Humic Acid Substances in Aquaculture for Fish Disease and Treatments

What is Humic Acid?

Humic substances are the main constituents of natural organic substances that arise from the physical, chemical, and microbiological transformation of biomolecules. These substances are found in debris and soil. It is stated that approximately 95% of dissolved organic matter in water is composed of humic substances [9, 10, 11, 12], and those substances constitute nitrogen (N) and carbon (C) reserves [13, 14].

Humic substances; are divided into three major fractions which are; humic acid, fulvic acid, and humin [9, 15]. The distinction in these substances is resulting from the differences in pH levels and molecular weights [9, 16]. While humic acids dissolve at higher pH, they do not dissolve when pH level is below two. It is also stated in the literature that fulvic acids dissolve at all pH values, whereas humine does not dissolve at any value [13, 14]. Furthermore, humic acids differ from substances such as fulvic acid and huminic in that they dissolve in alkali environments, partially dissolve in water, and are insoluble in acidic environments [17].

Benefits of Humic Acid

Humic acids prevent the increment of pathogen presence by optimizing the pH in the digestive tract, inhibit the adherence of viral particles to the cell by antiviral action, promote the utilization of calcium and trace elements, and reduce the toxic effect of heavy metals [18, 19, 20]. It is also reported that those are also effective in recovering the damages arising from bacterial and parasitic sources, especially from fungal pathogens [5]. Humic acids are known as important antioxidants due to their powerful properties on free radical scavenging activities [4]. They are also expressed to stabilize the intestinal flora, thus enable efficient use of nutrients in animal feed [21]. Considering humic acids are part of the natural environment of the fish and the defense systems of fish are accustomed to these natural xenobiotics, those can be ideal candidates for use in aquaculture [9].

As well as regulating immunity and blood sugar [18, 22], the humic substances are stated to have antiviral, antimicrobial, antibacterial, antioxidant, antitumor, anti- inflammatory, anticlastogenic, antiallergic, antitoxic, antiulcerogenic, antirheumatismal, antiangiogenic, antipyretic, antimutagenic, and analgesic properties. Taking into account the abovementioned features, together with their anti-HIV and immuno-stimulatory properties, it seems viable to use the humic acids as feed additives in the diets of aquatic organisms [4]. No allergic, anaphylactogenic, teratogenic, embryotoxic, or carcinogenic effects are reported as humic substances are used in recommended doses as well as they do not have harmful effects on humans and animals [12, 23, 24].

The Use of Humic Acids in Aquaculture

Humic acids can be used as alternative natural feed additives due to their positive effects on the parameters such as larvae and egg yield, growth and feed intake, disease resistance, survival rate, suppression of secondary infections, detoxification of harmful metals and chemicals in water, and rapid recovery from ectoparasites [18, 21, 25]. Humic acids; can increase the physiological condition of the organisms, reduce physiological and histological damages caused by stress, and quickly repair damages caused by fish pathogens. Since humic acids have different advantages over stress, their ecophysiological relationship in aquaculture is emphasized [5].

In the present work, some of the studies investigating the effects of humic compounds on aquatic organisms were listed (Table 1) and reviewed briefly.

The effects of different humic substances in carp (Cyprinus carpio) and channel catfish (Ictalurus punctatus) were investigated [22]. According to the results, potassium humate did not show a significant effect on the intestinal microbial flora of fish. The antimicrobial effect of Pseudomonas and Aeromonas cultures and ten bacterial strains isolated from fish has been investigated and silver humate has been reported to be the most effective bactericide.

In a study with common carp (Cyprinus carpio), four different diets were prepared using humic acid, which incorporated into diets at 0.0%, 0.4%, 0.8%, and 1.0% [26]. Fish were fed with those diets for 45 days; the growth performance, immune response, and disease resistance against Aeromonas hydrophila were evaluated at the end of the study. It was stated that the weight gain, specific growth rate, and lysozyme activity were found to be significantly higher in the 0.4% and 1% humic acid supplemented groups compared to the control group. According to challenge test results, the highest mortality rate was reported to be in the control group while the lowest mortality was in the group fed with 0.8% and 1% humic acid supplemented feeds.

