Estuaries can be areas of rapid environmental change as physico-chemical variables such as salinity and temperature may vary within tidal scales, localized seasonal weather patterns and inter-annual climatic shifts (Wallace et al. 1984, James et al. 2013, Potts et al. 2015). This is notwithstanding the further variability caused by anthropogenic drivers such as freshwater abstraction, impoundments, pollution and climate change (Koehn et al. 2011, James et al. 2013, Adams et al. 2016). Hypersaline conditions arise when salinity exceeds that of seawater through excess evaporation exceeding freshwater inflow during droughts, or through physical modifications to the estuary, with the biogeochemistry of the catchment area also playing a role (Potter et al. 2010, Gonzalez 2012, Strydom 2015). For example, the intermittently open Gqutywa Estuary in the Eastern Cape, South Africa, becomes hypersaline at times due to its closed nature and catchment geochemistry (Teske and Wooldridge 2001, Strydom et al. 2003).

Hypersaline conditions may result in several physiological challenges to fish species (Brauner et al. 2012). The difference in osmotic pressure between the internal structures and the external environment of teleost fish, such as the common bream Rhabdosargus holubi (family Sparidae), results in a loss of water, which must be mitigated by drinking (Brauner et al. 2012). This results in salt absorption across the gut, which must be excreted to prevent elevated internal levels and osmotic disturbances (Bœuf and Payan 2001, Brauner et al. 2012). A generalized trend for the impact of osmoregulation on fish respiration and growth is unclear (Bœuf and Payan 2001, Gonzalez 2012). Previous studies have shown that the impact of osmoregulation on the energy expenditure of fish may be higher than 50% or lower than 10%, varying considerably between species (Bœuf and Payan 2001, Gonzalez 2012, Ern et al. 2014). Thus, it is possible that changing salinity may have species-specific effects on the physiology of fish (Bœuf and Payan 2001). This may be especially true for euryhaline species such as R. holubi, which is an estuarine-dependent marine species that is endemic and common in South African estuaries, where salinities may vary considerably (Blaber 1973a, Whitfield 1998, Whitfield et al. 2006).

Hypersalinity can cause osmotic stress in organisms living in estuaries, resulting in potentially lower growth rates, skeletal deformities and mortality at extreme levels (Bœuf and Payan 2001, Gonzalez 2012, Boglione et al. 2013b). These kinds of skeletal deformities are prevalent in aquaculture conditions, where even small changes to salinity may impact on the survival and growth of larvae and juvenile fish (Boglione et al. 2013a, b). Skeletal anomalies usually occur during skeletogenesis, which persists into the juvenile phase, but skeletal and fin anomalies can also occur in smaller-sized juveniles (Boglione et al. 2009, 2013b, 2014).

Larvae and small juveniles of R. holubi migrate into estuaries at a size of less than 30 mm and grow for about a year and a half to over 140 mm before migrating back to the ocean to spawn (Whitfield 1998, Götz and Cowley 2013). Juvenile R. holubi are usually highly resident, preferring to stay within habitats such as Zostera capensis seagrass beds within the estuary (Edworthy and Strydom 2016, Grant et al. 2017, Muller and Strydom 2017). R. holubi is a strong osmoregulator able to maintain its internal osmotic environment up to 65 (Blaber 1973a, 1974b). Previous investigations of R. holubi tolerance to high salinity carried out in the laboratory have indicated a maximum threshold of 70 after a 16-hour exposure (Blaber 1973b). However, R. holubi individuals have been recorded alive in the wild at salinity levels up to 90, and it is one of the few species capable of surviving under intense drought conditions (Whitfield 1998, Whitfield et al. 2006). This makes it a euryhaline species able to tolerate a large variation in salinity, as opposed to stenohaline species that can tolerate only minor changes (Whitfield et al. 1981, Brauner et al. 2012, Kültz 2015). Spinal and fin deformities can still be observed in euryhaline fishes such as Atherina lagunae due to the potential combined effects of temperature, pollution, and oxygen deficiency (Ayed et al. 2008). Despite the high tolerances reported in the literature, the physiological effects of R. holubi living under high salinities for long periods are unknown and there are still potential effects to external structures, such as fin abrasions or swimbladder anomalies due to buoyancy changes and bacterial infections (Latremouille 2003, Boglione et al. 2013a, b). This study aims to assess the impact of natural in situ and laboratory-manipulated hypersaline conditions on the growth and frequency of skeletal anomalies of juvenile R. holubi.

