This is the first study determining the effects of bath exposure to fulvic acid, a humic substance, on the skin mucosal immunity of rainbow trout (Oncorhynchus mykiss). Humic substances have recently been gaining attention for their increasing concentrations in aquatic ecosystems and their use as supplements in sustainable aquaculture. This study demonstrated that water exposure to fulvic acid at concentrations of 5 mg C/L and 50 mg C/L increased lysozyme and alkaline phosphatase activities in the mucus by approximately 2 -fold and 2.5 to 3.2 -fold, respectively. Furthermore, exposure to 50 mg C/L resulted in a 77.0% increase in mucosal immunoglobulin concentrations compared to the other groups. Importantly, all mucus samples demonstrated significant antibacterial activity against Yersinia ruckeri, with control mucus reducing bacterial growth by 44.5% and exposure to fulvic acid increasing this effect to 26.3%. Although these modulations show promise for application in aquaculture, alterations of the beneficial microbiota from long-term exposure in natural waters can be expected. Monitoring the rising concentrations of humic substances in natural water bodies is therefore urgently needed. Overall, this study represents the first investigation revealing the ability of humic substances to modulate skin mucosal immunity and the capacity to combat microorganisms.

Peracetic acid (PAA) has a long history as an efficacious and eco-friendly disinfectant. It was first synthesised in 1902, and since then a wide range of applications has been developed in various industries. Aquaculture is a more recent industry wherein the potential of PAA is significant. As the global demand for sustainable development increases, there has likewise been growing interest in using PAA in aquaculture as an alternative to less environmentally friendly practices. PAA has no carcinogenic risk to humans (unlike formalin), has negligible harmful by-products (unlike chlorine-based disinfectants) and with appropriate precautions, the risks of causing severe human health damage is easier to control than ozone. Fish show strong physiological recovery and adaptation to PAA, whereas susceptible life stages of pathogens are highly vulnerable, enabling a safe and efficacious disinfection of the entire culture water and not the flow-restricted disinfection by such processes as ultraviolet radiation or ozone. The effective concentration of PAA against many fish pathogens is usually below 2 mg L-1, which is tolerable for most fish, and it has very low environmental risk due to rapid degradation. However, such degradation and the hydrodynamics in production-scale aquaculture systems complicate the practical use of PAA. In this review, we summarise key results of safe concentrations of PAA and its effectiveness specifically for fish farmers. We also outline major difficulties and possible solutions for practical uses of PAA. We intend to bring global attention to this compound and inspire future possibilities for its sustainable use as a water disinfectant in aquaculture.

Objective The antiparasitic effect of peracetic acid (PAA) was evaluated against an infestation of the protozoan Trichodina spp. in naturally infected juvenile domestic seventh-generation (F7) Striped Bass Morone saxatilis. Methods Replicated treatments (n = 3) consisted of 1 and 2 mg/L PAA and a control; treatments were applied every other day for three treatments (30-min static exposure). Infestation intensity was enumerated 20-24 h after each treatment by counting the number of Trichodina present in a wet mount of excised gill tissue. Result Neither treatment rate was able to completely eradicate the parasite; however, 2 mg/L PAA resulted in a statistically significant reduction, which equated to 75% reduction of observed parasites. Conclusion The 2-mg/L PAA treatment regimen in the present study is proposed as a safe, environmentally friendly, and effective method for reducing the intensity of Trichodina infestations in Striped Bass.

The generation of quinones in activated lignin allows crosslinking with multi-thiols via thiol-catechol-connectivities (TCCs), leading to a high-performance 2K adhesive that tolerates seawater and is biofriendly and useful for setting corals.