The effects of dietary humic acid on the growth performance of silver catfish (Rhamdia quelen) juveniles were tested at different pH levels (5.5 and 6.5) [27]. For this purpose four diets were prepared to contain humic acid at 0.0, 10.0, 25.0, and 50.0 mg L-1, and fed the fish for 40 days. At the end of the trial, the authors reported humic acid to be highly detrimental in the tested concentrations for juvenile silver catfish growth as it lowers the weight gain and the feed intake of fish.

In an experiment conducted to determine the effects of fulvic acid on growth performance and intestinal health of juvenile loach Paramisgurnus dabryanus, fish were fed with fulvic acid supplemented feeds which are incorporated into the diet at 0.0%, 0.5%, 1%, 1.5%, and 2% for 60 days [28]. It was stated that an elevating effect of fulvic acid was achieved on growth rates of fish with increasing doses and the maximum weight gain and specific growth rates were observed to be in the fish fed with the 1.5% fulvic acid supplemented diet. It was also reported that the fulvic acid supplementation decreased the presence of pathogenic bacteria Serratia, Acinetobacter, Aeromonas, and Edwardsiella, whereas an increase in the abundance of beneficial Lactobacillus was achieved in the intestine. The authors concluded that the fulvic acid might be an immune enhancer on loaches considering it promotes growth performance and enhances intestinal wellness of juvenile loach.

The effects of sodium humate on pathogen-exposed goldfish (Carassius auratus) and carp (Cyprinus carpio) was investigated [29]. It was stated at the end of the experiment that the sodium humate treatment reduced the mortality rate of infected fish and the fish treated with sodium humate showed significant improvement in skin, fins, and gills.

Six different diets were formulated to contain %0.0, 0.2%, 1.0%, 5.0%, 10.0%, and 20.0% humus extract for common carp Cyprinus carpio fry [30]. The fish were infected with Aeromonas salmonicida and fed with experimental feeds 30 days prior to the bacterial challenge and 22 days immediately after the challenge. At the end of the experiment, it was reported that the mortality and development of skin lesions such as ulcer and hemorrhages were significantly suppressed in the groups fed with the diets containing 1.0%, 5.0%, and 10.0% humus extract whereas the survival rates were higher in the fish fed with 5.0% and 10.0% humus extract supplemented feed. The authors have concluded that the humus extract treatment has been found to be effective to prevent A. salmonicida infection.

The preventive effects of orally administered humus extract on Ayu Fish (Plecoglossus altivelis) against the bacteria Flavobacterium psychrophilum was investigated [31]. For this purpose, the humus extract was incorporated into the diet at %0.0, 1.0%, 5.0%, 10.0%, and fed to fish for 30 days prior to the challenge and 21 days afterward. The feeds containing humus extract at 1.0%, 5.0%, 10.0% significantly suppressed the death rates, hemorrhages, and other skin lesions of fish challenged with F. psychrophilum. The mechanism of action on the protective feature of the extract was not expressed as the direct killing of bacteria or antibiotic activity, considering no distinct reduction in the bacterial count was achieved. It was reported at the end of the study that the humus extract was an effective feed additive against cold water disease.

In another study the effects of humate compounds on the control of Saprolegniosis, which leads to significant economic losses in fish farming were tested on Nile tilapia [32]. In the trial, fish were subjected to 5%, 10%, and 15% humate, and it was reported that the treatment of humate at 5% increased survival rates of the fish.

Dietary humic acid was incorporated into diets of common carp (Cyprinus carpio) whether it has an impact on growth performance and histopathology (gill, liver, and kidney) of fish [33]. At the end of the study, it was reported that despite dietary supplementation of humic acid (180 and 360 mg.kg-1) slightly promoted growth and feed utilization of fish; its detrimental effect on the tissues was found to be more evident.

Five different diets were supplemented with humic acid (HA) at 0.00%, 0.25%, 0.50%, 0.75%, and 1.50% and fed to sea bass Dicentrarchus labrax for 15 days to investigate the effects of humic acid on some immunological and histological parameters of fish [18]. For this purpose the parasite Amyloodinium sp. was introduced to the culture water. It was reported that the hemoglobin, hematocrit levels, and NBT activity increased in the group of fish fed with 0.50% HA supplemented feed. It was also stated that the serum NBT and LA significantly decreased in the fish infested with Amyloodinium sp. while MPO activity did not change. According to histopathological results, 0.50% HA supplemented feed caused an improvement in gill filaments compared to other experimental groups against Amyloodinium sp. infestation (Figure 1) [18]. It was concluded that the HA has positive effects on some health- promoting parameters of Dicentrarchus labrax juveniles challenged with Amyloodinium sp.

