crossed /min) crossed (gridlines LMV LMV (gridlines crossed /min) crossed (gridlines LMV p p p 0 00 0 ASTM 1 2 3 4 5 6 7 8 9 10 11 12 ASTMASTMASTM11 212 323 434 545 656 767 878 989 10109 111011 121112 12 Sites of Tocantins Araguaia hydrographic region SitesSites of Sitesof Tocantins Tocantins of Tocantins Araguaia Araguaia Araguaia hydrographic hydrographic hydrographic region region region

Water 2021, 13, 1077 8 of 13

since muscle contraction can be employed [50]. In addition, it is important to note that the feeding activity of planarians is dependent on their locomotor ability to capture prey, and also related to the nervous system functioning, such as movement and muscle action, the perception of chemical stimuli, as well as the extra-corporal digestion of prey [49,51,52]. The lower sensitivity of the feeding test, compared to pLMV, may be related to the short exposure time and period of feeding post-exposure. Although future studies are needed to elucidate this, we hypothesized that a longer exposure period of planarians (>96 h) and a shorter post-period of feeding (<12 h) would better reflect the increased energy required for maintenance and defense mechanisms [53]. Although in our study planarians have been exposed to TAHR water samples for 4 days (96 h static exposure), there still seems to be no consensus on the specific number of days planarians should be exposed in static or semi-static conditions—usually from 8 days of exposure up to 14 days [35–37]. Feeding tests reported in the most recent scientific literature generally occur for a period of 3 h, when organisms are exposed to a specific contaminant [35–37]. According to our results, it seems that the feeding activity of planarians is dependent on the locomotor activity and feeding requirement of exposed planarians: (i) planarians exposed to water of less contaminated sites showed a decreased locomotor activity and probably a low requirement of food; (ii) planarians exposed to highly contaminated water also showed a decreased locomotor activity, but an increased requirement for food to deal with deleterious effects of contaminants. Therefore, those differences seem to be more evident in feeding tests with long post-exposure periods. Various studies have reported the toxicological effects of one or more compounds in organisms [37,54]. However, studying the synergism or antagonism that may occur with the presence of contaminants and environmental parameters (pH, dissolved oxygen, turbidity) in organisms is complex [55]. Prolonged exposure to these metals can cause cell death and alterations in DNA, lipids, proteins, enzymes, and homeostasis of calcium and sodium ions [56]. For example, 40 mg L−1 Fe3+ has been shown to cause 100% mortality in Dugesia japonica at 25 ◦C; additionally, the low temperature could slow down the effect of Fe3+ on planarian toxicity, and at a suitable temperature, the toxic effects of Fe3+ on planarian can be accelerated [57]. Heavy metals, such as, Zn, Cu, and Fe, are necessary for the proper functioning of various enzymes and proteins. However, when the same metals accumulate above the threshold, they become toxic. This induces the generation of reactive nitrogen and oxygen species (RNS; ROS), which results in the peroxidation of lipids in the plasma membrane [23,58]. Reports show heavy metal bioaccumulation in organisms through food chains [59,60]. It has been shown that the enzymatic activity of superoxide dismutase and catalase can play an important role on glutathione peroxidase in the antioxidant defense system of Dugesia japonica in response to exposure to copper [38]. Another study conducted with Girardia schubarti suggests that copper could modulate the genotoxic effects associated with the exposure of complex mixtures in the environment [39]. Furthermore, in our work we found concentrations of up to 0.88 mg L−1 of dissolved iron with the temperature in the Tocantins state fluctuating between 30 and 38 ◦C. In the field conditions, temperatures above 27 ◦C could cause mortality and slow movements in G. tigrina; consequently, the species releases mucus to change the surrounding pH of its external environment to maintain its physiological function [23]. High levels of dissolved aluminum (point 7), total chlorine (points 4 and 11), and zinc (point 5) were found in water samples of TAHR, which may be associated with the intensive and periodic use of fertilizers and agricultural correctives (in the case of aluminum), whereas it is known that freshwater planarians can present different responses to heavy metals. The high toxicity of aluminum, if compared to chromium and cadmium, causes uncoordinated writhing in exposed planarians, suggesting that neurotoxic effects were produced in intact and regenerating Dugesia estrusca [40]. Furthermore, chlorine plays a key role in disinfecting drinking water and wastewater; on the other hand, it represents a Water 2021, 13, 1077 9 of 13

