Determination of critical processes underlying the resilience of aquatic microbial communities under distinct resource availabilities

Freshwater systems have always been under the most variate disturbance regimes. However, the number of multi-stressors and the frequency/intensity of stochastic pulse perturbations they will face in the future is predicted to increase. Stochastic pulse perturbations such as storms and floods are well known to temporarily disrupt ecosystem services that are fundamental for global existence within short time scales - as drinking water supply, irrigation, and food provision. Eutrophication - one of the main freshwater press perturbations deteriorating water quality - may enhance harmful cyanobacterial blooms, modify the structure of food webs, and alter long-term ecological stability, resulting in substantial ecological, social, and economic losses. Despite all our knowledge on the effects of stochastic events and eutrophication operating in isolation, we know little about how their interactions can shape the resilience of aquatic ecosystems. The aim of this thesis is to identify general processes of recovery and response pathways taking place after mortality pulse perturbations and assess how resource availability can modify them. For that, the existing literature was reviewed, and experiments were executed using aquatic systems of different ecological complexity. Chapter 2 starts with a global review of how climate change may modify in-lake processes that are prone to escalate into feedback loops of relevance to freshwater biogeochemistry (i.e., mixing regimes modifying nutrient upwelling). Chapter 3 explored if Early- Warning Signals of Regime Shift (EWS) could produce sufficient mechanistic empirical evidence to be used as a reliable methodological framework for quantifying Ecological Resilience. For that, we reviewed the literature for aquatic experiments that have designed interactions between pulse and press perturbations. Using the lessons learned from both reviews, the experiment of Chapter 4 used an eco-evolutionary microcosm with complex intraspecific interactions to focus on methodological constraints of using recovery rate and time for addressing engineering and ecological resilience. Also, this experiment investigated the role of resource availability on the processes ruling the recovery from pulse perturbations at a community-alike level. Last, in chapter 5, ecological complexity was scaled up using mesocosms filled with natural lake water to identify key processes involving the effect of nutrient availability on the microbial community response to pulse perturbations. Here the focus was on how eutrophication changes the phytoplankton resistance and resilience to mortality events that cause internal nutrient turnover. Together, these four chapters provided the core of the thesis. Overall, we identified that climate change can indeed create feedback loops capable of altering the nutrient dynamics in lakes, as well as the direction of change depending on the lake characteristics. We scrutinized processes that can lead to an increase of total nutrients in the epilimnion in the long run while creating longer periods of dissolved nutrient limitation within a season. Those processes are related to In-lake mixing regimes, which are susceptible to atmospheric conditions and responsible for the internal mixing of nutrients in lakes. Therefore, the presence of feedback loops in lake dynamics might become increasingly relevant under ongoing climate change. When reviewing the reliability of EWS to address the loss of ecological resilience in aquatic systems, we observed that the field is still at the stage of proof-of-concept. Complex experiments designed to assess the interaction between pulse and press perturbations were virtually absent. Most experiments considered either a pulse or a press perturbation affecting the system, and often without a mechanistic understanding of why an EWS was observed or not. Amongst all EWS, recovery rate or time were the most capable of bringing together how long-term changes in the system affect the response to stochastic pulse perturbations. Recovery rates showed reliable results when predicting population collapses; however, the translation of individual populational-level recovery rates to more complex community-based ecosystem dynamics is far from consolidated. Moving on into process-based approaches, our experiments highlighted that the effects of resource availability can be decomposed into two distinguished processes of recovery. One is the capability of the system to fully recover from disturbance. Another is the recovery pattern that the system