Hypersaline conditions may potentially place a significant impact on the physiology of estuarine fish during drought conditions or freshwater impoundments, thus affecting their ecology (Bœuf and Payan 2001, Harrison and Whitfield 2006, Gonzalez 2012). This study was aimed at assessing the growth and skeletal anomalies of R. holubi juveniles growing at different salinities in situ and in the laboratory using clearing and staining techniques (Taylor and Van Dyke 1985) to determine the impact of hypersaline conditions over the long term. It was found that hypersaline conditions with a salinity of 50 for two months did not significantly impact the growth of R. holubi for most of the duration of the experiment, compared with control samples kept at salinity levels around 30, similar to those in their natural habitat. It was found that skeletal anomalies were rare, occurring in less than 20% of all skeletal samples surveyed. The highest incidences of anomalies were found in R. holubi juveniles from the naturally hypersaline Gqutywa Estuary, particularly affecting their fin rays. Conversely, vertebra-related anomalies, i.e. those in vertebra, spines, arches and crania, did not differ significantly between treatments.

The degree of metabolic activity due to osmoregulation varies widely in the literature, ranging from less than 10% to more than 50% of the total energy cost (Bœuf and Payan 2001, Gonzalez 2012, Ern et al. 2014). Generally, when fish are gradually acclimated, tolerance to hypersaline conditions increases and may also lead to lower metabolic costs (Gonzalez et al. 2005, Whitfield et al. 2006, Gonzalez 2012). Fish may respond to increased solute concentrations by increasing salt excretion rates and reducing gill epithelial permeability (Gonzalez et al. 2005, Gonzalez 2012). Growth rates in many fish species, such as the marine estuarine-opportunist Mugil cephalus, are shown to decrease at high salinities due to the compromise of reducing oxygen intake while decreasing gill permeability to prevent the absorption of solutes (Bœuf and Payan 2001). Additionally, many other metabolic pathways are impacted during changing salinity, including changing gill ventilation, acid-base regulation and hormonal regulation (Claireaux and Lagardère 1999, Deane and Woo 2009, Ern et al. 2014). In some species, growth may even increase at high salinities. In the euryhaline milkfish Chanos chanos for example, swimming activity declines at a salinity of 55 compared with 35, leading to a higher growth rate over a period of four weeks (Swanson 1998). Thus, the lack of effect of salinity on growth may be due to the offset of osmoregulatory metabolic demands by a reduction in movement. This was seen anecdotally in the laboratory and also evidenced by a decrease in respiration rates at high salinities found in pilot studies (Kisten 2018). In an ecological context, these results show why R. holubi is able to tolerate and proliferate under high-salinity conditions (Whitfield et al. 2006) and can be the most populous marine estuarine-dependant species in intermittently open estuaries during the closed mouth state (James et al. 2007, Whitfield et al. 2017).

