DOI: 10.31038/AFS.2021354

Introduction

Edwardsiella tarda is one of the most significant bacterial pathogens to fish aquaculture. This Gram-negative, intracellular bacterium is the causal agent of the “edwardsiellosis” which cause generalized septicemia and lesions on internal organs. It has been reported worldwide in economically important fish species, including Japanese eel (Anguilla japonica), red sea bream (Pagrus major), yellowtail (Seriola quinqueradiata), channel catfish (Ictalurus punctatus), turbot (Scophthalmus maximus) and tilapia (Oreochromis sp.) [1,2]. This infection leads to enormous economic losses in cultured seawater and freshwater fish [1].

Different strategies have been attempted to treat and prevent E. tarda infections, including antibiotics and chemicals. However, overuse or abuse of these can result in the selection and development of drug resistant pathogens [3,4]. As a promising alternative, immunostimulants may represent a safe and effective treatment to enhance an antimicrobial response for preventing fish infection by a wide range of pathogens.

Synthetic growth hormone (GH) secretagogues (GHSs) consist of a family of ligands, initially termed GH-releasing peptides (GHRPs) [5] which are recognized by the GHS receptor (GHS-R). GHS-R is expressed predominantly in the brain and pituitary gland, and in immune system cells from mammals and fish, suggesting a relation between ligands for these receptors and the immune system [6]. Growth Hormone-Releasing Peptide 6 (GHRP-6) is a six amino acid synthetic peptide with demonstrated stimulatory effects on innate and adaptive immune system in teleost fish [7,8]. The aim of this study was to assess the antimicrobial effects of GHRP-6 against bacterial infection in tilapia, as well as to measure gene expression of some immune-related genes before and after E. tarda infection.

Materials and Methods

Fish and Peptide Source

Juvenile tilapias (Oreochromis niloticus, male, ~100 g) were obtained from the Aquaculture Research Station at the Center for the Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Fish were kept alive in aerated freshwater in 120 L tanks under a 12 h light: 12 h dark photoperiod. They were fed commercial dry diet for fish (CENPALAB, Habana, Cuba). Water temperature was maintained at 26-28°C. Fish were acclimated for one week before each experiment. All animal experiments were previously approved by the Ethics Committee of the Center for Genetic Engineering and Biotechnology, Havana, Cuba. The described work has been carried out in accordance with EU Directive 2010/63/EU for animal experiments.

GHRP-6 (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) with a purity > 99% was provided by Sigma-Aldrich, USA.

Fish Treatment with GHRP-6

Fish (26 tilapias per group) were injected by intraperitoneal route (i.p) during 7 days. One group of tilapias received GHRP-6 (0.2 µg/body weight (bwg)) in a total volume of 100 µL of PBS. The Control group was injected with the same volume of PBS. The dose employed for GHRP-6 was chosen based in previous experiments performed in the laboratory [9]. Spleen samples (n=6) were taken at 24 and 48 hours after the last injection. Tissue samples were stored in Ambion RNAlater (AppliedBiosystems, USA) at -20°C until use for RNA isolation.

Also, at 48 hours after the last peptide injection, a challenge was performed using an E. tarda strain by immersion bath as previously described by [10], with minor adjustments. Edwardsiella tarda strain was obtained from glycerol stock cultures stored at -80°C. Briefly, Nile tilapia juveniles (n=13 per group) were exposed to 108 CFU/mL of E. tarda (diluted in sterile PBS) by immersion bath during 1 h in a total volume of 40 L with aeration. After immersion bath, both groups were moved to 250 L aerated freshwater tanks in an isolated water flow system and kept in those conditions until sampling of gills and spleen (8 fish per group). The rest of the animals were daily checked until day 5th day when mortality started to occur. Two additional tanks with fish (n=7 per group: PBS and GHRP-6) were used as non-challenge controls of mortality. After the challenge concluded, waste water was treated with chlorine to avoid the releasing of live bacteria.

Spleen was sampled at 24 and 48 hours after challenge (hac) for gene expression analysis, as described above. To evaluate the antimicrobial effects of GHRP-6 on gills, samples were taken at 48 hours after the challenge (hac), under aseptic conditions and washed in 1 mL of 0.9% NaCl. Afterwards, this wash was serially diluted in sterile PBS, plated in tryptic soy agar (TSA) (Merck) and grown overnight at 37°C. The identity of the colonies grown in specific culture medium was confirmed by an API20E test (Biomérieux) and Gram staining.

Gene Expression Analysis

Tissue samples were processed on a Tissue Lyser unit (Qiagen, Hilden, Germany), extracting total RNA with RNeasy® Plus (QIAGEN GmbH, Germany) using the Quiacube platform. RNA purity and yield were determined using a NanoDrop Spectro-photometer (NanoDrop Technologies, USA). First-strand complementary DNA (cDNA) was synthesized from 1 μg total RNA using Superscript III First-Strand Synthesis Supermix for RT-PCR (Invitrogen Technologies, Carlsbad, California, USA) following fabricant ́s instructions.

Transcripts levels in the sampled organs were assessed by qPCR analysis. Gene’s sequences were obtained from the US National Center for Biotechnology Information database (NCBI). Specific primers were designed, using the web application Primer3 [11]. Each experimental sample was run in triplicate on 96-well plates and qPCR reactions were completed on the LightCycler® 480 II (Roche). All PCR reactions were of 20 µL volume and contained: 10 µL of LightCycler® 480 SYBR Green I Master 2x (Roche, Germany), 300 nM each primer (10 pmol/µL) and 4 µL of cDNA (50 ng/µL diluted 1:25 in RNase free water). For each plate, triplicate wells of a calibrator and RNA only control were also present. The program used for all qPCR reactions was as follows: pre-incubation at 95°C for 5 min followed by 50 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 s and extension at 72°C for 30 s. A melting curve was completed for every run from 72 to 95°C with a read every 5 s. Product specificity was determined through single PCR melting peaks. All qPCR data was analyzed using REST 2009 v2.0.13 [12]. Differences were expressed as fold changes. Gene expression was normalized to the references genes EF-1α and β-actin.

Statistical Analysis

The effects of GHRP-6 on the number of CFU in gills, was assessed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA). The normal distribution of data was analyzed with D’Agostino Pearson’s test and the variance homogeneity with Bartlett’s test. A Student’s t parametric test was performed as data followed normal distribution for comparisons in each time point. For qPCR analysis, reactions efficiency was calculated using LinReg software and data were analyzed using REST 2009 software v2.0.13 [12,13]. This software calculates the relative expression to a control condition after normalize with the efficiency of the selected references genes. Differences were expressed as fold changes relative to Control group. A significance level of 0.05 was used for all analyses.

Results

After the last peptide injection with GHRP-6, the transcripts levels of IL-1β relative to Control group were up-regulated in spleen both at 24 (2.37 ± 0.58) and 48 (2.27 ± 1.64) hours. Also, Oreochromicin III was significantly up-regulated at 48 hours after treatment (5.99 ± 3.48). However, no significant differences were observed for Oreochromicins I and II and Granzyme transcripts (Figure 1).

Figure 1: Gene expression analysis in spleen of tilapia (Oreochromis sp.) after treatment with GHRP-6 during seven consecutive days by i.p injection. Samples were taken at 24 and 48 hours after the last peptide injection. Fold change (FC) with respect to Control group were calculated by REST 2009 (FC>1 up-regulated; FC<1 down-regulated) and were expressed as mean ± SD (n=6). The asterisks indicate statistically significant differences relative to Control group, *(p < 0.05).

Once confirmed the positive modulation of a pro-inflammatory cytokine and an antimicrobial peptide in spleen, we evaluated the antimicrobial effect promoted by GHRP-6 after a challenge trial with E. tarda. With this aim, fish were challenged 48 hours after the peptide treatment with an E. tarda strain. Spleen samples were collected at 24 and 48 hours after challenge, while gills washes and were taken at 48 hours.

GHRP-6 treatment exerts a differential modulation over Oreochromicins, Granzyme and IL-1β mRNA expression profiles in spleen after E. tarda infection. Oreochromicin I was up-regulated (7.49 ± 3.32) at 48 hours, while, Oreochromicins II and III were down-regulated at 24 hours (0.12 ± 0.17 and 0.09 ± 0.07, respectively). On the other hand, IL-1β was statistically down-regulated (0.067 ± 0.002) at 48 hours (Figure 2).

Figure 2: Gene expression analysis in spleen of tilapia (Oreochromis sp.) after treatment with GHRP-6 (during 7 consecutive days by i.p injection) and challenge with an Edwardsiella tarda strain. Twenty-four and 48 hours after the last peptide injection, fish from Control and GHRP-6 treated groups (13 fish per group) were exposed for one hour to 1 × 10⁸ CFU/mL of E. tarda by immersion bath. Gene transcription was analyzed by qPCR in spleen sampled at 24 and 48 h after challenge. Fold change (FC) with respect to Control group were calculated by REST 2009 (FC>1 up-regulated; FC<1 down-regulated) and were expressed as mean ± SD (n=8). The asterisks indicate statistically significant differences relative to Control group, *(p < 0.05), **(p < 0.01) and ***(p < 0.001).

In gills washes, there were statistically significant lower CFU/mL of bacteria in group treated with GHRP-6 (6.8 x 104 ± 8.6 x 103 CFU/mL) compared to the Control group (3.9 x 105 ± 6.4 x 104 CFU/mL) (Figure 3). We observed clinical signs of the disease in some animals of the Control group such as loss of pigmentation and petechial hemorrhage in fin and skin, aspects reviewed by [1] as evidence of the infection. We started to observed mortality in Control group of infected fish since the 5th day, being 4/7 in Control group and 0/7 in GHRP-6 treated group.

Figure 3: Gills’ bacterial load in Control and GHRP-6 treated groups after treatment with GHRP-6 (during 7 consecutive days by i.p injection) and challenge with an Edwardsiella tarda strain. Forty-eight hours after the last peptide injection, fish from Control and GHRP-6 treated groups (13 fish per group) were exposed for one hour to 1 × 10⁸ CFU/mL of E. tarda by immersion bath. Total number of CFU/mL in gills washes were counted at 48 h after challenge. Data represent the mean ± SD (n=8) of three independent experiments. The analysis of data was performed using a Student’s t test. The asterisks represent statistically significant differences between groups, *(p < 0.05).

Discussion

Edwardsiella tarda is a Gram-negative intracellular pathogen which causes enormous economic losses in cultured seawater and freshwater fish [14]. Infection by E. tarda may produce severe lesions in internal organs such as spleen [15-17]. Gills constitutes one of the entry routes of E. tarda infections in fish [18], thus, the use of immunostimulants with antimicrobial effects for preventing infestations could be promising.

Previously, it has been demonstrated that GHRP-6 is a growth hormone secretagogue that improve different innate and adaptive immune system parameters in aquatic organisms [7,8,19]. More recently, we provided new evidence of a direct link between GHRP-6 treatment and enhanced antimicrobial peptides transcription in tilapia [20]. In this study we shed light over the effect of this peptide as stimulator of the antimicrobial immune response in tilapia with and without the presence of a challenge with E. tarda.

Spleen constitutes one of the major lymphoid tissues in fish and also is affected by the infection with bacterial pathogens, such as E. tarda [21]. GHRP-6 statistically up-regulated the mRNA levels of IL-1β in tilapia spleen. Our results are in agreement with previous studies where IL-1β was up-regulated after the administration of immunostimulants [22,23].This cytokine is a critical mediator of the inflammatory response against microbial invasion and can stimulate the immune response by activating lymphocytes or by inducing the release of other cytokines capable of triggering macrophages, NK cells and lymphocytes (Yuan et al., 2008; [18,24]. Also, Oreochromicin III was up-regulated after the treatment with the GHS. Previous studies have demonstrated that this antimicrobial peptide has immunomodulatory effect in mammals. Oreoch III stimulated the release of IFN-γ from mice splenocytes [25].

In the current study, Oreochromicins I, II and III were differentially regulated by GHRP-6 in spleen after the challenge with E. tarda. Therefore, Oreochromicins could be involved in the elimination of this pathogen in tilapia spleen and other tissues. These antimicrobial peptides belong to the piscidin-like family and are expressed in different cell types such as neutrophils, granular eosinophils, monocytes and macrophages [26]. [27] demonstrated strong antimicrobial activity of Oreochromicin II against E. tarda in vitro. Recent studies revealed that GHRP-6 stimulates the antimicrobial immune response against Pseudomonas aeruginosa in tilapia and up-regulated the transcription of Oreochromicins as well in the presence of this pathogen [20].

