Highly sensitive multi-residue analysis of veterinary drugs including coccidiostats and anthelmintics in pond water using UHPLC-MS/MS : application to freshwater ponds in Flanders, Belgium

Veterinary drugs, such as coccidiostats and anthelmintics are routinely administered in extensive animal husbandry, finding their way into the aquatic environment through urine and/or feces of treated animals kept outdoors or by the application of contaminated liquid manure on agricultural fields and subsequent mechanisms of surface run-off, leaching and drift. Several of these compounds are known to exert acute and chronic toxicity effects on aquatic organisms, and can lead to changes in biodiversity and ecosystem functioning. The overall objective of this research was to develop, validate and apply a highly sensitive, multi-residue SPE-UHPLC-MS/MS method for the determination of 12 coccidiostats, registered as a feed supplement or veterinary medicine in Europe and three regularly used anthelmintics, in pond water, often functioning as amphibian habitat. Sample extraction was optimized using a fractional factorial resolution design. Pond water filtration efficiency (i.e.80-118%, <= 25% RSD) and matrix effects (i.e.72-119%, <= 39% RSD) were evaluated using water from respectively 3 and 20 different ponds in Flanders. By incorporating internal standards, overall results improved and adequate precision values (i.e.<= 15%) were obtained according to the EMA guidelines. Acceptable within-run and between-run apparent recoveries, satisfactory precision as well as good linearity were demonstrated according to the CD 2002/657/EC, SANTE/12682/2019 and VICH 49 guidelines, except for robenidine for which the between-day precision was between 21.0 and 34.5%. Sample storage stability studies indicated that storage at 4 degrees C and analysis performed within 96 hours after sampling was sufficient to avoid loss by degradation for all compounds, excluding robenidine. Values for the limit of detection (LOD) and quantification (LOQ) were in nanograms per liter, which was essential for the environmental application of this novel method. The method was successfully applied on grab water samples from the water surface of 18 different ponds across Flanders, Belgium, detecting amprolium and levamisole at concentrations below the LOQ of 2.5 ng L(-1)and at 250.0 ng L(-1)or below the LOQ of 250.0 ng L-1, respectively. In conclusion, our newly developed method may provide insights about the contamination status of amphibian breeding ponds

Water quality assessment of streams and wetlands in a fast growing East African city

The combination of rapid urbanization, industrialization, population growth, and low environmental awareness poses a major threat to worldwide valuable freshwater resources, which provide important ecosystem services to humans. There is an urgent need to monitor and assess these resources, as this information is indispensable for sustainable decision -making and management. In this context, we analyzed the chemical and ecological water quality of the riverine environment of a fast growing city in Southwest Ethiopia for which we proposed possible remediation options that were evaluated with an empirical model. The chemical and ecological water quality was assessed at 53 sampling locations using the oxygen Prati index and the ETHbios, which is a biotic index based on macroinvertebrates. In addition, a microbiological analysis was performed to estimate the degree of fecal contamination. Finally, we analyzed the relationship between the oxygen content and the organic pollution to simulate the effect of organics removal from waste streams on the chemical water quality. Our results showed that the average values for dissolved oxygen (4.2 mg DO.L-1) and nutrients (0.9 mg oPO(4)(3-) . L-1 and 12.8 mg TAN.L-1) exceeded international standards. Moreover, high turbidity levels revealed that land erosion is a severe problem in the region. Along the rivers, a significant increase in oxygen consumption and in nutrient concentrations was observed, indicating organic pollution originating from different diffuse and point sources of pollution. The lack of proper sanitation also led to exceedingly high abundances of fecal coliforms in the surface water (>320 MPN. mL(-1)). However, fecal contamination was strongly reduced (>92%) after the polluted river water passed Boye wetland, indicating the purification potential of natural wetlands and the importance of conserving and protecting those ecosystems. The simulation results of the model showed that water quality could be substantially improved if municipal, industrial, and institutional wastewater was efficiently collected and transported to a treatment facility. Waste stabilization ponds and constructed wetlands are highly promising techniques, as they provide a cheap, effective, reliable, and sustainable way to purify wastewater. It is advised that the environmental awareness of the people via sensitization, education, and law enforcement is increased, as this is essential for sustainable development

Multi-class analysis of 46 antimicrobial drug residues in pond water using UHPLC-Orbitrap-HRMS and application to freshwater ponds in Flanders, Belgium