Aquaculture water disinfection with around 1 mg/L peracetic acid (PAA) was proved in various studies to effectively impair microbial proliferation and reduce pathogen-caused fish mortality. In the present study, we monitored the antibacterial effect of biweekly water disinfection with 1 mg/L PAA at a local flow-through fish farm for 8 weeks. Results from replicate water samples showed inconsistency of antibacterial efficacy assessed with colony forming units (CFU) of total culturable bacteria and a commercial enzyme activity assay. Single PAA disinfection caused up to 90% reduction of CFU in water. In contrast, the microbial hydrolase activity was hardly affected or even enhanced. Both methods have limitations and can't truly represent viable bacteria. We further monitored the temporal response of two Yersinia ruckeri isolates in suspensions to single PAA disinfection via high-throughput flow cytometry. We found out that the bacterial cell membrane damage appeared not instantly but with a lag phase of at least 2 h post disinfection at 4 degrees C. The abundance of cell membrane damage, considered as cumulative bacterial mortality, kept rising and maximized at 48 h post disinfection. The survived cells were more likely to enter the viable but non-culturable (VBNC) state at higher PAA concentrations. One isolate was more prone to enter the VBNC state than the other. Neither Y. ruckeri isolate showed measurable reaction with the enzyme substrate of the commercial microbial hydrolase activity assay. Our findings refresh knowledge about the dynamics of bacterial cell mortality post PAA disinfection (probably also similar non-thermal disinfection methods). Aquaculture practice should be aware of the delayed disinfection outcomes and pay attention to the dominance of VBNC state.

Inflammation is an essential process as a reaction towards infections or wounding. Exposure to hazardous environmental pollutants can lead to chronic inflammations, where the resolving phase is delayed or blocked. Very contradictory studies have been reported on the pro- and anti-inflammatory effects of humic substances (HSs) leading to significant disagreements between researchers. To a certain extent, this can be attributed to the chemical heterogeneity of this group of xenobiotics. Here we show for the first time that pro- and anti-inflammatory effects can occur by one HSs. We adapted an assay that uses green fluorescence-labeled zebrafish larvae and CuSO4 to indue an inflammation. In wild-type larvae, exposure to 50 µM CuSO4 for 2 h activated the production of reactive oxygen species, which can be monitored with a fluorescence dye (H2DCFDA) and a microplate reader. This allows not only the use of wild-type fish but also a temporal separation of copper exposure and inflammatory substance while retaining the high throughput. This modified assay was then used to evaluate the inflammatory properties of a fulvic acid (FA). We found, that the aromatic structure of the FA protects from inflammation at 5 and 50 mg C/L, while the persistent free radicals enhance the copper-induced inflammation at ≥ 300 mg C/L.

The prophylactic use of peracetic acid (PAA)-based disinfectants is becoming more popular in aquaculture due to rising concerns regarding sustainability, fish welfare and food safety. However, specific and effective PAA dosing protocols have not been developed to guide the aquaculture industry under diverse production conditions. In the present study, the effect of water hardness/alkalinity and humic substances (HS) on the toxicity of PAA to zebrafish Danio rerio embryos and the efficacy of PAA against the in vitro growth of Yersinia ruckeri and Saprolegnia parasitica was investigated. PAA concentrations that were safe to fish embryos demonstrated strong bactericidal, but limited fungistatic properties. In higher hardness/alkalinity water, or when HS was added, the same concentration of PAA resulted in a smaller pH decrease accompanied by a smaller increase of oxidation-reduction potential (ORP), and showed lower toxicity and weaker antimicrobial effects than in lower hardness/alkalinity waters. We suggest the determining factor of PAA toxicity and its antimicrobial capacity was likely ORP. At low hardness/alkalinity conditions, strong pH reduction (resulting in pH<5) was the dominant role in PAA toxicity to D. rerio embryos. In aquaculture settings, lower PAA doses should be used under lower hardness/alkalinity conditions. Addition of HS under lower hardness/alkalinity conditions can assist with reducing toxicity and the risk to fish. Finally, we determined that repeated PAA disinfection is necessary to achieve a sustained prophylaxis, and we caution that the instant formation of aggregates by HS at high hardness conditions and the subsequent attachment of bacteria may reduce their susceptibility to PAA disinfection.