Our results revealed that the treatment with GHRP-6 statistically decreased the bacterial load of E. tarda in gills washes 48 hours after the challenge as we observed a significant up-regulation of the mRNA levels of Oreochromicin I in spleen. Fish´s gills possess different cells populations and a mucus layer associated to it, which contains different antimicrobial molecules that contribute to the bacterial clearance [28,29]. The obtained results suggested that GHRP-6 may stimulate the antimicrobial response which limits the growth of the bacteria in gills, and thus could restrict the bacterial infection. Previous reports have shown that AMPs can protect against infections by E. tarda in fish [30]. Further studies will be conducted in order to gain knowledge at the histological level. In addition, after the challenge, we observed a down-regulation of IL-1β. Previous studies have demonstrated that when the expression of this cytokine decreases significantly at 48 hours, it is possible that this has increased within a few hours of completing the challenge. For example, [31], showed that in a challenge with Aeromonas salmonicida by intraperitoneal injection, IL-1β levels in spleen were up-regulated at 2 and 6 hours after the infection, but at 24 hours did not showed significant differences. Moreover, in a challenge carried out with E. tarda by intramuscular injection, an increase in IL-1β expression was obtained at 6 and 24 hours, in animals previously immunized with the flagellar protein FlgD by intramuscular injection [32-34].

The results of this trial suggest that the induction of IL-1β could be transient and that other effector components of innate immunity are participating in the protection of the organism. Various authors showed that when the expression of this cytokine decreases significantly at 48 hours, it is possible that this has increased within a few hours of completing the challenge.

In summary, our results suggested novel roles of GHRP-6 in tilapia as possible immunostimulant, which is able to promote the antimicrobial activity against some Gram-negative intracellular bacterial infections, such as E. tarda. Currently we are planning to evaluate the protective effect of this GHS at the histological level when administered in the presence of pathological bacteria such as E. tarda. Also, we are developing an oral formulation in order to evaluate its immunostimulant effects in long term experiments as part of the diet and its effects on guts bacterial load after a bacterial challenge. These studies will allow us to gain a deeper knowledge about the molecular basis of GHRP-6 mechanism in relation with antibacterial response in fish for its potential application in aquaculture.

Author Contributions

LH performed majority of experiments, contributed to experimental design, wrote the first draft and reviewed the final version of the manuscript. HC, ANR, DP, AM, LB, FH, OR, SP contributed with the development of the experiments. JV contributed with the development of the experiments and reviewed the final version of the manuscript. ME, contributed to experimental design and reviewed the final version of the manuscript. RM contributed to experimental design, wrote the first draft of the manuscript and reviewed the final version of the manuscript.

Data Availability

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Keywords

GHRP-6, Antimicrobial activity, Edwardsiella tarda, Tilapia

References

1. Park Bin S, Aoki T, Jung TS (2012) Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish. Veterinary Research 43: 1-11.
2. Baba E, Acar Ü, Öntaş C, Kesbiç OS, Yılmaz S (2016) Evaluation of Citrus limon peels essential oil on growth performance, immune response of Mozambique tilapia Oreochromis mossambicus challenged with Edwardsiella tarda. Aquaculture 465: 13-18.
3. Cha YJ, Lee CR., Kwon JY, Kang YJ (2017) Protective effects of CpG-ODN 2007 administration against Edwardsiella tarda infection in olive flounder (Paralichthys olivaceus). Fish and Shellfish Immunology 68: 327-331. [crossref]
4. Na-Phatthalung P, Teles M, Voravuthikunchai SP, Tort L, Fierro-Castro C (2018) Immune-related gene expression and physiological responses in rainbow trout (Oncorhynchus mykiss) after intraperitoneal administration of Rhodomyrtus tomentosa leaf extract: A potent phytoimmunostimulant. Fish and Shellfish Immunology 77: 429-437. [crossref]
5. Momany FA, Bowers CY, Reynolds GA, Chang D, Hong A, et al. (1981) Design, Synthesis, and Biological Activity of Peptides which Release Growth Hormone in Vitro. Endocrinology 108: 31-39.
6. Hattori N (2009) Growth Hormone & IGF Research Expression, regulation and biological actions of growth hormone ( GH ) and ghrelin in the immune system. Growth Hormone & IGF Research 19: 187-197. [crossref]
7. Martinez R, Carpio Y, Morales A, Lugo JM, Herrera F, et al. (2016a) Oral administration of the growth hormone secretagogue-6 (GHRP-6) enhances growth and non-specific immune responses in tilapia (Oreochromis sp.). Aquaculture 452: 304-310.
8. Martínez R, Hernández L, Gil L, Carpio Y, Morales A, et al. (2017a) Growth hormone releasing peptide-6 enhanced antibody titers against subunit antigens in mice (BALB/c), tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus). Vaccine 35: 5722-5728. [crossref]
9. Martinez R, Núñez de Villavicencio-Díaz T, Sánchez A, Noda J, Gil L, et al. (2016b) Comparative proteomics analysis of growth hormone secretagogue A233 treatment effect in J774A.2 murine macrophages cells. Biochem Biophys Rep 5: 379-387. [crossref]
10. Velázquez J, Acosta J, Herrera N, Morales A, González O, et al. (2017) Novel IFNγ homologue identified in Nile tilapia (Oreochromis niloticus) links with immune response in gills under different stimuli. Fish and Shellfish Immunology 71: 275-285. [crossref]
11. Rozen S, Skaletsky H (2000) Primer3. Methods Mol Biol 132: 365-386.
12. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST(C)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30: 36. [crossref]
13. Camacho-Rodiguez H, Guillén-Pérez IA, Roca-Campaña J, Baldomero-Hernández JE, Tuero-Iglesias, et al. (2018) Heberprot-P´s Effect on Gene Expression in Healing Diabetic Foot Ulcers. MEDICC Review 20: 10-14. [crossref]
14. Mohanty BR, Sahoo PK (2010) Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection. Fish and Shellfish Immunology 28: 613-621. [crossref]
15. Mohanty BR, Sahoo PK (2007) Edwardsiellosis in fish: a brief review. Journal of Biosciences 32: 1331-1344. [crossref]
16. Zhou Z, Sun L (2016) Edwardsiella tarda-Induced Inhibition of Apoptosis: A Strategy for Intracellular Survival. Frontiers in Cellular and Infection Microbiology 6: 76. [crossref]
17. Lee W, Ahn G, Young J, Min S, Kang N, et al. (2016) A prebiotic effect of Ecklonia cava on the growth and mortality of olive flounder infected with pathogenic bacteria. Fish and Shellfish Immunology 51: 313-320. [crossref]
18. Pressley ME, Phelan PE, Eckhard Witten P, Mellon MT, Kim CH (2005) Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Developmental and Comparative Immunology 29: 501-513. [crossref]
19. Martínez R, Carpio Y, Arenal A, Lugo JM, Morales R, et al. (2017b) Significant improvement of shrimp growth performance by growth hormone-releasing peptide-6 immersion treatments. Aquaculture Research 48: 4632-4645.
20. Hernández L, Camacho H, Nuñez-Robainas A, Palenzuela DO, Morales A, et al. (2021) Growth hormone secretagogue peptide-6 enhances oreochromicins transcription and antimicrobial activity in tilapia (Oreochromis sp.). Fish and Shellfish Immunology 119: 508-515. [crossref]
21. Velázquez J, Acosta J, Lugo JM, Reyes E, Herrera F, et al. (2018) Discovery of immunoglobulin T in Nile tilapia (Oreochromis niloticus): A potential molecular marker to understand mucosal immunity in this species. Developmental and Comparative Immunology 88: 124-136. [crossref]
22. Nootash S, Sheikhzadeh N, Baradaran B, Oushani AK, Maleki Moghadam MR, et al. (2013) Green tea (Camellia sinensis) administration induces expression of immune relevant genes and biochemical parameters in rainbow trout (Oncorhynchus mykiss). Fish and Shellfish Immunology 35: 1916-1923. [crossref]
23. Huang X, Hu B, Yang X, Gong L, Tan J, et al. (2019) The putative mature peptide of piscidin-1 modulates global transcriptional profile and proliferation of splenic lymphocytes in orange-spotted grouper (Epinephelus coioides). Fish and Shellfish Immunology 86: 1035-1043. [crossref]
24. Mehrabi Z, Firouzbakhsh F, Rahimi-Mianji G, Paknejad H (2019) Immunostimulatory effect of Aloe vera (Aloe barbadensis) on non-specific immune response, immune gene expression, and experimental challenge with Saprolegnia parasitica in rainbow trout (Oncorhynchus mykiss). Aquaculture 503: 330-338.
25. Acosta J, Carpio Y, Valdés I, Velázquez J, Zamora Y, et al. (2014) Co-administration of tilapia alpha-helical antimicrobial peptides with subunit antigens boost immunogenicity in mice and tilapia (Oreochromis niloticus). Vaccine 32(2): 223-229. [crossref]
26. Valero Y, Saraiva-fraga M, Guardiola FA (2018) Antimicrobial peptides from fish : beyond the fight against pathogens. Reviews in Aquaculture 12: 1-30.
27. Acosta J, Montero V, Carpio Y, Velázquez J, Garay HE, et al. (2013) Cloning and functional characterization of three novel antimicrobial peptides from tilapia (Oreochromis niloticus). Aquaculture 372-375: 9-18.
28. Koppang EO, Kvellestad A, Fischer U (2015) Mucosal Health in Aquaculture Fish mucosal immunity: gill. In Mucosal Health in Aquaculture. Elsevier.
29. Cabillon N, Lazado C (2019) Mucosal Barrier Functions of Fish under Changing Environmental Conditions. Fishes 4: 2.
30. Liang Y, Guan R, Huang W, Xu T (2011) Isolation and identification of a novel inducible antibacterial peptide from the skin mucus of Japanese Eel, Anguilla japonica. Protein Journal 30: 413-421. [crossref]
31. Feng CY, Johnson SC, Hori TS, Rise M, Hall JR, et al. (2009) Identification and analysis of differentially expressed genes in immune tissues of Atlantic cod stimulated with formalin-killed, atypical Aeromonas salmonicida. Genomics 37: 149-163. [crossref]
32. Zhang M, Wu H, Li X, Yang M, Chen T, et al. (2012) Edwardsiella tarda flagellar protein FlgD: A protective immunogen against edwardsiellosis. Vaccine 30: 3849-3856. [crossref]
33. Costa AA, Leef MJ, Bridle AR, Carson J, Nowak BF (2011) Effect of vaccination against yersiniosis on the relative percent survival, bactericidal and lysozyme response of Atlantic salmon, Salmo salar. Aquaculture 315: 201-206.
34. Xu T, Zhang XH (2014) Edwardsiella tarda: An intriguing problem in aquaculture. Aquaculture 431: 129-135.

DOI: 10.31038/AFS.2021353

Abstract

Physico-chemical parameters, mollusc distribution and diversity in Shiroro Lake, Minna, Niger State was investigated between the months of March and August, 2017 spanning through the wet and dry seasons. Water samples for physico-chemical parameters and snails samples were collected and identified on monthly basis following standard methods. From the results; the temperature ranged from 26.5-32°C. The pH was all basic throughout the period of the study which ranged from 8.5-9.2 across all the stations. Dissolved Oxygen concentration ranged from 2.6-5.2 mg/L, while the BOD ranged from 2.2-4.9 mg/L. Nitrate level (0.43-0.83 mg/L) was high, while phosphate concentration (0.24-0.42 mg/L) was relatively low in all the sampling stations. A total number of 7 snail species were encountered. Station 3 had the highest number (403), station 2 with 363 species and station 1 had the lowest with 345 species. The family Viviparidae and Bithyniidae has 2 species each. The Bellamya phthinotropis has the highest number of species followed by Bellamya capillata; and the lowest was recorded among the species of Gebtella barthi. In general, the abundance of snails was higher during the rainy season than during the dry season. Taxa richness determined as Margalef index showed significant difference among the sampling stations. Similarly, diversity indices (Shannon Wiener, Simpson, dominance) also showed significant difference among the sampling stations. Lower values of diversity and Eveness indices were recorded at station 1. Station 2 had the highest record of diversity and eveness indices. Shiroro Lake is moderately organically polluted and adequate measures should be taken to check-mate this.

