Intermittent Rivers as a Challenge for Freshwater Ecosystems Quality Evaluation: A Study Case in the Ribeira de Silveirinhos, Portugal

Abstract

Intermittent rivers and streams are aquatic ecosystems that lose hydrological connectivity during drought periods. Anthropogenic pressures combined with climate change accelerate the spatial and temporal intermittency of these ecosystems, promoting alterations in ecology and ecosystem functions. This study aimed to assess the ecological status of Ribeira de Silveirinhos, located in the municipalities of Valongo and Gondomar (north of Portugal), using the metrics described in the Water Framework Directive. Thus, five sampling sites were selected along Ribeira de Silveirinhos. Sampling was done in three different periods, spring of 2019 and 2020, and autumn of 2019. At each site, physical and chemical water parameters were measured, and the benthic macroinvertebrates community was characterized. Physical and chemical parameters showed low pH values at upstream sites (where the dry phase occurs and is surrounded by Eucalyptus monoculture) and high concentrations of phosphorus at the downstream sites (subjected to several anthropogenic activities, iron waters, and agriculture). The macroinvertebrate community revealed low values of specific abundance and richness, especially during the dry period. Sensitive taxa (Ephemeroptera, Plecoptera, and Trichoptera) were negatively correlated with flow intermittency. Results showed that Ribeira de Silveirinhos is classified between “poor” and “moderate” in terms of ecological status. Intermittent streams are undervalued, so their protection is at risk. Thus, it is important to consider the specific ecological functioning of these ecosystems and to adjust the planning and management models in order to guarantee ecological quality and conservation processes.

1. Introduction

Intermittent rivers are a type of fluvial ecosystems poorly understood but whose importance has already been recognized [1,2,3]. These aquatic ecosystems are defined as watercourses that at some point in time and space, naturally cease their flow [3,4]. Its regime alternates between wet and dry periods over a hydrologic annual cycle and may leave some pools between dry sections of the riverbed [5]. This type of river is frequent in the Mediterranean region, as well as in arid and semi-arid regions, where there is great seasonal variability in temperature and precipitation over a year [4]. Intermittent rivers represent a dynamic ecosystem, with a high spectrum of variations in terms of duration, predictability, and volume as well as the extent of interruption of the drought period [6,7]. They are endowed with a remarkable hydromorphological diversity since the river goes through periods of droughts and floods that alter the abiotic and biotic conditions of the ecosystem. Flow variability has been identified as an important factor affecting other abiotic and biotic factors that regulate processes in the ecosystem [1,8]. Several studies show that during the drought period, with the decrease of the water surface area, chain reactions are triggered in the physical and chemical parameters, such as the increase in temperature and hypoxia/anoxia that negatively affect the aquatic biota [1]. Moreover, the duration of the drought is crucial to determine the diversity in intermittent rivers [4]. Indeed, Sabater et al. [9] stated that the flow variation had a significant and more relevant influence on the macroinvertebrate community than on pollutants. Drying the riverbed represents a loss of longitudinal connectivity, fragmentation into sections, and changes in the availability of habitat. These changes result in a high variety of biotic communities across intermittent gradients, for example, richness decreases as the severity of intermittency increases [10]. However, despite the decrease in lotic species, the presence of pools between the dry sections allows the occupation of communities characteristics of lentic systems and semi-aquatic communities. The wet and dry periods allow a unique combination of aquatic, amphibious, and terrestrial associations, with a succession of biota being observed during the transition between the two periods. The peculiar conditions of the intermittent rivers impose, however, a strong selective pressure for the evolution of characteristics of resistance and resilience of the biota [5]. The great variability of intermittent rivers allows them to function as a biodiversity hotspot, throughout their annual cycle, with diverse and unique communities [6]. Beside this importance, intermittent rivers are highly threatened ecosystems, due to their devaluation, lack of information, and protection legislation [10].

The Water Framework Directive (WFD—Directive 2000/60/EC) of the European Parliament and of the Council of 23 October 2000 was created to establish a framework for community action in the field of water policy with the objective of the good ecological status of all water bodies of the European Union [11]. The ecological status of surface waters (e.g., rivers) is defined by the WFD as “the expression of the structural and functional quality of aquatic ecosystems associated with surface waters” [11]. The assessment of ecological status is carried out using three different types of quality elements: biological (e.g., benthic macroinvertebrates, fish fauna, aquatic flora), physical and chemical (e.g., thermal conditions, oxygen conditions, salinity, acidification status, nutrient concentrations) and hydromorphological (e.g., hydrographic regime, river continuity, morphological conditions). The classification of ecological status is defined based on the deviation from the natural conditions, called environmental quality standards (EQS) [12]. The water bodies are grouped based on their hydrological and geographical characteristics so that the environmental quality standards (EQS) provide a comparable ecological status [13].

