Click detection rate variability of central North Pacific sperm whales from passive acoustic towed arrays.

Passive acoustic monitoring (PAM) is an optimal method for detecting and monitoring cetaceans as they frequently produce sound while underwater. Cue counting, counting acoustic cues of deep-diving cetaceans instead of animals, is an alternative method for density estimation, but requires an average cue production rate to convert cue density to animal density. Limited information about click rates exists for sperm whales in the central North Pacific Ocean. In the absence of acoustic tag data, we used towed hydrophone array data to calculate the first sperm whale click rates from this region and examined their variability based on click type, location, distance of whales from the array, and group size estimated by visual observers. Our findings show click type to be the most important variable, with groups that include codas yielding the highest click rates. We also found a positive relationship between group size and click detection rates that may be useful for acoustic predictions of group size in future studies. Echolocation clicks detected using PAM methods are often the only indicator of deep-diving cetacean presence. Understanding the factors affecting their click rates provides important information for acoustic density estimation.

Narwhal (Monodon monoceros) echolocation click rates to support cue counting passive acoustic density estimation.

Estimating animal abundance is fundamental for effective management and conservation. It is increasingly done by combining passive acoustics with knowledge about rates at which animals produce cues (cue rates). Narwhals (Monodon monoceros) are elusive marine mammals for which passive acoustic density estimation might be plausible, but for which cue rates are lacking. Clicking rates in narwhals were investigated using a dataset from sound and movement tag records collected in August 2013-2016 and 2019 in East Greenland. Clicking rates were quantified for ∼1200 one-second-long systematic random samples from 8 different whales. Generalized additive models were used to model (1) the probability of being in a clicking state versus depth and (2) the clicking rate while in a clicking state, versus time and depth. The probability of being in a clicking state increased with depth, reaching ∼1.0 at ∼500 m, while the number of clicks per second (while in a clicking state) increased with depth. The mean cue production rate, weighted by tag duration, was 1.28 clicks per second (se = 0.13, CV = 0.10). This first cue rate for narwhals may be used for cue counting density estimation, but care should be taken if applying it to other geographical areas or seasons, given sample size, geographical, and temporal limitations.

A sperm whale cautionary tale about estimating acoustic cue rates for deep divers.

Passive acoustic density estimation has been gaining traction in recent years. Cue counting uses detected acoustic cues to estimate animal abundance. A cue rate, the number of acoustic cues produced per animal per unit time, is required to convert cue density into animal density. Cue rate information can be obtained from animal borne acoustic tags. For deep divers, like beaked whales, data have been analyzed considering deep dive cycles as a natural sampling unit, based on either weighted averages or generalized estimating equations. Using a sperm whale DTAG (sound-and-orientation recording tag) example we compare different approaches of estimating cue rate from acoustic tags illustrating that both approaches used before might introduce biases and suggest that the natural unit of analysis should be the whole duration of the tag itself.

Implications of porpoise echolocation and dive behaviour on passive acoustic monitoring.

Harbour porpoises are visually inconspicuous but highly soniferous echolocating marine predators that are regularly studied using passive acoustic monitoring (PAM). PAM can provide quality data on animal abundance, human impact, habitat use, and behaviour. The probability of detecting porpoise clicks within a given area (P̂) is a key metric when interpreting PAM data. Estimates of P̂ can be used to determine the number of clicks per porpoise encounter that may have been missed on a PAM device, which, in turn, allows for the calculation of abundance and ideally non-biased comparison of acoustic data between habitats and time periods. However, P̂ is influenced by several factors, including the behaviour of the vocalising animal. Here, the common implicit assumption that changes in animal behaviour have a negligible effect on P̂ between different monitoring stations or across time is tested. Using a simulation-based approach informed by acoustic biologging data from 22 tagged harbour porpoises, it is demonstrated that porpoise behavioural states can have significant (up to 3× difference) effects on P̂. Consequently, the behavioural state of the animals must be considered in analysis of animal abundance to avoid substantial over- or underestimation of the true abundance, habitat use, or effects of human disturbance.

Spatio-temporal changes in chimpanzee density and abundance in the Greater Mahale Ecosystem, Tanzania.