Aquaculture plays a pivotal role in covering dietary animal protein demands and restocking endangered fish populations. However, high mortality takes place at the earliest life stages: prior and immediately after hatching. Improving growth and health parameters by immunostimulants is widely used in older fish, but rarely studied in larvae. Fulvic acids (FAs) are natural substances found in soil and water. Using zebrafish as a model organism, we evaluated the effects of exposure to a FA at concentrations ranging from 1 to 500 mg C/L (mg dissolved organic carbon per liter) on embryonic development. Furthermore, the concentration of reactive oxygen species (ROS) inside the larvae as well as the molecular mechanisms involved in growth, immune response, and antioxidative protection were determined at 5, 50, and 500 mg C/L. 20 to 200 mg C/L accelerated the hatching, which was mediated by increased expression of ifg-1, gh, and he1-α. Furthermore, lyz and mpx were significantly increased at 5 and 50 mg C/L. A concentration of 500 mg C/L induced genes involved in the protection against ROS (nrf-2, keap-1, cat, sod-1), increased the concentration of ROS inside the larvae and caused tissue damage and mortality. Interestingly, 50 mg C/L activated ROS protection as well (nrf-2, sod-2), while no increase of ROS was found in the larvae. Our results show, that FA at low to medium concentrations can increase the health of larvae, but becomes detrimental at higher concentrations.

Aquaculture has become imperative to cover the demands for dietary animal protein. Simultaneously, it has to overcome prejudices from excessive use of antibiotics and environmental impacts. Natural supplements are traditionally applied orally. In this study, we demonstrated another pathway: the gills. Humic substances are immunostimulants and a natural part of every aquatic ecosystem, making them ideal to be used as bath stimulants. Five and 50 mg C/L of a fulvic acid-rich humic substance was added for 28 days to the water of juvenile rainbow trout ( Oncorhynchus mykiss ). This fulvic acid is characterized by a high content of phenolic moieties with persistent free radicals and a high electron exchange capacity. The high concentration of the fulvic acid significantly increased growth and reduced the food conversion ratio and the response to a handling-stressor. Phagocytosis and potential killing activity of head kidney leukocytes were increased, as well as the total oxyradical scavenging capacity (TOSC) and lysozyme activity in the gills. In conclusion, immunostimulation via gills is possible with our fulvic acid, and the high phenolic content improved overall health and stress resistance of fish.

Secondary salinization of freshwater ecosystems is of increasing global concern. One of the main causes are the effluents of the potash mining industry containing high concentrations of major ions (Cl-, Na+, Mg2+, K+). In Germany, the ongoing discharge of effluents into the River Werra led to a strong impoverishment of the biodiversity and abundance of local species. Young cohorts of many freshwater fish are completely absent suggesting reproductive failure under these conditions. Therefore, the aim of the study was to experimentally investigate the effects of high concentrations and imbalances of ions that are prevalent in potash mining effluents on reproductive traits of native freshwater teleosts. Sperm motility parameters of the common roach, Rutilus rutilus, and European perch, Perca fluviatilis, were assessed as well as fertilization rate, egg size, hatching, malformations and mortality of embryonic and larval stages of roach. Concentrations of the permitted thresholds (HT) and future thresholds (LT) as well as three ion solutions containing high Mg2+ (Mg), high K+ (K) and both in combination (Mg + K) were tested. Curvilinear velocity and linearity of perch spermatozoa were elevated with potentially adverse effects on fertilization success. Sperm motility parameters and fertilization rate of roach were not affected. However, egg sizes of roach were increased in all groups due to the osmotic action of ions and in LT, premature hatch was observed. Furthermore, all groups comprised a higher number of malformations including pericardial edema and spine curvatures and group HT exhibited a higher mortality rate compared to control. The results clearly demonstrated that particularly the sum of high concentrations of ions, as prevalent in HT and LT, rather than individual ion species exerts detrimental effects on early development of roach potentially increasing overall mortality under natural conditions. These results emphasize that currently permitted and future thresholds are exceeding tolerated ion concentrations.

