Small-bodied Fish Species Research Articles

Skin swabbing protocol to collect DNA samples from small-bodied fish species

Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, and impact the fish’s behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of analgesics, anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that gene expression and behaviour data collected from swabbed fish were less variable than similar data collected from fish that had been fin clipped. This potentially allows smaller sample sizes in experimental groups to be used after skin swabbing, thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs.

Gut Microbial Composition of Cyprinella lutrensis (Red Shiner) and Notropis stramineus (Sand Shiner): Insights from Wild Fish Populations

The gut microbiome is a highly intricate ecosystem that exerts a pivotal influence on the host’s physiology. Characterizing fish microbiomes is critical to understanding fish physiology and health, but little is known about the ecology and colonization dynamics of microorganisms inhabiting fish species. In this study, we investigated the bacterial communities of two small-bodied fish species, Cyprinella lutrensis (red shiner) and Notropis stramineus (sand shiner), two fish species where gut microbiomes have not been investigated previously and surrounding waters, collected from rivers in Nebraska, USA. Our study focused on evaluating microbial diversity in small-bodied fish and identifying autochthonous microbes present within these species irrespective of location to better understand bacterial community composition and possible roles of such bacterial species. Our results revealed that both red shiner and sand shiner exhibited gut bacterial communities dominated by typical bacterial phyla found in freshwater fish. The phylum Bacteroidota was minimally abundant in both species and significantly lower in relative abundance compared to the surrounding water microbial community. Furthermore, we found that the gut microbiomes of red shiner and sand shiner differed from the microbial community in the surrounding water, suggesting that these fish species contain host-associated bacterial species that may provide benefits to the host such as nutrient digestion and colonization resistance of environmental pathogens. The fish gut bacterial communities were sensitive to environmental conditions such as turbidity, dissolved oxygen, temperature, and total nitrogen. Our findings also show bacterial community differences between fish species; although they shared notable similarities in bacterial taxa at phyla level composition, ASV level analysis of bacterial taxa displayed compositional differences. These findings contribute to a better understanding of the gut bacterial composition of wild, freshwater, small-bodied fish and highlight the influence of intrinsic (host) and environmental factors on shaping the bacterial composition.

Analysis of Selenium in Fish Tissue: An Interlaboratory Study on Weight Constraints.

Environmental monitoring programs that target fish tissues for selenium (Se) analysis present unique sampling and analytical challenges. Se monitoring programs ideally focus on egg/ovary sampling but frequently sample multiple tissues with varying lipid content, often target small-bodied fish species due to their small home ranges, and require reporting in units of dry weight. Additionally, there is a growing impetus for non-lethal tissue sampling in fish monitoring. As a result, Se monitoring programs often generate low-weight tissue samples of varying lipid content, which challenges analytical laboratories to quantify tissue selenium concentrations accurately, precisely, and at desired detection limits. The objective of this study was to stress-test some conventional analytical techniques used by commercial laboratories in terms of their ability to maintain data quality objectives in the face of sample weight constraints. Four laboratories analyzed blind a suite of identical samples, and data were compared against a priori data quality objectives for accuracy, precision, and sensitivity. Data quality tended to decrease with decreasing sample weight, particularly when samples were less than the minimum weights requested by the participating laboratories; however, effects of sample weight on data quality were not consistent among laboratories or tissue types. This study has implications for accurately describing regulatory compliance in selenium monitoring programs, highlighting some important considerations for achieving high data quality from low-weight samples.

Balancing Physical Channel Stability and Aquatic Ecological Function through River Restoration