will embody after the pulse perturbation. In both experiments we observed the importance of biotic interaction for determining how the system responds to perturbations and the effect of resource availability on shaping the direction of those interactions. Competition, facilitation, intrinsic growth rates, population turnover, the complexity of ecological interactions, and rates of energy transfer, all played a role in the processes underlying 'resilience' (latu sensu). The eco-evolutionary microcosm taught us that resource availability modulates the speed of the recovery process (rate and time) but not necessarily the level of recovery. Systems with low resource availability showed weaker recovery rates and longer recovery times for both compositional and functional dimensions compared to when resources were high. However, all tested systems showed substantial plasticity in absorbing and recovering from a pulse perturbation (complete recovery). Therefore, the slower recovery process caused by low resource availability did not change “the measure of the persistence of the system” or “their ability to absorb change and disturbance and still maintain the same relationships between populations or state variables" – the formal definition of ecological resilience (Holling, 1973). When dealing with systems of different resource availability, ecological (sensu Holling) and engineering resilience (sensu Pimm) may potentially decouple in time. Therefore, we must be especially careful when using recovery from perturbation as a proxy of ecological resilience if resource availability is the main driver of ecosystem change. When scaling up the trophic complexity to a mesocosms level, we observed that pulse perturbations increased the amount of autochthonous nutrients in the system, selecting for 'small and fast-growers' at first. As more resources were allocated in the system before the pulse perturbation, more nutrients were released during the mortality event; and with more dissolved nutrients available for uptake, higher peaks of phytoplankton concentration were observed as a response to the pulse perturbation. However, the level of resource availability in the mesocosms did not change the resistance or the recovery from a mortality pulse perturbation. Together, the chlorophyll-a displacement declined after each sequential pulse perturbation despite increases in resource availability. The more perturbed the system was, the less responsive it became, and this can be interpreted as a possible community pathway towards stability. The mesocosms experiment indicated the capability of the system to adapt to sequential pulse perturbations in a way to reduce its impacts and that resource availability and sequential pulse perturbations are antagonistic forces shaping the response of the system to mortality events. Both experiments executed in this thesis presented a convergent story. Resource availability did not change the capability of the systems to recover from pulse perturbations, despite changing their compositional structure and process rates. However, the evidence that resource availability is unlikely to influence the capability of systems to recover should not lead to underestimations of the risks related to changing process rates in ecological systems. Systems under low resource availability might become more vulnerable to changes in the frequency of pulse perturbations, while systems with high resource availability may present stronger responses immediately after the pulse perturbation (i.e., peaks of Chl-a concentrations). This is an important consideration when managing waterbodies with strict regulations (i.e., drinking water provision) since systems with a surplus of resource availability may more easily trespass regulatory guideline values (e.g., maximum Chl-a threshold levels for safe water supply) following a pulse perturbation, while oligotrophic systems may become especially vulnerable to changes in the frequency of sequential weather-related pulse perturbations.Süßwassersysteme waren schon immer den unterschiedlichsten Störungsregimen ausgesetzt. Es wird jedoch vorhergesagt, dass die Anzahl der Multi-Stressoren und die Häufigkeit/Intensität der stochastischen Impulsstörungen, denen sie in Zukunft ausgesetzt sein werden, zunehmen werden. Es ist bekannt, dass stochastische Impulsstörungen wie Stürme und Überschwemmungen Ökosystemdienstleistungen, die für die globale Existenz von grundlegender Bedeutung sind, innerhalb kurzer Zeitspannen vorübergehend stören können - z. B. Trinkwasserversorgung, Bewässerung und Nahrungsmittelversorgung. Eutrophierung - eine der wichtigsten Störungen der Süßwasserpresse, die die