Species conservation and management require reliable information about animal distribution and population size. Better management actions within a species' range can be achieved by identifying the location and timing of population changes. In the Greater Mahale Ecosystem (GME), western Tanzania, deforestation due to the expansion of human settlements and agriculture, annual burning, and logging are known threats to wildlife. For one of the most charismatic species, the endangered eastern chimpanzee (Pan troglodytes schweinfurthii), approximately 75% of the individuals are distributed outside national park boundaries, requiring monitoring and protection efforts over a vast landscape of various protection statuses. These efforts are especially challenging when we lack data on trends in density and population size. To predict spatio-temporal chimpanzee density and abundance across the GME, we used density surface modeling, fitting a generalized additive model to a 10-year time-series data set of nest counts based on line-transect surveys. The chimpanzee population declined at an annual rate of 2.41%, including declines of 1.72% in riparian forests (from this point forward, forests), 2.05% in miombo woodlands (from this point forward, woodlands) and 3.45% in nonforests. These population declines were accompanied by ecosystem-wide declines in vegetation types of 1.36% and 0.32% per year for forests and woodlands, respectively; we estimated an annual increase of 1.35% for nonforests. Our model predicted the highest chimpanzee density in forests (0.86 chimpanzees/km2 , 95% confidence intervals (CIs) 0.60-1.23; as of 2020), followed by woodlands (0.19, 95% CI 0.12-0.30) and nonforests (0.18, 95% CI 0.10-1.33). Although forests represent only 6% of the landscape, they support nearly one-quarter of the chimpanzee population (769 chimpanzees, 95% CI 536-1103). Woodlands dominate the landscape (71%) and therefore support more than a half of the chimpanzee population (2294; 95% CI 1420-3707). The remaining quarter of the landscape is represented by nonforests and supports another quarter of the chimpanzee population (750; 95% CI 408-1381). Given the pressures on the remaining suitable habitat in Tanzania, and the need of chimpanzees to access both forest and woodland vegetation to survive, we urge future management actions to increase resources and expand the efforts to protect critical forest and woodland habitat and promote strategies and policies that more effectively prevent irreversible losses. We suggest that regular monitoring programs implement a systematic random design to effectively inform and allocate conservation actions and facilitate interannual comparisons for trend monitoring, measuring conservation success, and guiding adaptive management.

Modeling population effects of the Deepwater Horizon oil spill on a long-lived species.

The 2010 Deepwater Horizon (DWH) oil spill exposed common bottlenose dolphins (Tursiops truncatus) in Barataria Bay, Louisiana to heavy oiling that caused increased mortality and chronic disease and impaired reproduction in surviving dolphins. We conducted photographic surveys and veterinary assessments in the decade following the spill. We assigned a prognostic score (good, fair, guarded, poor, or grave) for each dolphin to provide a single integrated indicator of overall health, and we examined temporal trends in prognostic scores. We used expert elicitation to quantify the implications of trends for the proportion of the dolphins that would recover within their lifetime. We integrated expert elicitation, along with other new information, in a population dynamics model to predict the effects of observed health trends on demography. We compared the resulting population trajectory with that predicted under baseline (no spill) conditions. Disease conditions persisted and have recently worsened in dolphins that were presumably exposed to DWH oil: 78% of those assessed in 2018 had a guarded, poor, or grave prognosis. Dolphins born after the spill were in better health. We estimated that the population declined by 45% (95% CI 14-74) relative to baseline and will take 35 years (95% CI 18-67) to recover to 95% of baseline numbers. The sum of annual differences between baseline and injured population sizes (i.e., the lost cetacean years) was 30,993 (95% CI 6607-94,148). The population is currently at a minimum point in its recovery trajectory and is vulnerable to emerging threats, including planned ecosystem restoration efforts that are likely to be detrimental to the dolphins' survival. Our modeling framework demonstrates an approach for integrating different sources and types of data, highlights the utility of expert elicitation for indeterminable input parameters, and emphasizes the importance of considering and monitoring long-term health of long-lived species subject to environmental disasters. Article impact statement: Oil spills can have long-term consequences for the health of long-lived species; thus, effective restoration and monitoring are needed.

