Salinity is an influential abiotic environmental factor in aquatic species, specifically in freshwater, where salinization causes ecosystem degradation. Secondary salinization, that is increases in salinity due to anthropogenic activities, can affect both osmoregulation and behaviour in freshwater fishes. It is generally believed that invasive species handle climatic change and environmental degradation better than native species, which is one reason for their invasion success. However, how invasive and native species cope with salinity changes remains little understood. Therefore, we investigated how low (500 µS/cm) and high salinity (2000 µS/cm) conditions affected oxygen consumption and behaviour in the invasive round goby (Neogobius melanostomus) and the native European perch (Perca fluviatilis). Our results showed that in round goby oxygen consumption increased and swimming and non-swimming movements changed in response to salinity increments, whereas European perch was not affected by salinity. Thus, it seems as if the invasive round goby is more sensitive to changes in salinity than the native European perch. Our results fit with the minority of studies indicating invasive species being less tolerant than some native species to environmental changes. This finding could be explained by the adaptation of round goby to low salinity due to its long establishment in River Rhine. Further, our results are also confirming that the effect of salinity is species-specific. In addition, European perch and round goby show diametrically different behavioural response to disturbance which could be an effect of holding different ecological niches as well as their anatomical differences.

European perch, exploratory behaviour, general activity, metabolic rate, risk behaviour, round goby

Salinization is one of the major causes of biological changes in river ecosystems (Vander Laan et al. 2013). Salinity is a very important abiotic environmental factor influencing aquatic species, to the extent that aquatic species are normally divided into groups based on living environment concerning salinity, such as freshwater, brackish water, or seawater species. Increases in salinity can occur via natural accumulation of salts. This is called primary salinization and the time-scale is typically very long (~100 000 years with some variances) (Herbert et al. 2015). Secondary salinization, on the other hand, is caused by anthropogenic activities such as vegetation clearance, intensive irrigation, river regulation, mining and extraction, and de-icing salts, and for secondary salinization the time-scale is much shorter than for primary salinization (decades or shorter) (Herbert et al. 2015).

Secondary salinization can have adverse effects on aquatic animals because changing salinities could affect the metabolic cost of the organism (Hart et al. 1991). For instance, many freshwater fish species have their optimal growth and typically lower metabolic rates when exposed to intermediate salinities, while increased salinities seem to reduce food intake and growth in fish (Bœuf and Payan 2001). However, fish condition has been observed to increase in spite of the reduced food consumption rate (Hintz et al. 2017). Increased riverine salinities as a result of road salt have been shown to be toxic for fathead minnows (Pimephales promelas) (Corsi et al. 2010) and anthropogenic increases of salinity in the Great Menderes Basin in Turkey have led to the extinction of the previously most abundant fish, namely carp (Cyprinus carpio) and also Wels catfish (Silurus glanis) (Koç 2008).

Various behavioural traits of freshwater fish are affected by salinity, although no pattern seems apparent. For instance, with increasing salinity Eastern mosquitofish (Gambusia holbrooki) decreased their aggressive behaviour and needed more time to capture prey (Alcaraz et al. 2008). By contrast, in an invasive cichlid aggression increased when exposed to brackish water compared to freshwater (Lorenz et al. 2016). Exposure to increased salinity increased swimming activity in pikeperch (Sander lucioperca) (Scott et al. 2008), and reduced anti-predator responses in fathead minnows (Hoover et al. 2013). However, there are studies showing no effects of salinity upon behaviour in other freshwater fish, such as in the Iberian barbel (Luciobarbus bocagei) (Leite et al. 2019).

In general, it is considered that invasive aquatic species can handle environmental change better than native species in freshwater ecosystems (Bates et al. 2013), with invasive species being more tolerant to temperature fluctuation. The pattern seems to be true for temperature tolerance in the River Rhine, because native fishes in the Rhine seem to be more negatively affected by the temperature changes during the last century (lower minimum and higher maximum) than invasive fishes (Leuven et al. 2011). Salinity sensitivity difference between invasive and native freshwater fishes has not been studied extensively. Invasive freshwater suckermouth armoured catfish (Loricariidae: Pterygoplichthys) has been shown to tolerate brackish water in south-eastern Mexico (Capps et al. 2011), indicating that it could spread to a larger area. There are also some reports concerning invasive species being less tolerant to salinity than native species. For instance, the Aphanius iberus seems to handle salinity better than Eastern mosquitofish, but it is however also sensitive and grows worse in higher salinity environment (Sgarzi et al. 2020).