The different environmental tolerances between species and their responses to disturbances can be used to infer environmental conditions in a specific ecosystem [14,15,16,17]. Various indices have been developed to describe community responses and establish the ecological status of water bodies. The WFD recommends the use of multimetric approaches to discriminate disturbances [18], and for benthic macroinvertebrates, several indices are used (richness; diversity), including the Biological Monitoring Working Party (BMWP) index. This index was adapted for the Iberian Peninsula resulting in the IBMWP index (Iberian BMWP), where each family is assigned a score representative of pollution tolerance [15]. Portugal developed a similar index to assess water quality regarding macroinvertebrate communities: the IPtIN or IPtIS (Índice Português de Invertebrados do Norte (Equation (1)) or do Sul, respectively), applied according to the location of the different rivers. Its use makes it possible to assess the water quality of rivers to accomplish the WFD. It also describes the gradients of degradation of water bodies and discriminates as a final value quality class expressed in Ecological Quality Ratio (EQR) [13].

Based on previous findings, this study intends to characterize and evaluate the ecological conditions along a small and intermittent river (Ribeira de Silveirinhos, north of Portugal). This evaluation followed the guidelines of the WFD for lotic systems to perceive the effectiveness of physical and chemical parameters as well as the macroinvertebrate evaluation for this particular aquatic ecosystem.

2. Materials and Methods

2.1. Study Area

The watercourse evaluated was Ribeira de Silveirinhos, a small stream with a 4 km length, located in the north of Portugal. It is within the limits of Parque das Serras do Porto (PSeP—a regional protected landscape), inserted in the municipalities of Valongo and Gondomar (Figure 1). This aquatic ecosystem is a small intermittent mountain brook in slate and quartzite terrains. It flows into Rio Ferreira in Gondomar, being its main effluent. It belongs to the Douro Hydrographic Basin and it is part of an area covered by the Natura 2000 network classified as PTCON0024 (“Valongo”, included at the Sites of Community Importance) [19,20]. This region, with protected landscape status, highlights the landscape and heritage values present. The occurrence of significant natural ecosystems associating with a vast floristic and fauna biodiversity, a relevant geological and cultural heritage as well its location in the Metropolitan area of Porto (AMP) are some of the factors that recognize this area with high environmental development potential [20].

Ribeira de Silveirinhos is located in an area with a very mild climate, with a moderate thermal amplitude, which is expected given the proximity of the coast. Inserted in a northwestern Portuguese region, it is subject to the influence of the Atlantic regime, so there are relatively frequent rains [20,21]. However, an absence of precipitation during the summer period causes water stress on aquatic and terrestrial ecosystems. Therefore, the Ribeira de Silveirinhos has a temporary drought in its flow, being designated as an intermittent river [7]. In the area surrounding of this watercourse, the extractive activity dates to the Roman era, with the extraction of gold (Au) and iron (Fe) observed, and which is reflected in the observed water color/characteristics (Figure S1—supplementary material). This activity left significant marks on the landscape, which persist over time; one of them is the presence of very iron waters with a brownish-yellow hue [20,22].

2.2. Sampling Sites and Fieldwork

This study was carried out in the spring and autumn of 2019 (Sp19; Au19) and spring of 2020 (Sp20). In the autumn of 2019 sampling, the stream was dry at points RS_1, RS_2, and RS_3; therefore, no samples were collected, or ecological status evaluated. Five sampling sites were selected along Ribeira de Silveirinhos (Figure 1) and the site selection took into consideration the various human activities practiced in the surrounding areas along the watercourse to understand their impacts on the quality of the aquatic system.

RS_1 and RS_2 surrounding areas are characterized by forest landscape elements, essentially Eucalyptus (Figure 1; Figure S1—supplementary material). At site RS_3, the Eucalyptus monoculture was the predominant flora, however, some species of ferns were already visible, in scattered spots. From this site on, a brownish-yellow hue of water began to be noticed, coming from the runoff of iron water from the old mining operations (Figure S1—supplementary material). RS_4 is characterized by three distinct elements that are dominated by the landscape, forests, surface water bodies, and bushes (Figure 1; Figure S1—supplementary material). At this site, the Eucalyptus forest area is reduced, and an increase in the diversity of plant species around the stream was observed. At this sampling site, a brownish-yellow hue in the water was recorded. The RS_5 site was characterized by a mixed elements forest, surface water bodies, and agriculture in the surrounding area where agriculture has the highest representativeness (Figure 1; Figure S1—supplementary material). RS_5 also shows a brownish-yellow hue, and signs of anthropogenic pollution are visible (garbage like plastics, and tires). The deposition of residues recorded is related to the presence of houses, a bridge, and a road representing easier access to the area.

