Assessing the Ecotoxicity of Pharmaceutical Residues: A Multi-Tissue Study on the Toxicological Effects of the Nitroimidazole Antibiotic Metronidazole in Goldfish (Carassius auratus)

Assessing the Ecotoxicity of Pharmaceutical Residues: A Multi-Tissue Study on the Toxicological Effects of the Nitroimidazole Antibiotic Metronidazole in Goldfish (Carassius auratus)

Slide 1: Title

Good morning everyone.

I am Swapnil Pal from the Department of Zoology, University of Calcutta.

Today I am going to present my dissertation entitled "Assessing the Ecotoxicity of Pharmaceutical Residues: A Multi-Tissue Study on the Toxicological Effects of the Nitroimidazole Antibiotic Metronidazole in Goldfish (Carassius auratus)."

This work was carried out under the supervision of Professor (Dr.) Sajal Ray in the Aquatic Toxicology Laboratory.

Slide 2: What are Drugs?

Before discussing antibiotics, let us first understand what a drug is.

A drug is any chemical substance, other than a nutrient, that is introduced into the body to prevent, diagnose, or treat diseases or to modify physiological functions.

Drugs can enter the body through injection, inhalation, ingestion, skin absorption, or sublingual administration.

Unfortunately, after use, many pharmaceuticals enter rivers and lakes through human excretion, improper disposal, and industrial discharge. These pharmaceutical residues can persist in aquatic ecosystems and cause endocrine disruption, kidney damage, oxidative stress, and antibiotic resistance in aquatic organisms.

Slide 3: Major Classes of Drugs

There are several important classes of drugs.

These include antidepressants, antibiotics, anticoagulants, antifungal drugs, antidiabetic drugs, and beta-blockers.

Among these, antibiotics are particularly important because they are extensively used worldwide and are frequently detected in aquatic environments.

Since antibiotics are biologically active even at low concentrations, they can negatively affect non-target aquatic organisms.

Slide 4: Classification of Antibiotics

Antibiotics are classified according to their chemical structure and mechanism of action.

Major classes include:

Beta-lactams

• Macrolides

• Tetracyclines

• Quinolones

• Aminoglycosides

• Sulphonamides

• Glycopeptides

• Oxazolidinones

• Nitroimidazoles

• Amphenicols

• Lincosamides

• Lipopeptides

Each class inhibits bacterial growth through different molecular mechanisms and therefore produces different toxic effects in aquatic animals.

Slide 5: Beta-lactam Antibiotics

Beta-lactam antibiotics inhibit bacterial cell wall synthesis by binding to penicillin-binding proteins.

Examples include Penicillin, Aztreonam, and Meropenem.

In fish, exposure causes delayed embryonic development, spinal curvature, tail deformities, pigmentation disorders, heart abnormalities, and sometimes death.

Slide 6: Macrolides

Macrolides interfere with intracellular calcium regulation and important signalling pathways including M.A.P.K and N.F kappa B.

Examples include Erythromycin, Azithromycin, and Clarithromycin.

Their toxic effects include oxidative stress, D.N.A damage, liver degeneration, cardiovascular toxicity, delayed hatching, and reduced growth.

Slide 7: Tetracyclines

Tetracyclines bind to the 30 S ribosomal subunit and inhibit bacterial protein synthesis.

Examples are Oxytetracycline, Chlortetracycline, and Lymecycline.

In fish they induce D.N.A damage, chromosome abnormalities, reduced haemoglobin and R.B.C count, gill degeneration, liver oxidative stress, and delayed hatching.

Slide 8: Quinolones

Quinolones inhibit D.N.A gyrase and topoisomerase I.V, producing irreversible D.N.A breaks.

Examples include Norfloxacin, Ofloxacin, and Cinoxacin.

These antibiotics increase oxidative stress, impair embryo development, reduce hatching rate, and produce cardiac abnormalities.

Slide 9: Aminoglycosides

Aminoglycosides bind strongly to bacterial ribosomes and disrupt protein synthesis.

Examples are Streptomycin, Neomycin, and Tobramycin.

In fish they generate reactive oxygen species, destroy sensory hair cells, delay hatching, and may even cause renal damage.

Slide 10: Sulphonamides

Sulphonamides inhibit folic acid synthesis by blocking dihydropteroate synthase.

Examples include Sulfamethoxazole, Sulfadimethoxine, and Trimethoprim.

Their toxic effects include oxidative stress, immune suppression, delayed hatching, increased heart rate, and mortality.

Slide 11: Glycopeptides

Glycopeptides inhibit bacterial cell wall synthesis by binding to D-Ala-D-Ala residues.