Increasing anthropogenic pressure and agricultural pollution raises concerns regarding antimicrobial resistance and biodiversity loss in aquatic environments. In order to protect and restore water resources and biodiversity, antimicrobial drug residues should be monitored in all aquatic environments including pond water. Consequently, the objective of this research was to develop and validate a novel multi-residue method for the simultaneous quantification of 46 targeted human and veterinary antimicrobial drugs in pond water. A suitable extraction method based on solid-phase extraction (SPE) was developed, assisted by a fractional factorial design. A broad polarity range of compounds was covered (log P from -4.05 to 4.38), including major representatives of the following classes: sulfonamides, tetracyclines, quinolones, macrolides, lincosamides, nitrofurans, penicillins, cephalosporins, diaminopyrimidines, pleuromutilins and phenicols. All analytes were separated using ultra-high performance liquid chromatography (UHPLC) and detected in full-scan by Orbitrap high resolution mass spectrometry (Orbitrap-HRMS). Good linearity was obtained for all compounds with R-2 >= 0.993 and goodness-of-fit coefficient (g) <= 11.56%. Method detection limits ranged from 10 to 50 ng L-1 and method quantification limits were 50 ng L-1 for all compounds. Acceptable values were obtained for within-day and between-day apparent recoveries (i.e. between 50 and 120%), precision (< 30% and < 45%) and measurement uncertainty (< 50%). Targeted analysis of 18 freshwater ponds throughout Flanders was performed to demonstrate the applicability of the newly developed UHPLC-HRMS method. Overall, 20 antimicrobial drugs were detected with highest concentrations observed for tetracyclines and their transformation products ranging between 51 and 248 ng L-1. Finally, suspect screening was performed suggesting the presence of 14 additional pharmaceuticals including 3 antimicrobial degradation products (e.g. apo-oxytetracycline, amoxicillin penicilloic acid and penilloic acid) and 11 pesticides

Small patches of riparian woody vegetation enhance biodiversity of invertebrates

Patches of riparian woody vegetation potentially help mitigate environmental impacts of agriculture and safeguard biodiversity. We investigated the effects of riparian forest on invertebrate diversity in coupled stream-riparian networks using a case study in the Zwalm river basin (Flanders, Belgium). Agriculture is one of the main pressures in the basin and riparian forest is limited to a number of isolated patches. Our 32 study sites comprised nine unshaded “unbuffered” sites which were paired with nine shaded “buffered” sites on the same stream reach, along with five ‘least-disturbed’ sites and nine downstream sites. We sampled water chemistry, habitat characteristics and stream and riparian invertebrates (carabid beetles and spiders) at each site. Three methods were used to quantify riparian attributes at different spatial scales: a visually-assessed qualitative index, quantitative estimates of habitat categories in six rectangular plots (10 × 5 m) and geographic information system (GIS)-derived land cover data. We investigated relationships between invertebrates and riparian attributes at different scales with linear regression and redundancy analyses. Spiders and carabids were most associated with local riparian attributes. In contrast, aquatic macroinvertebrates were strongly influenced by the extent of riparian vegetation in a riparian band upstream (100–300 m). These findings demonstrate the value of quantifying GIS-based metrics of riparian cover over larger spatial scales into assessments of the efficacy of riparian management as a complement to more detailed local scale riparian assessments in situ. Our findings highlight the value of even small patches of riparian vegetation in an otherwise extensively disturbed landscape in supporting biodiversity of both terrestrial and freshwater invertebrates and emphasize the need to consider multiple spatial scales in riparian management strategies which aim to mitigate human impacts on biodiversity in stream-riparian networks

A Bayesian Belief Network learning tool integrates multi-scale effects of riparian buffers on stream invertebrates

Riparian forest buffers have multiple benefits for biodiversity and ecosystem services in both freshwater and terrestrial habitats but are rarely implemented in water ecosystem management, partly reflecting the lack of information on the effectiveness of this measure. In this context, social learning is valuable to inform stakeholders of the efficacy of riparian vegetation in mitigating stream degradation. We aim to develop a Bayesian belief network (BBN) model for application as a learning tool to simulate and assess the reach-and segment-scale effects of riparian vegetation properties and land use on instream invertebrates. We surveyed reach-scale riparian conditions, extracted segment-scale riparian and subcatchment land use information from geographic information system data, and collected macroinvertebrate samples from four catchments in Europe (Belgium, Norway, Romania, and Sweden). We modelled the ecological condition based on the Average Score Per Taxon (ASPT) index, a macroinvertebrate-based index widely used in European bioassessment, as a function of different riparian variables using the BBN modelling approach. The results of the model simulations provided insights into the usefulness of riparian vegetation attributes in enhancing the ecological condition, with reach-scale riparian vegetation quality associated with the strongest improvements in ecological status. Specifically, reach-scale buffer vegetation of score 3 (i.e. moderate quality) generally results in the highest probability of a good ASPT score (99-100%). In contrast, a site with a narrow width of riparian trees and a small area of trees with reach-scale buffer vegetation of score 1 (i.e. low quality) predicts a high probability of a bad ASPT score (74%). The strengths of the BBN model are the ease of interpretation, fast simulation, ability to explicitly indicate uncertainty in model outcomes, and interactivity. These merits point to the potential use of the BBN model in workshop activities to stimulate key learning processes that help inform the management of riparian zones