If fish are able to manage the solute levels in their internal environment, it follows that damage to, or malformations of, internal structures such as the skeleton may be minimized (Gonzalez 2012, Boglione et al. 2013b). The ability of the euryhaline R. holubi to maintain its internal osmotic environment near isosmotic levels at salinities up to 65 (Blaber 1974b) may explain the lack of incidence and difference between treatments of vertebral anomalies obtained in this study. Further, skeletal anomalies tend to be more pronounced if they develop during earlier stages in the life cycle (Boglione et al. 2001, 2013b, Alarape et al. 2015). This may explain the low incidence of skeletal anomalies in juveniles, especially in the aquarium samples, as the early preflexion larval stages occur in the relatively stable marine environment. This may also explain why wild hypersaline juveniles exhibited anomalies while aquariums samples did not, as individuals in the Gqutywa Estuary may have been exposed to high salinities and other factors at a younger age and for a longer period. However, due to a combination of pollution and other environmental variables, skeletal anomalies such as those observed in Atherina lagunae (Ayed et al. 2008) can also occur in early juvenile euryhaline fish species. Lordosis (abnormal upward concave curvature) and vertebral spine/arch malformations were some of the most common vertebral-related anomalies present in anomalous samples. Lordosis of the haemal and caudal regions can be attributed to high water current, forced swimming and high temperatures (Divanach et al. 1996, 1997, Boglione et al. 2013b), which may explain why smaller individuals in aquaria exhibited this in both salinity treatments used in this study, as the other variables were kept constant. Anomalies affecting neural and haemal spines and arches are considered less severe and less likely to impact the performance of fish (Boglione et al. 2013b), especially at the low levels observed in this study. These anomalies may also be linked to other environmental variables, and particularly a nutrient-deficient diet rather than environmental salinity (Divanach et al. 1996, Izquierdo et al. 2010, Boglione et al. 2013b). As such it is difficult to conclude that a hypersaline environment may have any effect on the internal structures of R. holubi at the salinity levels and exposure durations tested in this study, particularly since other factors such as temperature were kept constant in the laboratory but would vary considerably in the wild.

Fin rays were the most significantly affected structures found in hypersaline conditions both in situ in the Gqutywa Estuary and in the laboratory at a salinity of 50 (Fig. 2). Fin ray anomalies are common in many species in both the wild and in aquaculture conditions (Divanach et al. 1996, Boglione et al. 2013b). However, the impact on fin rays, especially the erosions exhibited in this study, may not be related to physiology, skeletal growth and developmental anomalies (Boglione et al. 2013b). Rather, fin erosions may be caused by factors such as abrasions, intraspecific aggression, nutritional deficiencies and bacterial infections (Latremouille 2003). Overall, the stress of unfavourable environments may also lead to fin erosion, as in the case of juvenile cobia Rachycentron canadum reared at suboptimal low salinities of 5 (Denson et al. 2003). The presence of eroded fins does affect mobility and results in changes to swimming patterns that may result in skeletal deformities over the long term (Divanach et al. 1996, Latremouille 2003, Boglione et al. 2013b). Ecologically, fin damage and pathological infections may make fish more vulnerable to predators potentially impacting on population size (Latremouille 2003, Hostetter et al. 2012).

In an ecological context, the results of this study indicate that R. holubi juveniles are resilient to hypersaline conditions, enabling them to persist in a saline-tolerant food web. Juvenile R. holubi depend on vegetated refugia for food and protection from piscivores (Blaber 1974a, Edworthy and Strydom 2016, Nel et al. 2017). This refuge is particularly important if mobility is reduced, due to the potential reduction in activity and fin erosion seen in this study. Conversely, the persistence of R. holubi populations together with other halotolerant species ensures the availability of food for halotolerant piscivores if planktivorous fish prey items decline during hypersaline conditions (Whitfield et al. 2006, Durand 2015). Thus, both the resilience to, and potential impacts of, hypersaline conditions seen in this study enable R. holubi to buffer food webs during hypersaline conditions. Droughts and hypersaline conditions may become more frequent and intense, especially during the dry phase of ENSO in the future (Gillanders et al. 2011, Koehn et al. 2011, James et al. 2013). This is further exacerbated by freshwater reduction practices in rivers and estuaries (Turpie et al. 2002, Adams et al. 2016). The potential synergistic effect of other factors such as increased temperature, ocean acidification, heavy metal pollution and salinity on the skeletal development of larvae and juveniles is yet unknown and may be an important aspect for study in the future.