Vortex rock weirs (VRW) are often used in natural channel design applications to maintain channel form and function, provide physical channel stability, and enhance aquatic habitats. A balanced approach is required to address (often) conflicting goals of VRWs, which include providing erosion protection and grade control while facilitating fish passage for target species. This research evaluated a sequence of modified VRWs in a small-scale watercourse in Southern Ontario, Canada. To determine passage suitability for the target fish species, the water level, water temperature, and channel geometries at 10 VRWs and 11 adjacent pools were monitored under different water level conditions. The structural dimensions and velocity at each VRW were compared to the burst swim speed of local small-bodied fish species to determine fish passage suitability and identify the best practices for VRW design and construction. The results concluded that VRWs provided suitable passage for small-bodied fish species through gap and over-weir flow pathways, particularly during low water level conditions. Further, appropriate design considerations based on the VRW gradient, VRW width, keystone size, and pool length contributed to 100% fish ‘passability’ under all water level conditions. The methodology is provided for predicting the velocity and small-bodied fish passage suitability through VRWs, informing the best practices for VRW design and construction while balancing the requirements for channel stability and fish passage, and contributing to fish population management strategies.

Unravelling Complex Interaction among Coastal Management and Marine Biodiversity: A Case Study in Southern Spain

In this work, we use an integrated modelling approach to explore the complex relationships and interconnections in marine ecosystems among anthropogenic pressures, biodiversity loss, delivery of ecosystem services (ES), and implemented conservation and management strategies. We selected 60 indicators at regional (34), national (12) and international (14) scales that provided long-term information during the 1985–2019 time frame. The results show a decline in marine biodiversity and its associated provisioning services despite the increasing number of responses delivered by a society which are not enough and/or need more time to exert their effects and highlight the pressure on exploited species of unknown conservation status. The decline in Provisioning ES is explained by the decrease in the overall biomass of the captures, mostly large and carnivorous commercially-targeted species and the increase in the number of small-bodied fish species included in the IUCN Red List. The degradation of ecosystem integrity and the continuing loss of biodiversity affect the ability of the ecosystem to provide Regulating ES. The Cultural ES delivery, related to artisanal fisheries, is better preserved in the Gulf of Cádiz. We conclude how the implementation of new management regulations is needed and should be developed through participatory processes to protect and improve marine ecosystem status.

A rapid assessment of non-native fish distributions in two English river basins using environmental DNA

Environmental DNA (eDNA) surveys are increasingly used to inform management decisions for non-native species, for example, by detecting the presence and plotting distributions of species that may be in too low abundance for easy detection by conventional means. A recently-developed nested PCR protocol was used to assess the distributions of three non-native fish species in two river basins of southern England (River Test, Hampshire; River Ouse, Sussex). These river basins were known to contain three non-native fishes, either in the recent past or currently: two invasive small-bodied fish species (topmouth gudgeon Pseudorasbora parva, sunbleak Leucaspius delineatus), as well as a currently non-invasive species predicted to become invasive under future climate conditions, pumpkinseed Lepomis gibbosus. Water samples were collected at locations from headwater streams to estuary. Pumpkinseed and sunbleak were both detected downstream of an angling venue in the Sussex Ouse catchment known to contain those species, with an upstream expansion of sunbleak suggested by the detection of eDNA at a few upstream locations. Neither sunbleak nor topmouth gudgeon was detected in water samples from the River Test catchment, suggesting that neither species has persistent populations in that river catchment.

Downstream fish passage at small-scale hydropower plants: Turbine or bypass?

Introduction: Hydropower plants are frequently equipped with physical and behavioral fish protection barriers to prevent downstream moving fish from harmful turbine passage and to guide them to alternative bypasses. As not only diadromous but also potamodromous fish species migrate and inevitably have to pass hydropower plants, knowledge on corridor usage for a wide range of species is important to identify potential deficits and to improve bypass efficiency.Methods: In this study, the corridor usage of downstream moving fish (6,646 individuals from 42 species) was investigated at four small-scale hydropower plants with different concepts to prevent turbine entrainment and to bypass fish.Results: Despite existing bypasses and fine screens with 15 mm and 20 mm bar spacing to prevent turbine entrainment, a large proportion of fish (35%–88%) still passed the turbines. The mainly poor efficiency of the investigated bypasses was probably due to low discharge and unfavorable bypass location or detectability. The various bypass types were used by a different range of fish species and sizes due to species-specific behavior and differing fish communities between sites. The effectiveness of the investigated downstream corridors was positively correlated with the share of discharge.Discussion: To reduce the negative ecological impacts of hydropower plants on downstream moving fish, well-performing bypasses are required that consider not only current requirements regarding design, dimensioning and location, but also the site-specific fish community. Thus, bypasses should function for the widest possible range of species, which can be achieved through less selective bypass types such as full-depth bypasses, or a combination of different bypass systems. Moreover, less harmful turbine technologies and more effective fish protection systems need to be implemented, since fine screens with 15 mm and 20 mm bar spacing cannot prevent small-bodied fish species and juvenile fish <20 cm from turbine entrainment.

