Utilization of Biosurfactants in common carp (Cyprinus carpio) Semi-Artificial Fish Reproduction: Toxicity, Antimicrobial Activity, and Broodstock condition.

Biosurfactants are surface-active compounds produced by a variety of microorganisms, including Bacillus, Pseudomonas, and Candida. Their biodegradability, low toxicity, and functional stability have made them attractive alternatives to synthetic surfactants in biomedical, aquaculture, pharmaceutical, and environmental applications (Banat et al., 2010). While biosurfactants are generally considered safe, it is essential to evaluate their potential toxic effects on aquatic organisms, especially fish larvae, which are highly sensitive to chemical alterations in their environment. Cyprinus carpio (common carp) is widely used as a bioindicator species in ecotoxicological studies due to its sensitivity, easy availability, and ecological relevance in freshwater systems. Assessing the toxicity of partially purified biosurfactants on carp larvae helps determine their environmental safety and suitability for applications such as bioremediation in aquatic environments, aquafeed enhancement, or pathogen control in aquaculture settings (OECD, 2013). Toxicity parameters such as mortality, behavioural changes, LC50 values, and developmental alterations provide crucial insights into biosurfactant biocompatibility. In addition to toxicity assessment, biosurfactants possess significant antimicrobial properties due to their ability to disrupt microbial membranes and inhibit biofilm formation. These properties have encouraged their application against multidrug-resistant (MDR) clinical pathogens, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans (Gudiña et al., 2016). Evaluating antimicrobial activity helps establish the therapeutic potential of biosurfactants for use in pharmaceutical formulations, wound care, disinfectants, and medical device coatings.

Therefore, simultaneous evaluation of environmental safety (through toxicity on Cyprinus carpio larvae) and therapeutic potential (through antimicrobial analysis) provides a comprehensive understanding of biosurfactant applicability. This dual assessment supports sustainable utilization, ensuring that biosurfactants are both effective against pathogens and safe for aquatic ecosystems.

MATERIAL AND METHODS

Collection of common carp brooder

Climate is a major factor influencing fish breeding cycles, particularly in carps, where temperature, rainfall, and photoperiod regulate gonadal maturation and spawning behavior. Cyprinus carpio is known to breed naturally during the monsoon season under favorable environmental conditions; however, under controlled hatchery conditions, breeding can be induced throughout the year, ensuring continuous seed availability (Jhingran & Pullin, 1985; Bromage & Roberts, 1995). In contrast, Indian Major Carps (IMC) show strong seasonal dependence on monsoon-related environmental cues, and induced breeding outside the natural breeding season often results in reduced fertilization rates and lower hatching success (Nandeesha & Singh, 1990). At Poongar farm, brooders were collected using the drag net operation method, a standard aquaculture practice for safe and efficient broodstock harvesting in pond systems (Lucas & Southgate, 2012).

 Condition of brooder fish

Preparation of safety gadgets like, glass, gloves. About 10 to 12 experienced persons  get into the one corner of the pond and spread the net in horizontal direction with floats at the top and sinkers at the bottom. They put their one of their leg into the holes in the bottom of the drag net so that drag net  spreads along the vertical direction to catch the fishes. For each species they use different kinds of mesh in size. Finally, they drag to the opposite direction and brooders were collected upto 95%.Using the drag net, they made into pouch at one corner so that brooders were collected using the hand net.Sex separation  Male and female fishes were kept in indivually.

Age and Weight of Brood Fish

Typically, Cyprinus carpio matures earlier than other Indian Major Carps, which generally attain sexual maturity at around two years of age under culture conditions, whereas common carp may reach sexual maturity as early as six months depending on environmental and nutritional factors (Jhingran & Pullin, 1985; Nandeesha & Singh, 1990). In this study, both females and males were weighed to determine the appropriate hormone dosage required for induced breeding. The success of induced breeding largely depends on administering the correct hormone dose based on the body weight of the fish, as improper dosage may reduce fertilization and hatching rates (Bromage & Roberts, 1995; Aanand & Rajeswari, 2018).

Hormone Administration (Aanand and Rajeswari, 2018)

The hormone used for induced breeding was a synthetic Gonadotropin-Releasing Hormone analogue (SGnRH), commercially available as WOVA-FH. In induced breeding protocols for Cyprinus carpio, brood fish are commonly selected in the evening hours and administered the hormone through intramuscular injection below the dorsal fin region. The hormone dosage is calculated based on the body weight of the fish to ensure optimal ovulation and spermiation response (Nandeesha & Singh, 1990; Bromage & Roberts, 1995; Aanand & Rajeswari, 2018).

