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.

Ion-rich potash mining effluents affect sperm motility parameters of European perch, Perca fluviatilis, and impair early development of the common roach, Rutilus rutilus.

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.

Potash mining effluents induce moderate effects on histopathological and physiological endpoints of adult zebrafish (Danio rerio).

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.

Potash mining effluents and ion imbalances cause transient stress in adult common roach, Rutilus rutilus.

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.

Potash mining effluents and ion imbalances cause transient osmoregulatory stress, affect gill integrity and elevate chronically plasma sulfate levels in adult common roach, Rutilus rutilus.

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 (24 h) 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-.

The evolutionary legacy of size-selective harvesting extends from genes to populations.

Size-selective harvesting is assumed to alter life histories of exploited fish populations, thereby negatively affecting population productivity, recovery, and yield. However, demonstrating that fisheries-induced phenotypic changes in the wild are at least partly genetically determined has proved notoriously difficult. Moreover, the population-level consequences of fisheries-induced evolution are still being controversially discussed. Using an experimental approach, we found that five generations of size-selective harvesting altered the life histories and behavior, but not the metabolic rate, of wild-origin zebrafish (Danio rerio). Fish adapted to high positively size selective fishing pressure invested more in reproduction, reached a smaller adult body size, and were less explorative and bold. Phenotypic changes seemed subtle but were accompanied by genetic changes in functional loci. Thus, our results provided unambiguous evidence for rapid, harvest-induced phenotypic and evolutionary change when harvesting is intensive and size selective. According to a life-history model, the observed life-history changes elevated population growth rate in harvested conditions, but slowed population recovery under a simulated moratorium. Hence, the evolutionary legacy of size-selective harvesting includes populations that are productive under exploited conditions, but selectively disadvantaged to cope with natural selection pressures that often favor large body size.

Growth inhibition of Aeromonas salmonicida and Yersinia ruckeri by disinfectants containing peracetic acid.

Peracetic acid (PAA) is a therapeutic agent used for disinfection in aquaculture, but it must be investigated thoroughly in order to mitigate diseases without harming the fish. Successful disinfectants (like PAA) should not leave dangerous residues in the environment in order to successfully contribute to sustainable aquaculture. The aim of our study was to compare the effectiveness of 6 commercial PAA products with different molecular PAA:H2O2 ratios to reduce bacterial growth of Aeromonas salmonicida and Yersinia ruckeri and to determine effective concentrations and exposure times. All products reduced colony-forming units (CFUs) of A. salmonicida and Y. ruckeri. Products with higher molecular PAA:H2O2 ratios inhibited growth better than products with lower molecular PAA:H2O2 ratios at the same PAA concentration; this indicates that H2O2 is not the driving force in the reduction of A. salmonicida and Y. ruckeri growth by PAA in vitro. The practical application of the products with high molecular PAA:H2O2 ratios should be prioritized if these pathogens are diagnosed.

Evaluation of continuous 4-day exposure to peracetic acid as a treatment for Ichthyophthirius multifiliis.

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The most effective treatment, malachite green, has been banned in Europe and North America for use in food fish production. Peracetic acid (PAA) was found to be toxic to I. multifiliis theronts at low concentrations. I. multifiliis-infested carp were exposed to 1 mg/l PAA in a dynamic exposure by means of peristaltic pumps. Five days after infestation, gills, tail fins, and skin below the dorsal fin were observed microscopically for I. multifiliis abundance. After PAA exposure, PAA-treated fish showed lower infestation of I. multifiliis in all investigated tissues than the unexposed control fish. The infestation increased in the control group whereas the infestation in the PAA-exposed groups significantly decreased (p = 0.0083, Bonferroni correction). The fish in the two exposure groups showed a slight reinfestation with I. multifiliis. This might be caused by a peroxide degradation (hydrolysis) and/or reduction of the delivered PAA concentration. Thus, PAA concentrations were possibly too low to be effective on the released trophonts and/or the infective theronts. This hypothesis is corroborated by the fact that the I. multifiliis in the gills, skin, and fins of the PAA-exposed carp were in an early developmental stage.

Acute toxicity of peracetic acid (PAA) formulations to Ichthyophthirius multifiliis theronts.

Peracetic acid (PAA) is an antimicrobial disinfectant used in agriculture, food processing, and medical facilities. It has recently been suggested as a means to control infestations of Ichthyophthirius multifiliis. The purpose of this study was to determine the acute toxicity of two products containing 4.5% and 40% PAA to I. multifiliis theronts from two geographically separate isolates. Theronts were exposed to concentrations of PAA in 96-well plates containing groundwater at 23 degrees C. Acute toxicity was observed over a 4-h period. No significant difference in the median lethal concentration (LC(50)) estimates was evident between the two isolates at 4 h with the 4.5% PAA product (0.146 versus 0.108 mg/l PAA), while there was a statistical difference between the 4 h LC(50) with the 40% PAA product (0.274 versus 0.158 mg/l PAA). These results suggest that PAA is toxic to I. multifiliis theronts at low concentrations and that one of the isolates was more resistant to this compound.