Based on this background, we wanted to investigate how an invasive fish and a native fish from River Rhine responded to different salinities. The species, the native European perch (Perca fluviatilis) and the invasive round goby (Neogobius melanostomus), were chosen based on their prevalence in the Rhine, where secondary salinization via mine water emission is extant (Wisotzky et al. 2018; Schulz and Cañedo-Argüelles 2019). Earlier studies have shown that both European perch (Overton et al. 2008; Christensen et al. 2019a) and round goby have a wide range of salinity tolerance (Karsiotis et al. 2012; Hempel and Thiel 2015). Further, European perch has a higher standard metabolic rate, that is minimum metabolic rate needed to sustain life for a specified temperature, (Ern et al. 2014; Christensen et al. 2019b), and round goby has a lower standard metabolic rate (Behrens et al. 2017) in brackish water compared to fresh water. Thus, we hypothesized that round goby would be less sensitive than European perch to an environmentally relevant increase of salinity. Specifically, we tested if oxygen consumption and/or exploratory behaviour, general activity and risk behaviour of both species were affected by salinity increments that could occur both naturally and following anthropogenic influences in the Rhine River.

Based on previous research, with lower standard metabolic rate in round goby (Behrens et al. 2017) and higher standard metabolic rate in European perch (Ern et al. 2014; Christensen et al. 2019b) in brackish water compared to fresh water, we had expected that the invasive round goby would be less sensitive to a higher salinity condition than the native European perch. However, round goby in high salinity condition showed an increase in oxygen consumption as well as changed behaviour compared to low salinity condition, and none of these differences were apparent in European perch. Thus, it seems as if the invasive round goby in the Rhine River system is more sensitive to increases in salinity than the native European perch.

Interestingly, it seems as if invasive species can differ in behaviour depending on time since colonization. For instance, in cane toad (Rhinella marina) anti-predatory responses differ between their native and invasive range with invasive toads being less likely to flee (Hudson et al. 2017). In addition, individuals from the invasive front were less likely to flee compared to toads from long-colonized areas and individuals even differed in morphology and locomotory traits between the invasive front and long-colonized areas (Hudson et al. 2017; Hudson et al. 2020). Further, native cane toad had higher stress responses than invasive cane toad at a similar climate (Kosmala et al. 2020a), and invasive cane toad had adapted to invasive areas abiotic challenges such as temperature (Kosmala et al. 2018), as well as moisture (Kosmala et al. 2020b). Round goby has also been reported to differ in several traits between the invasion front and long-colonized areas. Round goby at the invasion front were bolder, had a higher dispersal potential and higher resting metabolic rate (RMR) than gobies from long-colonized areas (present for approximately 10 years) (Myles-Gonzalez et al. 2015). Further, some studies also indicate that round goby have rapid adaptive traits in novel environments. For instance, it seems as if sperm velocity is adapting rapidly to novel salinity levels (Green et al. 2020). It has also been reported that genetic differentiation happened in fewer than ten generations of round goby in the southern Baltic Sea (Björklund and Almqvist 2010). Further, there is evidence that round goby differs in demography, morphology and feeding behaviour between an invasion front and a long colonised area (Brandner et al. 2013). The sampling site for round gobies of this study (Rhine-km ~590) can be considered a long-colonised area, because this species had been detected in the Dutch Rhine delta 2004 and from higher up in Rhine (Rhine-km 718) in 2008 (Borcherding et al. 2011). Consequently, the round goby used in this study might be more sensitive to salinity because they are long established and therefore could have adapted to the environment as previously shown in cane toad. There are some studies indicating that round goby could be sensitive to freshwater salinity levels. For instance, heat shock protein 70 (hsp70) expression was highest in round goby in freshwater (0‰) compared to 10 and 30‰ salinities (Puntila-Dodd et al. 2021). However, it is still perplexing that European perch from the same area seem to be more tolerant towards salinity, but other factors than sensitivity to abiotic factors can also be important for the establishment of invasive species, such as sensory biology (Abrahams et al. 2017).

Our results that round goby increased oxygen consumption following high salinity condition were unexpected. Earlier studies have shown that round goby has a wide spectrum of tolerance for salinity (0–20‰ without problems) (Karsiotis et al. 2012) and growth rate did not change within the range of 0.1 to 15‰ (Hempel and Thiel 2015). Additionally, in contrast to our results, Behrens et al. (2017) reported that standard metabolic rate (SMR) was lower at 10‰ compared to 0‰ salinity, but found no differences in maximal metabolic rate (MMR) and aerobic scope (AS). There are, however, several differences between this study and our study concerning methodology. For instance, we used salinities of 500 and 2000 µS/cm, which are ~0.25‰ and 1.00‰ respectively, which led to a lesser difference between salinity conditions. Further, for practical reasons, we did not measure SMR or MMR, rather something between routine metabolic rate (RMR) and active metabolic rate (AMR) (White et al. 2016). Additionally, in our experiment, the gobies were acclimated for a short time (3–5 days) whereas in Behrens et al. (2017) the gobies were acclimated for at least 20 days. Thus, some of the different results could be attributed to methodological differences. However, in the desert goby (Chlamydogobius eremius) oxygen consumption was higher for gobies in higher salinities (35‰ and 70‰) compared to gobies in 0‰ (Thompson and Withers 2002), which is similar to our results.