At each sampling site, general physical and chemical parameters of water were measured in situ: temperature conditions (°C), acidification status (pH), conductivity (µS/cm), oxygenation conditions (% saturation and mg/L), and total dissolved solids (TDS) [13], using a multi-parameter WTW probe, model Multi 350i. Additionally, at each sampling site, a water sample was collected in a 1.5 L plastic bottle (transported at 4 °C and under dark conditions) to quantify other parameters in the laboratory: the 5-day Biochemical Oxygen Demand (BOD₅) and nutrient concentrations (nitrates, ammoniacal nitrogen, and total phosphorus).

The sampling of the benthic macroinvertebrate community was carried out according to the INAG 2008 protocol [23]. A hand net with a mesh of 0.5 mm and 25 cm in width was used, which was dragged over the substrate; the riverbed was not actively disturbed by hand or foot in front of the net. At each site (see nature of the riverbed and general habitat at each site, in Figure S1—supplementary material), a composite sample [three drags were performed along one meter of the substrate (essentially sands, gravel, and rocks) and vegetation] was collected with the guarantee that all the habitats recorded were sampled. The benthic macroinvertebrates samples were preserved in 4% formaldehyde [23] and properly labeled and packaged in plastic bottles.

2.3. Laboratory Procedures

In the laboratory, on the day the water samples were collected, the BOD₅ was determined. Water samples from each site were placed in 250 mL amber glass bottles and the dissolved oxygen concentration (mg/L) was measured. Subsequently, a drop of a nitrification inhibitor (Allylthiourea 98%) was placed in each bottle and then these were filled and closed, ensuring the absence of air bubbles inside. The samples were incubated at 20 °C in the dark, for five days. At the end of this period, the dissolved oxygen concentration (mg/L) was again measured with a multiparametric probe, and BOD₅ was calculated considering the difference between the dissolved oxygen concentration at the beginning, and after 5 days of incubation, in the dark conditions. For the quantification of nutrients, the concentration of nitrates (NO₃, mg/L), ammoniacal nitrogen (NH₄, mg/L), and total phosphorus (P, mg/L) were determined on a Spectroquant Multy Colimeter bench photometer using standardized procedures (method 321; method 383; method 33, respectively).

Regarding the samples of the benthic macroinvertebrates’ community, the composite sample was washed with a 0.5 mm mesh sieve to remove all the fine sediment present in the sample. The material retained in the sieve was placed in trays and sorted, to separate the macroinvertebrates from the remaining debris. The sampled organisms were preserved in small bottles properly labeled in 70% alcohol, for later identification. The identification was performed to the family level in almost all taxa, consistent with the taxonomic level used for specific indexes in Europe [24,25,26]. Oligochaeta was identified to the subclass [25].

2.4. Data Analysis

2.4.1. Physical and Chemical Elements

To carry out a multivariate analysis, a data matrix was built with the values of the environmental variables measured in situ and in the laboratory, for each sampling site. The values were previously normalized using the equation x’ = (x − µ)/σ. A principal component analysis (PCA), calculated using CANOCO for Windows version 4.0, was used to summarize the physical and chemical parameters, allowing us to analyze the relationship between the different sampling sites and to identify the existence of gradients [27].

2.4.2. Biological Quality Elements

The index used for the aquatic ecosystem quality evaluation regarding macroinvertebrates community was the Portuguese Northern Invertebrate Index (IPtlN). The values of composition and abundance of benthic macroinvertebrates were analyzed to describe gradients of general degradation and to discriminate quality classes, using a set of metrics [13].

$$\mathrm{IPtIN}=\mathrm{N}°\mathrm{Taxa}\times 0.25+\mathrm{EPT}\times 0.15+\mathrm{Evenness}\times 0.1+\left(\mathrm{IASPT}-2\right)\times 0.3+\mathrm{Log}\left(\mathrm{Sel}.\mathrm{ETD}+1\right)\times 0.2$$

(1)

where:

EPT—N° of families belonging to the orders Ephemeroptera, Plecoptera, Trichoptera.

Evenness—Additionally, called the Pielou Index or Equitability, it is calculated as:

E = H/ln S

(2)

where H is Shannon-Wiener diversity, S = the number of taxa present, ln = natural or Neperian logarithm.

The Shannon-Wiener index is calculated using the expression:

H = − ∑ pilnpi

(3)

where pi = ni/N, that is, the number of individuals of each taxon i (ni) divided by the total number of individuals (N) present in the sample.