Keywords

Shiroro Lake, Physico-chemical parameters, Molluscs, Pollution and water quality

Introduction

Seasonal variations, pollution and its effects have over the years influenced the chemical contents of water and the survival of biotas in the aquatic environment. This is why it is difficult to understand the biological phenomenon fully because the chemistry of water levels tells much about the metabolism of the ecosystem and explains the general hydro-biological relationship (Basavaraja et al., 2011). Due to the pressures of increasing population and developing economy all over the world, the present situation of water quality management is far from satisfactory (Sawaya et al., 2003). Organic pollution is rampant in municipal water bodies posing health hazards to the neighbouring communities. Also, faecal pollution of drinking water causes water borne disease which has led to the death of millions of people [1]. The quality of drinking water is a complex issue and vital elements of public health. Poor water quality is responsible for the death of an estimated few million children annually (Holgate, 2000). The interaction of physical and chemical properties of water have a significant role in the composition, distribution and abundance of aquatic organisms which are therefore, used to determine the water quality and structural composition of aquatic community (Youne et al., 2003). Some of the major physico-chemical parameters that indicate water quality are the dissolved oxygen (DO) and biological oxygen demand (BOD). The dissolved oxygen is important in the natural self-purification capacity of the river; and the BOD is often used as a measurement of pollutants in natural and waste waters and to assess the strength of waste, such as sewage and industrial effluent waters [2]. BOD is an important parameter of water indicating the health scenario of freshwater bodies [3].

Gastropods are single-valve, soft-bodied class of animals in the phylum Mollusca. It is the largest, extremely diverse taxa that includes over 40,000 species of which 5,000 are fresh water snails found in wetlands like lakes, ponds and streams worldwide. Freshwater snails are an important food source for many fish, turtles, and other species of wildlife. As a result of their sensitivity to certain chemicals, many species are excellent water-quality indicators. Over the years, conservation and recovery efforts for freshwater snails include artificial culture or heliculture, water pollution control, and most importantly, habitat protection and restoration. Cleaning waterways not only improves the habitat for snails and other aquatic life, but it also improves the quality and supply of water for human consumption. Dam construction and other channel modifications, siltation, and industrial and agricultural pollution have all degraded the river habitats on which most species depend. As a result, the species richness and the abundance of freshwater snails have declined. Likewise, anthropogenic activities around the reservoir are on the increase, further compounding the problems and effects of pollution on the biota living within the aquatic habitat.

Shiroro Lake was constructed for domestic consumption within the Shiroro Local Government Area of Niger State and its environment. But there is little or no information about the snail’s distribution, diversity, and some selected physico-chemical parameters. This present research would serve as baseline information on the distribution, diversity of snails and changes in some physico-chemical parameters of the lake.

Materials and Methods

Description of the Study Area

Shiroro Lake was created with major objective of providing domestic water to Shiroro Local Government and its environment; however, fishing and irrigation have become other established uses of the reservoir. The area has a tropical climate with mean annual temperature, relative humidity and rainfall. The climate presents two distinct seasons: a rainy season and a dry season. The vegetation in the area is typically grass dominated savanna with scattered trees. The people are mostly engaged in agriculture, trading, artisanship and civil service for their living. Shiroro Lake was created in May, 1984 by damming the Kaduna River at Shiroro village, Niger State, Nigeria. Its coordinate is: Latitude 9.9724, Longitude 6.83532. The reservoir has an estimated surface area of 312 km² and a mean depth of 22.4 meters and continues to grow. It is now the second largest man-made lake in Nigeria followed by Jebba. The Shiroro Lake, like most other large man-made lakes in Nigeria and throughout the tropics, was expected to provide favourable conditions for large scale fish production and fishery development in Nigeria.

Water Sampling

Monthly sampling of the three study stations were carried-out from March to August, 2017 during the period of wet and dry seasons. Water samples were collected using 1 L plastic container from three sampling sites and transported to the Laboratory of the Department of Water, Aquaculture and Fishery Technology (WAFT), Federal University of Technology, Minna. Parameters like temperature, total dissolved solid, dissolved oxygen, pH and electrical conductivity were measured immediately from the sampling sites (Figure 1).

Figure 1: Map of the sampling stations of the Shiroro Lake, Niger State, Nigeria.

Snails Collection

Snails were collected from the study areas in the three sampling sites with the use of scoop net. Collected snails were transferred to a 1 L well labeled sample bottle with a cork and was preserved in 10% formalin. The samples were taken to the laboratory, viewed with the hand lens and subsequently identified using modified keys as described by [4] and pictorial diagrams.

Physico-chemical Parameters Analyses

Water Temperature

Water temperature at various sites were determined using mercury-in-glass thermometer which was immersed in water 6 cm below the water surface and left to stabilize for about two-five minutes before the readings were taken and recorded in °C.

DO, BOD, TDS, pH and EC Determination

Dissolved oxygen was determined with Hanna instrument (Hanna microprocessor pH /EC/TDS, P.R. (1970). This was done in duplicate for each site and each month of the sampling periods. The BOD was also determined using Hanna instrument after the samples had been incubated in the dark at laboratory temperature for five days. These were measured in mg/L. The Total Dissolved Solids (TDS in mg/L) and Electrical Conductivity (EC in µS/cm) were determined by the same instrument after rinsing in distilled water each time. The probe meter was standardized with buffer solutions of pH 4.0, 7.0, 9.0 before taking the reading for the pH in each station.

Sodium

Stock sodium was prepared by measuring 2.542 g of dried NaCl which was then diluted with a litre of distilled water. Intermediate sodium solution was prepared from stock solution by diluting 10 ml of stock solution with 100 ml distilled water. Standard sodium solution was then prepared from the intermediate sodium solution by diluting 10 ml of intermediate sodium solution with 100 ml distilled water. This was then used to prepare various concentrations in the range of 0.1 to 1.0 mg/L. The emission of these various concentrations was determined with a flame photometer at 589 nm. The Na content of the water sample was determined by measuring their emission with the flame photometer.

Potassium

Stock solution was prepared by dissolving 1.907 g of dried KCl crystal in distilled water which was then made up to 1 litre. Intermediate potassium solution was prepared by diluting 10 ml of stock potassium solution with 100 ml distilled water. Standard potassium solution was prepared by mixing 10 ml of intermediate potassium with 100 ml distilled water. Various concentrations in the range of 0.1 to 1.0 mg/L were prepared from the standard and were measured with flame photometer at 768 nm.

Nitrate

The nitrate concentration in the sampled water was determined using phenol disulphonic acid method. This method was carried out using spectrophotometer, laboratory glassware, hot water bath and reagents. Phenol disulphonic acid; 25 g of white phenol was dissolved in 150 ml (concentrated) and 85 ml of concentrated sulphuric acid was further added. The solution was heated until it dried out. 25 ml of water sample was placed in a porcelain basin and was evaporated to dryness on a hot water bath. 0.5 ml of phenol disulphonic acid (reagent 1) was then added to the residue and stirred with glass spatula. 5 ml of distilled water was added and 1.5 ml of potassium hydroxide solution (reagent 2) was also added. The mixture was thoroughly stirred by mixing. It turned yellow indicating the presence of Nitrate. The absorbance was read using spectrophotometer at 410 nm [5].

Phosphate

The concentration of phosphate in the water sample was determined by placing 25 ml of water sample in an Erlenmeyer flask and evaporated to dryness. The residue was cooled and dissolved in 1 ml of 70% perchloric acid (reagent 1). The flask was heated gently until the contents became colourless. It was cooled and 10 ml of distilled water was added together with 2 drops of Phenolphtalein indicator (reagent 2)_ this was prepared from 1.0 g of phenolphthalein dissolved in 100 ml of ethyl alcohol and 100 ml of distilled water. The above was titrated against sodium hydroxide solution (reagent 3) which was prepared by dissolving 4.0 g of the sodium hydroxide in 100 ml distilled water until there was an appearance of a slight pink colour. Volume was made up to 25 ml by adding distilled water; 1 ml of ammonium molybdate solution was added (reagent 4 made from 62 ml concentrated sulphuric acid and 80 ml distilled water and allowed to cool. 5 g of ammonium molybdate was then dissolved separately in 35 ml of distilled water and mixed with the sulphuric acid solution to 200 ml). 3 drops of stannous chloride solution (reagent 5_this was made from 0.5 g stannous chloride and dissolved in 2 ml hydrochloric acid and then, diluted to 20 ml with distilled water and used fresh). A blue colour indicates the presence of phosphate. The absorbance was recorded on spectrophotometer 690 nm after 10 minutes.

Data Analysis

One way Analysis of Variance (ANOVA) was used to determine the monthly variations in physico-chemical parameters using SPSS IBM (Version 20 for window) statistical package at P<0.05 level of significance. Taxa richness (Margalef and Menhnick indices), Diversity (Shannon and Simpson dominance indices), Eveness indices and Huctchenson T-test for inter-site comparison were determined using the computer basic programme SP DIVERS.

Results and Discussions

Monthly Variation in Physico-chemical Parameters of the Sampling Stations of Shiroro Lake, Minna Niger State

Water Temperature

The highest water temperature was recorded in station 1 with 30.5°C in the month of May and the lowest was recorded at station 1 in the month of August (Table 1).

DO. The highest dissolved oxygen was recorded in station 1 with 5.4 mg/L in the month of March and the lowest was recorded at station 2 in the month of August (Table 1).

BOD. The highest BOD was recorded at station 1 with 5.2 mg/L in the month of March and the lowest was recorded at station 2 in the month of August (Table 1).

TDS. The highest TDS was recorded in station 1 with 108 mg/L in the month of June and the lowest was recorded at stations 2 and 3 in the month of August (Table 1).

pH. The highest pH was recorded in station 1 with 10 in the month of March and the lowest was recorded at station 3 in the month of August (Table 1).

Electrical Conductivity. The Electrical conductivity for the three stations was the same through-out except for the Electrical conductivity of station 1 which increased slightly from 0.03 to 0.04 µS/cm in June (Table 1).

Sodium. The highest Sodium concentration was recorded in station 1 with 8.83 mg/L in the month of July and the lowest was also recorded at station 1 in the month of May (Table 1).

Potassium. The highest potassium concentration was recorded in station 3 with 4.11 mg/L in the month of March and the lowest was recorded at station 2 in the month of April (Table 1).

NO₃. The highest Nitrate concentration was recorded in station 3 with 0.83 mg/L in the month of July and the lowest was recorded at station 2 in the month of March (Table 1).

PO₄. The highest phosphate concentration was recorded in station 3 with 4.11 mg/L in the month of March and the lowest was recorded at station 2 in the month of April (Table 1).

Table 1: Summary of the physico- chemical parameters of the study stations in Shiroro Lake, Niger State from March to August, 2017. The highest and lowest values obtained during the sampling periods are indicated in parenthesis.

Abundance and diversity of snail species collected from Shiroro Lake. Total number of 7 species of snails was encountered during the study period. 2 species of Melanoides polymorpha were found in stations 2 and 3 but none in station 1. Ballamya phthinotropis was the major species with relative aboundance of 413 species (37%) followed by Ballamya capillata with 346 species (31%), Afrogyrus rodriguezensis with the total number of 178 species (16%) and Melanoides polymorpha with the lowest number of species (0.4%) (Table 2). Shannon index and Eveness index analyses showed that there were significant differences (P< 0.05) among the stations (Table 3).

Table 2: Spatial variations in snail species collected from Shiroro Lake, Niger State from March to August, 2017.

Table 3: Taxa richness, Diversity, Eveness and Dominance Indices of Snail species collected from Shiroro Lake, Niger State from March to August, 2017.

Taxa-S, Dominance_ D, Simpson_1-D, Shannon_H and Eveness_e^H/S stand for Taxa richness, Dominance Diversity, Simpson indices, Shannon indices and Eveness indices respectively.

