Fish Biomonitoring Research Articles

Choice of primer pairs and PCR polymerase affect the detection of fish eDNA

Efficient biomonitoring is essential for fish protection and management. Environmental DNA (eDNA) has become a promising tool for fish surveys, and its accuracy and robustness are closely related to the primer pairs and DNA polymerases, especially for different environmental samples. However, there is still a lack of sufficient efforts to assess the effects of both two factors on fish biomonitoring. Here, we selected ten primer pairs in the mitochondrial 12S rRNA gene region and three commercial DNA polymerases and analyzed their effects on fish eDNA monitoring in surface water and sediment samples of Dianchi Lake. We found that primer pairs and DNA polymerases significantly affected fish biomonitoring in surface water and sediments of Dianchi Lake. First, there were significant variations in annotated fish eDNA sequences in different groups of primer pairs and DNA polymerases, the percentage of fish sequences amplified by the groups related to primers Riaz-12S and 12S-V5 was more than 90% of the total sequences. Second, the composition of different classification levels of fish taxa varied considerably across groups of primer pairs and DNA polymerases, and the groups related to primers Riaz-12S (i.e., Taq Master‒Riaz-12S, Rapid Taq‒Riaz-12S) and 12S-V5 (i.e., Taq Master‒12S-V5, Rapid Taq‒12S-V5) identified more taxa than other groups. Third, primer pairs had greater impacts on the structure of fish communities than DNA polymerases, and the interactions between two factors had more significant effects than any single one. This study highlights that primer pairs and DNA polymerases play critical roles in fish biomonitoring, and this work aimed to provide methodological guidance for assisting the design of the fish eDNA survey scheme.

Bioaccumulation and fate of pharmaceuticals in a Mediterranean coastal lagoon: Temporal variation and impact of a flash flood event

Coastal ecosystems are particularly vulnerable to terrestrial inputs from human-impacted areas. The prevalence of wastewater treatment plants, unable to remove contaminants such as pharmaceuticals (PhACs), leads to their continuous input into the marine environment. In this paper, the seasonal occurrence of PhACs in a semi-confined coastal lagoon (the Mar Menor, south-eastern Spain) was studied during 2018 and 2019 by evaluating their presence in seawater and sediments, and their bioaccumulation in aquatic organisms. Temporal variation in the contamination levels was evaluated by comparison to a previous study carried out between 2010 and 2011 before the cessation of permanent discharges of treated wastewater into the lagoon. The impact of a flash flood event (September 2019) on PhACs pollution was also assessed. A total of seven compounds (out of 69 PhACs analysed) were found in seawater during 2018–2019, with a limited detection frequency (<33%) and concentrations (up to 11 ng/L of clarithromycin). Only carbamazepine was found in sediments (ND–1.2 ng/g dw), suggesting an improved environmental quality in comparison to 2010–2011 (when 24 and 13 compounds were detected in seawater and sediments, respectively). However, the biomonitoring of fish and molluscs showed a still remarkable accumulation of analgesic/anti-inflammatory drugs, lipid regulators, psychiatric drugs and β-blocking agents, albeit not higher than in 2010. The flash flood event from 2019 increased the prevalence of PhACs in the lagoon, compared to the 2018–2019 sampling campaigns, especially in the upper water layer. After the flash flood the antibiotics clarithromycin and sulfapyridine yielded the highest concentrations ever reported in the lagoon (297 and 145 ng/L, respectively), alongside azithromycin in 2011 (155 ng/L). Flash flood events associated with sewer overflows and soil mobilisation, which are expected to increase under climate change scenarios, should be considered when assessing the risks posed by pharmaceuticals to vulnerable aquatic ecosystems in the coastal areas.