IASPT—ASPT Iberic, which corresponds to the BMWP Iberic [23] divided by the number of families included in the calculation of the BMWP Iberic;

Log (Sel. ETD + 1)—Log10 of 1 + sum of the abundances of individuals belonging to the families Heptageniidae, Ephemeridae, Brachycentridae, Goeridae, Odontoceridae, Limnephilidae, Polycentropodidae, Athericidae, Dixidae, Dolichopodidae, Empididae, and Strididae.

The final IPtIN value is the result of the sum of the weighted metrics. Two normalization steps are performed, before the multiplication of the metrics by the weighting factor and after the sum of the weighted metrics, reaching the final value expressed in the Ecological Quality Ratio (EQR) [13,28]. The normalizations are computed through the quotient between the obtained value and the EQS value for that type of river. The Ribeira de Silveirinhos falls within the categorization of small rivers in the North, less than 100 km² [13,28].

The final water ecological classification system [by EQR] is divided into five quality classes, according to a scale of values between 0 and 1. The range of values for each class differs according to the type of river. For small northern rivers (e.g Ribeira de Silveirinhos), five classes are used [28]:

EQR

High ≥0.87

Good [0.68–0.87]

Moderate [0.44–0.68]

Poor [0.22–0.44]

Bad [0–0.22]

The class reached will represent the degree of change in the ecosystem, due to the anthropogenic pressures to which the surface water is subjected.

Additionally, a data matrix with the values of macroinvertebrates abundance was built to conduct a multivariate analysis (using CANOCO for Windows version 4.0) between species and environmental variables. A DCA analysis showed a length of a gradient of 5.95, and afterward, a canonical correspondence analysis (CCA) was calculated to extract the association of benthic macroinvertebrates with environmental factors.

3. Results and Discussion

3.1. Physical and Chemical Elements

Table 1. Results of physical and chemical parameters: temperature (T), conductivity (Cond), total dissolved solids (TDS), pH, dissolved oxygen (O₂), nitrates (NO₃), ammoniacal nitrogen (NH₄), and total phosphorus (P) and biochemical oxygen demand (BOD₅), for each sampling site for the three sampling periods. Environmental quality standards (EQS) values indicated by the WFD to achieve “Good ecological status” in small northern rivers were also included. Bold values show those that exceed the EQS stipulated for the respective parameter.

Sampling Sites	Season	T (°C)	Cond. (µS/cm)	TDS (mg/L)	pH	O2 (mg/L)	O2 (%)	NO3 (mg/L)	NH4 (mg/L)	P (mg/L)	BOD5 (mg/L)
EQS [13]					≥6 to ≤9	≥5	≥60 and ≤120	≤25	≤1	≤0.10	≤6
RS_1	Sp19	15.4	52.6	53	5.56	8.44	85.7	Φ	0.02	Φ	0.27
	Au19	No water
	Sp20	16.1	55.9	56	5.17	6.35	74.7	Φ	Φ	Φ	0.47
RS_2	Sp19	14.7	53.3	53	6.05	9.72	96.8	Φ	0.02	Φ	0.20
	Au19	No water
	Sp20	17.9	55.6	56	5.91	7.69	82.2	Φ	Φ	Φ	0.57
RS_3	Sp19	15.1	75.7	76	6.41	9.41	93.4	Φ	0.02	0.15	0.47
	Au19	No water
	Sp20	17.6	107.7	108	6.60	7.05	74.6	Φ	Φ	Φ	0.98
RS_4	Sp19	16.5	391.0	392	6.77	9.15	93.5	Φ	Φ	0.16	1.46
	Au19	17.7	1091.0	1091	6.77	6.81	70.8	Φ	0.42	0.23	3.34
	Sp20	18.8	910.0	854	6.93	8.11	87.5	Φ	0.03	0.25	3.54
RS_5	Sp19	17.3	449.0	449	7.17	9.45	97.5	Φ	0.05	0.16	0.49
	Au19	15.6	950.0	950	7.72	9.70	96.7	Φ	0.20	Φ	2.06
	Sp20	20.1	774.0	774	7.35	8.41	93.0	0.50	Φ	0.02	0.67

Φ—Below the detection limit of the equipment: concentration of nitrates (NO₃) < 0.5 mg/L; concentration of ammoniacal nitrogen (NH₄) < 0.03 mg/L and concentration of total phosphorus (P) < 0.01 mg/L.

shows the results of the physical and chemical parameters evaluated during the three sampling periods (Sp19, Au19, and Sp20). Temperature, conductivity, and Total Dissolved Solids (TDS) do not present a limit value (EQS) for small northern rivers, according to WFD. Despite the first three sampling sites being dry in the autumn sampling (Au19; Figure S1—supplementary material), higher values of conductivity and TDS were observed at the downstream sites (RS_4 and RS_5).