Many classical therapeutants are going to be banned in Europe, and an urgent need for alternatives is emerging. This issue can be exemplified by one major parasitic disease in aquaculture and ornamental fish breeding: velvet disease. This disease, caused by dinoflagellates of the genera Amyloodinium and Piscinoodinium, is an important infection affecting cultured freshwater and marine ornamental and food fish, and consistently causes great financial loss to the associated industries. Therapeutants available contain copper, malachite green, or methylene blue, and which can be toxic to non-target organisms in the surrounding environment. As a result, these chemicals are banned for use by the aquaculture industry in several countries, and a prohibition for commercial ornamental fishkeeping is likely to follow in most countries. Increasing development of resistance to therapeutants, and growing public awareness for animal welfare and environmental protection, have prompted research in the areas of alternative treatment options and immunostimulants. Hydrogen peroxide and peracetic acid are possible 'green' therapeutants which do not contribute residues to the environment. Natural feed supplements such as pre- and probiotics can increase animal welfare and prevent stress and/or infections. Humic substances are another promising, natural immunostimulants which will be considered in depth. The aim of this review is to provide an overview of risks and benefits of current treatment options and new approaches to replace harmful therapeutants and minimize the number of toxic residues discharged into the environment. Treatments will be discussed on various parasitic infections and focus, where available, on Amyloodinium and Piscinoodinium.

Peracetic acid (PAA)-based disinfectants are considered as sustainable alternatives in aquaculture due to their harmless residues from spontaneous decay. The key components of PAA-based disinfectants are PAA and hydrogen peroxide (H2O2). Little is known if the exposure to exogenous PAA and H2O2 from PAA-based disinfectants interferes with endogenous reactive oxygen species (ROS) regulation and innate immunity in fish. In the present study, rainbow trout (Oncorhynchus mykiss) were exposed to a PAA-based disinfectant for 6 weeks in flow-through systems by either periodic (pulse) or continuous modes. For the periodic exposure, a single dose of 1 mg L-1 PAA (and 1.4 mg L-1 H2O2) was applied biweekly and spontaneously degraded within hours. For the continuous exposure, a constant drip was applied which aimed to maintain 0.2 mg L-1 PAA (and 0.28 mg L-1 H2O2) in the inflow water but was counteracted by escalating degradation by the second week. The exogenous PAA/H2O2 elevated the level of endogenous total free radicals (TFR), probably through diffusion at the gill, as well as stress-activated endogenous generation (by periodic exposure only at the higher PAA/H2O2 dose). In response, the total antioxidant capacity (TAC) in gill and serum was significantly elevated. Liver showed no significant changes in the levels of TFR and TAC. Epidermal mucous cell density was significantly lower in response to persistent scarce exogenous PAA/H2O2 of the continuous exposure compared to the control. This sensitive response was absent in the periodic exposure probably due to rapid epidermal recovery during the exposure intervals. In contrast, the branchial structural adjustment of trout in response to exogenous ROS was less sensitive. Minimal hyperplasia of lamellae was present only in the periodic exposure. Enzymes responsible for innate cutaneous and humoral immunity including lysozyme, alkaline phosphatase, myeloperoxidase and esterase were not influenced by either exposure. However, ceruloplasmin was highly sensitive to exogenous PAA/H2O2; it was significantly elevated in fish skin, independent of the exposure modes, suggesting potential antioxidant protection of ceruloplasmin in fish skin. In serum, ceruloplasmin reduction was positively correlated to the reduction of antiprotease activity which indicates that ceruloplasmin may have anti-proteolytic function in fish blood. In conclusion, both exposure modes of the PAA-based disinfectant triggered mild antioxidant defenses in rainbow trout. To minimize the risk of oxidative damage while applying PAA-based disinfectants, sufficient intervals between periodic applications might be a better option than the persistent low PAA/H2O2 concentration of the continuous application.