Wasserqualität verschlechtert - kann schädliche Cyanobakterienblüten fördern, die Struktur von Nahrungsnetzen verändern und die langfristige ökologische Stabilität beeinträchtigen, was zu erheblichen ökologischen, sozialen und wirtschaftlichen Verlusten führt. Trotz all unserer Kenntnisse über die Auswirkungen von stochastischen Ereignissen und Eutrophierung, die isoliert voneinander auftreten, wissen wir nur wenig darüber, wie ihre Wechselwirkungen die Widerstandsfähigkeit aquatischer Ökosysteme beeinflussen können. Ziel dieser Arbeit ist es, allgemeine Erholungsprozesse und Reaktionswege zu identifizieren, die nach Störungen durch Mortalitätsimpulse ablaufen, und zu bewerten, wie die Ressourcenverfügbarkeit diese verändern kann. Zu diesem Zweck wurde die vorhandene Literatur gesichtet, und es wurden Experimente mit aquatischen Systemen unterschiedlicher ökologischer Komplexität durchgeführt. Kapitel 2 beginnt mit einem globalen Überblick darüber, wie der Klimawandel Prozesse im See verändern kann, die sich zu Rückkopplungsschleifen entwickeln können, die für die Biogeochemie des Süßwassers von Bedeutung sind (z. B. Mischungsregime, die den Nährstoffauftrieb verändern). In Kapitel 3 wurde untersucht, ob Frühwarnsignale für Regimeverschiebungen (Early-Warning Signals of Regime Shift, EWS) genügend mechanistische empirische Beweise liefern können, um als zuverlässiger methodischer Rahmen für die Quantifizierung der ökologischen Widerstandsfähigkeit verwendet werden zu können. Zu diesem Zweck haben wir die Literatur zu aquatischen Experimenten durchgesehen, bei denen Wechselwirkungen zwischen Impuls- und Druckstörungen untersucht wurden. Auf der Grundlage der Erkenntnisse aus beiden Übersichten wurde im Experiment in Kapitel 4 ein ökoevolutionärer Mikrokosmos mit komplexen intraspezifischen Interaktionen verwendet, um die methodischen Einschränkungen bei der Verwendung von Erholungsrate und -zeit für die Untersuchung der technischen und ökologischen Belastbarkeit zu untersuchen. Außerdem untersuchte dieses Experiment die Rolle der Ressourcenverfügbarkeit für die Prozesse, die die Erholung von Impulsstörungen auf einer gemeinschaftsähnlichen Ebene bestimmen. In Kapitel 5 schließlich wurde die ökologische Komplexität mit Hilfe von Mesokosmen, die mit natürlichem Seewasser gefüllt waren, erhöht, um die Schlüsselprozesse zu identifizieren, die die Auswirkungen der Nährstoffverfügbarkeit auf die Reaktion der mikrobiellen Gemeinschaft auf Impuls-Störungen betreffen. Hier lag der Schwerpunkt darauf, wie die Eutrophierung die Resistenz und Widerstandsfähigkeit des Phytoplanktons gegenüber Mortalitätsereignissen verändert, die einen internen Nährstoffumsatz verursachen. Zusammen bilden diese vier Kapitel den Kern der Arbeit. Insgesamt haben wir festgestellt, dass der Klimawandel in der Tat Rückkopplungsschleifen schaffen kann, die die Nährstoffdynamik in Seen verändern können, und dass die Richtung der Veränderung von den Eigenschaften des Sees abhängt. Wir haben Prozesse untersucht, die langfristig zu einem Anstieg der Gesamtnährstoffe im Epilimnion führen können und gleichzeitig längere Perioden der Begrenzung gelöster Nährstoffe innerhalb einer Saison verursachen. Diese Prozesse hängen mit dem Durchmischungsregime im See zusammen, das von den atmosphärischen Bedingungen abhängt und für die interne Durchmischung von Nährstoffen in Seen verantwortlich ist. Daher könnte das Vorhandensein von Rückkopplungsschleifen in der Seedynamik bei fortschreitendem Klimawandel zunehmend an Bedeutung gewinnen. Bei der Überprüfung der Verlässlichkeit von EWS im Hinblick auf den Verlust der ökologischen Widerstandsfähigkeit aquatischer Systeme haben wir festgestellt, dass sich das Feld noch im Stadium des Proof-of-Concept befindet. Komplexe Experimente zur Bewertung der Wechselwirkung zwischen Impuls- und Pressestörungen waren praktisch nicht vorhanden. In den meisten Experimenten wurde entweder eine Impuls- oder eine Druckstörung untersucht, die das System beeinflusste, oft ohne ein mechanistisches Verständnis dafür, warum ein EWS beobachtet wurde oder nicht. Von allen EWS waren die Erholungsrate oder die Zeit am ehesten in der Lage zu erfassen, wie sich langfristige Veränderungen im System auf die Reaktion auf stochastische Impulsstörungen auswirken. Erholungsraten zeigten verlässliche Ergebnisse bei der Vorhersage von Populationszusammenbrüchen; die Übertragung von individuellen Erholungsraten auf Populationsebene auf komplexere gemeinschaftsbasierte Ökosystemdynamiken ist jedoch noch lange nicht gefestigt. Unsere Experimente haben gezeigt, dass die Auswirkungen der Ressourcenverfügbarkeit in zwei unterschiedliche Erholungsprozesse zerlegt werden