Fish Biomonitoring and Ecological Assessment in the Dianchi Lake Basin Based on Environmental DNA

Fish are among the main target groups when conducting biomonitoring and ecological assessment of aquatic environments. Environmental DNA (eDNA) metabarcoding provides a unique opportunity to enhance biomonitoring throughput, precision, and standardization. However, few fish monitoring studies based on eDNA metabarcoding have been conducted at the basin scale. Taking Dianchi Lake and the three inflowing rivers (Panlong River, Baoxiang River, and Chai River) as research objects, this study monitored fish diversity via eDNA metabarcoding, and the Fish Index of Biological Integrity (F-IBI) was constructed and applied for the ecological assessment of the aquatic environment. Here, we detected 41 species belonging to 9 orders, 15 families, and 35 genera in the sampling areas, including 17 native fish species such as Hemiculter leucisculus, Cyprinus carpio, and Silurus soldatovi. Distinct diversity patterns were observed among Dianchi Lake and the three inflowing rivers. Particularly, the fish diversity of Panlong River was richer than that of Dianchi Lake, Baoxiang River, and Chai River. Water temperature (WT), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) significantly influenced the distribution of fish communities in most sampling sites of the Dianchi Lake basin (p &lt; 0.05). Moreover, according to the F-IBI assessment system, 25% of the sampling sites were defined as ‘fine’ or above, and 75% were ‘impaired’ or below. This study provided insights into the ecological restoration and precise management of the aquatic environment of the Dianchi Lake basin.

Experimental assessment of downstream environmental DNA patterns under variable fish biomass and river discharge rates

AbstractThe development of environmental DNA (eDNA) methods toward implementation as a cost‐effective, nonlethal tool for fish biomonitoring in lotic environments requires insights on the temporal and spatial distribution of eDNA in river systems. Yet, little is known on how downstream eDNA dispersal is affected by the combination of river discharge and source biomass effects. In this study, we aimed at unraveling the effect of source‐ and system‐specific processes on the stream reach of eDNA. We used a longitudinal cage study in two river sections characterized by a significantly different discharge rate, where two invasive and two native fish species were introduced under two contrasting biomass treatments. Using droplet digital PCR (ddPCR) analyses, we found that eDNA concentrations become strongly reduced 2 km downstream from the source, an effect that is strengthened with increasing river discharge and coinciding dilution effects. Higher discharge rates resulted in equal or even higher detection probabilities at increasing distance from the source. The introduction of high fish stock biomass resulted in an increase of eDNA concentrations, as well as detection probabilities and in parallel reduced the stochasticity of the measurements. A peak in eDNA concentrations at a downstream distance ranging between 300 m and 2 km confirms the complexity of plume‐shaped downstream eDNA patterns. Our results showed interspecific variation in eDNA emission and suggest species‐specific differences in eDNA persistence. This study underlines the impact of both river discharge rate and source biomass on downstream eDNA detection and dispersal patterns.

Challenges in assessing the Neotropical fishes using DNA metabarcoding

Species richness is a metric of biodiversity usually used in fish community assessment for monitoring programs. This metric is often obtained using traditional fisheries methods that rely on capture of target organisms, resulting in underestimation of fish species. DNA metabarcoding has been recognized as a powerful noninvasive alternative tool for fish biomonitoring and management. Despite the increasing popularity of this method for the assessment of aquatic megadiverse ecosystems, its implementation for studying the highly diverse Neotropical ichthyofauna still presents some challenges. One of them is to devise what primer set could reliably amplify the DNA of all fish species from a megadiverse river basin and have enough resolution to identify them. In order to identify and overcome these drawbacks, we have investigated the efficiency of the metabarcoding approach on Neotropical fishes using a mock sample containing genomic DNA of 18 fish species from the Jequitinhonha River basin, Eastern Brazil. We compared three primer sets targeting the 12S rRNA gene: two universal and widely used markers for fish metabarcoding [MiFish (~170bp) and Teleo_1 (~60bp)], and NeoFish (~190bp), recently developed by our research group specifically for the identification of Neotropical fishes (Milan et al., 2020). Two samples amplified using three primers were sequenced in a single multiplexed Illumina MiniSeq run, using normalized and non-normalized pools. Bioinformatic analyses were performed using a DADA2/Phyloseq based pipeline to perform filtering steps and to assign Amplicon Sequence Variants (ASVs). We used a custom 12S reference sequence database that included 190 specimens representing 101 species and 70 genera from the Jequitinhonha and São Francisco river basins. A total of 187 ASVs were recovered: 79, 66 and 42 for NeoFish, MiFish and Teleo_1, respectively. ASVs of unexpected species were identified for both pools (Fig. 1), though each of these ASVs had an abundance of less than 50 copies. In addition, species of the Hoplias and Prochilodus genera could not be identified at the species level, due to identical sequences within each genus, possibly because of the insufficient variation within the 12S region recovered by these primers’ amplicons. Unexpectedly, although a single individual of each species was placed in the pools, more than one ASV was identified for some species, likely caused by PCR biases. Overall, all primer sets displayed similar taxonomic resolution for the DNA pools and recovered all species, except for NeoFish, which could not detect Steindachneridion amblyurum due to an incompatibility in the 3’ of the NeoFish forward primer and Teleo_1, which could not identify Steindachnerina elegans. These results highlight the need of reliable databases in order to enable the full assignment of ASVs and OTUs to species level, and the importance of calibrating the DNA metabarcoding approach with mock samples to identify weaknesses and pivotal steps prior to the application on large scale DNA based biodiversity evaluation, that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Using Environmental DNA to Monitor the Reintroduction Success of the Rhine Sculpin (Cottus rhenanus) in a Restored Stream