As a group, oomycete pathogens are associated with enormous economic losses worldwide in both plant and animal agriculture, including aquaculture (Wawra et al., 2012). Oomycetes of the genus Saprolegnia are particularly problematic for numerous species of wild and farmed fish (van den Berg, McLaggan, Dieguez-Uribeondo, & van West, 2013) and are the causal agents of the fish disease known as saprolegniasis (or, colloquially, 'fungus'). There are over 500 identified species of oomycetes (Walker & van West, 2007), with a range of Saprolegnia oomycete species considered ubiquitous in freshwater environments (Jiang et al., 2013) and known to be associated with disease in salmonid aquaculture (Sandoval-Sierra, Latif-Eugenin, Martin, Zaror, & Dieguez-Uribeondo, 2014). It is recognized that Saprolegnia spp. have been responsible for more than 10% of annual economic losses in the salmonid aquaculture industry, with previous annual production losses up to 50% in certain cases (Bly et al., 1994; van West, 2006). Methods to control saprolegniasis in farmed settings have included the use of malachite green and formalin. Many aquaculture regions around the world, however, have banned malachite green due to its toxic and carcinogenic properties (Culp & Beland, 1996). Likewise, formalin use has been reduced due to similar human safety and environmental concerns. Therefore, new control methods are currently needed to counter the impact of saprolegniasis on farmed fish production (van den Berg et al., 2013). Over the past decade, there has been a significant research has been devoted to developing methods to control saprolegniasis in aquaculture, including the use of immunostimulants in feed (e.g. Saha, Pal, Sahu, & Saha, 2016), novel medicated feeds (e.g. fluconazole – Saha, Pal, Sahu, Saha, & Goswami, 2017), water ozonation (Hamad & Mustafa, 2018) and vaccine development (e.g. Minor et al., 2014). At present, however, the need for effective control methods for saprolegniasis remains high. In general, fish become susceptible to infection with Saprolegnia spp. when injured, stressed or infected with other pathogen(s) (Duan et al., 2018; Pickering & Willoughby, 1982), while healthy, uninjured fish are normally at low risk for developing clinical saprolegniasis (van den Berg et al., 2013). In Atlantic salmon Salmo salar culture, specific life stages (e.g. fry stage, smoltification phase) or routine events (e.g. physical handling) are known to be associated with the development of saprolegniasis. A major risk period for saprolegniasis is during the weeks following vaccination (Greg Lambert, Cooke Aquaculture, personal communication), and a procedure carried out when juvenile salmon are typically in freshwater systems. Both the stress involved, and the tissue damage associated with intracoelomic vaccine injection, favour opportunistic Saprolegnia spp. to establish infection and cause clinical disease. In the Atlantic salmon industry, there has been a significant shift in recent years away from traditional flow-through systems and towards producing smolts in land-based, closed-containment facilities utilizing water recirculation aquaculture system (RAS) technologies (Summerfelt, Mathisen, Holan, & Terjesen, 2016). One of the challenges to raising fish in RAS is that therapeutants administered to diseased populations will also circulate through the biofilter and thus have the potential to impact beneficial organisms responsible for the critical process of nitrification. Past research has focused on various therapeutants and their effects on biofilter function (Noble & Summerfelt, 1996). More recent studies have focused on applying low-dose therapeutants to RAS to develop treatment protocols that are both efficacious against pathogens and not harmful to biofilter microbial communities involved in nitrification (Pedersen & Pedersen, 2012; Pedersen, Pedersen, Nielsen, & Nielsen, 2009, 2010). Determining an effective preventive treatment for saprolegniasis during high-risk periods in the production cycle, while simultaneously maintaining RAS biofilter integrity, would be very beneficial to numerous aquaculture facilities. We therefore sought to determine whether varying (low) doses of peracetic acid (PAA), a relatively novel therapeutant, could prevent saprolegniasis during the period following vaccination while assessing potential impacts on RAS biofilter function. Approximately 2,400 juvenile Atlantic salmon (94 ± 3 g) originating from the US Department of Agriculture (Agriculture Research Service) National Cold Water Marine Aquaculture Center were randomly allocated to 12 small (0.5 m3) circular tanks that had been retrofitted from flow-through to experimental-scale RAS using miniature fluidized sand biofilters (American Aquarium Products), degassing columns, and submerged pumps to create and maintain water recirculation. Make-up (new) water originated from an on-site freshwater spring and was provided at approximately 4 L/min for a total system hydraulic retention time of approximately 100 min. Initial mean fish stocking density was 49.8 kg/m3. Fish were maintained on constant photoperiod and were fed a commercial salmon diet via automated feeders at hourly intervals using a standard feeding chart. Mean water temperature over the study period was 13.9°C. After 1 week of acclimation, all fish were anaesthetized with 75 mg/L tricaine methanesulfonate (Western Chemical, Inc.) and individually vaccinated via intracoelomic injection, following vaccine manufacturer protocols, with Forte Micro (Elanco Canada Ltd.), a salmonid vaccine containing formalin-inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, V. ordalii and V. salmonicida. Following vaccination, four treatment groups were randomly allocated among the 12 study tanks (n = 3) for daily pulse treatments of: (a) 0.2 mg/L PAA, (b) 0.5 mg/L PAA, (c) 1.0 mg/L PAA and (d) 5 ml deionized water (control). The PAA used was VigorOx® SP-15 (PeroxyChem), a commercial product containing 15% PAA and 10% H2O2. By providing vaccination stress, we attempted to induce clinical saprolegniasis by creating conditions that permit ubiquitous Saprolegnia spp. to cause disease. The study period lasted for 6 weeks post vaccination, with daily mortalities noted to assess post-vaccination survival. Biofilter function was assessed through total ammonia nitrogen (TAN) removal efficiency, such that mean TAN removal rates were assessed at 1, 3 and 6 weeks after treatment onset. Feeding and flow rates remained constant throughout the study. Minimum and maximum mean TAN levels entering the biofilters were 0.240 and 0.353 mg/L respectively. At study's end, length and weight data were collected from a representative sample of fish from each tank, and health and welfare were assessed. Three fish per tank were humanely euthanized with 200 mg/L tricaine methanesulfonate, and representative samples of gill, spleen and kidney tissue were fixed in 10% formalin solution for histopathological processing and evaluation. Additionally, 20 fish per tank were euthanized and evaluated for the presence or absence of fin erosion, external haemorrhage and/or visible Saprolegnia spp. infection (which was supported via skin scrapes and wet-mount microscopy to observe the characteristic morphology and hyphae of Saprolegnia spp. oomycetes; subsequent culture and sequencing identified the agent as Saprolegnia australis). Survival, growth performance and biofilter TAN removal efficiencies were analysed via ANOVA followed by the Tukey procedure to determine significant (p < .05) differences among treatment groups. Survival and TAN removal efficiency percentages were first arcsine-transformed prior to ANOVA. Fin erosion, haemorrhage and external saprolegniasis data were analysed via logistic regression, reporting odds ratios. All statistical procedures were performed in STATA (StataCorp). Overall, survival during the 6-week study period was significantly higher in all three PAA treatment groups relative to the control group (Figure 1), although elevated mortality due to saprolegniasis was not observed in the control group despite the vaccination stressor. Conversely, mean Atlantic salmon weight was significantly higher in the control fish compared to all three treatment groups (Figure 1). Whether this growth difference was a consequence of an inhibitory effect of PAA requires further investigation, as previous research with PAA and rainbow trout Oncorhynchus mykiss has not demonstrated an inhibitory growth effect at low (0.2 mg/L) doses, either as pulse or continuous PAA application (Liu, Straus, Pedersen, & Meinelt, 2017). Despite the observed differences in growth performance, histopathology of the specific tissues sampled was unremarkable overall, with minimal lesions observed and no relationships between treatment groups determined. It is therefore likely that physical damage associated with PAA treatment did not impact growth performance; however, other tissue types (e.g. skin, liver, gastrointestinal tract.) should be evaluated in future PAA studies for confirmation. Recent research (Soleng et al., 2019) has demonstrated that Atlantic salmon mount both systemic and mucosal stress responses following exposure to PAA (both at 0.6 and 2.4 mg/L), which could result in energy diversion away from somatic growth and thereby account for the growth differences observed in the present study. Fish were fed at equal rates throughout this experiment; however, due to the nature of the study tanks (i.e. no sump in which to observe wasted feed) and the 24-hr feeding period, it was not possible to compare actual feed consumption among treatment groups. Therefore, feed intake might have been lowered in response to PAA exposure, and this possibility requires examination in future studies. While fin erosion and external signs of haemorrhage were not associated with any particular treatment group(s) (Table 1), it is interesting to note that control fish were significantly more likely to exhibit external saprolegniasis compared to PAA treated fish; that is, with an odds ratio of 0.074, the odds of saprolegniasis significantly decreased with increasing PAA dosage. Finally, biofilter TAN removal efficiency was not significantly impacted by PAA treatment (Figure 1), although broad standard errors in two of the four treatment groups suggest that a higher power study is required to confirm these findings. Further research should also focus on potential effects of PAA on other biofilter types, for example moving bed, microbead and fixed bed biofilters (Malone & Pfeiffer, 2006; Pedersen, Oosterveld, & Pedersen, 2015). In conclusion, the principal findings of this study were as follows: (a) at the dosages tested, PAA significantly reduced observable external saprolegniasis following vaccination and led to overall higher survival rates, and (b) biofiltration did not appear to be impacted by PAA exposure at the dosages, and treatment regimens tested. These results are promising with regard to the development of efficacious protocols to reduce post-vaccination saprolegniasis in Atlantic salmon RAS. Future research should expand on these findings and address the study limitations discussed. Special thanks are extended to Karen Schroyer, Susan Glenn and Christina Russell for water chemistry analysis, to Josh Kretzer for transportation of the research fish, to Jennifer Lipscomb for assistance with vaccinations and to Cindy Ledbetter for Saprolegnia spp. culture. This research was supported by the USDA Agricultural Research Service under Agreement Nos. 59-1930-0-046 and 59-8082-5-001. All experimental protocols and methods were in compliance with the US National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by The Conservation Fund Freshwater Institute's Institutional Animal Care and Use Committee prior to study commencement. All authors listed have substantially contributed to this research according to journal standards for authorship qualification. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The authors do not have any conflicts of interest to declare. The Conservation Fund and USDA are equal opportunity employers and providers. Data are available upon request to the corresponding author.