risk to the freshwater ecosystem when used for a longer period [28]. These authors report the chronic effect of chlorine on Girardia tigrina with observed effect concentrations (LOEC) of 210 µg/L for feeding and pLMV, using the same methodology of our assays, as well as LOEC of 168 µg/L for head regeneration and 263 µg/L for reproduction (fertility and fertility). This is worrying from an environmental point of view, since at the sample of point 11 of TAHR, we found concentrations of total chlorine of 70 µg/L—the same order of magnitude as the LOEC values reported by Rodrigues Macêdo and co-authors [28]. On the other hand, zinc exposures have been reported to cause oxidative damage in aquatic organisms, for example, in the shrimp Atyaephyra desmarestii, while the feeding rate of amphipod Echinogammarus meridionalis was severely reduced with zinc exposures [60]. The authors report that a decrease in feeding rates reduces the total energy available, and the processes of metabolism and detoxification are also likely to be reduced. We did not measure oxygen levels during experimental feeding and locomotion tests, since the exposure occurred in 96 h in a shallow, flat vessel (Petri dish). In spite of this, the results of the analysis of the samples collected in the field revealed low concentrations of dissolved oxygen, mainly in the water samples of points 5 and 11. Although we cannot say that the effects on the feeding of planarians are related to low concentrations of oxygen, we speculate whether the effects observed in pLMV could be related, among other factors, to low oxygen concentrations in samples 5 and 11. It has been reported that low respiration rates can affect the physiological performance of freshwater invertebrates and have been associated with changes in behavior [37,61,62]. During moderate stress, maintenance costs increase to meet the additional energy demands for stress protection and damage repair, or metabolism and/or food assimilation is affected by the stressor. As a result, the aerobic range decreases [53,63]. Increases in temperature (as occur in TAHR-tropical climate) can lead to an increased metabolic rate and O2 consumption by aquatic organisms and the consequent production of reactive oxygen species (ROS) [64,65]. For example, studies in Girardia dorotocephala and Schmidtea mediterranea show that as the temperature increases, oxygen consumption will also increase [66]. Studies in G. tigrina show that it decreases pLMV when exposed to xenobiotics, for example, pLMV displayed a dose-dependent negative correlation with scopolamine concentrations from 0.001 to 1.0 mM, and a further increase in scopolamine concentration to 2.25 mM did not further decrease pLMV [67]. In another study, galantamine showed high anticholinesterasic activity when compared to the other drugs, with a reduction in pLMV, presenting screw-like movement and hypokinesia, with a pLMV of 65 crossed lines during 5 min [68]. The pLMV seems to be a good indicator when exposed to xenobiotics, as shown by experiments carried out where, as the concentration increases, the locomotion speed of the planaria decreases [35–37,54]. This is also demonstrated in our study, where pLMV decreases in local (2–5, 7, 10–11) sites with the presence of xenobiotics. It should be considered that alterations in the behavior of the planarian can affect the food chain. The stability of a species has been shown to be highest when it is at the top of the food chain, and lowest when it is just below the top level, and it exhibits a switching pattern at intermediate levels [69]. Consequently, we could mention that the decrease in the feeding rate and the pLMV could be a consequence of the reallocation of energy to the stress response and the maintenance of homeostasis in planarian. The study of these biomarkers of behavior in the planarian suggests that the trophic chains in the freshwater ecosystems of the TAHR are being altered, since these organisms play a fundamental role in these ecosystems. Likewise, the results of this study alert us to the need to monitor the consequences of anthropic actions and agricultural pressure in the state of Tocantins—Brazil. At the same time, behavioral tests used with planarians seem to be fast screening tools that contribute to the biomonitoring of freshwater systems and further conservation measures to be taken in the Tocantins-Araguaia hydrographic system. In sum, the tributaries of the Tocantins-Araguaia hydrographic region, comprising the municipalities of Formoso do Araguaia, Lagoa da Confusão, Gurupi, and Porto Nacional, presented contamination by heavy metals and other pollutants, which caused deleterious Water 2021, 13, 1077 10 of 13

effects in behavioral responses of the freshwater planarian G. tigrina. Finally, this research provides an important approach to assess the ecological effects of contaminants in lotic ecosystems, using the behavior of planarians to assess water quality in tropical aquatic systems subjected to anthropic pressure. We aimed to use a behavioral tool that could predict deleterious effects of water samples from the environment even without knowing the physical or chemical properties of water. This would help to identify the impacted hotspots in a fast and cost-effective way. Obviously, further biomonitoring would be necessary on those hotspots for the identification of the environmental problems. Briefly, our goal was to use a behavioral biomarker to assess the sublethal effects of water contamination. The use of a behavioral test presents the advantage of being cumulative and integrative, i.e., it is caused by insufficient energy allocation for behavior due to its use for other processes altered by stress conditions, and that might be a chemical compound, an abiotic factor, or even the conjugation of several factors.

5. Conclusions The development of agriculture in the hydrographic Tocantins-Araguaia region con- tributes to the increased concentrations of metals, causing deleterious effects on benthic organisms, such as G. tigrina, that potentially affect various organizational levels of the trophic chain. The exposure in the laboratory of planarians to water of different sampling points in the hydrographic region Tocantins-Araguaia caused a reduction in the feeding rate and pLMV. The use of these cumulative and integrative behavioral biomarkers showed high sensitivity to contaminants and abiotic factors of water samples. These results emphasize the importance of evaluating biomarkers of environmental contamination, using the behavioral parameters of G. tigrina as fast screening tools in order to help the development and ecological relevance of biomonitoring programs in contaminated watersheds.

Author Contributions: Conceptualization, R.A.S. and A.M.V.M.S.; methodology, A.M.C.L. and A.d.S.S.; software, A.M.C.L. and A.d.S.S.; validation, C.G., R.A.S. and A.M.V.M.S.; formal analysis, C.G.; investigation, A.M.C.L. and A.d.S.S.; resources, R.A.S. and A.M.V.M.S.; data curation, A.d.S.S.; writing—original draft preparation, A.M.C.L. and A.d.S.S.; writing—review and editing, all authors; visualization, C.G.; supervision, R.A.S. and A.M.V.M.S.; project administration, R.A.S.; funding acquisition, R.A.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, Brazil (Edital 71/2013–Programa Ciência Sem Fronteiras–Modalidade Pesquisador Visitante Especial-PVE–Projeto: A058_2013). Thanks are also due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020 + UIDB/50017/2020), through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. Renato A. Sarmento received a scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Brazil (Produtividade em Pesquisa-Projeto: 306652/2018-8). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study is available in the current manuscript, raw data is available on request from the corresponding author. Acknowledgments: The authors thank the Universidade Federal do Tocantins—Campus Gurupi, Brazil for support and the Instituto Federal de Educação, Ciência e Tecnologia Goiano—Campus Campos Belos, Brazil for partnership. Conflicts of Interest: The authors declare no conflict of interest. Water 2021, 13, 1077 11 of 13

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