The remaining physical and chemical parameters evaluated show an EQS for establishing the good ecological status, in this study for small rivers of the northern type (Table 1) [28]. It should be noted that only the pH and the phosphorus concentration values were outside of the EQS values for establishing good ecological status. Regarding the low pH values at upstream sites (RS_1 at Sp19 and Sp20; RS_2 at Sp20), they are below the limits defined by WFD (≥6 and ≤9). In natural waters, the pH depends on the geology of the area, the absorption of atmospheric gases, and the oxidation of organic matter, but its value can also be changed by anthropogenic factors such as the dumping of domestic and industrial waste [29]. The low pH values here obtained can be explained by a reduced flow upstream, the physical–chemical component being potentially influenced by soil properties. Land occupation in the surrounding area can influence the quantified pH (more acidic in RS_1 and RS_2), due to the high expression of Eucalyptus monoculture (Figure 1), which can promote bacterial diversity and richness, which is closely related to pH, namely acid. This fact may also be due to the existence of a reduced environment result of mine activities, characteristic of the occurrence of iron waters [30,31], as observed in Figure S1—supplementary material.

The results of phosphorus concentration also exceeded the WFD environmental quality standard of ≤0.10 mg/L [13] in almost all determinations. In Table 1 the highest phosphorus concentrations were observed in the RS_3 and RS_5 in Sp19, and RS_4 in all sampling periods having reached a value of 0.25 mg/L in the RS_4 in Sp20. These sites are in the downstream area of the stream, characterized by an increase in agriculture and anthropogenic activities (see surrounding area characterization in Figure 1 and Figure S1—supplementary material) which suggests that these values may be related to the proximity of these activities.

The results of the physical and chemical elements of general water support do not show negative impacts, although a few variations in some parameters have been detected. However, the occurrence of river intermittency during the dry period must be considered when observing these results. Flow variability has been identified as an important factor affecting abiotic factors [32]. Boulton and Lake [33] carried out studies on the temporal changes in environmental characteristics in intermittent rivers demonstrating that the amplitudes in physical and chemical conditions far exceed those in perennial rivers. They observed that in rivers that dried completely, the tendency was for an increase in conductivity and temperature, with a decrease in pH and dissolved oxygen when the flow decreased. Intermittent rivers show high sensitivity to nutrients and other pollutants that increase the risk of eutrophication, especially in low-flow conditions, and can have high impacts on water quality and the biota [34].

In order to perceive if a natural gradient occurs in Ribeira de Silveirinhos, a Principal Component Analysis (PCA) was carried out between the physical and chemical parameters (in situ and the laboratory) and the sampling sites (Figure 2). In Sp19 and Sp20, a gradient along the sampling sites was observed with an increase in oxygen and pH values. This oxygen gradient increases towards the downstream in both spring campaigns, and the Sp19 sampling revealed higher values than the Sp20 sampling sites. On the other hand, a relationship between the high pH and conductivity values was recorded, where a gradient of Sp20 was more evident. This phenomenon can be justified by the presence of iron waters (Figure S1—supplementary material) that manifest themselves more strongly in the same direction. No association with any of the studied parameters was recorded in the autumn sampling (Au19), however, in this sampling period, only 2 sites out of 5 were sampled (Figure S1—supplementary material).

3.2. Biological Parameters—Benthic Macroinvertebrates

Table 2. Abundances of macroinvertebrates (number per sampling effort) at each site and a sampling period of Ribeira de Silveirinhos.