Further, our results show that high salinity condition does not seem to have any significant effect on oxygen consumption in European perch. This result was unexpected because earlier reports have shown higher oxygen consumption, either in standard metabolic rate (SMR) or maximal metabolic rate (MMR), in perch exposed to higher salinities. For instance, European perch had a higher SMR in brackish water (10‰) compared to fresh water (0‰) (Ern et al. 2014). Further, SMR seems to be increased in 15‰ compared to 0 and 10‰ at 10 °C and 20 °C, but MMR seems to be lower at 5 °C in 15‰ compared to 0 and 10‰ (Christensen et al. 2017), however this was in perch from brackish water. Salinity did not affect MMR in European perch from low respectively high saline background (Christensen et al. 2019b), but SMR was higher at 10‰ than in 0‰ in perch from high saline background (Christensen et al. 2019b). There are again several differences between these studies and our study. For instance, we used a lesser difference between salinity conditions compared to the other studies. As mentioned above, we measured something between routine metabolic rate (RMR) and active metabolic rate (AMR) (White et al. 2016). This could make comparisons difficult. However, we suggest that our results with no difference between treatments could be attributed to the freshwater origin of the perch as well as not measuring SMR, and thus fitting in with perch from low saline background showing no differences in SMR and MMR between 0‰ and 10‰ salinity (Christensen et al. 2019b).

In our study, we showed that oxygen consumption decreased during the exposure time in both European perch and round goby. This indicates that the initiation of the procedure, netting the fish from the aquarium and putting it into the respirometer chamber, was stressful and that the fishes acclimated to the situation over time. Our set-up was similar to White et al. (2016), which also had preliminary data suggesting the first measurements were higher based on a stress response following the transfer to the respirometer chamber. That respirometry can induce a stress response has also been reported in rainbow trout (Onchorhynchus mykiss) (Murray et al. 2017). Further, the respirometry chamber could also be considered to induce stress of confinement (Ellis et al. 2012). Thus, we believe that we have measured an initial stress response following the short chasing as well as confinement, but that the fishes acclimated and therefore decreased their oxygen consumption to normal levels.

We had expected that both species would change their behaviour following salinity increment. However, while in our study elevated salinity seemed to have no effect on behaviour in European perch, in round goby there were several significant differences between the salinity treatments. In general, the activity was increased at elevated salinity in round goby. This could be an increase of activity to avoid the salinity by changing location. Further, round goby has been proposed to use risky strategies during starvation and winter conditions (Fortes Silva et al. 2019). Consequently, it seems possible that they would also be using riskier strategy when exposed to elevated salinity, and thus have an overall increased activity. There are several studies indicating behavioural effects of salinity in freshwater fishes. For instance, male desert goby exert less aggressive behaviour but do not change courtship behaviour at elevated salinity (Lehtonen et al. 2016). Further, fathead minnows exposed to 8000 ppm salinity spent less time moving compared to 4000 and 1000 ppm salinity (Hoover et al. 2013). Additionally, the fathead minnows exposed to 4000 and 8000 ppm salinity could not distinguish between low and high alarm cues shown through anti-predator behaviour, which minnows exposed to 1000 ppm salinity could (Hoover et al. 2013). Furthermore, sailfin molly (Poecilia latipinna) prefer to remain in freshwater with predator cue in comparison to salt water (Tietze and Gerald 2016). Elevated salinity in combination with elevated temperature negates the increased interspecific aggression following only elevated temperature in Eastern mosquitofish and Australian bass juveniles (Macquaria novemaculeata) (Lopez et al. 2018). However, some fish do not change behaviour when exposed to salinity such as Iberian barbel (Leite et al. 2019). Thus, our results are confirming that the effect of salinity in freshwater fishes do give disparate results, and that each species should be studied to be able to predict how salinity would affect them, and that the specific salinities should also be considered thoroughly.

There were also distinct differences between European perch and round goby in their behavioural response to disturbance i.e., comparing risk behaviour with general activity. European perch increased their swimming and tended to rest less after disturbance. On the other hand, round goby hid more and tended to swim less after disturbance. Thus, it seems as if the responses to disturbance between the two species are diametrically different, and could be interpreted as European perch trying to flee the disturbance and round goby trying to hide from the disturbance. This could be an effect of their differences in ecology such as being a benthivore and a piscivore, as well as their anatomical differences.

We want to thank Steffen Wieland and Christian von Landwüst of the German Federal Institute of Hydrology for providing fish for the experiment. We also would like to thank Michael Götten, Anika Leyendecker, Jan Neuser, and Barbara Nuyken for helping out with some of the practical elements and Dirk Hübner for supervising the animal trials. Further, we would like to thank Susanne Worischka for commenting on the manuscript. Further, we would like to thank two anonymous reviewers, whose suggestions helped improve and clarify this manuscript. The project was funded by Forum Bergbau Wasser (T0518/33421/2019).