können. Der eine ist die Fähigkeit des Systems, sich vollständig von einer Störung zu erholen. Der andere ist das Erholungsmuster, das das System nach der Impulsstörung verkörpern wird. In beiden Experimenten konnten wir beobachten, wie wichtig biotische Interaktionen sind, um zu bestimmen, wie das System auf Störungen reagiert, und wie sich die Verfügbarkeit von Ressourcen auf die Richtung dieser Interaktionen auswirkt. Konkurrenz, Erleichterung, intrinsische Wachstumsraten, Populationsumsatz, die Komplexität ökologischer Interaktionen und die Energieübertragungsraten spielten alle eine Rolle bei den Prozessen, die der "Resilienz" (latu sensu) zugrunde liegen. Der ökoevolutionäre Mikrokosmos lehrte uns, dass die Ressourcenverfügbarkeit die Geschwindigkeit des Erholungsprozesses (Geschwindigkeit und Zeit), aber nicht unbedingt das Ausmaß der Erholung beeinflusst. Systeme mit geringer Ressourcenverfügbarkeit wiesen sowohl bei der Zusammensetzung als auch bei den funktionalen Dimensionen geringere Erholungsraten und längere Erholungszeiten auf als Systeme mit hoher Ressourcenverfügbarkeit. Alle getesteten Systeme zeigten jedoch eine beträchtliche Plastizität bei der Absorption und Erholung von einer Impulsstörung (vollständige Erholung). Der langsamere Erholungsprozess, der durch die geringe Ressourcenverfügbarkeit verursacht wurde, änderte daher nicht "das Maß für die Persistenz des Systems" oder "seine Fähigkeit, Veränderungen und Störungen zu absorbieren und dennoch die gleichen Beziehungen zwischen Populationen oder Zustandsvariablen aufrechtzuerhalten" - die formale Definition der ökologischen Resilienz (Holling, 1973). Bei Systemen mit unterschiedlicher Ressourcenverfügbarkeit können sich ökologische (im Sinne von Holling) und technische Belastbarkeit (im Sinne von Pimm) möglicherweise zeitlich entkoppeln. Daher müssen wir besonders vorsichtig sein, wenn wir die Erholung von Störungen als Indikator für die ökologische Resilienz verwenden, wenn die Ressourcenverfügbarkeit die Haupttriebkraft für Veränderungen im Ökosystem ist. Beim Hochskalieren der trophischen Komplexität auf die Ebene der Mesokosmen konnten wir beobachten, dass die Impulsstörungen die Menge an autochthonen Nährstoffen im System erhöhten, wobei zunächst kleine und schnell wachsende Arten bevorzugt wurden. Da vor der Impulsstörung mehr Ressourcen im System vorhanden waren, wurden während des Mortalitätsereignisses mehr Nährstoffe freigesetzt; und da mehr gelöste Nährstoffe zur Aufnahme zur Verfügung standen, wurden als Reaktion auf die Impulsstörung höhere Spitzenwerte der Phytoplanktonkonzentration beobachtet. Das Niveau der Ressourcenverfügbarkeit in den Mesokosmen änderte jedoch nichts an der Widerstandsfähigkeit oder der Erholung von einem Mortalitätsimpuls. Insgesamt nahm die Chlorophyll-a-Verschiebung nach jeder aufeinanderfolgenden Impulsstörung ab, obwohl die Verfügbarkeit der Ressourcen zunahm. Je stärker das System gestört wurde, desto weniger reagierte es, was als möglicher Weg der Gemeinschaft zur Stabilität gedeutet werden kann. Das Mesokosmen- Experiment zeigte, dass das System in der Lage ist, sich an sequenzielle Impulsstörungen so anzupassen, dass die Auswirkungen reduziert werden, und dass Ressourcenverfügbarkeit und sequenzielle Impulsstörungen antagonistische Kräfte sind, die die Reaktion des Systems auf Mortalitätsereignisse bestimmen. Beide Experimente, die im Rahmen dieser Arbeit durchgeführt wurden, zeigten eine konvergente Geschichte. Die Ressourcenverfügbarkeit änderte nichts an der Fähigkeit der Systeme, sich von Impulsstörungen zu erholen, obwohl sich ihre Zusammensetzungsstruktur und Prozessraten änderten. Der Nachweis, dass die Ressourcenverfügbarkeit keinen Einfluss auf die Erholungsfähigkeit von Systemen hat, sollte jedoch nicht dazu führen, dass die Risiken im Zusammenhang mit veränderten Prozessraten in ökologischen Systemen unterschätzt werden. Systeme mit geringer Ressourcenverfügbarkeit könnten anfälliger für Änderungen der Häufigkeit von Impulsstörungen werden, während Systeme mit hoher Ressourcenverfügbarkeit unmittelbar nach der Impulsstörung stärkere Reaktionen zeigen können (z. B. Spitzenwerte der Chl-a-Konzentration). Dies ist eine wichtige Überlegung bei der Bewirtschaftung von Gewässern mit strengen Vorschriften (z. B. bei der Trinkwasserversorgung), da Systeme mit einem Überschuss an Ressourcenverfügbarkeit nach einer Impulsstörung leichter die vorgeschriebenen Richtwerte (z. B. maximale Chl-a-Grenzwerte für die sichere Wasserversorgung) überschreiten können, während oligotrophe Systeme besonders anfällig für Änderungen der Häufigkeit aufeinander folgender wetterbedingter Impulsstörungen sein können

