This study presents a multi-compound method for the determination of 20 pharmacologically active dyes from 5 different chemical classes in environmental water samples. These compounds, including triphenylmethane dyes (malachite green, crystal violet, brilliant green, ethyl violet, methyl violet 2B, pararosaniline, victoria blue B, victoria blue R, victoria pure blue BO), phenothiazine dyes (methylene blue, azure A, azure B, azure C, new methylene blue, thionine), phenoxazine dye (nile blue A), acridine dyes (acriflavine, proflavine) and xanthene dyes (rhodamine B, rhodamine 6G) constitute pharmacologically active substances (PASs). For the optimisation of sample preparation, different solid-phase extraction (SPE) sorbents and a wide range of pH (from 2 to 12) of water samples were tested. Finally, water samples were preconcentrated and cleaned up on diol SPE cartridges. Extracts were analysed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) operating in the positive electrospray ionisation (ESI+) mode. The chromatographic separation of the 20 pharmacologically active dyes was achieved within 5 min by using a pentafluorophenyl (F5) analytical column and mobile phases of ammonium acetate buffer (0.05 M, pH = 3.5) and acetonitrile with gradient elution. The developed method was validated proving to be suitable for the determination of all tested compounds. Limits of quantification were 0.01–0.1 μg/l, are sensitive enough to quantify very low concentration levels of the dyes in environmental water samples. The obtained recovery values for all tested analytes were between 71.2 and 104.9% with a good RSD, less than 14 % at all fortification levels. The application of the developed method to water samples allows the detection of dyes such as crystal violet, rhodamine B, and methyl violet in two wastewater samples in concentration range from 0.017 to 0.0043 μg/l).

The information about mtDNA methylation is still limited, thus epigenetic modification remains unclear. The lack of comprehensive information on the comparative epigenomics of mtDNA prompts comprehensive investigations of the epigenomic modification of mtDNA in different species. This is the first study in which the theoretical CpG localization in the mtDNA reference sequences from various species (12) was compared. The aim of the study was to determine the localization of CpG sites and islands in mtDNA of model organisms and to compare their distribution. The results are suitable for further investigations of mtDNA methylation. The analysis involved both strands of mtDNA sequences of animal model organisms representing different taxonomic groups of invertebrates and vertebrates. For each sequence, such parameters as the number, length, and localization of CpG islands were determined with the use of EMBOSS (European Molecular Biology Open Software Suite) software. The number of CpG sites for each sequence was indicated using the newcpgseek algorithm. The results showed that methylation of mtDNA in the analysed species involved mitochondrial gene expression. Our analyses showed that the CpG sites were commonly present in genomic regions including the D-loop, CYTB, ND6, ND5, ND4, ND3, ND2, ND1, COX3, COX2, COX1, ATP6, 16s rRNA, and 12s rRNA. The CpG distribution in animals from different species was diversified. Generally, the number of observed CpG sites of the mitochondrial genome was higher in the vertebrates than in the invertebrates. However, there was no relationship between the frequency of the CpG sites in the mitochondrial genome and the complexity of the analysed organisms. Interestingly, the distribution of the CpG sites for tRNA coding genes was usually cumulated in a larger CpG region in vertebrates. This paper may be a starting point for further research, since the collected information indicates possible methylation regions localized in mtDNA among different species including invertebrates and vertebrates.

This is the first study in which the Daphnia magna (D. magna) nuclear genome (nDNA) obtained from the GenBank database was analyzed for pseudogene sequences of mitochondrial origin. To date, there is no information about pseudogenes localized in D. magna genome. This study aimed to identify NUMTs, their length, homology, and location for potential use in evolutionary studies and to check whether their occurrence causes co-amplification during mitochondrial genome (mtDNA) analyses. Bioinformatic analysis showed 1909 fragments of the mtDNA of D. magna, of which 1630 were located in ten linkage groups (LG) of the nDNA. The best-matched NUMTs covering >90% of the gene sequence have been identified for two mt-tRNA genes, and they may be functional nuclear RNA molecules. Isolating the total DNA in mtDNA studies, co-amplification of nDNA fragments is unlikely in the case of amplification of the whole tRNA genes as well as fragments of other genes. It was observed that TRNA-MET fragments had the highest level of sequence homology, thus they could be evolutionarily the youngest. The lowest homology was found in the D-loop-derived pseudogene. It may probably be the oldest NUMT incorporated into the nDNA; however, further analysis is necessary.