Discussion

During the dry season the water temperature recorded was high in March, April, and May, 2017. This may be due to the increase in solar radiation (which is a usual phenomenon during dry season) during the period of the study. The mean water temperature obtained in this study was typical of tropical inland fresh water and river. This is in line with the findings of [6]. This is also in agreement with the studies of [7], [8] and [9] who also recorded high temperatures during the dry season. In addition, the relatively low temperature recorded in the months of June, July, August, 2017 may be due to the onset of the raining season and increasing water volume. This finding is also in agreement with the reports of [7] and [8].

The mean dissolved oxygen values obtained during the period of the study for the stations were low. The low level of DO probably indicated polluted nature of the water body. Similar low level of DO was reported in Dal Lake, Kashmir [10]. Monthly variations in DO for all the three stations were fluctuating. This fluctuation in the levels of DO could be as a result of differential influx of pollutants as run-offs from the neighbouring communities. It could also be due to rainfall regime pattern.

The BOD values indicate the extent of organic pollution in the aquatic system which affects the water quality [10]. Based on the BOD classification of [5], the mean BOD values in station 1 was high (2.7 mg/L), while stations 2 and 3 were low (2.4 mg/L) and (2.2 mg/L), respectively compared to that of station 1. The fluctuations in the BOD of each station and month probably indicate that the water in these stations was moderately polluted. This may be due to anthropogenic activities around the lake and the presence of the market at the lake side which may have led to organic pollution.

The monthly variations in Nitrate level were relatively high in all the sampling stations with the highest value of 0.83 mg/L in the month of July. Slightly higher range of values was reported in the studies of [12] in Bahir Dar Gulf of lake Tana, Ethiopia and [13] in a perturbed Tropical Stream in the Niger Delta, Nigeria; and [9] in River Galma, Zaria with 0.92-4.18 mg/L, 0.22-2.87 mg/L, 0.03 ± 0.0-1.11 ± 0.04 mg/L, respectively). Furthermore, the mean phosphate concentration ranged from 0.24-0.42 mg/L. This is higher than the mean values of 0.01 ± 0.01-0.20 ± 0.01 mg/L reported by [9]. Lower levels of phosphates, sulphates, nitrates indicate low level of organic pollution. The highest sodium content was recorded in July with 8.83 mg/L. This is probably because the most common source of elevated sodium levels in the lake water are: erosion of salt deposit and sodium bearing rock minerals, infiltration of surface water contaminated by road salt, irrigation and precipitation of leachate from landfill or industrial sites [14]. And during the rainy season there was influx of run-offs from the surrounding environment into the water body.

The EC in this research was very low (0.03 μs/cm) and would probably not pose any threat to the biota of the lake. The FEPA acceptable limit for conductivity in domestic water supply is 70 μs/cm [15]. Higher values were recorded from the same study site by [16]. This is also in contrast to the findings of [9] who reported a range mean value of 69.20 ± 3.12-157.80 ± 24.69 μS/cm in River Galma, Zaria. Likewise, the TDS are very low in comparison with previous studies. This may have arisen from improved maintenance of the lake.

The snail species in Shiroro Lake is highly diverse with Bellamya phthinotropis dominating the Shiroro Lake. The numerical density and species richness of snails were higher in the raining season than in the dry season. The snail species may have responded to changes in the water quality parameters as it was observed in changes in composition of species assemblage and abundance in the various sites. They may also have came out of their hiding places since there are now increased vegetation cover that could portend improved feeding and fertile ground for reproduction. Similar studies by [17] reported 29.06% relative abundance of mollusc in Obazuwa Lake, Benin city and attributed the abundance of the molluscs and oligochaetes in all the stations to the non-occurrence of habitat restriction in the study areas.

Conclusions and Recommendations

Anthropogenic activities and changes in season influenced the environmental conditions of the Shiroro Lake, thus affecting the snail species composition in each station. Overall results showed that changes in water quality of the Lake have significant effects on the structure, abundance and diversity of the snail species that were found. Station 3 had the highest number (403), station 2 with 363 species and station 3 had the lowest with 345 species. The family Viviparidae and Bithyniidae has 2 species each, the Bellamya phthinotropis has the highest number of individual species followed by Bellamya capillata and the lowest was recorded among the species of Gebtella barthi. In general the abundance of Snail was higher during the rainy season than during the dry season.

The highest DO was observed in the month of March with 5.4 mg/L, the highest Electrical conductivity was observed in the month of April with 0.4 (µS/cm), the highest TDS was observed in the month of April with 108 mg/L, the highest BOD was observed in the month of March with 5.2 mg/L. The variations in the phosphate, nitrates, sodium and potassium indicate that Shiroro Lake is organically polluted but do not pose any serious danger to the survival and adaptation of the biota.

More care should be taken to minimize the entry of pollutants into the water body so that the snails species and other biota of the Lake observed in this water body can be conserved.

References

1. Adefemi SO, Awokunmi EE (2010) Determination of physico-chemical parameters and heavy metals in water samples from Itaogbolu area of Ondo-State, Nigeria. African Journal of Environmental Science and Technology 4: 145-148.
2. Zeb BS, Malik AH, Waseem A, Mahmood Q (2011) Water quality assessment of Siran River, Pakistan. International Journal of Physical Sciences 6: 7789-7798.
3. Bhatti MT, Latif M (2011) Assessment of water quality of a river using an indexing approach during the low-flow season. Irrigation Drainage 60: 103-114.
4. Brown DS (2005) Freshwater snails of Africa and their medical importance. 3rd edn. London: Taylor and Francis e-Library 12-41.
5. American Public Health Association (APHA) (2005) Standard Methods for the Examination of Water and Waste Waters. 20th edn. Washington DC 1134.
6. Arimoro FO, Iwegbue CMA, Osiobe O (2008) Effects of industrial Waste Water on the physical and chemical characteristics of Warri River, a coastal water in the Niger Delta, Nigeria. Research Journal of Environmental Science 2: 209-220.
7. Ezra AG (2000) Planktonic Algae in relation to the Physico chemical Properties of some Fresh Water Ponds in Bauchi, Nigeria. Nigeria Journal of Experimental and Applied Biology 1: 19-26.
8. Ibrahim S (2009) A survey of zooplankton Diversity of Challawa River, Kano and Evaluation of some physic chemical condition. Bayero Journal of Applied Sciences 2: 19-2.
9. Samuel PO, Adakole JA, Suleiman B (2015) Temporal and Spatial Physico-Chemical Parameters of River Galma, Zaria, Kaduna State, Nigeria. Resources and Environment 5: 110-123.
10. Iqbal PJM, Pandit AK, Jaceel JA (2006) Impact of sewage waste from Human settlement om physic-chemical characteristics of Dal Lake, Kashmir. Journal of Research Development 6: 81-85.
11. Jonnalagadda SB, Mhere G (2001) Water Quality of Odizi River in Eastern Highland of Zimbabwe. Water research 35: 2371-2376. [crossref]
12. Imoobe TOT, Akoma AC (2008) Assessment of Zooplankton Communitiy Structure of the Bahir Dar gulf of Tana, Ethiopia. Journal of Environmental Studies and Management 1: 36-34.
13. Arimoro FO, Oganah AO (2010) Zooplankton community response in a Perturbed Tropical Stream in the Niger Delta, Nigeria. The Open Environmental & Biological Monitoring Journal 3: 1-11.
14. Butkus SN, Hermanson RE (2007) Washington State University Extension Sodium Content of your Drinking Water.
15. DWAF (1996) South African Water Quality Guidelines. Domestic Uses. 2nd. Ed. Department of Water Affairs and Forestry, Pretoria 1.
16. Kolo RJ, Oladimeji AA (2004) Water quality and some nutrient levels in Shiroro Lake Niger State. Nigeria. Journals of Aquatic Sciences 19: 99.
17. Olomukoro JO, Oviojie EO (2015) Diversity and Distribution of Benthic Macroinvertebrate Fauna of Obazuwa Lake in Benin city, Nigeria. Journal of Biology, Agriculture and Health care 5: 94-100.

DOI: 10.31038/AFS.2021351

Abstract

Aquaculture is a growing industry with a high demand mainly due to its significant contribution to the food sector. One of the main challenges of aquaculture is the prevention and treatment of fish diseases through the extensive application of antibiotics. However, information on the various consequences of pharmaceuticals in fish farms remains limited to this day. Based on the existing scientific literature, this report aims to give an overview of the most commonly used antibiotics in aquaculture, which belong to the groups of quinolones, sulfonamides, tetracyclines, amphenicols and macrolides. This review paper summarizes the information available on the characterization, ecotoxicology and application of florfenicol, erythromycin, furazolidone, oxolinic acid, ciprofloxacin, ofloxacin, sulfadiazine, sulfadimethoxine, sulfamethoxazole, oxytetracycline and tetracycline.

Keywords

Veterinary drugs, Antibiotics, Characterization, Ecotoxicity, Treatment

Introduction

Aquaculture is the food production sector with the strongest growth, as farming, trading, and processing of marine products are of major social, economic, and environmental importance. Global fish production has reached 179 million tons in 2018, where aquaculture accounted for 46% of the total production. China is by far the major fish producer with 35% of the worldwide production [1]. The recent expansion of aquaculture raises concerns related to the destruction of natural habitat, the utilization of potentially harmful synthetic compounds, the effect of escapees on wild stocks, wasteful production of fishmeal and fish oil, and social and cultural effects on aquaculture laborers and communities. Fish farming requires extensive use of veterinary drugs, including antibiotics. Some of these pharmaceuticals are only partially metabolized, and then excreted, entering the aquatic environment in their native form or as metabolites and showing considerable persistence [2]. Quinolones, amphenicols, macrolides, tetracyclines, and sulfonamides are the most widely used groups of antibiotics [3-6]. The 10 drug molecules that are the most commonly used to prevent and treat fish diseases are: florfenicol, erythromycin, furazolidone, oxolinic acid, ciprofloxacin, ofloxacin, sulfadiazine, sulfadimethoxine, sulfamethoxazole, oxytetracycline and tetracycline [7]. Due to their variety and accumulation in the environment, aquatic organisms are exposed to various antibiotics that can be found as mixtures with potentially enhanced “cocktail effect”, which have not been studied in-depth to this day. The main concerns associated to the use of veterinary drugs are the development of antibiotic resistance, mutagenicity, and inhibition or acute toxicity of aquatic organisms [8]. In natural settings, prolonged exposure to low doses of antibiotics can lead to the selective proliferation of resistant bacteria, which can transfer their resistance genes to other bacterial species, including pathogenic bacteria [9]. Despite their low concentrations, the bioaccumulation and biomagnification of pharmaceuticals can have a significant impact on the aquatic ecosystem and even human health (Pan et al., 2019). Furthermore, additional sources of pharmaceuticals have to be considered for extensive environmental evaluation and implementation of possible control measures. These sources include pharmaceutical facilities, hospitals, and households, as a significant percentage of drugs is not fully eliminated by conventional water treatment technologies [10]. In fish farming, drugs are usually coated (mostly with fish oil, gelatin, or vegetable oil) and gradually added to the fish food. The coating agent helps to prevent the drug from entering the water in the fish farm [11], but effluents containing unconsumed food pellets with high levels of antibiotics are released into the aquatic environment without any treatment [12]. There is a limited knowledge about the fate and consequences of antibiotics once they reach the environment. This report aims at compiling information on available analytical methods to detect and identify low concentrations of antibiotics in the environment, summarize potential ecotoxicological risks, and ultimately to propose possible treatment technologies to effectively eliminate them.

This review paper is divided into four parts. The first one describes the most commonly employed molecules, their use and the associated drawbacks. The second part is devoted to their ecotoxicity. The third part reviews the current methods used for the analysis of these pharmaceuticals in various environmental matrices. The last part discusses the water treatment processes currently applied to remove and/or degrade these compounds.

Main Drugs Used in Fish Farming

This part reviews the six main families of drugs usually used in fish farming, their main properties and their mode of administration: cyclins, amphenicols, sulfonamides, quinolones, macrolides and nitrofurans.