Freshwaters face some of the highest rates of species loss, caused by strong human impact. To decrease or even revert this strong impact, ecological restorations are increasingly applied to restore and maintain the natural ecological status of freshwaters. Their ecological status can be determined by assessing the presence of indicator species (e.g., certain fish species), which is called biomonitoring. However, traditional biomonitoring of fish, such as electrofishing, is often challenging and invasive. To augment traditional biomonitoring of fish, the analysis of environmental DNA (eDNA) has recently been proposed as an alternative, sensitive approach. The present study employed this modern approach to monitor the Rhine sculpin (Cottus rhenanus), a fish species that has been reintroduced into a recently restored stream within the Emscher catchment in Germany, in order to validate the success of the applied restorations and to monitor the species’ dispersal. We monitored the dispersal of the Rhine sculpin using replicated 12S end-point nested PCR eDNA surveillance at a fine spatial and temporal scale. In that way, we investigated if eDNA analysis can be applied for freshwater assessments. We also performed traditional electrofishing in one instance to validate our eDNA-based approach. We could track the dispersal of the Rhine sculpin and showed a higher dispersal potential of the species than we assumed. eDNA detection indicated the species’ dispersal across a potential dispersal barrier and showed a steep increase of positive detections once the reintroduced population had established. In contrast to that, false negative eDNA results occurred at early reintroduction stages. Our results show that eDNA detection can be used to confirm and monitor reintroductions and to contribute to the assessment and modeling of the ecological status of streams.

Alternatives for the biomonitoring of fish and phytoplankton in tropical streams

Biomonitoring programs need to balance accurate responses in assessments of changes in biological communities with sampling that is fast and low cost. In this study, we evaluated the concordance among fish and phytoplankton communities of streams. We tested the cross-taxa surrogacy, taxonomic, numerical resolution and ecological substitute group (habitat use and trophic guilds) resolution with Procrustes analyses aim of simplifying the biomonitoring process. We collect a total fish abundance of 8,461 individuals, represented by the ecological classes of habitat, including benthic, nektonic, nektobenthic, marginal and trophic guilds by detritivore, terrestrial invertivore, aquatic invertivore, piscivore, algivore and herbivore. We sampled a phytoplankton total density of 1,466.68 individuals/ml, represented by four Morphology-Based Functional Groups and nine Reynolds Functional Groups. Our results don’t support the use of substitute groups among fish and phytoplankton. For fish, habitat use and trophic guild are good surrogates for species-level data. Additionally, our results don’t support the use of functional groups as surrogates for phytoplankton. We suggest the use of higher taxonomic levels (genus and family) and record only the occurrence of species and/or genus for fish and phytoplankton. Our findings contribute to decreasing the costs and time of biomonitoring programs assessments and/or conservation plans on fish and phytoplankton communities of headwater streams.