Only a single dose is done to both the male and female brooders

•  0.3 ml/kg – Common carp.
•   0.2 ml/500kg – Common carp.
•   0.6 ml/ kg –Indian major carp.

For male:   mix WOVA-FH with saline water.

For female: only WOVA-FH.

Injecting the hormone at the end of dorsal fin region of both male and female fishes before introducing the fishes into the Chinese hatchery tank

The breeding sex ratiomaintained is 2:1(male: female).

Semi-Artificial Fertilization method:

After injecting the brood fishes with the required amount of hormone they were released into a happa (1 m x 1 m x 2 m) suspended inside the tank. They were provided with sufficient aeration. Some substrates were provided such as nylon mesh, banana, and mango leaves to create a suitable environment for spawning. After fertilization, the eggs were incubated till the development of eyes.

 They started feeding on natural and artificial feeds. Therefore, rotifers were given as a live feed which was collected from the natural pond using plankton net. Additionally, yeast was given as a supplementary feed. It was clearly observed that all larvae were swimming freely. A few larvae were selected and used for the toxicity assay.

 Spawning and collection of egg:

Common carp (Cyprinus carpio) are known to mate and spawn during late evening or overnight under suitable environmental conditions (Jhingran & Pullin, 1985). After spawning, the fertilized eggs are adhesive in nature and attach to aquatic vegetation such as the roots of Eichhornia (water hyacinth). The freshly fertilized eggs are typically orange to yellowish in color, indicating healthy embryonic development (Bromage & Roberts, 1995). Following spawning, brood fish are removed from the breeding or surfactant-treated pond to prevent egg predation and mechanical disturbance. Under optimal water temperature conditions (around 26–30°C), hatching generally occurs within 48–72 hours (Lucas & Southgate, 2012). After 2–3 days, the fry are collected in the hatchery tank. Fry collection is commonly performed by gently draining the water through an outlet covered with fine muslin cloth to prevent loss or injury of larvae, which is a standard hatchery management practice in carp seed production (Jhingran & Pullin, 1985).

SEED PRODUCTION

The production of Indian Major Carp (IMC) and common carp (CC) fish seeds with high quality depends on several environmental and management factors. One of the major factors influencing successful seed production is the availability of natural plankton in the source water, particularly in dam or reservoir-fed hatchery systems. Plankton serves as a primary natural food source for early larval stages and significantly enhances survival, growth rate, and overall seed quality (Jhingran & Pullin, 1985). Adequate plankton density improves larval nutrition and reduces mortality during the critical nursery phase (Nandeesha & Singh, 1990). The seeds were subsequently produced and reared to different growth stages under controlled conditions, as proper nursery and rearing management practices play a crucial role in determining seed performance, uniform growth, and survival rate (Lucas & Southgate, 2012).

Collection of Fish Larvae

Larvae were collected from the happa and transferred to small containers to prevent fungal contamination from dead eggs, as early-stage fish larvae are highly susceptible to fungal infections such as Saprolegnia under crowded or poorly managed conditions (Bromage & Roberts, 1995). The water in the containers was changed frequently and maintained under continuous aeration to ensure optimal dissolved oxygen levels and water quality, which are critical for larval survival and normal development (Lucas & Southgate, 2012). During early development, larvae exhibited vertical or upside-down swimming behavior until swim bladder inflation occurred. Proper swim bladder development is essential for buoyancy regulation and normal horizontal swimming (Lucas & Southgate, 2012). For the first three days post-hatching, larvae did not require external feeding as they depended entirely on endogenous nutrition from the yolk sac reserves, which supply energy for growth and organ development (Kamler, 2008). After yolk sac absorption and swim bladder inflation, the larvae began swimming horizontally and actively developed functional mouthparts, enabling exogenous feeding. At this stage, they were fed cultured rotifers, which are widely recognized as suitable first live feed due to their small size, slow movement, and high digestibility for carp larvae (Lavens & Sorgeloos, 1996; Lucas & Southgate, 2012).