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In a different study, humic-rich culture water and synthetic humic supplemented feed both reduced the ratio of infected fish (Poecilia reticulata) when challenged with parasites Gyrodactylus turnbulli and Dactylogyrus sp [34]. The authors have concluded that the humic-rich water and sludge derived from a recirculating aquaculture system has the ability to protect fish from ectoparasites, whereas humic compounds do not exhibit a toxic effect on the parasites used.

The effects of dietary humic acid on growth performance, haemato-immunological, and physiological parameters of rainbow trout juveniles were investigated in another experiment [4], in which, the authors fed the experimental fish with humic acid incorporated diets at 0.0%, 0.3%, 0.6%, and 1.2% for 60 days. At the end of the experiment, it was stated that the growth performance and haematological parameters of fish were not affected by humic acid supplemented diets whereas 0.6% humic acid supplementation significantly increased the activities of pepsin, trypsin, and lipase. After 20 days of exposure to the bacteria Yersinia ruckeri, to test the resistance of fish, significantly higher survival rates were reported to be in the 0.6% humic acid group. It was concluded that the dietary humic acid might be a good alternative to antibiotic treatments of Yersinia ruckeri infected rainbow trout (Oncorhynchus mykiss).

The effects of humic substances containing products FARMARIN®XP and INFISH-AQUA® on growth performances, immune responses, digestive enzyme activities, and disease resistance of juvenile rainbow trout challenged with the bacteria Yersinia ruckeri was investigated [35]. The diets were supplemented with FARMARIN®XP at 0.0%, 0.1%, 0.2%, 0.4% while a fifth experimental group was additionally formed and treated with INFISH-AQUA® (sulphadiazine 20% + trimethoprim 4%). The diets were given to the fish for 60 days and mortalities were recorded after the challenge test. It has been stated in the study that the dietary FARMARIN®XP may exhibit positive effects on immune and serum biochemical parameters of rainbow trout juveniles, increase the survival rates against Y. ruckeri, and therefore can be used as a non-antibiotic additive to prevent Yersiniosis disease.

In the fulvic acid study on red swamp crayfish (Procambarus clarkia), body weight gain and survival rate of crayfish were found to be higher in treatment groups according to the control group [36]. While protease, lipase, and amylase activities were significantly increased in the mid-dose group, the high dose did not affect these rates. As a result of the challenge of crayfish with Aeromonas hydrophila, the survival rate in all treatment groups was found to be higher than the control.

Fulvic acid incorporated diets were fed to Pacific white shrimp (Litopenaeus vannamei) at varying levels in a different study [37]. It was reported that the final wet weights were higher in moderate levels of fulvic acid (0.9% and 1.2%) than the control, whereas no statistically significant differences were observed at lower levels. Moreover, the HSP70 value in hemolymph and hepatopancreas reported to be decreased as the level of fulvic acid increased. According to the antioxidant parameters, SOD, CAT, and GSH values ​in the hepatopancreas and hemolymph was reported to be significantly higher in the treatment groups compared to the control.

The effects of humic acid and Yucca schidigera extract were examined whether they promote the quality of culture water under low water-exchange [38]. It was stated that the humic acid has a considerable effect on the availability of phosphorus compounds to benefit, and both substances were reported to reduce the effect of ammonia stress.

In a study conducted to evaluate possible control strategies against Salmonella hadar in local tambaqui farms of Brazil, fish were fed with humic acid and probiotic supplemented diets [39]. It was reported at the end of the experiment that after 20 days feeding with experimental diets caused a 50% reduction in Salmonella hadar shedding in guts.

Another study was carried out on Oreochromis niloticus in which dietary sodium humate was fed to fish [40]. Increasing levels of sodium humate were reported to possess a linear relationship with the hepatosomatic index (HIS) at the end of the study. No statistically significant differences in feed intake, protein efficiency ratio, and viscerosomatic index (VSI) were noticed, and the survival rates of treatment groups were reported to be higher than the control when challenged with Aeromonas hydrophila.