Tetracycline

Tetracyclines were discovered in the 1940s and showed activity against a wide range of microorganisms. They are inexpensive and applied for the treatment of human and animal infections as well as in animal feed to promote growth. Tetracycline (TET) is one of the most common type of antibiotics used in medicine, agriculture, and animal husbandry; its chemical structure comprises a fused linear tetracyclic nucleus to which various functional groups are attached as shown in Figure 1 [13]. Due to its widespread application, a growing number of pathogens are developing resistance to tetracycline, therefore decreasing its efficiency. Oxytetracycline (OTC) is a broad-spectrum antibiotic used in veterinary medicine to treat, among others, diseases in fish [8]. It has a role as an antibacterial and anti-inflammatory drug, protein synthesis inhibitor, and antimicrobial agent (ChEBI 27701). OTC is active against gram-positive bacteria, gram-positive bacilli, and gram-negative organisms [14]. In the past decades, the use of OTC increased with the development of aquaculture and livestock production [15] and thanks to its low cost and its broad-spectrum efficacy in treating infections [14]. There are three main ways to administer OTC to farmed fish: through the feed, bath treatment, and injection. Among these options, the incorporation of the antibiotic in food for oral administration is the most common and the one with the least risk in terms of environmental pollution [8]. OTC is only minimally metabolized and is mainly excreted through urine.

Figure 1: Chemical structures of tetracycline (left) and oxytetracycline (right).

Amphenicols

Amphenicols are important veterinary antibiotics with wide-spectrum antimicrobial activity. Florfenicol (FF, see Figure 2) is an antimicrobial agent, which is extensively used in fish farming. Grave et al. investigated the use of antimicrobial drugs in Norwegian aquaculture from 2000 to 2005 by analyzing prescription data, in close relation with the national data of the sold antimicrobial drugs. FF has long been the most abundant drug prescribed for halibut farming in Norwegian aquaculture [16]. In 2013, 0.3 tons and 300 tons of FF were used in fish farming in Norway and Chile, respectively. In 2016, it was reported that over 1000 tons of FF were used in China. Temperature, exposure time, coating agent, and pellet size are the important factors that influence release of FF into the water [11]. According to the EU Council directive, the maximum residue limit value of the sum of FF and florfenicol amine in muscle and skin in natural proportions of healthy fish was given as 1,000 μg/kg (Commission Regulation EU No 37/2010). FF is bacteriostatic, which means it prevents the bacteria from protein synthesis [11]; it is usually used for respiratory and intestinal infections [17].

Figure 2: Chemical structure of chloramphenicol.

Sulfonamides

Sulfonamides are used as chemotherapeutics to treat various bacterial infections in veterinary medicine [5]. Due to their broad-spectrum antimicrobial activity and low cost, they were among the most applied antibiotics and thus, commonly detected in aquaculture wastewater [18]. Currently, only a few drugs belonging to sulfonamides are used due to the developed resistance in previously susceptible microorganisms. Sulfadiazine (SDZ) and sulfadimethoxine (SDM) are the most used antibiotics in fish farming; their chemical structures are displayed Figure 3. When applied to animals, sulfadiazine, is excreted in its native form and its N₄-acetyl metabolite [19]; it is often considered as a representative antibiotic of sulfonamides due to its wide presence in the environment with low hydrophobicity and high water solubility in [20]. Sulfadimethoxine (SMX – Figure 4) is a broad-spectrum antibiotic used in human therapy, aquaculture, livestock, and veterinary medicine. Although its use is decreasing in humans, its low cost secures its popularity in veterinary medicine [21].

Figure 3: Chemical structures of sulfadiazine (left hand), sulfamethoxine (middle) and sulfamethoxazole (right hand).

Figure 4: Chemical structures of some quinolone drugs: oxolinic acid (OXO), ofloxacin (OFLO), ciprofloxacin (CIPX) and flumequine (FLU).

Quinolones

The antibiotics from the group of quinolones are widely used in human and veterinary medicines to treat infectious diseases and to promote livestock growth (Yang et al., 2020). They directly inhibit DNA replication by interacting with two enzymes. Oxolinic acid (OXA) is efficient against a gram-negative bacterium that causes diseases such as vibriosis, yersiniosis, and furunculosis. It has been administrated to farm fishes as a prophylactic and chemotherapeutic agent acting as anti-infective, antibacterial and enzyme inhibitor [22]. Its use in humans is now prohibited in several countries but is still frequently used in veterinary medicine to treat urinary infections, and it was detected in animal excreta [23]. Ofloxacin (OFLO) is a quinolone that was previously used for human health, for the therapy of mild to moderate bacterial infections, it has since been replaced by more potent and less toxic antibiotics, however it is still used in aquaculture. In the group of quinolones, ciprofloxacin (CIPX) is considered as a representative drug against gram-negative bacteria; it has been detected in marine environment, as well as in freshwater. In aquatic environments it affects the metabolism of some bacteria carbon sources and is toxic to fishes (Yang et al., 2020). Flumequine (FLU), as a first generation quinolone, is structurally related to nalidixic and oxolinic acid [24]. Oxidation experiments carried out on flumequine led to a total of 19 transformation products issued from hydroxylation, dehydrogenation, hydroxyl substitution, decarboxylation, demethylation, and ring opening transformation pathways [25].

Macrolides

The group of macrolides refers to macrocyclic lactone ring structures; they are used for their immunomodulatory and antibacterial functions (Yang et al., 2020). In addition to their antibacterial action, macrolides can have an anti-inflammatory effect by decreasing the activity of immune cells and altering bacterial cells. Erythromycin (ERY, see Figure 5) is efficient against gram-positive bacteria, as streptococcus species. However, most of the microorganisms that cause infection in fish are gram-negative, so this compound should only be utilized after fish culturing and sensitivity test results should affirm its viability. Additionally, erythromycin is not efficient in bath treatment and must be administered by injection or in the feed [26].

Figure 5: Chemical structure of erythromycin.

Nitrofurans

Nitrofurans are antimicrobial agents which have been used for animal production. They have strong impact on gram-negative and gram-positive bacteria and act against protozoa as well. In 1993, their application has been banned by EU due to their potential mutagenic effect. Furazolidone (FUR, see Figure 6) is a nitrofuran antibiotic widely used in human and aquaculture medicine against protozoal and Helicobacter pylori infections. Its biodegradation leads to 3-amino-2-oxazolidone and β-hydroxyethylhydrazine [27]. It is still utilized in developing countries, although it is banned in developed countries.

Figure 6: Chemical structure of furazolidone.

Ecotoxicology of the Main Drugs Used for Fish Farming

Table 1 presents a summary of the ecotoxicological data available for veterinary drugs mainly used in fish farming. The data are discussed below, for each pharmaceutical family.

Table 1: Ecotoxicology data for the veterinary drugs mainly used in fish farming

Tetracyclines

In aquaculture, oxytetracycline is one of the most used antibiotics that raises concerns due to its effects on human and animal health, and environmental pollution [8]. In the marine environment, oxytetracycline and quinolones are photochemically degraded and form divalent cationic complexes in the presence of Ca²⁺ and Mg²⁺ ions, causing a loss in antibacterial activity. In freshwater, the antimicrobial activity of tetracyclins is of bigger concern due to the development of antibiotic resistance [28]. OTC inhibits the growth of two species of algae: pseudokirchneriella subcapitata (the international standard species for evaluation of inhibition) and Ankistrodesmus fusiformis (native from Argentina). The former was the most sensitive with an EC₅₀ of 0.92±0.30 mg/L [29]. OTC can also affect nitrification since the main bacteria responsible for biofilters – nitrosomonas that promote the conversion of ammonia to nitrite and Nitrobacter that convert nitrite to nitrate – are gram-negative. Li et al. investigated the toxic effect of tetracycline and tetracycline hydrochloride on two model ciliates: Stentor coeruleus and Stylonychia lemnae. The 24 h-EC₅₀ of tetracycline for Stentor coeruleus and Stylonychia lemnae were 94.4 mg/L and 40.1 mg/L, respectively. For tetracycline hydrochloride, the EC₅₀ for Stentor coeruleus and Stylonychia lemnae were 8.39 mg/L and 14.0 mg/L, respectively [15]. This shows that tetracycline hydrochloride is more toxic than tetracycline in these test organisms. Both compounds deteriorate the ultra-cell structure and inhibit the cell growth rate [29].

Amphenicols

Florfenicol (FF) restrains the bacterial protein synthesis efficiency by binding to 50S subunit and 70S ribosome, and it is listed as the top-priority monitoring compounds in veterinary drugs in South-Korea [30]. FF also inhibits the hematopoietic system [31] and deteriorates the mobility, regeneration, and population increase of the tropical Cladocera silvestrii [32]. It also decreases egg hatchability and hinders the development of the cardiovascular system [33]. Guilhermino et al. showed that a FF concentration in water greater than 1.8 mg/L can inhibit the growth of the mollusk Corbicula fluminea [34].

Sulfonamides

The chronic and acute toxicity of sulfadiazine on the crustacean Daphnia magna was investigated and the LC50 value was determined as 1.884 mg/L [35]. The exposition of zebrafish to some sulfonamides, including sulfadiazine, caused an increased heart rate and abnormal swimming. This investigation also proved that sulfadiazine was a typical inducer of metabolic enzymes and suggested a potential ecotoxicological risk [36]. It is important to consider that sulfadiazine can have an impact on biological water treatment processes. Li et al. studied the effects of this antibiotic on a sequencing batch biofilm reactor (SBBR). They found that a sulfadiazine concentration higher than 6 mg/L inhibits COD (Chemical Oxygen Demand) and ammoniacal nitrogen removal within the SBBR when treating aquaculture waste [37]. Sulfadiazine promotes the secretion of extracellular polymeric substances by the microorganisms and impacts the biofilm composition, it has a direct impact on nitrification and the removal of organic matter. Sulfamethoxazole has been studied in a microcosmos composed of water, sediment and zebrafishes. The drug concentration decreased gradually in water while increasing over time in sediment and zebrafish. Bioaccumulation in zebrafish was reduced by 13-28% in the presence of sediment particles in the water; it was further reduced (24-33%) when increasing the water salinity [38]. In the study conducted by [39], sulfadimethoxine was considered to be moderately toxic with an EC₅₀ towards the green algae Chlorella vulgaris of 4.24 mg/L in fresh water, which was measured to be higher (11.2 mg/L) in salt water [39].

Quinolones

According to a study by Tkaczyk et al., oxolinic acid EC₅₀ was 4.6 mg/L EC₅₀ for Daphnia magna [40]. Sun et al. investigated the bioaccumulation of ofloxacin in crucian carp and showed that fluorine increases the bioaccumulation of the antibiotic; higher bioaccumulation potential appears at a low concentration of drug, mostly in the liver [41]. De Liguoro et al. evaluated the effect of continuous and alternate exposure of flumequine at 2 mg/L on Daphnia magna survival, growth, and reproduction. At this concentration, mortality was observed at a rate of 23  ± 14% across generation [42].

Macrolides

Erythromycin has shown medium risk for algae, but bacteria are the main target of antibiotics such as erythromycin. Erythromycin can cross the cell membrane of the bacteria and bind to 50S subunit ribosome [43]. It is also very persistent in the environment due to having aromatic rings, which makes it refractory [44].

Nitrofurans

Lewkowski et al. investigated the effect of furazolidone on growth of oat, radish Sativus, and Alivibrio fischeri bacteria. Their results indicated that furazolidone is very toxic to Alivibrio fischeri with an EC₅₀ of 2.05 mg/L [45]. The accumulation of furazolidone metabolites have been reported in the literature [5]; its use is prohibited by the Food and Drug Administration due to being a nitrofuran compound and inhibiting monoamine oxidase [46].

Methods for Characterization of Drugs Used in Fish Farming

Table 2 presents a summary of the analytical approaches employed for the detection and quantification of veterinary drugs mainly used in fish farming; protocols and methods are discussed below.