In vivo solid-phase microextraction sampling combined with metabolomics and toxicological studies for the non-lethal monitoring of the exposome in fish tissue

Various environmental studies have employed the biomonitoring of fish in their aquatic ecosystems in order to identify potential metabolic responses to the exposome. In this study, we applied in vivo solid-phase microextraction (SPME) to perform non-lethal sampling on the muscle tissue of living fish to extract toxicants and various endogenous metabolites. Sixty white suckers (Catastomus commersonii) were sampled from sites upstream, adjacent, and downstream from the oil sands development region of the Athabasca River (Alberta, Canada) in order to track their biochemical responses to potential contaminants. In vivo SPME sampling facilitated the extraction of a wide range of endogenous metabolites, mainly related to lipid metabolism. The obtained results revealed significant changes in the levels of numerous metabolites, including eicosanoids, linoleic acids, and fat-soluble vitamins, in fish sampled in different areas of the river, thus demonstrating SPME's applicability for the direct monitoring of exposure to different environmental toxicants. In addition, several classes of toxins, including petroleum-related compounds, that can cause serious physiological impairment were tentatively identified in the extracts. In vivo SPME, combined with the analysis of contaminants and endogenous metabolites, provided important information about the exposome; as such, this approach represents a potentially powerful and non-lethal tool for identifying the mechanisms that produce altered metabolic pathways in response to the mixtures of different environmental pollutants.

Predicting New Zealand riverine fish reference assemblages

Biomonitoring is a common method to monitor environmental change in river ecosystems, a key advantage of biomonitoring over snap-shot physicochemical monitoring is that it provides a more stable, long-term insight into change that is also effects-based. In New Zealand, the main biomonitoring method is a macroinvertebrate sensitivity scoring index, with little established methods available for biomonitoring of fish. This study models the contemporary distribution of common freshwater fish and then uses those models to predict freshwater fish assemblages for each river reach under reference conditions. Comparison of current fish assemblages with those predicted in reference conditions (as observed/expected (O/E) ratios) may provide a suitable option for freshwater fish biomonitoring. Most of the fish communities throughout the central North Island and lower reaches show substantial deviation from the modelled reference community. Most of this deviation is explained by nutrient enrichment, followed by downstream barriers (i.e. dams) and loss of riparian vegetation. The presence of modelled introduced species had relatively little impact on the presence of the modelled native fish. The maps of O/E fish assemblage may provide a rapid way to identify potential restoration sites.

Severity Multipliers as a Methodology to Explore Potential Effects of Climate Change on Stream Bioassessment Programs

Given the scientific consensus that climate change is impacting biodiversity, estimates of future climate change effects on stream communities and assessments of potential biases are necessary. Here, we propose a simple technique to approximate changes in invertebrate and fish biomonitoring results. Taxa lists for 60 (invertebrate) and 52 (fish) sites were each modified by 10 multipliers as stepwise 5% or 10% changes in abundances to simulate potential climate-change severity, reflecting increasing climate change effects. These 10 multipliers were based on the stream zonation preference for invertebrates and the Fish Region Index (FRI) values for fish, both reflecting the longitudinal gradient present in river ecosystems. The original and modified taxa lists were analyzed using the standard assessment software for the particular group, followed by analysis of key biomonitoring metrics. For invertebrates, our simulations affected small good quality streams more often negatively while large poor mountain streams showed a tendency to improve. Forty percent of the invertebrate data sites showed a change in the final ecological assessment class when using the multipliers, with the poor quality sites changing more often. For fish, metric changes were variable, but the FRI ratio showed mostly positive responses, i.e., a shift in FRI towards downstream communities. The results are discussed as an example that facilitates the interpretation of potential climate-change effects with varying severity. Further, we discuss the simplified approach and implications for assessment from climate change induced range shifts.

In vitro mucus transportability, cytogenotoxicity, and hematological changes as non-destructive physiological biomarkers in fish chronically exposed to metals

The biomonitoring of fish using biomarkers represents a useful tool for the assessment of aquatic pollution. This study evaluated the sublethal toxic effects of aquatic pollution on fish collected from a site contaminated by metals. Water and fish (Oreochromis niloticus) samples were collected from a pond in the Parque Ecológico do Tietê (PET) that lies along the Tietê River (São Paulo, Brazil), and from a control site (an experimental fish farm). The metal content of the water was evaluated, and fish were used to examine the properties of gill mucus and blood. The PET fish were evaluated for alterations in the in vitro transportability of mucus and changes in blood properties (e.g., cell volume, hemoglobin concentration, red blood cells, and white blood cell count). The results of the water analyzes indicated metal levels above the legal standards for Fe (0.71mg/L), Ni (0.06mg/L), Mn (0.11mg/L), and Pb (0.48mg/L). Compared to the controls, the hematologic parameter analyzes of PET fish revealed significantly higher numbers of erythrocytes (RBC), leukocytes (WBC), lymphocytes, erythroblasts, and Mean Corpuscular Volume (MCV); however, the hemoglobin content and Mean Corpuscular Hemoglobin Concentration (MCHC) values were significantly lower. The frequencies of nuclear abnormalities and micronuclei were significantly higher and the mucociliary transport was significantly lower in PET fish than in the controls. These results suggest that fish from the contaminated site exhibit a series of physiological responses, which probably indicate health disturbances. Furthermore, the results suggest that blood and mucus are promising, non-destructive targets for use in the monitoring of pollution.