Secondary salinization is a growing global ecological issue. One cause is the discharge of effluents by the potash mining industry into surface waters such as the River Werra in Germany. Increases of major ions require various physiological responses of freshwater organisms to maintain the hydromineral balance of body fluids. However, only little is known about the acute and chronic effects of high concentrations and imbalances of ions on osmoregulation in freshwater teleosts. The present study aimed to elucidate the effects of potash mining effluents and different cation ratios on the osmoregulatory capacity and gill histopathology of a native fish species. Individuals of Rutilus rutilus were exposed to the currently allowed (HT) and intended future (LT) thresholds as well as to high concentrations of Mg2+ (Mg), K+ (K), and Mg2+ and K+ (Mg + K) for a period of 24 h, 7 d, 21 d and 8 wk. Plasma osmolarity, [Na+], [Mg2+], [K+], [Ca2+], [Cl-] and [SO42-] and branchial Na+/K+-ATPase activity were determined. Moreover, histological gill alterations after 21 d and muscle water content after 8 wk were examined. HT transiently (24h) elevated plasma osmolarity, plasma [Na+] and [Ca2+], whereas [SO42-]was chronically increased even after 8 wk. Exposure to LT, Mg and Mg + K led to increased [SO42-] levels for at least 21 d. It seems that [SO42-] is mainly disturbed by multiple ions at high concentrations and long-term effects are unknown. Hydromineral homeostasis was maintained as indicated by unchanged Na+/K+-ATPase activity and muscle water content. However, mild structural alterations of the gills were observed in all exposure groups suggesting adaptational responses but with the potential to affect gas exchange capacity. Hence, the current thresholds for potash mining effluents affect osmomineral regulation in roach and further investigations should address potential impacts on reproduction in native fish species and physiological effects of SO42-.( )(C) 2019 Elsevier Ltd. All rights reserved.