	Order	Trichoptera				Hirudinea		Plecoptera		Dugesiidae	Oligochaeta	Odonata				Heteroptera						Gastropoda			Ephemeroptera				Diptera								Crustacea	Coleoptera
Family		Hydropsychidae (Hyp)	Leptoceridae (Lec)	Philopotamidae (Phi)	Polycentropodidae (Pol)	Glossiphoniidae (Glo)	Hirudidae (Hir)	Leuctridae (Leu)	Nemouridae (Nem)	Dugesiidae (Dug)	Oligochaeta (Oli)	Aeshnidae (Aes)	Cordulegastridae (Cor)	Gomphidae (Gom)	Libellulidae (Lib)	Aphelocheridae (Aph)	Gerridae (Ger)	Hydrometridae (Hym)	Nepidae (Nep)	Notonectidae (Not)	Veliidae (Vel)	Assimineidae (Ass)	Lymnaeidae (Lym)	Planorbidae (Pla)	Baetidae (Bae)	Leptophlebiidae (Lep)	Potamanthidae (Pot)	Siphlonuridae (Sip)	Athericidae (Ath)	Ceratopogonidae (Cer)	Chironomidae (Chi)	Culicidae (Cul)	Empididae (Emp)	Limoniidae (Lim)	Simuliidae (Sim)	Tipulidae (Tip)	Crangonycitidae (Cra)	Curculionidae (Cur)	Dryopidae (Dry)	Dytiscidae (Dyt)	Elmidae (Elm)	Gyrinidae (Gyr)	Hydrophilidae (Hyd)
Site	Season
RS_1	Sp19				1				4		8										4										373				3					19
	Au19
	Sp20										5							1	2		5					1					148									29
RS_2	Sp19		1	1		1		3	4		97		4							2	5					3					729		3	1	16		1			3			1
	Au19
	Sp20							59				1						1			1					12					355			1
RS_3	Sp19						1	12			8									1						9				4	126			3	19					1	9
	Au19
	Sp20							10			1			1			1								1	1			1	3	33			1	4					2
RS_4	Sp19					1					7		9		1	1	1	1		5	5					1			1	4	57							1		2	2
	Au19												1											1					1		5			3		2			1
	Sp20							1		1	4		1																1	3	103			1
	Sp19										8				1		1	7	1		22		1							12	41					2			7	1			1
RS_5	Au19	4			1					2	2		1				2	7			7		9						3		163				7	6			1			1	1
	Sp20							3										1					1							2	28		1			2							1

presents the abundances of macroinvertebrates at each site in the different sampling periods. There is a clear dominance of the family Chironomidae (Diptera) in the three sampling periods, showing that these organisms are less affected by environmental changes. Indeed, this family is characterized by being tolerant to high pollution and environmental impacts [15,35]. The EPT taxa (belonging to the order: Ephemeroptera, Plecoptera, and Trichoptera) were associated with the occurrence of flow, and all the families of Ephemeroptera and Plecoptera were only found in spring samples. Oligochaeta was found at all the sampling periods and sites, however, they are considered a generalist group that adapts to different conditions. During the drought period, an increase in individuals of Gastropoda was observed, which may indicate a greater resilience of these taxa to these environmental conditions. One of the families that was only found one time was Planorbidae (Pla, Gastropoda), in the Au19 sampling in RS_4, strongly associated with P concentration (Figure 3). This family, unlike most mollusks, has hemoglobin in their blood instead of hemocyanin. As a result, Planorbidae individuals can breathe oxygen more efficiently. This adaptation allows the storage of oxygen and therefore the occupation of habitats with variations in oxygen concentration and temperature [36].

The CCA analysis (Figure 3) showed that RS_5 is the site with the most distinguished macroinvertebrate community independently of the sampling season. It seems that its position is mainly related to pH, conductivity, and NH₄ mainly characterizing the 1st axis (see also Table 1). The position of RS_5 in Sp20 is influenced by temperature characterizing the 2nd axis (see also Table 1) related to a specific group of taxa (Ass—Gastropoda; Sip and Pot—Ephemeroptera; Cul—Diptera; Table 2). Moreover, RS_4 Au19 is related mainly by P characterizing the 1st axis (see also Table 1) and Cordulegastridae (see also Table 2). RS_4 and RS_5 Sp19 showed a strong relation with Dry (Dryopidae) organisms (taxa with a score of 5 in the IBMWP index, medium pollution tolerance) and conductivity (see Table 1). On the other hand, the remaining sites are centered in the CCA analysis, as well as the taxa association.

Several studies showed that macroinvertebrates communities are not significantly affected by hydric stress, despite their resilience [6,13]. Despite its resilience, significant changes in the structure and composition of these communities are observed. In the dry phase, the Plecoptera organisms decreased while an increase in Diptera is recorded [1]. On the other hand, Stubbington et al. [35] demonstrated that as flow velocities fall, rheophilic taxa (flow-loving) lose their habitat and most disappear after the flow ceases, including many species of EPT (Figure S1—Group B). These are replaced by macroinvertebrates characteristics of lentic habitats such as Odonata nymphs, diving beetles (Dysticidae family), Heteroptera, and Gastropoda (Figure S1—Group D). Other taxa such as fly larvae (Diptera) and Oligochaeta are generalist organisms and can persist in lotic and lentic conditions (Figure S1—Group E). According to Miliša et al. [3], fewer taxa are indicative of sites with higher flux intermittence, which they suggest as driven by river type, not climate. For example, indicators of Mediterranean intermittent rivers and ephemeral streams comprised Odonata, Coleoptera, and Heteroptera taxa, which are favored by lentic conditions [3]. The first organisms to reappear when the flow returns, are larvae of Simuliidae, filtering organisms that require the flow to feed, and nymphs of Plecoptera are also recorded as early colonizers.

