Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos.

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Assessment of toxicity potential of neglected Mithi River water from Mumbai megacity, India, in zebrafish using embryotoxicity, teratogenicity, and genotoxicity biomarkers.

The Mithi River begins at Vihar Lake and flows through the industrial hub of the city of Mumbai, India, and merges with the Arabian Sea at Mahim Creek. The current study was carried out to assess the ecotoxicological effects of the Mithi River surface water in zebrafish (Danio rerio) embryos. Water samples were collected from ten sampling sites (S1 to S10) located along the course of the Mithi River. The toxicity of water samples was assessed using a zebrafish embryo toxicity test (ZFET). Water samples were diluted from all sites at 1:0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 times. The lowest and highest LDil 20 values for 96 h were estimated as 9.16 and 74.18 respectively for the S2 and S5 sites. The results of embryotoxicity and teratogenicity assays indicated a significant difference (p < 0.0001) between embryos exposed to control and sampling sites (except S1) for various endpoints such as mortality, egg coagulation, pericardial edema, yolk sac edema, tail bend, and skeletal deformities. The histopathological analysis revealed various lesions, ascertaining the toxic effects of water samples. The comet assay revealed significantly higher DNA damage (except S1) in embryos exposed to sites S5 and S6 with OTM values of 4.46 and 2.48 respectively. The results indicated that the Mithi River is polluted with maximum pollution load at the middle stretches. The study further indicated that the pollutants in the Mithi River (except S1) could potentially be hazardous to the aquatic organisms; therefore, continuous biomonitoring of the river is needed for its revival.

Teleost Nonapeptides, Isotocin and Vasotocin Administration Released the Milt by Abdominal Massage in Male Catfish, Clarias magur.

In the present work the nonapeptides i.e., isotocin and vasotocin alone or in a combination were tested in C. magur to evaluate their effect on stripping by abdominal massage. Also, we used chitosan-carbon nanotube nanocomposites to conjugate the nonapetides isotocin (abbreviated as COOH-SWCNTCSPeP) and isotocin and vasotocin (COOH-SWCNTCSPePs) with the aim of sustaining the effect for a longer duration. The conjugation of nonapeptides with nanocomposites was confirmed by Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). Two experiments were conducted to study the effect of naked (without nanoparticles) and conjugated nonapeptides on the milt release by stripping. Both the experiments consisted of eight treatments which included four naked groups two nanoconjugated groups and two controls. Both naked and nonconjugated formulations were successful in stripping the male catfish. The mRNA expression of selected reproductive genes was analysed to decipher the effect of nanopeptides at the molecular level. Nonapeptide treatment either naked or nanoconjugated, resulted in the upregulation of the transcript level of genes. Histological analysis revealed the concentration of spermatozoa was more in peptide injected groups than in the controls. The synergistic effects of nonapeptides and Ovatide had a positive impact on GSI. Thus, the present formulations were successful in stripping the male catfish to obtain the milt with significant reproductive success. Even though the naked groups perform better but the number of males required to fertilize the eggs in nanoconjuagted groups was smaller making it worth using for the delivery of nonapeptides.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.