El derrame de petróleo Deepwater Horizon (DWH) en 2010 expuso gravemente a este hidrocarburo a los delfines (Tursiops truncatus) de la Bahía Barataria, Luisiana, causando un incremento en la mortalidad y en las enfermedades crónicas, y deteriorando la reproducción de los delfines sobrevivientes. Realizamos censos fotográficos y evaluaciones veterinarias durante la década posterior al derrame. Asignamos un puntaje pronóstico (bueno, favorable, moderado, malo, o grave) a cada delfín para proporcionar un indicador integrado único de la salud en general. También examinamos las tendencias temporales de estos puntajes. Usamos información de expertos para cuantificar las implicaciones de las tendencias para la proporción de delfines que se recuperaría dentro de su periodo de vida. Integramos esta información, junto con información nueva, a un modelo de dinámica poblacional para predecir los efectos sobre la demografía de las tendencias observadas en la salud. Comparamos la trayectoria poblacional resultante con aquella pronosticada bajo condiciones de línea base (sin derrame). Las condiciones de enfermedad persistieron y recientemente han empeorado en los delfines que supuestamente estuvieron expuestos al petróleo de DWH: 78% de aquellos evaluados en 2018 tuvieron un pronóstico moderado, malo o grave. Los delfines que nacieron después del derrame contaron con mejor salud. Estimamos que la población declinó en un 45% (95% CI 14-74) relativo a la línea base y tardará 35 años (95% CI 18-67) en recuperar el 95% de los números de línea base. La suma de las diferencias anuales entre el tamaño poblacional de línea base y el dañado (es decir, los años cetáceos perdidos) fue de 30,993 (95% CI 6,607-94,148). La población actualmente está en un punto mínimo de su trayectoria de recuperación y es vulnerable a las amenazas emergentes, incluyendo los esfuerzos de restauración ambiental planeada que probablemente sean nocivos para la supervivencia de los delfines. Nuestro marco de modelado demuestra una estrategia para la integración de diferentes fuentes y tipos de datos, resalta la utilidad de la información de expertos para los parámetros de aportación indeterminable, y enfatiza la importancia de la consideración y el monitoreo de la salud a largo plazo de las especies longevas sujetas a los desastres ambientales. Modelado de los Efectos Poblacionales del Derrame de Petróleo Deepwater Horizon sobre Especies Longevas.

Recovery of SARS-CoV-2 from large volumes of raw wastewater is enhanced with the inuvai R180 system.

Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a powerful tool to complement syndromic surveillance. Although detection of SARS-CoV-2 in raw wastewater may be prompted with good recoveries during periods of high community prevalence, in the early stages of population outbreaks concentration procedures are required to overcome low viral concentrations. Several methods have become available for the recovery of SARS-CoV-2 from raw wastewater, generally involving filtration. However, these methods are limited to small sample volumes, possibly missing the early stages of virus circulation, and restrained applicability across different water matrices. The aim of this study was thus to evaluate the performance of three methods enabling the concentration of SARS-CoV-2 from large volumes of wastewater: i) hollow fiber filtration using the inuvai R180, with an enhanced elution protocol and polyethylene glycol (PEG) precipitation; ii) PEG precipitation; and iii) skimmed milk flocculation. The performance of the three approaches was evaluated in wastewater from multiple wastewater treatment plants (WWTP) with distinct singularities, according to: i) effective volume; ii) percentage of recovery; iii) extraction efficiency; iv) inhibitory effect; and v) the limits of detection and quantification. The inuvai R180 system had the best performance, with detection of spiked control across all samples, with average recovery percentages of 68% for porcine epidemic diarrhea virus (PEDV), with low variability. Mean recoveries for PEG precipitation and skimmed milk flocculation were 9% and 14%, respectively. The inuvai R180 enables the scalability of volumes without negative impact on the costs, time for analysis, and recovery/inhibition. Moreover, hollow fiber ultrafilters favor the concentration of different microbial taxonomic groups. Such combined features make this technology attractive for usage in environmental waters monitoring.

Food talk: 40-Hz fin whale calls are associated with prey biomass.