Table 2: Analytical methods reported for the detection and quantification of the main antibiotic veterinary drugs used in fish farming

^a LC: liquid chromatography, HPLC: high performance liquid chromatography, UPLC: ultrahigh performance liquid chromatography, UVD: UV detector, FLD: fluorescence detector, PDA: Photodiode array detector, MS/MS: tandem mass spectrometry, HR-MS: high resolution mass spectrometry
^b Q: quadrupole, TQ: triple quadrupole, q: collision cell, TRAP: ion trap, LIT: linear ion trap, TOF: time-of-flight
^c LLE: liquid-liquid extraction, SPE: solid phase extraction, QuEChERS: sample preparation method (Quick, Easy, Cheap, Efficient, Rugged and Safe), MAE : microwave assisted extraction
^d ACN: acetonitrile, MeOH: methanol, EDTA: ethylenediaminotetraacetic acid, FA: formic acid, TFA: trifluoro acetic acid

The first step of the analytical process consists in sample preparation, which includes extraction of analytes from the matrix, purification and concentration. When imposed by the analytical technique, a derivation step is added at the end of the process. Solid phase extraction (SPE) has been extensively reported as a tool for choice for the extraction of the pharmaceuticals mainly used in fish farming because (i) it is suitable for very complex matrices, (ii) it is highly selective, and (iii) it permits significant pre-concentration. Furthermore, SPE can be installed on-line with a chromatographic system and automated, which allows high throughput analysis; this is the case for some studies reported in Table 2 [47]. Oasis HLB™ are the most used cartridges in the reported studies, as they are suitable for the simultaneous extraction of compounds from various families: sulfonamides (SMX, SDZ, SMZ), tetracyclines (TET, OTC), quinolones (OFLO, FLU, CPIX), macrolides (ERY), nitrofurans and phenicols (FF) [15,23,47]. Apart from SPE-based approach, various sample preparation processes have been reported, including liquid-liquid extraction (LLE) with or without sonication or microwave assistance (Norambuena et al., 2013; Prat et al., 2006). In a general way, acetonitrile appears as the solvent of choice for the extraction of these molecules.

In most of the studies devoted to drugs used in fish farming, analytes are separated using liquid chromatography. High-performance liquid chromatography (HPLC) is the most widely applied technique to detect veterinary drugs with great accuracy and reliability [48]; it is nevertheless more and more replaced by UPLC (Ultrahigh Performance Liquid Chromatography), which uses a new generation of columns filled with small particles of hybrid material (diameter at the μm scale) and operates with a much higher column pressure (up to 15,000 psi) than HPLC. UPLC considerably reduces runtime and thus increases sample throughput; it performs also much better in terms of resolution, sensitivity, and separation efficiency; it is almost always coupled with a mass spectrometer (see below). Li et al. developed a rapid, sensitive, and reliable UPLC-MS/MS method for the determination of 21 antibiotics belonging to 7 classes in different wastewater matrixes (Li et al., 2009). Yu et al. reported a selective and sensitive method to measure 11 antibiotics in water by UPLC-MS/MS; it was successfully used to identify ofloxacin in wastewater and sludge samples [49]. Dinh et al. used UPLC-MS/MS to find different veterinary drugs including furazolidone, with a limit of detection of 1.5-3 μg/kg for the later [5]. Whatever the chromatographic system (HPLC or UPLC), most of the studies were conducted using reverse phase chromatography. Many brands and geometries were tested but at the end the retained columns are almost all C18-types. The mobile phase is almost always a very classical one in the following type of configuration: a binary gradient made of an aqueous phase (ultrapure water) and an organic one (ACN or MeOH), both acidified with a small organic acid (acid formic, acetic, trifluoroacetic…) generally at 0.1%. Among all the studies listed in Table 2, the only one that uses a non C18 stationary phase (phenylpropylsilane phase) is that by Norambuena et al., devoted to the analysis of oxytetracycline, florfenicol, flumequine and oxolinic acid in marine sediments (Norambuena et al., 2013). In this work, HPLC was equipped with a PDA (photodiode array detector). HPLC with PDA detection was also used for the detection of florfenicol in water by Zhang et al. and that of sulfonamide residues by Won et al. [50,51]. HPLC-PDA has been also reported for the detection of sulfadiazine in South-Korean marine products, although SDZ was only identified in 2 of the 10 samples analyzed [51]. Han et al. reported a combination of UVD (UV detector) and FLD (fluorescence detector) for the analysis of tetracyclines in feeds [48]. Studies reporting the detection of flumequine and oxolinic acid by HPLC-FLD in sediments, soils and various types of water have been also reported [25]. In a general way “classical” HPLC detectors tend to be gradually replaced by mass spectrometers, which are today recognized as the best detectors in terms of sensitivity, specificity and selectivity.

In almost all of the studies conducted using LC-MS coupling, the liquid chromatograph (HPLC or UPLC) is coupled with a triple quadrupole (TQ) operated in the MRM (multiple reaction monitoring) mode on two transitions. Tandem mass spectrometry (MS/MS) is preferred to simple MS as it strongly reduces the risk of false negatives when performing analysis of complex matrices (Bouchonnet, 2013). Hybrid mass spectrometers associating a quadrupole, a collision cell, and an ion trap have been also reported; their principle of operation is quite similar to that of TQs except that the ion trap permits ion accumulation before detection [47,49]. Among all the studies listed in Table 2, only that by Goessens et al. refers to a high-resolution mass spectrometer (Orbitrap™), which has been employed to analyze 46 antimicrobial drug residues including sulfonamides, tetracyclines, quinolones, macrolides, nitrofurans and phenicols in aquatic matrices [23]. Since it allows the differentiation of isobaric ions, high-resolution mass spectrometry (HRMS) constitutes a tool of choice for structural elucidation of metabolites and by-products; it also significantly reduces interferences during routine analysis of very complex matrices. The use of MS/MS and/or HRMS enables the development and validation of multi-residue methods able to detect and quantify many molecules in one unique run. Even if the number of analytes remains limited in comparison with methods reported for pesticide residue dosages (hundreds of analytes), some of the methods reported for the analysis of veterinary antibiotics allows the simultaneous detection of tens of drugs. For instance, Gros et al. developed a LC-MS/MS method for the one run detection of 73 pharmaceuticals including tetracycline, oxytetracycline, sulfadiazine, sulfamethoxazole, ofloxacin, flumequine, ciprofloxacin and erythromycin in river water and WWTP influent and effluent while a LC-HRMS was developed by Goessens et al. for the analysis of 46 antimicrobial drug residues including sulfonamides, tetracyclines, quinolones, macrolides, nitrofurans and phenicols in aquatic matrices [23].

A few analytical approaches were developed apart from traditional separative processes such as liquid chromatography. For example, an aptaprobe was used for the detection of sulfadimethoxine; it simply and conveniently detects SDM with accuracy in the range of 94.2-113% in seawater and 104-118% in fish [52]. A dichromatic label-free aptasensor detects SDM presence through fluorescent emission and color changes of gold nanoparticles. This aptasensor can be applied to the rapid detection in fish and water samples with accuracies between 99.2 and 102% for fish, 99.5 and 100.5% for water [53]. Almeida et al. also developed a new low-cost plastic membrane electrode that detects low concentrations of SDM in aquaculture waters. To test this device, sulfadimethoxine was added to aquaculture waters and the results showed a good agreement between added and measured drug amounts with recoveries ranging from 96.8% to 101%, with a relative error between -0.7% and 3.5%, which suggests that it constitutes a good method that could be extended to the determination of other pharmaceuticals in water [54].

Treatment Processes

Once they reach the environment, micropollutants are subjected to three main degradation processes: biodegradation, hydrolysis, and photolysis [8]. Due to their own antibacterial activity, antibiotics are poorly or not degraded by natural biotic processes. Chemical and physical processes – natural or industrial – can be considered as a better option to remove these refractory veterinary drugs. As we will see below, many treatments have been considered for their degradation and/or removal from aqueous media; they include membrane anodic Fenton, advanced oxidation processes, heterogeneous photocatalysis, and electrocoagulation [55]. Table 3 lists some treatments applied for the removal of veterinary pharmaceuticals considered individually.

Table 3: Various treatment methods for the removing of the veterinary drugs mainly used in fish farming from aqueous media

Using a pilot drinking water treatment plant, Vieno et al. observed the elimination of some pharmaceuticals with a process consisting in ferric salt coagulation, rapid sand filtration, ozonation, and two-stage granular activated carbon filtration. Most pharmaceuticals ceased to be quantifiable at the end of ozonation; only ciprofloxacin passed all treatment steps almost unaffected [56]. No treatment appears as a universal solution at this day. For instance, classical wastewater treatments are known to be inefficient for the elimination of sulfonamides [47]. Tetracycline is hard to degrade using conventional wastewater treatments such as activated sludge, which might be due to tetracyclines’ strong hydrophilic properties related to their stable naphthalene ring structure (Zhao et al., 2020). The biological degradation of oxytetracycline is limited due to its broad-spectrum antimicrobial properties, which are considered responsible for the development of antibiotic resistance genes in the environment (Xie et al., 2016). OTC fails to be degraded into non-toxic transformation products by most abiotic processes; therefore sonocatalytic degradation has been proposed to form less toxic intermediates [57].

Most of the alternative approaches for the removal of the most resistant molecules are based on photocatalytic or electrochemical approaches. For instance, sulfamethoxazole is not efficiently removed in conventional wastewater treatment plants [47] but it is susceptible to photodegradation in aqueous solutions along several pathways [58]. UV-photolysis appears as a treatment of choice for many micropollutants, especially in the presence of a catalyst that increases both the kinetics and yields of the degradation reaction. UV-irradiation induces the formation of hydroxyl radicals from water and dissolved oxygen; these highly reactive radicals are responsible for the oxidation of micropollutants. The addition of hydrogen peroxide, alone or with a metal has been frequently reported. Zhao et al. evaluated the effect of H₂O₂/Fe addition to the UV treatment of tetracycline. They found out that optimum concentrations of H₂O₂ (0.5 mM) and Fe(II) (0.05 mM) promote the degradation of TET. Also, a higher pH level facilitated the UV-attenuation of TET (Zhao et al., 2020) while a lower pH helped its degradation under ozonation conditions. Ming Zheng et al. utilized CaO₂/UV as an advanced oxidation process to remove FF and other active pharmaceutical compounds from wastewater. CaO₂ is considered as the “solid form” of H₂O₂ with an advantage of being more stable than H₂O₂ in presence of base or catalyst. CaO₂ can produce ^.OH And O₂^. radicals and oxidize complex chemical compounds. The highest removal of FF happened at 0.1 g.L^-1 of CaO₂. The addition of CaO₂ also allows reducing the irradiation time and so decreasing the consumption of energy [31]. Titanium oxide appears as a widely used photocatalyst for the removal of pharmaceuticals. UV-irradiation with TiO₂ removed 99% of sulfadiazine under the following conditions: initial concentrations of sulfadiazine at 5.0 mg/L and TiO₂ at 0.08 g/L, pH = 7, radiation intensity of 1000 μw/cm² and reaction time of 50 min [59]. SDZ can be also effectively degraded using gamma irradiation, the elimination efficiency being improved under acidic conditions [60]. Degradation of sulfamethoxazole under ultraviolet light with TiO₂ reached 96%. TiO₂ is the most suitable catalyst due to its stability, non-toxicity, and high catalytic activity [59]. Oxolinic acid was degraded using heterogeneous photocatalysis with titanium dioxide suspended on particles. After 30 min under optimal conditions both the substrate and the microbial activity were eliminated [61] and the residual byproducts did not show antibacterial activity [62]. Organic matter can both hinder and activate the photodegradation. For instance, oxolinic acid persistence is lower in ultrapure water than in environmental water, especially in the presence of high salinity values. The presence of organic matter can decrease the photodegradation rate in freshwater by acting as a light filter and hydroxyl radicals scavenger [63]. An ultraviolet/peroxydisulfate system was reported for the degradation of ofloxacin in synthetic seawater and in synthetic marine aquaculture water; the global toxicity (including toxicities of reagents and by-products) induced by such a process is lower than that induced with traditional approaches using NaClO [64]. Traditional removal processes only based on chemical reactions tend to be replaced by the so-called AOPs (advance oxidation processes) but some of them remains in use. For instance, [21] demonstrated that sulfadimethoxine can be removed by potassium permanganate in water. The degradation is affected by the pH of the solution and a higher temperature is also beneficial for the removal [21]. The use of zero-valent iron-activated persulfate has been developed to remediate antibiotic-contaminated wastewater since it removes 69% and 74% of SDM from filtered and unfiltered discharge water, respectively [65]. It has to be kept in mind that in a general way catalysis is hardly applied to complex matrices (water containing high levels of dissolved organic matter for instance) as it becomes quite inefficient in the presence of large amounts of organic and inorganic species. Furthermore, altering the pH when necessary can be very costly.