The feeding habits of small-bodied fishes mediate the strength of top-down effects on plankton and water quality in shallow subtropical lakes

The proliferation of small-bodied fishes in lakes is often accompanied by deterioration of water quality and ecosystem function. However, the potential impacts of different types of small-bodied fish species (e.g., obligate zooplanktivores and omnivores) on subtropical lake ecosystems in particular have been overlooked mainly due to their small size, shorter life spans and lower economic value. Therefore, we conducted a mesocosm experiment to elucidate how plankton communities and water quality respond to different types of small-bodied fishes, including a common zooplanktivorous fish (thin sharpbelly Toxabramis swinhonis) and other small-bodied omnivorous fishes (bitterling Acheilognathus macropterus, crucian carp Carassius auratus and sharpbelly Hemiculter leucisculus). During the experiment, the mean weekly total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (CODMn), turbidity, chlorophyll-a (Chl.α) and trophic level index (TLI) values were generally higher in treatments where fish were present compared to treatments where fish were absent, but responses varied. At the end of the experiment, phytoplankton abundance and biomass and the relative abundance and biomass of cyanophyta were higher while the abundance and biomass of large-bodied zooplankton were lower in the fish-present treatments. Moreover, the mean weekly TP, CODMn, Chl.α and TLI values were generally higher in treatments with the obligate zooplanktivore, thin sharpbelly, when compared to treatments with omnivorous fishes. Also, the ratio of zooplankton to phytoplankton biomass was the lowest, and the ratio of Chl.α to TP was the highest in treatments with thin sharpbelly. Collectively, these general findings indicate that an overabundance of small-bodied fishes can have adverse effects on water quality and plankton communities and that small-bodied zooplanktivorous fishes likely induce stronger top-down effects on plankton and water quality than omnivorous fishes. Our results emphasise that small-bodied fishes should be monitored and controlled if overabundant when managing or restoring shallow subtropical lakes. From the perspective of environmental protection, the combined stocking of different piscivorous fish species that feed in different habitat types could be a way forward to control small-bodied fishes with different feeding habits, but more research is needed to assess the feasibility of this approach.

Validating skin swabbing as a refined technique to collect DNA from small-bodied fish species

DNA samples are often used to identify fish before they are utilised in other experiments. Our recent research has shown that skin swabbing can be used to collect DNA for genotyping, and that swabbing causes less harm to fish than fin clipping, another common technique. In this study we investigated potential refinements to the skin swabbing protocol by pre-treating fish with the analgesic lidocaine. We could not detect any differences in cortisol release, behaviour or expression of stress axis marker genes in skin swabbed sticklebacks or zebrafish regardless of lidocaine application. In contrast, fin clipping caused changes in cortisol release, gene expression and behaviour when analgesia was not used. These changes were rescued by pre-treatment with lidocaine confirming that analgesia was effective. The results demonstrates that skin swabbing is a refined technique for DNA collection that does not require analgesia.

Environmental DNA (eDNA) reveals potential for interoceanic fish invasions across the Panama Canal.