Animals use varied acoustic signals that play critical roles in their lives. Understanding the function of these signals may inform about key life-history processes relevant for conservation. In the case of fin whales (Balaenoptera physalus), that produce different call types associated with different behaviours, several hypotheses have emerged regarding call function, but the topic still remains in its infancy. Here, we investigate the potential function of two fin whale vocalizations, the song-forming 20-Hz call and the 40-Hz call, by examining their production in relation to season, year and prey biomass. Our results showed that the production of 20-Hz calls was strongly influenced by season, with a clear peak during the breeding months, and secondarily by year, likely due to changes in whale abundance. These results support the reproductive function of the 20-Hz song used as an acoustic display. Conversely, season and year had no effect on variation in 40-Hz calling rates, but prey biomass did. This is the first study linking 40-Hz call activity to prey biomass, supporting the previously suggested food-associated function of this call. Understanding the functions of animal signals can help identifying functional habitats and predict the negative effects of human activities with important implications for conservation.

Estimating acoustic cue rates in bowhead whales, Balaena mysticetus, during their fall migration through the Alaskan Beaufort Sea.

Eight years of passive acoustic data (2007-2014) from the Beaufort Sea were used to estimate the mean cue rate (calling rate) of individual bowhead whales (Balaena mysticetus) during their fall migration along the North Slope of Alaska. Calls detected on directional acoustic recorders (DASARs) were triangulated to provide estimates of locations at times of call production, which were then translated into call densities (calls/h/km2). Various assumptions were used to convert call density into animal cue rates, including the time for whales to cross the arrays of acoustic recorders, the population size, the fraction of the migration corridor missed by the localizing array system, and the fraction of the seasonal migration missed because recorders were retrieved before the end of the migration. Taking these uncertainties into account in various combinations yielded up to 351 cue rate estimates, which summarize to a median of 1.3 calls/whale/h and an interquartile range of 0.5-5.4 calls/whale/h.

Density and distribution of western chimpanzees around a bauxite deposit in the Boé Sector, Guinea-Bissau.

The Boé sector in southeast Guinea-Bissau harbors a population of western chimpanzees (Pan troglodytes verus) that inhabits a mosaic of forest and savanna. The Boé sector contains a substantial bauxite deposit in a region called Ronde Hill, and there are plans for the construction of a mine, which may endanger the chimpanzee population. In 1-week survey in May 2013, we used the standing crop nest counts method to obtain the number of chimpanzee nests and from that estimate the density and abundance of chimpanzees. We carried out five 1-km line transects that covered the bauxite deposit and surrounding valleys. We used density surface modeling to analyze habitat preferences, then predicted chimpanzee nest density and distribution based on environmental variables. We found the projected location of the mine partially coincides with an area of high predicted abundances of chimpanzee nests and is surrounded by highly suitable areas for chimpanzees (northeast and southwest). We conclude the mine could have significant direct and indirect effects on this population of chimpanzees whose impacts must be carefully considered and properly mitigated if the mine is built.

The Relevance of In Silico, In Vitro and Non-human Primate Based Approaches to Clinical Research on Major Depressive Disorder.

Major depressive disorder (MDD) is the most severe form of depression and the leading cause of disability worldwide. When considering research approaches aimed at understanding MDD, it is important that their effectiveness is evaluated. Here, we assessed the effectiveness of original studies on MDD by rating their contributions to subsequent medical papers on the subject, and we compared the respective contribution of findings from non-human primate (NHP) studies and from human-based in vitro or in silico research approaches. For each publication, we conducted a quantitative citation analysis and a systematic qualitative analysis of the citations. In the majority of cases, human-based research approaches (both in silico and in vitro) received more citations in subsequent human research papers than did NHP studies. In addition, the human-based approaches were considered to be more relevant to the hypotheses and/or to the methods featured in the citing papers. The results of this study suggest that studies based on in silico and in vitro approaches are taken into account by medical researchers more often than are NHP-based approaches. In addition, these human-based approaches are usually cheaper and less ethically contentious than NHP studies. Therefore, we suggest that the traditional animal-based approach for testing medical hypotheses should be revised, and more opportunities created for further developing human-relevant innovative techniques.

Spatio-temporal variation in click production rates of beaked whales: Implications for passive acoustic density estimation.