A present ecological issue causing secondary salinization in different countries is the discharge of effluents by the potash mining industry. In Germany, the River Werra is used as a sink for potash mining discharges containing high concentrations of ions, predominantly Cl-, K+, Na+, and Mg2+ resulting in a strong decline of the biodiversity and abundance of local species. However, hardly anything is known about the acute and chronic physiological effects of high concentrations and imbalances of ions being prevalent in potash mining effluents in fish. Therefore, the stress response and selected immune and growth parameters were investigated in standardized laboratory experiments. A native freshwater fish species, Rutilus rutilus, was exposed to concentrations of the high currently allowed (HT) and lowered future thresholds (LT) and three different ion solutions (containing high Mg2+ (Mg), high K+ (K) and high Mg2++K+ (Mg+K) concentrations) for four different exposure times (24 h, 7 d, 21 d, 8 wk). Tank water (additionally after 9 and 12 h) and plasma cortisol, glucose and protein, hematocrit and hemoglobin were determined after each exposure time. Furthermore, plasma lysozyme and head kidney leucocyte respiratory burst activity (only after 21 d) were evaluated as well as growth parameters. A transient stress response was induced in almost all groups. Tank water cortisol was elevated after 9 h in HT, LT and Mg+K and in HT after 12 h, whereas glucose concentrations increased after 24 h in all exposure groups except K. HT led to enhanced hematocrit and hemoglobin content after 24 h. Plasma protein, immune system and growth were not affected in any group. None of the ion solutions induced acute toxicity but most triggered typical acute stress reactions. Rather the sum of high ion concentrations than single ions challenged the fish. Even though the effects observed in adult roach were only transient and indicate acclimatization under laboratory conditions, adverse effects observed in the river are evident and further research on physiological endpoints including reproductive parameters and impacts on younger life stages seem to be needed to scientifically base protective thresholds.