Performance of one-dimensional hydrodynamic lake models during short-term extreme weather events

Numerical lake models are useful tools to study hydrodynamics in lakes, and are increasingly applied to extreme weather events. However, little is known about the accuracy of such models during these short-term events. We used high-frequency data from three lakes to test the performance of three one-dimensional (1D) hydrodynamic models (Simstrat, GOTM, GLM) during storms and heatwaves. Models reproduced the overall direction and magnitude of changes during the extreme events, with accurate timing and little bias. Changes in volume-averaged and surface temperatures and Schmidt stability were simulated more accurately than changes in bottom temperature, maximum buoyancy frequency, or mixed layer depth. However, in most cases the model error was higher (30-100%) during extreme events compared to reference periods. As a consequence, while 1D lake models can be used to study effects of extreme weather events, the increased uncertainty in the simulations should be taken into account when interpreting results

A preliminary study on the ecotoxic potency of wastewater treatment plant sludge combining passive sampling and bioassays

Highlights • The passive samplers collected bioavail able and bioaccessible chemicals from the WWTP sludge samples. • The sampler extracts were acutely and chronically toxic to water flea. • The sampler extracts were cytotoxic and genotoxic. • The sludge treatment such as composting and digesting diminished the toxicity. • Effect-based methods should be part of the risk assessment of sludge recycling.Sewage sludge is an inevitable byproduct produced in wastewater treatment. Reusing nutrient-rich sludge will diminish the amount of waste ending in soil dumping areas and will promote circular economy. However, during sewage treatment process, several potentially harmful organic chemicals are retained in sludge, but proving the safety of processed sludge will promote its more extensive use in agriculture and landscaping. Environmental risk assessment of sludge requires new methods of characterizing its suitability for various circu lar economy applications. Bioavailable and bioaccessible fractions are key variables indicating leaching, transport, and bioaccumulation capacity. Also, sludge treatments have a significant effect on chemical status and resulting environmental risks. In this study, the concentrations of polyaromatic hydrocarbons (PAHs), triclosan (TCS), triclocarban (TCC), methyl triclosan (mTCS), and selected active pharmaceutical ingredients (APIs) were deter mined in different sludge treatments and fractions. Passive samplers were used to characterize the bioavailable and bioaccessible fractions, and the sampler extracts along the sludge and filtrate samples were utilized in the bioassays. The TCS and PAH concentrations did not decrease as the sludge was digested, but the contents diminished after composting. Also, mTCS concentration decreased after composting. The API concentrations were lower in digested sludge than in secondary sludge. Digested sludge was toxic for Aliivibrio fischeri, but after composting, toxicity was not observed. However, for Daphnia magna, passive sampler extracts of all sludge treatments were either acutely (immobility) or chronically (reproduction) toxic. Secondary and digested sludge sampler extracts were cytotoxic, and secondary sludge ex tract was also genotoxic. The measured chemical concentration levels did not explain the toxicity of the samples based on the reported toxicity thresholds. Bioassays and sampler extracts detecting bioavailable and bioaccessible contaminants in sludge are complementing tools for chemical analyses. Harmonization of these methodswill help establish scientifically sound regulative thresholds for the use of sludge in circular economy applications