Passive acoustic monitoring has become an increasingly prevalent tool for estimating density of marine mammals, such as beaked whales, which vocalize often but are difficult to survey visually. Counts of acoustic cues (e.g., vocalizations), when corrected for detection probability, can be translated into animal density estimates by applying an individual cue production rate multiplier. It is essential to understand variation in these rates to avoid biased estimates. The most direct way to measure cue production rate is with animal-mounted acoustic recorders. This study utilized data from sound recording tags deployed on Blainville's (Mesoplodon densirostris, 19 deployments) and Cuvier's (Ziphius cavirostris, 16 deployments) beaked whales, in two locations per species, to explore spatial and temporal variation in click production rates. No spatial or temporal variation was detected within the average click production rate of Blainville's beaked whales when calculated over dive cycles (including silent periods between dives); however, spatial variation was detected when averaged only over vocal periods. Cuvier's beaked whales exhibited significant spatial and temporal variation in click production rates within vocal periods and when silent periods were included. This evidence of variation emphasizes the need to utilize appropriate cue production rates when estimating density from passive acoustic data.

Delphinid echolocation click detection probability on near-seafloor sensors.

The probability of detecting echolocating delphinids on a near-seafloor sensor was estimated using two Monte Carlo simulation methods. One method estimated the probability of detecting a single click (cue counting); the other estimated the probability of detecting a group of delphinids (group counting). Echolocation click beam pattern and source level assumptions strongly influenced detectability predictions by the cue counting model. Group detectability was also influenced by assumptions about group behaviors. Model results were compared to in situ recordings of encounters with Risso's dolphin (Grampus griseus) and presumed pantropical spotted dolphin (Stenella attenuata) from a near-seafloor four-channel tracking sensor deployed in the Gulf of Mexico (25.537°N 84.632°W, depth 1220 m). Horizontal detection range, received level and estimated source level distributions from localized encounters were compared with the model predictions. Agreement between in situ results and model predictions suggests that simulations can be used to estimate detection probabilities when direct distance estimation is not available.

Where to nest? Ecological determinants of chimpanzee nest abundance and distribution at the habitat and tree species scale.

Conversion of forests to anthropogenic land-uses increasingly subjects chimpanzee populations to habitat changes and concomitant alterations in the plant resources available to them for nesting and feeding. Based on nest count surveys conducted during the dry season, we investigated nest tree species selection and the effect of vegetation attributes on nest abundance of the western chimpanzee, Pan troglodytes verus, at Lagoas de Cufada Natural Park (LCNP), Guinea-Bissau, a forest-savannah mosaic widely disturbed by humans. Further, we assessed patterns of nest height distribution to determine support for the anti-predator hypothesis. A zero-altered generalized linear mixed model showed that nest abundance was negatively related to floristic diversity (exponential form of the Shannon index) and positively with the availability of smaller-sized trees, reflecting characteristics of dense-canopy forest. A positive correlation between nest abundance and floristic richness (number of plant species) and composition indicated that species-rich open habitats are also important in nest site selection. Restricting this analysis to feeding trees, nest abundance was again positively associated with the availability of smaller-sized trees, further supporting the preference for nesting in food tree species from dense forest. Nest tree species selection was non-random, and oil palms were used at a much lower proportion (10%) than previously reported from other study sites in forest-savannah mosaics. While this study suggests that human disturbance may underlie the exclusive arboreal nesting at LCNP, better quantitative data are needed to determine to what extent the construction of elevated nests is in fact a response to predators able to climb trees. Given the importance of LCNP as refuge for Pan t. verus our findings can improve conservation decisions for the management of this important umbrella species as well as its remaining suitable habitats.

New density estimates of a threatened sifaka species (Propithecus coquereli) in Ankarafantsika National Park.