Figure S2 (supplementary material), adapted from Stubbington et al. [35], shows the principal families that occur in the different phases in an intermittent river. Highlighted in green are the families that were found in the spring samples (Sp19 and Sp20: Leptophlebiidae, Simulidae and Dytiscidae) and in orange the families recorded in autumn samples (Au19: Planorbidae). An agreement in the results obtained was observed, with the occurrence of the family Planorbidae in the dry phase and the families Leptophlebiidae and Simulidae in the wet phase (Table 2). Chironomidae and Oligochaeta were always families that occurred (Table 2) and that are found in both phases. However, Dytiscidae is marked as a family that belongs to the dry phase, in the present study it was sampled in Sp19 and Sp20. The permanence of pools (see Figure S1—supplementary material) allows resilient beings to persist in refuges until the flow returns. However, if the pools dry, there is an elimination of taxa sensitive to desiccation, which causes marked reductions in the richness of the ecosystem [36].

Table 3. Values obtained and Environmental Quality Standards (EQS) of WFD stipulated for small rivers in the North that make up the IPtlN and the respective Ecological Quality Ratio (EQR) for each sampling site and period regarding the macroinvertebrate communities. Different colors stand for the EQR classification (derivation of EQR from IPtIN; see Section 2.4.2).

Sampling Sites	Season	Abundance	Diversity	Richness	Evenness	EPT	log (Sel. ETD + 1)	IASTP-2	IPtIN	EQR
EQS [18]				30	0.71	16	1.95	4.52	1.02
RS_1	Sp19	412	0.45	7	0.23	2	0.30	2	0.27	0.27
	Au19	No water
	Sp20	191	0.78	7	0.40	1	0.00	1.57	0.23	0.22
RS_2	Sp19	875	0.69	17	0.24	5	0.60	3.25	0.50	0.49
	Au19	No water
	Sp20	430	0.59	7	0.30	2	0.00	3.71	0.37	0.36
RS_3	Sp19	193	1.32	11	0.55	2	0.00	2.55	0.36	0.35
	Au19	No water
	Sp20	59	1.56	12	0.63	3	0.30	3.33	0.47	0.46
RS_4	Sp19	99	1.68	16	0.61	1	0.30	3	0.46	0.45
	Au19	14	1.73	7	0.89	0	0.30	3.29	0.43	0.42
	Sp20	115	0.52	8	0.25	1	0.30	3.50	0.37	0.37
RS_5	Sp19	105	1.84	13	0.72	0	0.00	1.54	0.31	0.31
	Au19	217	1.16	16	0.42	2	0.70	2.44	0.44	0.44
	Sp20	39	1.12	8	0.54	1	0.30	2.25	0.33	0.33

presents the results of the individual metrics for the calculation of the Portuguese Northern Invertebrate Index (IPtlN) and the value of the EQR, for the three sampling periods. Overall, Ribeira de Silveirinhos showed a moderate to a poor water quality due to the low values obtained for the individual metrics that went below the stipulated environmental quality standards (EQS) for this river type (small rivers in the North) [13]. Intermittent rivers are likely to have a lower diversity than permanently flowing waters (or at least a different composition) and this would be a reason why the EQS standard of 30 families (richness, Table 3) was not met. However, an increase was observed in the diversity and evenness at the downstream sites despite the season sampling. Despite a trend to increase richness to the downstream sites, low richness values were observed at all sites and sampling periods, varying from 7 in RS_1 to 17 in RS_2 in Sp19. These low values may be due to the low pH values recorded at the upstream sites (<6, Table 1). Extremely low values owned by EPT taxa were recorded, with the lowest values observed in RS_4 (Au19) and RS_5 (Sp19). These organisms are very sensitive to water pollution and these sites are subjected to iron water, agriculture leachates (increased P concentration values, Table 1), and a reduced flow due to the intermittent river characteristics of Ribeira de Silveirinhos (see nature of the riverbed and general habitat at each site, in Figure S1—supplementary material). Indeed, taxonomic richness and %EPT taxa were negatively correlated with flow intermittency as shown in the study of Datry et al. [37]. On the other hand, the lack of macrophytes and the type of substrate leads to the existence of poorer habitats that can influence organisms to settle in this area. Fluctuations in the water level concomitantly and the availability of diverse habitats are the factors that seem to influence these communities.