Electro-Fenton technology is also an advanced oxidation process that produces hydroxyl radicals to degrade refractory pollutants. However, this process may be inefficient due to the high dissociation energy of some chemical bonds, such as C-F in florfenicol. On the other hand, UV light has shown to be capable of cleaving such bonds, and the combination of Electro-Fenton with UV could be an efficient technology. Jiang et al. coupled the photoelectrochemical reaction in a sequential filtration system to degrade and mineralize FF. Their study revealed 78.1 ± 9.1% mineralization of FF at a low concentration of 14 µM [66]. Various technologies have been studied to remove furazolidone from wastewater samples; among them, Kong et al. used an electrochemical system to degrade FUR in a cathode compartment. Their study evaluated the effect of different cathode potentials, initial antibiotic concentration, and cathode buffer solution on FUR degradation. Different cathode potentials resulted in different degradation products, and different buffer solutions and initial concentrations of furazolidone had just an obvious effect on its removal efficiency [67].

Ozonation has also been reported as a powerful tool for micropollutants abatement. For example, it allowed the degradation of tetracycline at 99.5% with 40% of mineralization (C. Wang et al., 2020). Sulfadimethoxine showed to be completely removed from water by ozone bubbling within 20 minutes; this strong efficiency was partially explained by the presence of one or several aromatic rings on which the O₃ molecule can fix itself before hydrolysis and/or ring opening [68]. Ozonation at an application rate of 0.17 g O₃/min was able to remove some antibiotics in about 20 min, although the degradation of erythromycin was slower, and more effective at a high pH or with H₂O₂ addition [68].

As ciprofloxacin is particularly refractory to conventional wastewater treatments, special attention has been paid to its removal using alternative processes. Reverse osmosis was successfully tested for the removal (99.96% removal) of CPIX in seawater [69]. Additionally, ciprofloxacin removal by electrosorption has been successfully demonstrated using graphite felt electrodes [70].

Finally, and despite their microbial activity, some veterinary drugs were submitted to treatments involving biological processes. In two sewage treatment plants in Guangzhou, more than 85% of ofloxacin was removed in the effluents after activated sludge [71]. A novel microalgae biofilm membrane photobioreactor (MBMP) was developed for the cultivation of microalgae and the removal of sulfonamides from residual wastewater of aquaculture. The reduction of sulfadiazine in the MBMP during its stable operation was up to 61-79.2%. It can be considered that the performance of the MBMP is higher than the one achieved by traditional batch cultivation [18]. The removal of sulfamethoxazole by a batch culture of microalgae c. vulgaris was 34.07% after 12 days of concentration in marine aquaculture wastewater (against 3.33% without microalgae). Gurav et al. used a magnetic biochar to remove furazolidone from wastewater; they showed that the magnetic biochar had a higher surface area as compared to normal biochar, and possessed a much better removal efficiency [27].

Conclusion

Aquaculture is a booming industry, which by necessity uses drugs to prevent and treat diseases in fish farms. The current literature has been mainly focused on the possible adverse effects on human health due to the possible remnants of these drugs in fish. In recent years, the presence of drugs in environmental matrices has become more evident but the effects of aquaculture waste on the environment have been poorly documented. Thanks to the continuous improvement of analytical methods, it is now possible to successfully determine veterinary pharmaceuticals at trace amounts in complex matrices. The most common analytical processes rely on solid phase extraction and liquid chromatography – HPLC or UHPLC – coupled with mass spectrometry. Current efforts are aimed at developing multiresidue methods for the simultaneous analysis of various drugs from different families. In any case, the literature directed specifically to the water discarded by aquaculture is very scarce. Although most of the analytical methods are focused on separative processes, recent ventures successfully tested aptasensors and aptaprobes to quickly and efficiently measure low concentrations of antibiotics in water.

Waste from agriculture and aquaculture usually reaches the environment directly, without being previously treated. Currently, there is more knowledge about the toxic effects of pharmaceuticals in humans and animals than about their environmental impact. A good evaluation of their potential ecotoxicological impact should consider factors such as the presence of sediments, the stability of water and other substances with which the drugs can interact since all these parameters may affect the final result of the investigations [72]. It is known that contamination by antibiotics includes the development of resistance in the aquatic pathogens, direct toxicity to microflora and microfauna, and even possible risks to human health due to the consumption of non-target contaminated benthic fauna [73]. For this reason, ways to treat or eliminate these pollutants are still under investigation given that conventional wastewater treatment plants are not fully efficient – and sometimes quasi inefficient – for their removal. The antimicrobial activity of pharmaceuticals makes their treatment through biotic processes difficult and advanced oxidation processes appear as a tool of choice for their removal. Photocatalysis and electrochemistry are not really viable on a wide scale to this day but ozonation and UV/H₂O₂ oxidation are much more applicable. In a general way, it is of first importance to mitigate the ecotoxicological risks associated with aquaculture waste released into the sea, by (i) selecting the proper veterinary drugs, (ii) limiting the amount of waste release from fish farming and (iii) setting efficient solutions for the removal of pharmaceuticals once or before they reach the environment [74-77].