Determination of critical processes underlying the resilience of aquatic microbial communities under distinct resource availabilities

The number of multi-stressors that freshwater systems will face in the future is predicted to increase. Stochastic pulse perturbations such as storms and floods are well known to temporarily disrupt ecosystem services like drinking water supply, irrigation, and food provision. Eutrophication, as a press perturbation, may alter long-term ecological stability, resulting in substantial ecological, social, and economic losses. Despite all our knowledge on the effects of stochastic events and eutrophication in isolation, we know little about how their interactions can shape resilience of aquatic ecosystems. The aim of this thesis was to identify general processes of recovery and response pathways taking place after mortality pulse perturbations and assess how eutrophication can modify them. Experiments showed that eutrophication affects the type of ecological interactions unfolded after a stochastic pulse perturbation, determining the recovery pathway. Although, resource availability did not change the capability of the systems to resist or recover from pulse perturbations

Utilização de técnica de respirometria microbiana para avaliação de impacto ambiental : uma abordagem microbiológica para estudos em ecotoxicologia

O crescimento acelerado da população mundial vem aumentando a preocupação com as problemáticas ambientais. Técnicas vem sendo propostas na tentativa de analisar a qualidade dos ecossistemas e avaliar possíveis impactos causados por determinadas substâncias quando em contato com o meio ambiente. Neste cenário, os ensaios ecotoxicológicos rápidos vem surgindo como aliados em momentos onde a rápida tomada de decisões se vê necessária. Análises com base em respirometria do microrganismo Pseudomonas putida foram realizadas utilizando o equipamento Baroximeter®. Um total de 368 ensaios respirométricos foram realizados com água destilada em dois equipamentos em um intervalo de 11 dias para avaliar a repetibilidade e a confiabilidade da metodologia de respirometria proposta para futuras análises ecotoxicológicas. Foram utilizados cultivos de P. putida crescidos em meio nutriente LB em 4 fases fisiológicas distintas, ápice de fase exponencial, final de fase exponencial/inicio de fase estacionária, estacionária inicial e estacionária estabilizada para avaliação do melhor estágio de crescimento para padronização metodológica. Para as análises respirométricas os cultivos foram concentrados e alíquotados em eppendorfs de 2 ml conservados em geladeira a 4 0C até o uso. A viabilidade do cultivo microbiano foi avaliada diariamente pela técnica de plaqueamento em gota até o final dos experimentos. Como resultados foram observadas diferenças entre a média de leitura respirométrica entre os dois equipamentos e uma variabilidade aleatória na janela de resultados obtidos. Foi observada a necessidade de um grande número de repetições nas medições respirométricas para que houvesse a redução da variação dos resultados. Apenas as células em ápice de fase logarítmica apresentaram mortalidade durante o período analisado indicando a centrifugação como uma forma eficiente de conservação a frio por um curto intervalo de tempo.The rapid growth of the world population has been increasing the concern about environmental issues. Techniques have been proposed in an attempt to analyze the quality of ecosystems and evaluate possible impacts caused by certain substances when in contact with it. Under this scenario, the direct toxicity assessments come as an allied in situations when rapid decisions are needed. Assessments based on respirometric rates of the microorganism Pseudomonas putida were performed using Baroximeter®. A total of 368 respirometric tests were performed with distilled water in two devices during 11 days to assess the repeatability and reliability of the methodology proposed for future ecotoxicological analyzes. Cultures of P. putida were grown in LB nutrient medium in 4 distinct physiological stages, apex of exponential phase, late exponential phase/ early stationary phase, initial stationary phase and stabilized stationary phase to evaluate the best growth stage for methodological standardization. For respirometric analyzes, bacterial grown were concentrated and aliquoted (pellets) in 2 ml eppendorfs and stored under refrigeration at 40 C until use. The viability of the cells were evaluated daily using the method of drob plate counting until the end of the experiments. As a result were identified differences on the average respiration rates between the two respirometric equipment and random variability in the results obtained. We observed the need of a large amount of repetitions to reduce the variation of the results. Only the cells made from apex of exponential phase showed mortality during the period analyzed, indicating that centrifugation is an efficient storage method for short time conservation