Toxicity assessment of partially purified Biosurfactant

The toxicity assessment of the partially purified biosurfactant obtained from Serratia rubidaea was performed using common carp larvae (Cyprinus carpio) as a model organism. Fish larvae are highly sensitive biological indicators and are widely used in aquatic toxicology studies due to their rapid developmental stages and susceptibility to environmental contaminants (Organisation for Economic Co-operation and Development [OECD], 2013). The larvae were selected immediately after mouth development to ensure uniform physiological condition during exposure (OECD, 2013). Approximately 20 fish larvae were distributed into six plastic containers containing 100 ml of dechlorinated tap water. Various concentrations of biosurfactant ranging from 100 ppm to 600 ppm (100–600 mg/L) were added along with live feed (rotifers), and the experiment was conducted for 48–72 hours at room temperature. A control group containing rotifers without biosurfactant was maintained under identical conditions. Acute toxicity was determined based on cumulative mortality recorded after 72 hours, following standard fish acute toxicity testing guidelines (Organisation for Economic Co-operation and Development, 2019).

Behavioral responses such as erratic swimming, surface gulping, loss of balance, and reduced activity were monitored at 3-hour intervals, as behavioral endpoints are considered early indicators of toxic stress in fish larvae (Rand, 1995). All treatments were performed in triplicate to ensure statistical reliability and reproducibility of results (OECD, 2013).

Antimicrobial activity of partially purified Biosurfactant against Clinical pathogens

The antimicrobial activity of the partially purified biosurfactant against clinical pathogens was determined using the agar well diffusion method, a widely accepted technique for evaluating antimicrobial susceptibility (Clinical and Laboratory Standards Institute, 2022; Balouiri et al., 2016). The clinical pathogens used were Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, and Vibrio cholerae. Muller–Hinton agar plates were prepared and sterilized by autoclaving at 121°C for 15 minutes following standard microbiological procedures (CLSI, 2022). The 24-hour-old bacterial cultures were inoculated using the lawn culture method to ensure uniform microbial growth (Balouiri et al., 2016). Using a sterile well cutter (3 mm diameter), wells were bored into the agar plates at regular intervals.

About 1 mg and 10 mg of biosurfactant were dissolved separately in 1 ml of DMSO in sterile Eppendorf tubes. Aliquots of 25 μl, 50 μl, 75 μl, and 100 μl of the prepared samples were dispensed into the respective wells using a micropipette. Tween 80 (25 μl) was used as a control to verify that the solvent did not produce inhibitory effects (Gudiña et al., 2016). The plates were incubated at 37°C for 24 hours, and the zones of inhibition were measured in millimeters to determine antimicrobial activity (CLSI, 2022).

RESULT:

Collection of brooder:               

Fig.collection of comman carp (Cyprinus carpio)

CONDITION FOR BROODER FISH:

Table:1 Physical and Reproductive Condition of Brooder Fish – Common Carp (Cyprinus carpio)

TABLE :2 Age and Weight of Brooder Fish – Cyprinus carpio

  Semi artificial fertilization method

Fig 2.  Chinese hatchery tank prior to place the male and female brooders

  SPWANING:

Fig 3: common carp egg in monsoon season

TABLE 3: SEED GROWTH:

TABLE 4: Different stages of growth and their mean period.

Physical parameters of larvae tank  water.

Toxicity assessment of partially purified Biosurfactant in larvae tank

Antimicrobial activity of partially purified Biosurfactant against Clinical pathogens

The antimicrobial activity of the partially purified biosurfactant was assessed against selected clinical pathogens using the agar well diffusion method. The biosurfactant demonstrated inhibitory effects against all tested organisms, with the extent of inhibition varying depending on the concentration applied. Distinct zones of inhibition were observed around the wells containing biosurfactant volumes of 25 µl, 50 µl, 75 µl, and 100 µl. An increase in the diameter of the inhibition zone was noted with increasing biosurfactant concentration, indicating a clear dose-dependent antimicrobial response. Lower volumes (25 µl) produced minimal inhibition, whereas higher volumes (75 µl and 100 µl) resulted in pronounced and well-defined zones.

Among the Gram-positive bacteria tested, Staphylococcus aureus and Streptococcus pyogenes exhibited greater sensitivity to the biosurfactant, showing comparatively larger inhibition zones. Listeria monocytogenes displayed moderate susceptibility. In contrast, Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae showed moderate inhibition, while Pseudomonas aeruginosa and Vibrio cholerae were comparatively less sensitive, particularly at lower biosurfactant concentrations.

The standard control, Tween 80 (25 µl), did not produce any significant inhibitory effect against the tested pathogens, confirming that the antimicrobial activity observed

Fig 4: Inhibition of antimicrobial activity