Interoceanic canals can facilitate biological invasions as they connect the world's oceans and remove dispersal barriers between bioregions. As a consequence, multiple opportunities for biotic exchange arise and the resulting establishment of migrant species often causes adverse ecological and economic impacts. The Panama Canal is a key region for biotic exchange as it connects the Pacific and Atlantic Oceans in Central America. In this study, we used two complementary methods (environmental DNA (eDNA) metabarcoding and gillnetting) to survey fish communities in this unique waterway. Using COI (cytochrome oxidase subunit I) metabarcoding, we detected a total of 142 fish species, including evidence for the presence of sixteen Atlantic and eight Pacific marine fish in different freshwater sections of the Canal. Of these, nine are potentially new records. Molecular data did not capture all species caught with gillnets, but generally provided a more complete image of the known fish fauna as more small-bodied fish species were detected. Diversity indices based on eDNA surveys revealed significant differences across different sections of the Canal reflecting in part the prevailing environmental conditions. The observed increase in the presence of marine fish species in the Canal indicates a growing potential for interoceanic fish invasions. The potential ecological and evolutionary consequences of this increase in marine fishes are not only restricted to the fish fauna in the Canal as they could also impact adjacent ecosystems in the Pacific and Atlantic Oceans.

Critical thermal maxima of three small-bodied fish species (Cypriniformes) of different origin and protection status

Environmental changes related to global warming are both a threat to cold-water fishes and simultaneously create favourable conditions for the spread of eurythermic or warm-water species. In both cases, it is necessary to develop conservation strategies supported by precise ecological data, including thermal requirements. In this study, we determined the upper thermal tolerance thresholds and the critical maxima for three small, non-commercial Eurasian freshwater fish species; i.e. lake minnow Eupallasella (=Rhynchocypris) percnurus, sunbleak Leucaspius delineatus and topmouth gudgeon Pseudorasbora parva using the Critical Thermal Method at different acclimation temperatures, i.e. 18, 23, 28, and 33°C (highest treatment for topmouth gudgeon only). We hypothesized that lake minnow would have the lowest thermal tolerance and the topmouth gudgeon the highest. The response to temperature rise and the upper thermal limits were species-specific and correlated with the acclimation temperature, but not with fish length. Sunbleak showed the lowest thermal tolerance, though at 28°C both E. percnurus and L. delineatus reached a similar critical thermal limit. Topmouth gudgeon showed distinctly higher upper thresholds of thermal tolerance and at the highest acclimation temperature the critical upper limit for the species was close to 42°C. The results obtained for L. delineatus were surprising as we predicted that this leuciscid fish would be more tolerant of high temperatures than E. percnurus. We discuss the results in relation to the threat of extinction and the risk of species spreading beyond their natural range in the context of a warming environment.

Evaluating the risk of fish stranding due to hydropeaking in a large continental river

AbstractWith the continuous development of hydropower on a global scale, stranding of freshwater fishes is of growing concern, and an understanding of the mechanisms and variables affecting fish stranding in hydropeaking rivers is urgently needed. In particular, a methodology is required to identify the magnitude and timing at which fish stranding occurs in relation to environmental conditions. Here, we studied fish stranding in three reaches downstream of a hydropeaking generation station in the Saskatchewan River, Saskatchewan, Canada, using an innovative remote photography approach with 45 trail cameras and traditional transect monitoring, conducting 323 transects. We observed that juvenile sport and commercial fish species are stranding at a higher proportion than small bodied fish species. The remote photography approach provided more precise fish stranding timing and associated the environmental and physical conditions with a given stranding event, but captured fewer fish and only rarely allowed species identification. The comparison of the two methodologies resulted in similar stranded fish densities, but the remote photography allowed for continuous observations whereas the transect monitoring was limited by the observer availability in the field. Remote photography allowed for additional information on the scavenging of stranded fish, with scavenging occurring on average within 240 minutes of the fish being stranded. The probability of fish stranding increased significantly with increasing water temperature and substrate particle size resulted in greater stranding on finer substrates. Our findings have important implications for hydroelectric flow management by introducing an innovative, standardized method to study the effects of hydropeaking events on fish stranding that can be applied to increase our understanding of the impacts of hydropeaking on fish communities.