Utilização de técnica de respirometria microbiana para avaliação de impacto ambiental : uma abordagem microbiológica para estudos em ecotoxicologia

O crescimento acelerado da população mundial vem aumentando a preocupação com as problemáticas ambientais. Técnicas vem sendo propostas na tentativa de analisar a qualidade dos ecossistemas e avaliar possíveis impactos causados por determinadas substâncias quando em contato com o meio ambiente. Neste cenário, os ensaios ecotoxicológicos rápidos vem surgindo como aliados em momentos onde a rápida tomada de decisões se vê necessária. Análises com base em respirometria do microrganismo Pseudomonas putida foram realizadas utilizando o equipamento Baroximeter®. Um total de 368 ensaios respirométricos foram realizados com água destilada em dois equipamentos em um intervalo de 11 dias para avaliar a repetibilidade e a confiabilidade da metodologia de respirometria proposta para futuras análises ecotoxicológicas. Foram utilizados cultivos de P. putida crescidos em meio nutriente LB em 4 fases fisiológicas distintas, ápice de fase exponencial, final de fase exponencial/inicio de fase estacionária, estacionária inicial e estacionária estabilizada para avaliação do melhor estágio de crescimento para padronização metodológica. Para as análises respirométricas os cultivos foram concentrados e alíquotados em eppendorfs de 2 ml conservados em geladeira a 4 0C até o uso. A viabilidade do cultivo microbiano foi avaliada diariamente pela técnica de plaqueamento em gota até o final dos experimentos. Como resultados foram observadas diferenças entre a média de leitura respirométrica entre os dois equipamentos e uma variabilidade aleatória na janela de resultados obtidos. Foi observada a necessidade de um grande número de repetições nas medições respirométricas para que houvesse a redução da variação dos resultados. Apenas as células em ápice de fase logarítmica apresentaram mortalidade durante o período analisado indicando a centrifugação como uma forma eficiente de conservação a frio por um curto intervalo de tempo.The rapid growth of the world population has been increasing the concern about environmental issues. Techniques have been proposed in an attempt to analyze the quality of ecosystems and evaluate possible impacts caused by certain substances when in contact with it. Under this scenario, the direct toxicity assessments come as an allied in situations when rapid decisions are needed. Assessments based on respirometric rates of the microorganism Pseudomonas putida were performed using Baroximeter®. A total of 368 respirometric tests were performed with distilled water in two devices during 11 days to assess the repeatability and reliability of the methodology proposed for future ecotoxicological analyzes. Cultures of P. putida were grown in LB nutrient medium in 4 distinct physiological stages, apex of exponential phase, late exponential phase/ early stationary phase, initial stationary phase and stabilized stationary phase to evaluate the best growth stage for methodological standardization. For respirometric analyzes, bacterial grown were concentrated and aliquoted (pellets) in 2 ml eppendorfs and stored under refrigeration at 40 C until use. The viability of the cells were evaluated daily using the method of drob plate counting until the end of the experiments. As a result were identified differences on the average respiration rates between the two respirometric equipment and random variability in the results obtained. We observed the need of a large amount of repetitions to reduce the variation of the results. Only the cells made from apex of exponential phase showed mortality during the period analyzed, indicating that centrifugation is an efficient storage method for short time conservation

Early warning signals of regime shifts for aquatic systems: Can experiments help to bridge the gap between theory and real-world application?