Humic substances. Part 2: Interactions with organisms.

GOAL, SCOPE AND BACKGROUND: Freshwater bodies which chemistry is dominated by dissolved humic substances (HS) seem to be the major type on Earth, due to huge non-calcareous geological formations in the Northern Hemisphere and in the tropics. Based on the paradigm of the inertness of being organic, direct interactions of dissolved HS with freshwater organisms are mostly neglected. However, dissolved organic carbon, the majority of which being HS, are natural environmental chemicals and should therefore directly interact with organisms. Major results that widened our perspective on humic substance ecology come from experiments with the compost nematode, Caenorhabditis elegans, which behaved contradictorily to textbook knowledge and provoked an in-depth re-consideration of some paradigms. APPROACH: To overcome old paradigms on HS and their potential interactions with organisms, we reviewed recent international literature, as well as 'grey' literature. We also include results from own ongoing studies. RESULTS: This review focuses on direct interactions of dissolved HS with freshwater organisms and disregards indirect effects, such as under-water light quenching. Instead we show with some macrophyte and algal species that HS adversely interfere with photosynthesis and growth, whereby closely related algal species show different response patterns. In addition to this, HS suppress cyanobacteria more than eukaryotic algae. Quinones in the HS appear to be the effective structure. Furthermore, HS can modulate the offspring numbers in the nematode C. elegans and cause feminization of fish and amphibians--they possess hormone-like properties. The ecological consequences of this potential remain obscure at present. HS also have the potential to act as chemical attractants as shown with C. elegans and exert a mild chemical stress upon aquatic organisms in many ways: induction of molecular chaperons (stress proteins), induction and modulation of biotransformation and anti-oxidant enzymes. Furthermore, they produce an oxidative stress with lipidperoxidation as one clear symptom or even stress defense strategy. Stronger chemical stresses by HS may even lead to teratogenic effects as shown with fish embryos; all physiological responses to HS-mediated stress require energy, which were compensated on the expense of yolk as shown with zebra fish embryos. One Finnish field survey supports the view of a strong chemical stress, as the weight yield in fish species decreases with increasing HS content in the lakes. DISCUSSION: HS exert a variety of stress symptoms in aquatic and compost organisms. According to current paradigms of ecotoxicology, these symptoms have to be considered adverse, because their compensation consumes energy which is deducted from the main metabolism. However, the nematode C. elegans looks actively for such stressful environments, and this behavior is only understandable in the light of new paradigms of aging mechanisms, particularly the Green Theory of Aging. In this respect, we discuss the mild HS-mediated stress to aquatic and compost organisms. New empirical findings with HS themselves and HS building blocks appear to be consistent with this emerging paradigm and show that the individual lifespan may be expanded. At present the ecological consequences of these findings remain obscure. However, a multiple-stress resistance may be acquired which improves the individual fitness in a fluctuating environment. CONCLUSIONS: It appears that dissolved HS have to be considered abiotic ecological driving forces, somewhat less obvious than temperature, nutrients, or light. PERSPECTIVES: The understanding of the ecological control by dissolved humic substances is still fragmentary and needs to be studied in more details.

Humic substances. Part 1: Dissolved humic substances (HS) in aquaculture and ornamental fish breeding.

GOALS, SCOPE AND BACKGROUND: Changing environmental conditions and handling stress are well known to cause chronic or acute stress situations in fish with subsequent infections. These requires a therapy by means of antibiotics and chemicals. In contrast to the huge number of pathogens, only a few substances are permitted for application as therapeutics in German aquaculture. Hence, there emerges an urgent need for highly effective and residueless alternatives. MAIN FEATURES: The prophylactic stimulation and training of the defense system of fish by alternative approaches becomes increasingly necessary. One approach is the application of dissolved humic substances (HS) of natural or artificial origin. For example, there exist several reports on the positive effect of HS to fishes. These effects shall be considered in detail. Furthermore, the impact of HS on the constitution of parasites and pathogens will be displayed. The reports on this issue are diverse, if not inconsistent. We try to shed some light on these discrepancies. The last aspect covered by this review is the outdated paradigm that calcium ions act as antidotes. In the presence of HS, even the opposite effect may occur. APPROACH: To overcome old paradigms on HS and their potential interactions with fish and fish parasites, we reviewed recent international literature, as well as 'grey' literature. We also include results from own former and ongoing studies. RESULTS AND DISCUSSION: HS are able to increase the physiological condition of the individuals and to reduce adverse physiological and histological consequences caused by stress; the mechanism behind remains obscure. HS detoxify heavy metals and organic pollutants. Damages caused by several fish pathogens, such as bacteria and parasites, can be repaired more quickly in the presence of HS. Some parasites--mainly fungi--appear to be directly affected by HS. Comparing the fungicidal effects of HS from various sources, evidence is increasing that the aliphatic moiety may be the effective structures. However, further research is necessary to relate more physiological and anti-pathogenic effects to the chemical characteristics of HS. CONCLUSIONS: HS are not real alternatives to strong traditional therapeutics. However, they show different advantages in repairing secondary, stress induced damages in fish. The ecophysiological relevance of HS in either aquatic systems or aquaculture is getting conspicuously. PERSPECTIVES: The lack of therapeutic and antiparasitic substances in aquaculture requires new strategies and ways of thinking. The search for alternatives to the 'traditional' chemical therapeutics calls for the intensive research. Inevitably, this search will lead to an intensive contemplation on HS as 'health promoting substances' and/or even therapeutics. Basic research is needed to detect the functional groups of the HS responsible for the effects observed. Health promoting effects of first investigations made in vitro to affect pathogens via application of HS and several field studies with HS raises hopes for a broader utilisation of HS to reduce stress consequences in fish and fish pathogens residuelessly.