Barrier removal and dynamics of intermittent stream habitat regulate persistence and structure of fish community

Barrier effects observed in the presence of weirs are exacerbated by low water levels. We conducted a 10-year study to assess the ecological effects of stream restoration while analysing the possibility of a seasonal lack of hydrological continuity, with multiple measurements before and after restoring stream structural continuity. The research hypothesis assumes that in intermittent streams, there would be little or no change in the fish community downstream the barrier before vs. after barrier removal, and a significant change upstream the barrier before vs. after. Our results indicate, that by removing small barriers, their detrimental effects on the longitudinal passage of riverine fishes and fish assemblages can be rehabilitated. In the wet season, fish migrants from the mainstem river appeared in the downstream section of the stream. Stream intermittency, however, placed a habitat filter over the assemblage. Thus, after barrier removal, only two small-bodied fish species that tolerate periodic oxygen deficiencies and rising water temperatures gradually shifted upstream and formed stable populations. We emphasize, that we should not refrain from restoring the longitudinal continuity of intermittent streams, because they periodically provide fish valuable refugia and can also be a source of new generations and strengthen fish populations in mainstem river.

Skin swabbing protocol to collect DNA samples from small-bodied fish species.

Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, and impact the fish's behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of analgesics, anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that gene expression and behaviour data collected from swabbed fish were less variable than similar data collected from fish that had been fin clipped. This potentially allows smaller sample sizes in experimental groups to be used after skin swabbing, thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs.

Corrigendum: A New Method for Standardizing Inland Fish Community Surveys: Characterizing Habitat Associated With Small-Bodied Fish Species, Abundance, and Size Distribution in a Highly Modified Estuary

Corrigendum: A New Method for Standardizing Inland Fish Community Surveys: Characterizing Habitat Associated With Small-Bodied Fish Species, Abundance, and Size Distribution in a Highly Modified Estuary

A New Method for Standardizing Inland Fish Community Surveys: Characterizing Habitat Associated with Small-Bodied Fish Species, Abundance, and Size Distributions in a Highly Modified Estuary

Monitoring fish assemblages in estuary environments is often difficult to standardize due to habitat complexity and gear-specific selectivity. This is further compounded by conventional sampling methods which require fish handling that might cause stress or mortality. To ameliorate these issues, we developed the Single-Platform Aquatic Species and Habitat Sampling System (Platform), an integrated and mobile concentrator net and live box prototype, coupled with a range of physical monitoring equipment. The long-term goal of the Platform is to employ non-invasive sampling techniques such as video and eDNA; however, our aim in this study was to test the Platform’s ability to sample across different habitat types and detect differences in fish assemblages. We investigated the utility of the Platform over a short time period (2 mo) in a relatively small and complex embayment within the highly modified San Francisco Estuary, California. We were able to identify clear physical distinctions among estuary/delta habitat types and detect habitat segregation by ecological groups of sampled fishes with the Platform. The Platform also detected discernable ontogenetic shifts (i.e., size differences) within estuary habitats of commonly observed fish species. These initial results demonstrate the Platform’s ability to contrast fish density, size, and species diversity, which sets the stage to advance more passive monitoring techniques, including video and eDNA methods. The Platform has the potential to fill a methodological gap in non-invasive surveying of small-bodied fish across a range of estuarine habitats, warranting further investigation of potential applications.

Assessing Effects of Flow Regulation and an Experimental Flow Pulse on Population Size Structure of Riverine Fish with Contrasting Biological Characteristics.

Despite effects of dams and water extraction on river hydrology and consequences of aquatic ecosystems being broadly appreciated, empirical evidence is lacking for many regions (e.g. subtropics). Evidence is necessary to determine (i) the circumstances where environmental flows are necessary to protect or improve ecological processes and (ii) what hydrological events are required to achieve those ecological characteristics. Here, temporal variation in the size structure of two small-bodied fish species with contrasting ecological characteristics (Australian smelt, Cox's gudgeon) was compared between two pairs of unregulated and regulated rivers in subtropical Australia. Frequency of in-channel flow pulses in each regulated river was lower compared to paired unregulated rivers. An experimental flow pulse was delivered to one regulated river to assess the ecological outcomes of in-channel flow pulses and inform future decisions about the use of environmental water allocations. Temporal changes in the population size structure of both species were similar between unregulated and regulated rivers and showed no response to the experimental pulse. While the experimental flow briefly suppressed in-stream temperature, changes in temperature were not beyond the thresholds at which spawning occurs or the thermal tolerances for either species. Similar population structure between unregulated and regulated rivers can be attributed to the magnitude of flow regulation being insufficient to alter the physico-chemical conditions, habitat and trophic mechanisms supporting population dynamics during the study period. This suggests current regulated hydrology is protective of local populations of Australian smelt and Cox's gudgeon in these study rivers.

