Tim Reyns

Toxic textile dyes accumulate in wild European eel Anguilla anguilla

Dyes are used to stain inks, paints, textile, paper, leather and household products. They are omnipresent, some are toxic and may threaten our environment, especially aquatic ecosystems. The presence of residues of sixteen dyes (triarylmethanes, xanthenes, phenothiazines and phenoxazines) and their metabolites was analyzed in muscle tissue samples of individual yellow-phased European eels (Anguilla anguilla) from 91 locations in Belgian rivers, canals and lakes sampled between 2000 and 2009 using ultra performance liquid chromatography-tandem mass spectrometry. Eel was contaminated by dyes in 77% of the sites. Malachite Green, Crystal Violet and Brilliant Green were present in 25-58% of the samples. Dye occurrence was related to the distribution of textile and dye production industries. This field study is the first large-scale survey to document the occurrence of artificial dyes in wildlife. Considering the annual amounts of dyes produced worldwide and the unintentional spillage during their use, our observations warrant additional research in other parts of the world. The presence of these highly toxic dyes in the European eel may form an additional threat to this critically endangered species. The contaminated eels should be considered as not suitable for consumption.

Occurrence of antibiotics in pharmaceutical industrial wastewater, wastewater treatment plant and sea waters in Tunisia

Antibiotics are among the most commonly used group of pharmaceuticals in human medicine. They can therefore reach surface and groundwater bodies through different routes, such as wastewater treatment plant effluents, surface runoff, or infiltration of water used for agricultural purposes. It is well known that antibiotics pose a significant risk to environmental and human health, even at low concentrations. The aim of the present study was to evaluate the presence of aminoglycosides and phenicol antibiotics in municipal wastewaters, sea water and pharmaceutical effluents in Tunisia. All analysed water samples contained detectable levels of aminoglycoside and phenicol antibiotics. The highest concentrations in wastewater influents were observed for neomycin and kanamycin B (16.4 ng mL(-1) and 7.5 ng mL(-1), respectively). Chloramphenicol was found in wastewater influents up to 3 ng mL(-1). It was observed that the waste water treatment plants were not efficient in completely removing these antibiotics. Chloramphenicol and florfenicol were found in sea water samples near aquaculture sites at levels up to, respectively, 15.6 ng mL(-1) and 18.4 ng mL(-1). Also aminoglycoside antibiotics were found near aquaculture sites with the highest concentration of 3.4 ng mL(-1) for streptomycin. In pharmaceutical effluents, only gentamycin was found at concentrations up to 19 ng mL(-1) over a sampling period of four months.

Transsplenic portal catheterization combined with a jugular double-lumen catheter for pharmacokinetic and presystemic metabolization studies in pigs.

The reliability of a silicone double-lumen catheter implanted into the external jugular vein and tunnelled towards the neck region was investigated in eight pigs. Surgery was uneventful without interference with the normal homoeostasis during 8 days. After injection of amoxicillin/clavulanic acid through the distal port of the catheter, analysis of drug components in the simultaneous blood samples obtained by the proximal port and a Venoject system were comparable in one pig. Histological control of the catheterized jugular veins pointed to an acceptable tissue reaction while bacteriological examination of the tip of the catheters was negative in only three animals. A moulding of the intestinal veins was made in a pig cadaver to determine the optimal length of insertion of a silicone portal catheter from the splenic vein towards the portal vein. Surgery was straightforward in four pigs whereby the catheter was exteriorized towards the back region. No complications were encountered during and after surgery for 9 days. The technique of a double-lumen catheter placed into the jugular vein and a transsplenic portal catheter is a useful tool for the study of the pharmacokinetics and also the first-pass effect of drugs in experimental pigs.

Isolation and characterization of antibiotic-resistant bacteria from pharmaceutical industrial wastewaters.

Contamination of surface waters in underdeveloped countries is a great concern. Treated and untreated wastewaters have been discharged into rivers and streams, leading to possible waterborne infection outbreaks which may represent a significant dissemination mechanism of antibiotic resistance genes among pathogenic bacterial populations. The present study aims to determine the multi-drug resistance patterns among isolated and identified bacterial strains in a pharmaceutical wastewater effluent in north Tunisia. Fourteen isolates were obtained and seven of them were identified. These isolates belong to different genera namely, Pseudomonas, Acinetobacter, Exiguobacterium, Delftia and Morganella. Susceptibility patterns of these isolates were studied toward commonly used antibiotics in Tunisia. All the identified isolates were found to have 100% susceptibility against colistin sulfate and 100% resistance against amoxicillin. Among the 11 antibiotics tested, six patterns of multi-drug resistance were obtained. The potential of the examined wastewater effluent in spreading multi-drug resistance and the associated public health implications are discussed.