Assessing the drivers behind the structure and diversity of fish assemblages associated with rocky shores in the Galapagos archipelago

Oceanic islands harbor unique yet fragile marine ecosystems that require evidence-based environmental management. Among these islands, the Galapagos archipelago is well known for its fish diversity, but the factors that structure communities within and between its islands remain poorly understood. In this study, water quality, physical habitats and geographical distance were assessed as potential predictors for the diversity and structure of fish assemblages. Differences in the structure of fish assemblages of the two studied islands (Santa Cruz and Floreana) were most likely driven by temperature and nutrient concentrations. In the relatively highly populated island Santa Cruz, the structure of fish assemblages was more affected by water conditions than physical habitats while the contrary was true for the more pristine area of Floreana. A wide variety of species with different geographical origins were distributed over the different islands, which indicates that most fish species are able to reach the islands of the archipelago. However, temperature gradients and elevated nutrient levels cause large differences in the structure of local fish assemblages. In addition, in Santa Cruz nutrient concentrations were negatively correlated with α diversity. Since pollution is a clear pressure on the fish assemblages of oceanic islands, environmental management of the coastal areas is of paramount importance

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains

Wildlife diseases are contributing to the current Earth’s sixth mass extinction; one disease, chytridiomycosis, has caused mass amphibian die-offs. While global spread of a hypervirulent lineage of the fungus Batrachochytrium dendrobatidis (BdGPL) causes unprecedented loss of vertebrate diversity by decimating amphibian populations, its impact on amphibian communities is highly variable across regions. Here, we combine field data with in vitro and in vivo trials that demonstrate the presence of a markedly diverse variety of low virulence isolates of BdGPL in northern European amphibian communities. Pre-exposure to some of these low virulence isolates protects against disease following subsequent exposure to highly virulent BdGPL in midwife toads (Alytes obstetricans) and alters infection dynamics of its sister species B. salamandrivorans in newts (Triturus marmoratus), but not in salamanders (Salamandra salamandra). The key role of pathogen virulence in the complex host-pathogen-environment interaction supports efforts to limit pathogen pollution in a globalized world

Integrated ecological assessment and modelling of chytridiomycosis in support of sustainable farmland pond management

Amphibians across the globe are experiencing alarming declines, with about half of all known species vulnerable to extinction. One of the drivers is the parasitic chytrid species Batrachochytrium dendrobatidis, which is a major causal agent of the virulent, infectious disease chytridiomycosis. To counteract population losses, it is important to create endemic situations in which the host-pathogen interaction constitutes a co-existence in the absence of mass mortality events. Investigating the chytrid infection requires an integrated view as it is in influenced by a variety of intertwining factors. Models are useful tools to help understand the epidemiological processes. However, the level of integration in current infection models is limited. In this PhD research, we aimed to provide integrated ecological insights in chytrid infection via models. Farmland ponds were the ecosystems of interest because they are likely to play a key role in infection mitigation. First, we investigated the habitat requirements of the alpine newt, a major chytrid host. Both fish absence and low levels of eutrophication and erosion ensured the presence of the alpine newt. Second, we developed a model which allowed to make a prediction of the infection probability based on several infection-steering ecological variables. Infection risk reduced with low host density, periodic drying and, to a lesser extent, low total phosphorous levels and low water temperatures. This model did not incorporate any non-host species interactions. However, biological control is a promising infection mitigation strategy as it involves the reduction in infectious motile zoospores in the water column through direct consumption by zooplankton. In the third research topic, we modelled the predation behaviour of two common water fleas in farmland ponds. The models revealed that water fleas mutually interfered with each other whilst feeding on zoospores and ingestion rate was positively related to body size. The results of this PhD research provide useful insights and tools for sustainable farmland pond management