Propithecus coquereli is one of the last sifaka species for which no reliable and extensive density estimates are yet available. Despite its endangered conservation status [IUCN, 2012] and recognition as a flagship species of the northwestern dry forests of Madagascar, its population in its last main refugium, the Ankarafantsika National Park (ANP), is still poorly known. Using line transect distance sampling surveys we estimated population density and abundance in the ANP. Furthermore, we investigated the effects of road, forest edge, river proximity and group size on sighting frequencies, and density estimates. We provide here the first population density estimates throughout the ANP. We found that density varied greatly among surveyed sites (from 5 to ∼100 ind/km2) which could result from significant (negative) effects of road, and forest edge, and/or a (positive) effect of river proximity. Our results also suggest that the population size may be ∼47,000 individuals in the ANP, hinting that the population likely underwent a strong decline in some parts of the Park in recent decades, possibly caused by habitat loss from fires and charcoal production and by poaching. We suggest community-based conservation actions for the largest remaining population of Coquerel's sifaka which will (i) maintain forest connectivity; (ii) implement alternatives to deforestation through charcoal production, logging, and grass fires; (iii) reduce poaching; and (iv) enable long-term monitoring of the population in collaboration with local authorities and researchers.

Fin whale song evolution in the North Atlantic.

Animal songs can change within and between populations as the result of different evolutionary processes. When these processes include cultural transmission, the social learning of information or behaviours from conspecifics, songs can undergo rapid evolutions because cultural novelties can emerge more frequently than genetic mutations. Understanding these song variations over large temporal and spatial scales can provide insights into the patterns, drivers and limits of song evolution that can ultimately inform on the species' capacity to adapt to rapidly changing acoustic environments. Here, we analysed changes in fin whale (Balaenoptera physalus) songs recorded over two decades across the central and eastern North Atlantic Ocean. We document a rapid replacement of song INIs (inter-note intervals) over just four singing seasons, that co-occurred with hybrid songs (with both INIs), and a clear geographic gradient in the occurrence of different song INIs during the transition period. We also found gradual changes in INIs and note frequencies over more than a decade with fin whales adopting song changes. These results provide evidence of vocal learning in fin whales and reveal patterns of song evolution that raise questions on the limits of song variation in this species.

Estimating Florida manatee (Trichechus manatus latirostris) abundance using passive acoustic methods.

Manatees are difficult to detect, particularly cryptic populations that inhabit areas with limited water clarity. The effectiveness of using vocal detections to estimate manatee abundance was evaluated in a clear water spring where manatees congregate seasonally. Vocalizations were extracted by a detection classifier that clustered sounds with similar spectral properties. Vocalization counts from recordings in Blue Spring, FL, USA were strong predictors of manatee abundance. The link between independent visual counts and abundance estimates from passive acoustic monitoring was used to provide an estimate of 1.059 (95% confidence interval 0.963-1.127) vocalizations/manatee/5-min, which might be used elsewhere for cue counting of manatees.

Aeromonas spp. Prevalence, Virulence, and Antimicrobial Resistance in an Ex Situ Program for Threatened Freshwater Fish-A Pilot Study with Protective Measures.

Ex situ breeding programs are important conservation tools for endangered freshwater fish. However, developing husbandry techniques that decrease the likelihood of disease, antimicrobial resistance, and virulence determinants acquisition during this process is challenging. In this pilot study, we conducted a captivity experiment with Portuguese nase (Iberochondrostoma lusitanicum), a critically endangered leuciscid species, to investigate the influence of simple protective measures (i.e., material disinfection protocols and animal handling with gloves) on the dynamics of a potential pathogenic genus, Aeromonas, as well as its virulence profiles and antimicrobial resistance signatures. Our findings show that antimicrobial resistance in Aeromonas spp. collected from I. lusitanicum significantly increased during the extent of the assay (5 weeks), with all isolates collected at the end of the study classified as multidrug-resistant. Additionally, humans handling fishes without protective measures were colonized by Aeromonas spp. The use of protective measures suggested a decreasing trend in Aeromonas spp. prevalence in I. lusitanicum, while bacterial isolates displayed significantly lower virulence index values when virulence phenotypical expression was tested at 22 °C. Despite this study representing an initial trial, which needs support from further research, protective measures tested are considered a simple tool to be applied in ex situ breeding programs for aquatic animals worldwide. Furthermore, current results raise concern regarding antimicrobial resistance amplification and zoonotic transmission of Aeromonas spp. in aquatic ex situ programs.

Discrimination and surveillance of infectious severe acute respiratory syndrome Coronavirus 2 in wastewater using cell culture and RT-qPCR.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.