Reduction in vegetative growth of the water mold Saprolegnia parasitica (Coker) by humic substance of different qualities.

Humic substances (HS) account for 50-80% of the dissolved organic matter in non-eutrophicated freshwater ecosystems. HS are not inert, but are taken up by and may interact with aquatic organisms. However, at present no information is available on the interaction of HS with fungi, for instance, the fish-pathogenous species Saprolegnia parasitica. To fill this gap, we tested effects of HS on S. parasitica growth in-vitro using 25-500mgL(-1) carbon of HS on GY-agar. We investigated 20 HS including natural organic matter (NOM) samples, two lignite derived HS, and one synthetic HS. The aim was (1) to find out, if there are inhibiting effects and (2) if potential effects can be explained by humic matter properties by structure activity relationships. The growth of S. parasitica was related to the growth on HS-free agar controls. Characterization of HS and NOMs included elemental analysis, high-pressure size exclusion chromatography (HPSEC), UV/VIS, FTIR-, and EPR-spectroscopy in order to obtain information on elemental and structural composition including various metals, molecular weights of the HS fraction, aromaticity, free organic radicals, and functional groups. NOMs with high moieties of high-molecular carbohydrates supported the growth of S. parasitica, all other HS and NOMs reduced it. However, no inhibition of the development of the sporangia and primary zoospores was found. Therefore, the impact of the HS on S. parasitica has to be classified as fungistatic, rather than fungicidal. Synthetic and lignite-derived HS were among the most efficient HS sources. Growth inhibition was correlated (p<0.05) with the molecular weights of the HS-fraction, sUVa, COOH groups, C and H. Our results suggest that especially HS with higher molecular weights and aromaticity which contain a high number of organic radicals are the most efficient in reducing fungal growth. Furthermore, highly functionalized HS seem to be important for the observed effect. The development of internal oxidative stress could be a mechanism explaining the observed growth inhibition of S. parasitica.

Cadmium accumulation in zebrafish (Danio rerio) eggs is modulated by dissolved organic matter (DOM).

Experiments were conducted to investigate factors influencing the accumulation of cadmium (Cd(2+)) into zebrafish (Danio rerio) eggs. The accumulation of (109)Cd was affected by: (1) concentration, (2) time, (3) presence of dissolved organic material (DOM), (4) different origin of DOM and (5) different parts of fish eggs. Over a 5-h exposure, zebrafish eggs showed a steady increase in Cd-accumulation. DOM-concentrations over 15ppm carbon (C) decreased Cd-uptake significantly. Both samples of DOM, brown water marsh (LM) and a eutrophic pond (SP), at 16.9ppmC, reduced the Cd-accumulation in the chorion, perivitelline liquid and the embryo. Cd was mainly accumulated in the egg's outer shell chorion (61%) and only small amounts passed through the chorion into the perivitelline liquid (38%) and embryo (1%). In the presence of LM-DOM, the accumulation of Cd into the egg components was decreased by 43% (chorion), 52% (perivitelline liquid) and 52% (embryo), respectively, compared with the control group. Similarly, the presence of SP-DOM reduced the Cd-accumulation by 29% (chorion), 61% (perivitelline liquid) and 60% (embryo), respectively, compared with the controls. DOM-concentration should be taken into consideration when determining ecotoxicological effects of Cd on fish populations.

Hormonelike effects of humic substances on fish, amphibians, and invertebrates.

Humic substances comprise the majority of organic matter in freshwater ecosystems and were thought to be inert or refractory, except for photolytic degradation. However, evidence is increasing that humic substances interact with aquatic organisms similarly to weak anthropogenic chemicals with nonspecific and specific effects. One specific effect is a hormonelike effect, namely, modulation of the number of offspring, which was first described with the nematode Caenorhabditis elegans. Yet a hormonelike effect is not restricted to only the nematode. With the ornamental swordtail fish, Xiphophorus helleri, and the South African clawed frog, Xenopus laevis, we present phenomenological evidence that slight feminization occurred when these vertebrate species were exposed to a synthetic humic substance, a condensation product of polyphenols. The slight feminization was dose dependent.