Examples include Vancomycin, Telavancin, and Oritavancin.

Fish exposed to these antibiotics exhibit impaired nervous system development, reduced swimming ability, shortened body length, abnormal organ development, and death.

Slide 12: Oxazolidinones

Oxazolidinones inhibit formation of the bacterial initiation complex required for protein synthesis.

Examples are Linezolid, Delpazolid, and Radezolid.

These antibiotics can induce liver steatosis, tumour formation, and carcinogenic effects in fish.

Slide 13: Nitroimidazoles

Nitroimidazoles damage bacterial D.N.A directly.

Examples include Metronidazole, Tinidazole, and Secnidazole.

In fish they produce D.N.A damage, gill injury, liver degeneration, increased erythrocyte count, and digestive tract damage.

Since Metronidazole is one of the most widely used nitroimidazole antibiotics, it became the focus of this study.

Slide 14: Amphenicols

Amphenicols inhibit protein synthesis by affecting the 50 S ribosomal subunit.

Examples include Chloramphenicol, Florfenicol, and Thiamphenicol.

Exposure causes erratic swimming, hepatic necrosis, reduced R.B.C count, increased W.B.C count, and oxidative stress.

Slide 15: Lincosamides

Lincosamides also interfere with protein synthesis.

Examples are Lincomycin and Clindamycin.

Fish exposed to these antibiotics develop brain malformations, elevated acetylcholinesterase activity, increased oxidative stress, and abnormal heart rate.

Slide 16: Lipopeptides

Lipopeptides damage bacterial membranes by forming pores.

Examples include Daptomycin and Polymyxin B.

In fish they reduce food intake, disturb respiration, and may ultimately cause death.

Slide 17: Why Metronidazole?

Metronidazole was developed during the 1950s and entered commercial use in 1960.

It is listed as an essential medicine by the World Health Organization and is one of the most commonly prescribed antibiotics worldwide.

However, it is poorly biodegradable and frequently contaminates aquatic ecosystems, where it affects beneficial microorganisms and damages fish organs.

Its mechanism involves entering bacterial cells, generating free radicals, and directly damaging D.N.A.

Slide 18: Materials and Methods

The experiment was conducted using healthy goldfish.

Fish were exposed to different concentrations of Metronidazole for predetermined exposure periods.

Multiple tissues including liver, gills, intestine, and brain were collected.

Biochemical antioxidant assays and haematological analyses were then performed to evaluate oxidative stress and tissue damage.

Slide 19: Biomarkers Used

Four antioxidant enzymes were analysed.

Xanthine Oxidase or X.O is a marker of reactive oxygen species generation.

Glutathione S-transferase or G.S.T is involved in detoxification.

Superoxide Dismutase or S.O.D converts superoxide radicals into hydrogen peroxide.

Catalase or Cat removes hydrogen peroxide by converting it into water and oxygen.

Together, these biomarkers provide a comprehensive picture of oxidative stress.

Slides 20 to 23: Results

The results clearly demonstrate tissue-specific responses.

X.O activity increased significantly, indicating enhanced oxidative stress.

G.S.T activity initially increased as a defence response but later declined under severe stress.

S.O.D activity changed differently among tissues, suggesting different antioxidant capacities.

Catalase activity also varied with exposure concentration and duration, showing failure of antioxidant defence in severely affected tissues.

Overall, the liver showed strong detoxification ability initially, whereas the gills were highly vulnerable during prolonged exposure.

Slide 24: Haematological Alterations

Blood analysis revealed significant changes.

Red blood cell counts initially decreased due to oxidative damage of erythrocytes.

Later, R.B.C production increased as a compensatory response to hypoxia.

White blood cell counts increased continuously, indicating persistent inflammation and immune activation.

These findings confirm systemic physiological stress.

Slide 25: Conclusion

From this study we conclude that Metronidazole causes severe oxidative stress in multiple organs of goldfish.

It disrupts antioxidant defence mechanisms, damages blood cells, impairs organ function, and produces tissue-specific toxicity.

The liver remains relatively resilient, while the gills become highly susceptible during prolonged exposure.

Overall, Metronidazole represents an important environmental pollutant capable of threatening aquatic ecosystem health.

Slide 26: Future Directions

Future research should investigate long-term exposure at environmentally relevant concentrations.

D.N.A damage studies should be incorporated to better understand neurotoxicity.

Combined toxicity with heavy metals should also be evaluated.

Finally, advanced wastewater treatment technologies should be developed to effectively remove Metronidazole before it reaches aquatic ecosystems.

Final Slide: Thank You

Thank you all for your kind attention.

I would be happy to answer any questions.

You have reached the end of the document.