Early-warning signals of a regime shift (EWS) indicate, for a wide range of systems, if a tipping-point is being approached. In ecology, EWS are well established from a theoretical perspective but are far from unequivocal when applied to field data. The gap between theory and application is caused by a set of limitations, like the lack of coherence between different EWS, data acquisition issues, and false results. Experiments assessing EWS may provide an empirical mechanistic understanding of why an EWS was observed (or failed to be observed), which often cannot be elucidated by simple computational modeling or pure environmental data. Here we focused on aquatic experiments to explore to what extent the existing EWS experiments can bridge the gap between the theory and real-world application. For that, we used the Thomson-ISI Web of Science© database to retrieve EWS experiments executed before early-2020, detailing their experimental designs and each EWS assessed. Success rates - correct anticipation of tipping points – were around 70% for the most used EWS (assessment of autocorrelation, variance, recovery, and shape of the distribution using abundance, Chlorophyll-a, Phycocyanin, and dissolved oxygen data). Yet, no EWS showed to be 100% reliable, and their use demands cautious interpretation. As a rule, we observed that experiments were not designed to tackle issues encountered in real-world situations. They lack a deep mechanistic understanding of why, when, and how an EWS was observed or not. When experiments did aim to assess issues encountered in the real world, the experimental designs were often of low ecological significance. We also investigated the relationship between sampling and the success rate of EWS, observing that the sampling regime might have to be tailor-made towards specific monitoring objectives. Moreover, experiments have taught us that the use of EWS can be more versatile than expected, going from monitoring the extinction of single populations to the anticipation of transient regime shifts. Most of the experiments presented here supported empirical proof of the existence of EWS in aquatic systems. Still, to bridge the gap between theory and application, experiments will have to move closer to real-world conditions and better support a mechanistic understanding of why EWS may succeed or fail to anticipate a regime shift. For that, we provide six elements to take into account when designing experiments that could enhance the capabilities of EWS to go beyond the stage of proof-of-concept

Phytoplankton responses to repeated pulse perturbations imposed on a trend of increasing eutrophication

While eutrophication remains one of the main pressures acting on freshwater ecosystems, the prevalence of anthropogenic and nature‐induced stochastic pulse perturbations is predicted to increase due to climate change. Despite all our knowledge on the effects of eutrophication and stochastic events operating in isolation, we know little about how eutrophication may affect the response and recovery of aquatic ecosystems to pulse perturbations. There are multiple ways in which eutrophication and pulse perturbations may interact to induce potentially synergic changes in the system, for instance, by increasing the amount of nutrients released after a pulse perturbation. Here, we performed a controlled press and pulse perturbation experiment using mesocosms filled with natural lake water to address how eutrophication modulates the phytoplankton response to sequential mortality pulse perturbations; and what is the combined effect of press and pulse perturbations on the resistance and resilience of the phytoplankton community. Our experiment showed that eutrophication increased the absolute scale of the chlorophyll‐a response to pulse perturbations but did not change the proportion of the response relative to its pre‐event condition (resistance). Moreover, the capacity of the community to recover from pulse perturbations was significantly affected by the cumulative effect of sequential pulse perturbations but not by eutrophication itself. By the end of the experiment, some mesocosms could not recover from pulse perturbations, irrespective of the trophic state induced by the press perturbation. While not resisting or recovering any less from pulse perturbations, phytoplankton communities from eutrophying systems showed chlorophyll‐a levels much higher than non‐eutrophying ones. This implies that the higher absolute response to stochastic pulse perturbations in a eutrophying system may increase the already significant risks for water quality (e.g., algal blooms in drinking water supplies), even if the relative scale of the response to pulse perturbations between eutrophying and non‐eutrophying systems remains the same. Press (eutrophication) and repeated pulse perturbations are antagonistic forces shaping the microbial response to stochastic perturbations. Eutrophication increases the absolute response of the phytoplankton community to pulse perturbations, but does not change the proportionality of response compared to the pre‐event condition. Resistance increase, and recovery decrease after each pulse perturbation independently of the eutrophication process

Stelzer et al., (2017) Data Analysis.pdf

Statistical Analysis from the article "<i>Is it Fish Embryo Test (FET) according to OECD 236 sensible enough for delivering quality data for effluent risk assessment?</i>"<br