References

1. FAO (2020) The State of World Fisheries and Aquaculture 2020. FAO.
2. Castilla-Fernández D, Moreno-González D, Bouza M, Saez-Gómez A, Ballesteros E, et al. (2021) Assessment of a specific sample cleanup for the multiresidue determination of veterinary drugs and pesticides in salmon using liquid chromatography/tandem mass spectrometry. Food Control 130: 108311.
3. Chokmangmeepisarn P, Thangsunan P, Kayansamruaj P, Rodkhum C (2021) Resistome characterization of Flavobacterium columnare isolated from freshwater cultured Asian sea bass (Lates calcarifer) revealed diversity of quinolone resistance associated genes. Aquaculture 544: 737149.
4. Pastor-Belda M, Campillo N, Arroyo-Manzanares N, Hernández-Córdoba M, Viñas P (2020) Determination of amphenicol antibiotics and their glucuronide metabolites in urine samples using liquid chromatography with quadrupole time-of-flight mass spectrometry. Journal of Chromatography B 1146: 122122.
5. Dinh QT, Munoz G, Vo Duy S, Tien Do D, Bayen S, et al. (2020) Analysis of sulfonamides, fluoroquinolones, tetracyclines, triphenylmethane dyes and other veterinary drug residues in cultured and wild seafood sold in Montreal, Canada. Journal of Food Composition and Analysis 94: 103630.
6. Du J, Li X, Tian L, Li J, Wang C, et al. (2021) Determination of macrolides in animal tissues and egg by multi-walled carbon nanotube-based dispersive solid-phase extraction and ultra-high performance liquid chromatography–tandem mass spectrometry. Food Chemistry 365: 130502.
7. Lulijwa R, Rupia EJ, Alfaro AC (2019) Antibiotic use in aquaculture, policies and regulation, health and environmental risks: A review of the top 15 major producers. Reviews in Aquaculture 12: 640-663.
8. Leal JF, Santos EBH, Esteves VI (2018) Oxytetracycline in intensive aquaculture: Water quality during and after its administration, environmental fate, toxicity and bacterial resistance. Reviews in Aquaculture 11: 4.
9. Archundia D, Duwig C, Lehembre F, Chiron S, Morel MC, et al. (2017). Antibiotic pollution in the Katari subcatchment of the Titicaca Lake: Major transformation products and occurrence of resistance genes. Science of The Total Environment 576: 671-682.
10. Krzeminski P, Tomei MC, Karaolia P, Langenhoff A, Almeida CMR, et al. (2019) Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: A review. Science of The Total Environment 648: 1052-1081. [crossref]
11. Barreto FM, da Silva MR, Braga PAC, Bragotto APA, Hisano H, et al. (2018) Evaluation of the leaching of florfenicol from coated medicated fish feed into water. Environmental Pollution 242: 1245-1252. [crossref]
12. Kim W, Lee Y, Kim SD (2017) Developing and applying a site-specific multimedia fate model to address ecological risk of oxytetracycline discharged with aquaculture effluent in coastal waters off Jangheung, Korea. Ecotoxicology and Environmental Safety 145: 221-226. [crossref]
13. Chopra I, Roberts M (2001) Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews 65: 232-260. [crossref]
14. Lee PT, Liao ZH, Huang HT, Chuang CY, Nan FH (2020) β-glucan alleviates the immunosuppressive effects of oxytetracycline on the non-specific immune responses and resistance against Vibrio alginolyticus infection in Epinephelus fuscoguttatus × Epinephelus lanceolatus hybrids. Fish & Shellfish Immunology 100: 467-475. [crossref]
15. Li Z, Qi W, Feng Y, Liu Y, Ebrahim S, et al. (2019) Degradation mechanisms of oxytetracycline in the environment. Journal of Integrative Agriculture 18: 1953-1960.
16. Grave K, Hansen MK, Kruse H, Bangen M, Kristoffersen AB (2008) Prescription of antimicrobial drugs in Norwegian aquaculture with an emphasis on “new” fish species. Preventive Veterinary Medicine 83: 156-169. [crossref]
17. Wang Y, Li X, Fu Y, Chen Y, Wang Y, et al. (2020) Association of florfenicol residues with the abundance of oxazolidinone resistance genes in livestock manures. Journal of Hazardous Materials 399: 123059.
18. Peng YY, Gao F, Yang HL, Wu HWJ, Li C, et al. (2020) Simultaneous removal of nutrient and sulfonamides from marine aquaculture wastewater by concentrated and attached cultivation of Chlorella vulgaris in an algal biofilm membrane photobioreactor (BF-MPBR). Science of The Total Environment 725: 138524. [crossref]
19. Al-Ansari MM, Benabdelkamel H, Al-Humaid L (2021) Degradation of sulfadiazine and electricity generation from wastewater using Bacillus subtilis EL06 integrated with an open circuit system. Chemosphere 276: 130145. [crossref]
20. Cheng L, Chen Y, Zheng YY, Zhan Y, Zhao H, et al. (2017) Bioaccumulation of sulfadiazine and subsequent enzymatic activities in Chinese mitten crab (Eriocheir sinensis). Marine Pollution Bulletin 121: 176-182. [crossref]
21. Zhuang J, Wang S, Tan Y, Xiao R, Chen J, et al. (2019) Degradation of sulfadimethoxine by permanganate in aquatic environment: Influence factors, intermediate products and theoretical study. Science of the Total Environment 671: 705-713. [crossref]
22. (2020) Oxolinic acid. National Center for Biotechnology Information.
23. Goessens T, Huysman S, De Troyer N, Deknock A, Goethals P, et al. (2020) Multi-class analysis of 46 antimicrobial drug residues in pond water using UHPLC-Orbitrap-HRMS and application to freshwater ponds in Flanders, Belgium. Talanta 220: 121326.
24. Barmpa A, Frousiou O, Kalogiannis S, Perdih F, Turel I, et al. (2018). Manganese (II) complexes of the quinolone family member flumequine: Structure, antimicrobial activity and affinity for albumins and calf-thymus DNA. Polyhedron 145: 166-175.
25. Qi, Y, Qu R, Liu J, Chen J, Al-Basher G, et al. (2019) Oxidation of flumequine in aqueous solution by UV-activated peroxymonosulfate: Kinetics, water matrix effects, degradation products and reaction pathways. Chemosphere 237: 124484.
26. Yanong RPE (2019) Use of Antibiotics in Ornamental Fish Aquaculture. Fisheries and Aquatic Sciences.
27. Gurav R, Bhatia SK, Choi TR, Park YL, Park JY, et al. (2020) Treatment of furazolidone contaminated water using banana pseudostem biochar engineered with facile synthesized magnetic nanocomposites. Bioresource Technology 297: 122472. [crossref]
28. Sreejith S, Shajahan S, Prathiush PR, Anjana VM, Viswanathan A, et al. (2020) Healthy broilers disseminate antibiotic resistance in response to tetracycline input in feed concentrates. Microbial Pathogenesis 149: 104562. [crossref]
29. Magdaleno A, Carusso S, Moretton J (2017) Toxicity and Genotoxicity of Three Antimicrobials Commonly Used in Veterinary Medicine. Bulletin of Environmental Contamination and Toxicology 99: 315-320. [crossref]
30. Zhang Y, Zhang X, Guo R, Zhang Q, Cao X, et al. (2020) Effects of florfenicol on growth, photosynthesis and antioxidant system of the non-target organism Isochrysis galbana. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 233: 108764. [crossref]
31. Zheng M, Daniels KD, Park M, Nienhauser AB, Clevenger EC, et al. (2019) Attenuation of pharmaceutically active compounds in aqueous solution by UV/CaO2 process: Influencing factors, degradation mechanism and pathways. Water Research 164: 114922.
32. Freitas EC, Rocha O, Espíndola ELG (2018) Effects of florfenicol and oxytetracycline on the tropical cladoceran Ceriodaphnia silvestrii: A mixture toxicity approach to predict the potential risks of antimicrobials for zooplankton. Ecotoxicology and Environmental Safety 162: 663-672.
33. Hu D, Meng F, Cui Y, Yin M, Ning H, et al. (2020) Growth and cardiovascular development are repressed by florfenicol exposure in early chicken embryos. Poultry Science 99: 2736-2745. [crossref]
34. Guilhermino L, Luís R Vieira, Diogo Ribeiro, Ana Sofia Tavares, Vera Cardoso (2017) Uptake and effects of the antimicrobial florfenicol, microplastics and their mixtures on freshwater exotic invasive bivalve Corbicula fluminea. The Science of the Total Environment 622-623: 1131-1142. [crossref]
35. Duan Y, Deng L, Shi Z, Liu X, Zeng H, et al. (2020) Efficient sulfadiazine degradation via in-situ epitaxial grow of Graphitic Carbon Nitride (g-C3N4) on carbon dots heterostructures under visible light irradiation: Synthesis, mechanisms and toxicity evaluation. Journal of Colloid and Interface Science 561: 696-707.
36. Lin T, Yu S, Chen Y, Chen W (2014) Integrated biomarker responses in zebrafish exposed to sulfonamides. Environmental Toxicology and Pharmacology 38: 444-452. [crossref]
37. Li Z, Chang Q, Li S, Gao M, She Z, et al. (2017) Impact of sulfadiazine on performance and microbial community of a sequencing batch biofilm reactor treating synthetic mariculture wastewater. Bioresource Technology 235: 122-130.
38. Chen Y, Zhou JL, Cheng L, Zheng YY, Xu J (2017) Sediment and salinity effects on the bioaccumulation of sulfamethoxazole in zebrafish (Danio rerio). Chemosphere 180: 467-475. [crossref]
39. Borecka M, Białk-Bielińska A, Haliński ŁP, Pazdro K, Stepnowski P, et al. (2016) The influence of salinity on the toxicity of selected sulfonamides and trimethoprim towards the green algae Chlorella vulgaris. Journal of Hazardous Materials 308: 179-186. [crossref]
40. Tkaczyk A, Bownik A, Dudka J, Kowal K, Ślaska B (2021) Daphnia magna model in the toxicity assessment of pharmaceuticals: A review. Science of The Total Environment 763: 143038. [crossref]
41. Sun Y, Zhang L, Zhang X, Chen T, Dong D, et al. (2020) Enhanced bioaccumulation of fluorinated antibiotics in crucian carp (Carassius carassius): Influence of fluorine substituent. Science of The Total Environment, 748, 141567. [crossref]
42. De Liguoro M, Maraj S, Merlanti R (2019) Transgenerational toxicity of flumequine over four generations of Daphnia magna. Ecotoxicology and Environmental Safety 169: 814-821. [crossref]
43. Sendra M, Moreno-Garrido I, Blasco J, Araújo CVM (2018) Effect of erythromycin and modulating effect of CeO2 NPs on the toxicity exerted by the antibiotic on the microalgae Chlamydomonas reinhardtii and Phaeodactylum tricornutum. Environmental Pollution 242: 357-366.
44. Hua T, Li S, Li F, Ondon BS, Liu Y, et al. (2019) Degradation performance and microbial community analysis of microbial electrolysis cells for erythromycin wastewater treatment. Biochemical Engineering Journal 146: 1-9.
45. Lewkowski J, Rogacz D, Rychter P (2019) Hazardous ecotoxicological impact of two commonly used nitrofuran-derived antibacterial drugs: Furazolidone and nitrofurantoin. Chemosphere 222: 381-390. [crossref]
46. Sun Y, Waterhouse GIN, Xu L, Qiao X, Xu Z (2020) Three-dimensional electrochemical sensor with covalent organic framework decorated carbon nanotubes signal amplification for the detection of furazolidone. Sensors and Actuators B: Chemical 321: 128501.
47. García-Galán MJ, Díaz-Cruz MS, Barceló D (2011) Occurrence of sulfonamide residues along the Ebro river basin: Removal in wastewater treatment plants and environmental impact assessment. Environment International 37: 462-473. [crossref]
48. Han J, Jiang D, Chen T, Jin W, Wu Z, et al. (2020) Simultaneous determination of olaquindox, oxytetracycline and chlorotetracycline in feeds by high performance liquid chromatography with ultraviolet and fluorescence detection adopting online synchronous derivation and separation. Journal of Chromatography B 1152: 122253. [crossref]
49. Yu R, Chen L, Shen R, Li P, Shi N (2020) Quantification of ultratrace levels of fluoroquinolones in wastewater by molecularly imprinted solid phase extraction and liquid chromatography triple quadrupole mass. Environmental Technology & Innovation 19: 100919.
50. Zhang Q, Guo R, Zhang Y, Zhang X, Mostafizur RMD, et al. (2020) Study on florfenicol-contained feeds delivery in Dicentrarchus labrax seawater recirculating and flowing aquaculture systems. Aquaculture 526: 735326.
51. Won SY, Lee CH, Chang HS, Kim SO, Lee SH, et al. (2011) Monitoring of 14 sulfonamide antibiotic residues in marine products using HPLC-PDA and LC-MS/MS. Food Control 22: 1101-1107.
52. Chen XX, Lin ZZ, Hong CY, Yao QH, Huang ZY (2020) A dichromatic label-free aptasensor for sulfadimethoxine detection in fish and water based on AuNPs color and fluorescent dyeing of double-stranded DNA with SYBR Green I. Food Chemistry 309: 125712. [crossref]
53. Chen XX, Lin ZZ, Yao QH, Huang ZY (2020) A practical aptaprobe for sulfadimethoxine residue detection in water and fish based on the fluorescence quenching of CdTe QDs by poly(diallyldimethylammonium chloride). Journal of Food Composition and Analysis 91: 103526.
54. Almeida SAA, Montenegro MCBSM, Sales MGF (2013) New and low cost plastic membrane electrode with low detection limits for sulfadimethoxine determination in aquaculture waters. Journal of Electroanalytical Chemistry 709: 39-45.
55. Nariyan E, Aghababaei A, Sillanpää M (2017) Removal of pharmaceutical from water with an electrocoagulation process; effect of various parameters and studies of isotherm and kinetic. Separation and Purification Technology 188: 266-281.
56. Vieno NM, Härkki H, Tuhkanen T, Kronberg L (2007) Occurrence of pharmaceuticals in river water and their elimination in a pilot-scale drinking water treatment plant. Environmental Science & Technology, 41: 5077-5084. [crossref]
57. Kumar Subramani A, Rani P, Wang P.-H, Chen B.-Y, Mohan S et al. (2019) Performance assessment of the combined treatment for oxytetracycline antibiotics removal by sonocatalysis and degradation using Pseudomonas aeruginosa. Journal of Environmental Chemical Engineering 7: 103215.
58. Moore DE (1998) Mechanisms of photosensitization by phototoxic drugs. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 422: 165-173.
59. He Z, Cheng X, Kyzas GZ, Fu J (2016) Pharmaceuticals pollution of aquaculture and its management in China. Journal of Molecular Liquids 223: 781-789.
60. Guo Z, Zhou F, Zhao Y, Zhang C, Liu F, et al. (2012) Gamma irradiation-induced sulfadiazine degradation and its removal mechanisms. Chemical Engineering Journal 191: 256-262.
61. Giraldo AL, Peñuela GA, Torres-Palma RA, Pino NJ, Palominos RA, et al. (2010) Degradation of the antibiotic oxolinic acid by photocatalysis with TiO2 in suspension. Water Research 44: 5158-5167. [crossref]
62. Pereira JHOS, Reis AC, Queirós D, Nunes OC, Borges MT, et al. (2013) Insights into solar TiO2-assisted photocatalytic oxidation of two antibiotics employed in aquatic animal production, oxolinic acid and oxytetracycline. Science of The Total Environment 463-464: 274-283. [crossref]
63. dos Louros VL, Silva CP, Nadais H, Otero M, Esteves VI, et al. (2020) Oxolinic acid in aquaculture waters: Can natural attenuation through photodegradation decrease its concentration? Science of The Total Environment 749: 141661. [crossref]
64. Zhu Y, Wei M, Pan Z, Li L, Liang J, et al. (2020) Ultraviolet/peroxydisulfate degradation of ofloxacin in seawater: Kinetics, mechanism and toxicity of products. Science of The Total Environment 705: 135960. [crossref]
65. Chokejaroenrat C, Sakulthaew C, Angkaew A, Satapanajaru T, Poapolathep A, et al. (2019) Remediating sulfadimethoxine-contaminated aquaculture wastewater using ZVI-activated persulfate in a flow-through system. Aquacultural Engineering 84: 99-105.
66. Wen-Li J, Yang-Cheng D, Muhammad Rizwan H, Han JL, Liang B, et al. (2019) A novel TiO2/graphite felt photoanode assisted electro-Fenton catalytic membrane process for sequential degradation of antibiotic florfenicol and elimination of its antibacterial activity. Chemical Engineering Journal 391: 123503.
67. Kong D, Liang B, Yun H, Ma J, Li Z, et al. (2015) Electrochemical degradation of nitrofurans furazolidone by cathode: Characterization, pathway and antibacterial activity analysis. Chemical Engineering Journal 262: 1244-1251.
68. Lin AYC, Lin CF, Chiou JM, Hong PKA (2009) O3 and O3/H2O2 treatment of sulfonamide and macrolide antibiotics in wastewater. Journal of Hazardous Materials 171: 452-458. [crossref]
69. Alonso JJS, El Kori N, Melián-Martel N, Del Río-Gamero B (2018) Removal of ciprofloxacin from seawater by reverse osmosis. Journal of Environmental Management 217: 337-345. [crossref]
70. Divyapriya G, Mohanalakshmi J, kumar KV, Nambi IM (2020) Electro-enhanced adsorptive removal of ciprofloxacin from aqueous solution on graphite felt. Journal of Environmental Chemical Engineering 8: 104299.
71. Peng X, Wang Z, Kuang W, Tan J, Li K (2006) A preliminary study on the occurrence and behavior of sulfonamides, ofloxacin and chloramphenicol antimicrobials in wastewaters of two sewage treatment plants in Guangzhou, China. Science of The Total Environment 371: 314-322. [crossref]
72. Isidori M, Lavorgna M, Nardelli A, Pascarella L, Parrella A (2005) Toxic and genotoxic evaluation of six antibiotics on non-target organisms. Science of The Total Environment 346: 87-98.
73. Rigos G, Nengas I, Alexis M, Troisi GM (2004) Potential drug (oxytetracycline and oxolinic acid) pollution from Mediterranean sparid fish farms. Aquatic Toxicology 69: 281-288. [crossref]
74. Barmpa A, Frousiou O, Kalogiannis S, Perdih F, Turel I, et al. (2018). Manganese (II) complexes of the quinolone family member flumequine: Structure, antimicrobial activity and affinity for albumins and calf-thymus DNA. Polyhedron 145: 166-175.
75. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin.
76. Drzymała J, Kalka J (2020) Ecotoxic interactions between pharmaceuticals in mixtures: Diclofenac and sulfamethoxazole. Chemosphere 259: 127407. [crossref]
77. Guo H, Li Z, Lin S, Li D, Jiang N, et al. (2021) Multi-catalysis induced by pulsed discharge plasma coupled with graphene-Fe3O4 nanocomposites for efficient removal of ofloxacin in water: Mechanism, degradation pathway and potential toxicity. Chemosphere 265: 129089.