As stated by [10], intermittent rivers are characterized by low abundance and species richness, due to the short connectivity, resulting in agreement with the results here-obtained. However, there are a few exceptions to this pattern, Bonada et al. [4] recorded comparable values of richness in perennial and intermittent locations in a network of Mediterranean rivers. It is verified the disappearance of taxa is sensitive to desiccation progressively, but the temporal variation in habitats promotes occupation by other taxa at different times [7,35]. Moreover, Sánchez-Motoya et al. [38] showed that intermittent rivers had metrics with low values compared to other types of rivers, which can be attributed to differences in macroinvertebrate communities.

3.3. Ecological Status

Table 4. Classification of physical and chemical elements; biological quality elements (benthic macroinvertebrates) and determination of the ecological status of each sampling point in the three sampling periods, according to the WFD classes (different colors excellent, good, moderate, poor, bad). The ecological status is determined based on the element with the worst classification between the elements of general physical and chemical quality and the elements of biological quality.

Sampling Sites	Season	Physical and Chemical Elements	Biological Elements	Ecological Status
RS_1	Sp19	Moderate	Poor	Poor
	Au19	No water
	Sp20	Moderate	Poor	Poor
RS_2	Sp19	Good	Moderate	Moderate
	Au19	No water
	Sp20	Moderate	Poor	Poor
RS_3	Sp19	Moderate	Poor	Poor
	Au19	No water
	Sp20	Good	Moderate	Moderate
RS_4	Sp19	Moderate	Moderate	Moderate
	Au19	Moderate	Poor	Poor
	Sp20	Moderate	Poor	Poor
RS_5	Sp19	Moderate	Poor	Poor
	Au19	Good	Poor	Poor
	Sp20	Good	Poor	Poor

shows the ecological status of each sampling site along Ribeira de Silveirinhos, for the three sampling periods (Sp19, Au19, Sp20). Ecological status is the result of the combination of the worst-rated element between physical and chemical elements and biological elements. Thus, it was found that the biological elements studied, benthic macroinvertebrates community, are the element with the worst classification; therefore, the ecological status shows this classification. These results showed that this biological element is sensitive for the assessment of water quality also in intermittent rivers, and overall aquatic ecosystem health since they respond to all water components and conditions. These findings are corroborated by the study of Pinto et al. [39].

A moderate ecological status was observed only in three situations (RS_2 and RS_4 in Sp19; RS_3 in Sp20) while nine samples showed a poor ecological status (Table 4). Degradation of the water body at RS_2 and RS_4 has been visible since spring 2019 with the assessment of a moderate ecological status (Sp19) to a poor ecological status (Au19 and Sp20). The increase in P concentration and the low diversity and abundance recorded (Table 1 and Table 3) already demonstrated that ecological status would be of low quality.

4. Conclusions

The different tools used in this study (followed the WFD metrics), although they have proven to be effective in assessing water systems, were not created to assess intermittent rivers. The results of this work allowed us to draw some conclusions about the ecological quality of Ribeira de Silveirinhos, and can, therefore, be used as a valuable monitoring tool in assessing freshwater aquatic environments. Diptera dominate the fauna, probably because they are more tolerant to drought conditions and have more efficient recolonization mechanisms. According to the WFD methodology, for lotic systems, and the assessment of the physical and chemical parameters as well as the benthic macroinvertebrate communities, the conclusion is that the entire stream needs interventions to improve its ecological status and achieve the classification of “good”. However, it was found that the community of macroinvertebrates in intermittent rivers has high structural variability. Mechanisms of resistance and resilience allow species and communities to persist in intermittent rivers during the dry phases and recolonize quickly once the flow returns. The EQS for biological parameters, namely community of benthic macroinvertebrates, should be reconsidered, mainly as they are likely to have a lower diversity than permanent waters (or at least a different composition), and this would be one reason why the EQS standard of 30 families was not met in this work.

We think that the roles that rivers play in maintaining biodiversity and controlling material fluxes will change substantially when intermittent rivers are fully integrated into regional and global analyses. To investigate the dynamics of intermittent river populations, communities, flow regimes, and biogeochemistry and to identify climatic gradients and anthropogenic effects on these dynamics, data sets must be compiled from multiple sources, integrated, processed and analyzed. Indeed, evaluation models targeted to assess intermittent rivers are scarce, namely taking into account their temporal variability and peculiar ecology. Nowadays, when we face the challenge of climate change with the greatest occurrence of extreme events, rivers are fragile ecosystems and are susceptible to several threats. According to this, it is expected that the number of intermittent rivers will increase worldwide, especially in regions where severe climatic droughts occur.

Future works to continue the scientific research on these types of ecosystems, to better understand their ecological standards and their functioning are necessary, in order to find an adequate assessment for the ecological classification of intermittent rivers and, consequently, their conservation and improve management measures.