Fulvic acid modulates mucosal immunity in fish skin: Sustainable aquaculture solution or environmental risk factor?

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.

Modification of the chemically induced inflammation assay reveals the Janus face of a phenol rich fulvic acid.

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.

Fulvic acid accelerates hatching and stimulates antioxidative protection and the innate immune response in zebrafish larvae.

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.

Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture.

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.

Overlooked Risks of Biochars: Persistent Free Radicals trigger Neurotoxicity in Caenorhabditis elegans.

In recent years, biochars have gained increasing interest in mitigating climate changes and revitalizing contaminated or drained soil. Studies determining their impact on the ecosystem, especially on soil invertebrates, however, are still scarce and the neurotoxic potential of biochars has never been evaluated before. Using the model organism Caenorhabditis elegans we determined the neurotoxic effect of biochar produced from rice straw by pyrolysis at 500 °C at concentrations ranging from 0 to 2000 mg C·L-1. Biochar had a hormetic effect on locomotion behavior. Furthermore, high concentrations impaired defecation as well as the recognition and response to a chemical attractant. None of the potential toxic chemicals in the biochar had sufficient high concentrations to explain the detected neurotoxic effect. Using electron paramagnetic resonance (EPR) spectroscopy, we detected free radicals in the biochar. Detrimental reaction of free radicals with biotic macromolecules can induce oxidative stress responses and are a potential reason for the evaluated neurotoxic effect of biochar. Overall, we were able to prove that biochars have the potential to act as weak neurotoxins to soil organisms and effects of persistent free radicals should be investigated further.

Adsorbable organic bromine compounds (AOBr) in aquatic samples: a nematode-based toxicogenomic assessment of the exposure hazard.

Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode Caenorhabditis elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa-enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the upregulation of cyp- and the downregulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth, and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely, the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR.

Natural Marine and Synthetic Xenobiotics Get on Nematode's Nerves: Neuro-Stimulating and Neurotoxic Findings in Caenorhabditis elegans.

Marine algae release a plethora of organic halogenated compounds, many of them with unknown ecological impact if environmentally realistic concentrations are applied. One major compound is dibromoacetic acid (DBAA) which was tested for neurotoxicity in the invertebrate model organism Caenorhabditis elegans (C. elegans). This natural compound was compared with the widespread synthetic xenobiotic tetrabromobisphenol-A (TBBP-A) found in marine sediments and mussels. We found a neuro-stimulating effect for DBAA; this is contradictory to existing toxicological reports of mammals that applied comparatively high dosages. For TBBP-A, we found a hormetic concentration-effect relationship. As chemicals rarely occur isolated in the environment, a combination of both organobromines was also examined. Surprisingly, the presence of DBAA increased the toxicity of TBBP-A. Our results demonstrated that organohalogens have the potential to affect single organisms especially by altering the neurological processes, even with promoting effects on exposed organisms.

Two organobromines trigger lifespan, growth, reproductive and transcriptional changes in Caenorhabditis elegans.

Organobromines of natural and artificial origin are omnipresent in aquatic and terrestrial environments. Although it is well established that exposure to high concentrations of organobromines are harmful to vertebrates, few studies have investigated the effect of environmentally realistic concentrations on invertebrates. Here, the nematode Caenorhabditis elegans was challenged with two organobromines, namely dibromoacetic acid (DBAA) and tetrabromobisphenol-A (TBBP), and monitored for changes in different life trait variables and global gene expression patterns. Fifty micromolar DBAA stimulated the growth and lifespan of the nematodes; however, the onset of reproduction was delayed. In contrast, TBBP changed the lifespan in a hormetic fashion, namely it was stimulated at 0.1 µM but impaired at 50 µM. The reproductive performance was even impaired at 2 µM TBBP. Moreover, DBAA could not reduce the toxic effect of TBBP when applied as a mixture. A whole-genome DNA microarray revealed that both organobromines curtailed signalling and neurological processes. Furthermore on the transcription level, 50 µM TBBP induced proteolysis and DBAA up-regulated biosynthesis and metabolism. To conclude, even naturally occurring concentrations of organobromines can influence the biomolecular responses and life cycle traits in C. elegans. The life extension is accompanied by negative changes in the reproductive behaviour, which is crucial for the stability of populations. Thus, this paper highlights that the effects of exposure to moderate, environmentally realistic concentrations of organobromines should not be ignored.

Neurotoxic evaluation of two organobromine model compounds and natural AOBr-containing surface water samples by a Caenorhabditis elegans test.

Brominated organic compounds are known as disinfection byproducts. Very recently, however, even natural brominated organic compounds (analyzed as adsorbable organic bromine; AOBr) have been found in decaying freshwater cyanobacteria blooms. Among the identified compounds was dibromoacetic acid (DBAA), which has proven to be neurotoxic at rather high concentrations in mammalian assays. Currently it is open how single compounds as well as complex mixtures impact organisms at environmentally realistic concentrations. Furthermore, it is also unclear how natural organic matter, mainly humic substances (HS), which are present in all freshwater systems, modulates the toxic impact of AOBr. Therefore, two AOBr compounds (DBAA and tetrabromobisphenol-A; TBBP-A) and AOBr-containing water samples were tested using a Caenorhabditis elegans neurotoxicity assay that measured autonomic and sensory functions. TBBP-A had an impact on three response variables of C. elegans and can be classified neurotoxic. In contrast to our expectations, DBAA led to neurostimulation of two autonomic functions, but had a temporary impact on the defecation interval. All surface water samples contained measurable amounts of AOBr. Exposure of C. elegans to concentrated water samples - one in particular - increased three of the four locomotion traits and left defecation activity and both sensory variables unchanged. This stimulation might be due to unidentified compounds in the samples or to a hormetic effect of the AOBr compounds. Thermotactic behavior was characterized by a temporary preference for the colder environment, indicating a temporary mild neurotoxicity. Overall, the set of relative simple phenotypic tests used in the current study revealed a meaningful neurotoxic or neurostimulative profile in response to chemical compounds or natural samples. Furthermore, it shows that the resulting response to natural AOBr compounds at environmentally realistic concentrations was not necessarily adverse, but instead, that the mixtures of natural AOBr were neurostimulatory.