Effects of Fish Populations on Pacific Loon (<i>Gavia pacifica</i>) and Yellow-billed Loon (<i>G. adamsii</i>) Lake Occupancy and Chick Production in Northern Alaska


 Predator populations are vulnerable to changes in prey distribution or availability. With warming temperatures, lake ecosystems in the Arctic are predicted to change in terms of hydrologic flow, water levels, and connectivity with other lakes. We surveyed lakes in northern Alaska to understand how shifts in the distribution or availability of fish may affect the occupancy and breeding success of Pacific (Gavia pacifica) and Yellow-billed Loons (G. adamsii). We then modeled the influence of the presence and abundance of five fish species and the physical characteristics of lakes (e.g., hydrologic connectivity) on loon lake occupancy and chick production. The presence of Alaska blackfish (Dallia pectoralis) had a positive influence on Pacific Loon occupancy and chick production, which suggests that small-bodied fish species provide important prey for loon chicks. No characteristics of fish species abundance affected Yellow-billed Loon lake occupancy. Instead, Yellow-billed Loon occupancy was influenced by the physical characteristics of lakes that contribute to persistent fish populations, such as the size of the lake and the proportion of the lake that remained unfrozen over winter. Neither of these variables, however, influenced chick production. The probability of an unoccupied territory becoming occupied in a subsequent year by Yellow-billed Loons was low, and no loon chicks were successfully raised in territories that were previously unoccupied. In contrast, unoccupied territories had a much higher probability of becoming occupied by Pacific Loons, which suggests that Yellow-billed Loons have strict habitat requirements and suitable breeding lakes may be limited. Territories that were occupied had high probabilities of remaining occupied for both loon species.

Novel hydraulic guidelines can assist upstream fish passage through smooth box culverts

ABSTRACT Culverts are important hydraulic structures for delivering a range of valuable socioeconomic services; however, they are also known to have negative impacts on freshwater and estuarine river systems, including the blockage of upstream fish passage, particularly small-bodied fish species. Herein new guidelines are proposed for fish-friendly multi-cell smooth box culvert designs, with a focus on small-bodied Australian native fish species and juveniles of larger fish. The new set of hydraulic engineering design guidelines, in the process of being implemented in New South Wales (Australia), optimise culvert design for fish passage for water discharges Q < QT = 0.1× Qdes, for which the reduced-velocity zone (RVZ) represents 15% of the flow area where 0 < Vx < Ufish, with Vx the local time-averaged longitudinal velocity component and Ufish the maximum prolonged swimming speed of target fish species. After a presentation of the basic concepts, a design application is developed based upon a real-world case study in north-eastern New South Wales (Australia). The novel approach relies upon a sound understanding of the velocity flow in the box culvert barrel at less-than-design discharges to accurately characterise the RVZ next to the barrel walls and corners, combined with a detailed understanding of the swimming performance and behaviour of native fish species.

Hybrid modelling of low velocity zones in box culverts to assist upstream fish passage

A culvert is a covered channel designed to pass water through an embankment. The recognition of the adverse ecological impacts of culverts on upstream fish passage is driving the development of new culvert design guidelines, with a focus on small-bodied fish species seeking low velocity zones to minimise energy expenditure. Herein a hybrid modelling technique was applied, combining physical modelling, one-dimensional theoretical calculation and three-dimensional computational fluid dynamics modelling. The results reveal fundamental turbulent processes that may affect small-body-mass fish navigability and provide new insights for the development of standard box culvert design guidelines. Systematic validations were performed to a wide range of initial conditions and smooth barrel geometries. A physical relationship was derived from numerical and experimental data of past and present studies, correlating the dimensionless flow area with a normalised local velocity V/V.