Cd, Pb and Hg Biomonitoring in Fish of the Mediterranean Region and Risk Estimations on Fish Consumption

Cadmium (Cd), lead (Pb) and mercury (Hg) are toxic metals with increasing interest due to their tendency to bioaccumulate in fish tissue which may pose a threat to human health via fish consumption. This review of the recent literature on Cd, Pb, Hg levels summarizes data of fish biomonitoring studies in the Mediterranean Sea in order to determine potential risks due to dietary intake of metals. The analytical methods applied are described, with Atomic Absorption Spectroscopy and Inductively Coupled Plasma Mass Spectroscopy being the most popular. Most of the literature reviewed is focused on the Eastern Mediterranean. Results from the studies indicate that metals mostly accumulate in liver, followed by muscle. Although there are few studies reporting metal levels in fish exceeding the maximum residue levels (MRLs), the bulk of the studies cite levels below the MRLs. The hazard index (HI) of fish consumption, namely the ratio of estimated weekly intake to provisional tolerable weekly intake (EWI/PTWI) was estimated for adult consumers and no risk emerged. The EWI/PTWI ratios of lead and mercury for Italy (0.14 and 0.22 respectively) represent the highest HI levels estimated. In view of maximizing the benefits while minimizing the risks of fish consumption, a more detailed fish-specific database on intakes for consumers is required and extended bimonitoring in as many regions as possible.

Validation of methodology for determining As, Pb and Cd in fish by using ICP-MS: Preliminary studies

The progress of industry has led to the increased emission of pollutants into ecosystems, and fish consumption has been a remarkable source of human exposure to toxic metals. Arsenic, cadmium and lead constitute a potentially significant threat to human health because they are associated with several adverse health effects. Therefore, fish biomonitoring has been a crucial tool for assessing the environmental exposure to contaminants. This study aimed at determining arsenic, cadmium and lead contents in fish samples from a Proficiency Testing for Metals conducted by the Nuclear and Energy Research Institute (IPEN), in order to develop a routine analytical methodology using ICP-MS. The methodology was evaluated by using the certified reference oyster tissue material, which indicated good agreement between the certified and the determined concentrations for As, Cd and Pb. The report on the Proficiency Test performance evaluation, based on the z-score index, evidenced satisfactory results in both samples of the analyzed elements.

Comparison of Mercury Distribution Between Liver and Muscle - A Biomonitoring of Fish from Lightly and Heavily Contaminated Localities.

Tissue samples from 1,117 fish of 25 species were collected from 1991 through 1996 at 13 locations along the River Elbe. The principal indicator species were perch (Perca fluviatilis) (n=118), chub (Leuciscus cephalus L.) (n=113) and roach (Rutilus rutilus) (n=138). Mercury (Hg) concentrations in muscle and liver were determined by atomic absorption spectrometry. The liver/muscle index in three indicator species from heavily contaminated and lightly contaminated localities were significantly different. In fish from heavily contaminated localities, Hg was deposited preferentially in the liver (the depository for inorganic and organic forms of Hg), while in lightly contaminated areas, it was deposited preferentially in muscle.

Industrial effluent monitoring incorporating a recent automated fish biomonitoring system

An automated, continuous biological monitoring facility which is in operation at a U.S. Army ammunition manufacturing plant is described. The system monitors the ventilatory behavior of bluegill sunfish,Lepomis macrochirus Raf, to assess the quality of two wastewater discharges with the aid of an on-line minicomputer system. Certain chemical/physical parameters are also monitored automatically on a continuous basis. The capability for detecting unapproved discharges is discussed. The overall concept of this system and discussion of its potential for economically monitoring water and wastewater discharges, both automatically and continuously, are presented.