Rapid method for the confirmatory analysis of chrysoidine in aquaculture products by ultra-performance liquid chromatography-tandem mass spectrometry.

A sensitive and fast method for the quantification of the illegal dye chrysoidine in aquaculture products with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is presented. Muscle tissues were made alkaline with sodium hydroxide and extracted with ethyl acetate. After evaporation and subsequent defatting with n-hexane, extracts were directly injected onto the UPLC-column. Chromatography was performed on a C(18) column using 0.1% formic acid in water and an acetonitrile gradient within 6 min. Mass spectrometric analysis was performed in the positive electrospray MS/MS mode. The limit of quantification was 0.25 ng/g, which was 30 times lower than the only previously published method with gas chromatographic detection. A complete validation according to the scientific literature and as defined by the European Union was performed. The applicability of the method was shown in the analysis of more than 50 unknown samples in the framework of a monitoring program.

Multi-dye residue analysis of triarylmethane, xanthene, phenothiazine and phenoxazine dyes in fish tissues by ultra-performance liquid chromatography-tandem mass spectrometry.

Beside the possible illegal use of malachite green in aquaculture, other familiar dyes could also been applied by fraudulent producers due to their antiseptic and antibacterial activity. In this contribution, a new sensitive multi-residue method was developed to determine triarylmethane, xanthene, phenothiazine and phenoxazine dyes in fish by ultra-performance liquid chromatography-tandem mass spectrometry. Samples were extracted with acetonitrile, followed by an oxidation step using 2,3-dichloro-5,6-dicyanobenzoquinone. Further clean-up was performed by tandem solid phase extraction using weak and strong cation exchange cartridges. Extracts were analysed by UPLC-MS(n) operating in the positive electrospray ionisation mode (ESI+). The fourteen dyes were separated within only 12min on a C18 BEH column using 1mM ammonium acetate in water at pH 4.5 and acetonitrile as mobile phases at a flowrate of 0.4mLmin(-1). The presented method was validated as defined by the European Union and scientific literature. Good linearity (R ≥0.99 and goodness-of-fit (g) ≤10%) was achieved over the tested concentration range (0.25-2ngg(-1)). Limit of quantification was 0.25ngg(-1) for all dyes, with a signal-to-noise ratio of at least 10/1. This is at least 5 to 10 times lower than previous published methods. Limits of detection were all <0.1ngg(-1). Precision and trueness fell within the criteria requested by the EC requirements for this concentration range. Decision limit (CCα) and detection capability (CCβ) were all <1 and <0.25ngg(-1), respectively. Due to background levels of the xanthene dyes, the two rhodamine dyes could only be determined above 0.75ngg(-1). For these dyes, the method can only be used for screening purposes. To show the applicability of the method, a monitoring study was performed to investigate the occurrence of artificial dyes in wildlife European eel in Flemish rivers.

Ecotoxicological potential of antibiotic pollution–industrial wastewater: bioavailability, biomarkers, and occurrence in Mytilus galloprovincialis

Environmental pollution by pharmaceutical residues has become a major problem in many countries worldwide. However, little is known about the concentrations of pharmaceuticals in water sources in Tunisia. Residues in the natural environment have been of increasing concern due to their impact on bacteria resistance development and toxicity to natural communities and ultimately to public health. In this work, we collected the wastewater sample from a pharmaceutical industry, which specializes in the antibiotics manufacture, during the years 2014–2015. Generally, this effluent is discharged into the marine environment and causes environmental problems. The Mediterranean mussel Mytilus galloprovincialis was commonly used as a model organism for its peculiar morphofunctional properties which also make it an excellent marine environmental biomonitoring species. The histological sections of mussel, which are exposed at different dilutions of pharmaceutical wastewater (PW), indicate a large pathological power revealed on the gills. On the other hand, genotoxicity of the studied effluent was evaluated using comet assay for quantification of DNA fragmentation in gill cells. Results show that PW exhibited a statistically significant (p < 0.001) genotoxic effect in a dose-dependent manner. However, the toxic effects of PW decreased significantly after its treatment with Bacillus atrophaeus. Toxicities can be imputed to the presence of antibiotics. In fact, chemical analysis of the gills of mussel M. galloprovincialis using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) showed the presence of some antibiotic residues. These concentrations decrease to half in mussels treated with PW biodegraded by B. atrophaeus.