Stress in fish can be caused by a variety of factors and has the potential to evoke stress responses leading to a reduction of physical condition and of health. The river Werra (Germany) presents a severe case of secondary salinisation caused by potash mining activities. The model organism Danio rerio was exposed to different ion-concentrations depicting current (HT) and future (LT) threshold values of the Werra, as well as to solutions with single-exceeding ions (Mg2+ + K+ (KMg), Mg2+ (Mg) and K+ (K)). After a six-week exposure period, cortisol levels, growth and weight were measured, gills and gonads were histologically analysed and mRNA expression of follicle stimulating hormone (FSH), luteinising hormone (LH), growth hormone (GH) and prolactin (PRL) were determined. Cortisol was still elevated in fish in the HT and K group, indicating moderate stress. However, gills revealed structural changes in zebrafish in all exposure groups, size of oocytes differed in the LT and K group, male FSH mRNA levels were elevated in the HT and LT group whereas PRL mRNA levels were lower in HT and LT for both, male and female fish. These results suggest that ion-stress induces moderate effects on a variety of biological parameters that mainly serve to adapt to elevated ion concentrations. For these reasons current and even future thresholds should be reconsidered, including thresholds for total as well as single ion concentrations. Future research looking at the effects on local fish species is needed, along with regular and long-term monitoring of environmental conditions, species abundance and diversity.

Peracetic acid (PAA) is considered an eco-friendly alternative to other antimicrobial agents of common use in aquaculture. The literature suggests that fish can habituate to PAA exposure based on a reduction of the fish corticosteroid response to PAA administration after repeated exposures. If that is true, PAA would also be a good option from the point of view of fish physiology. However, stronger evidence is needed to confirm that the use of PAA is welfare-friendly to fish. Besides habituation, other hypothetical factors such as desensitization, physiological exhaustion or PAA-mediated endocrine disruption could potentially explain the reduction in the corticosteroid response after repeated/prolonged PAA exposure. In this study, rainbow trout that had been exposed to PAA for several weeks were challenged with a secondary chasing stressor: fish were pursued with a dipnet for 1 min and their acute response was evaluated by measuring plasma cortisol, plasma glucose, plasma lactate and brain serotonergic activity. All fish were equally able to mount a normal physiological stress response to the secondary stressor independent of previous exposure to PAA. This suggests that the decrease in the cortisol response after repeated exposure to PAA, as seen in previous studies, is a true habituation to PAA administration, which supports the use of PAA as a welfare-friendly antimicrobial agent in aquaculture.