Spatial and seasonal distribution of cyanobacteria Moorea species in coastal waters of Tanzania.

This study aimed at identifying the presence of harmful cyanobacteria, detecting potential harmful algae toxins and their distribution in three seasons: December to February (hot dry season), March to May (rainy season), and June to November (cool dry season) of 2016. The samples were collected in five study sites in Tanzania: Tumbe, Chwaka, Paje, Bweleo in Zanzibar islands and Songosongo Island, mainland Tanzania, where skin irritation problems were observed in seaweed workers in an earlier study. The cyanobacteria from the Moorea genus were microscopically detected in the seawater, with highest concentrations in the months with the highest seawater temperature or hot dry season, than in the other two seasons. The concentration of Moorea species was significantly higher in Songosongo, Tanzania mainland than in Zanzibar Islands in all three seasons, corresponding to the higher level of nutrients of nutrients (PO43-, NO3- and NH4+) in the prior season. However, the concentrations were considered relatively low and thus not collected during an ongoing algal bloom. This is one of the first studies that detect Moorea sp. in Tanzanian seawater, and complementary studies including genome sequencing to characterize the species are warranted.

The gill epithelial cell lines RTgill-W1, from Rainbow trout and ASG-10, from Atlantic salmon, exert different toxicity profiles towards rotenone.

In order to ensure the proper use and interpretation of results from laboratory test systems, it is important to know the characteristics of your test system. Here we compare mitochondria and the handling of reactive oxygen species (ROS) in two gill epithelial cell lines, the well-known RTgill-W1 cell line from Rainbow trout and the newly established ASG-10 cell line from Atlantic salmon. Rotenone was used to trigger ROS production. Rotenone reduced metabolic activity and induced cell death in both cell lines, with RTgill-W1 far more sensitive than ASG-10. In untreated cells, the mitochondria appear to be more fragmented in RTgill-W1 cells compared to ASG-10 cells. Furthermore, rotenone induced mitochondrial fragmentation, reduced mitochondria membrane potential (Δψm) and increased ROS generation in both cell lines. Glutathione (GSH) and catalase is important to maintain the cellular oxidative balance by eliminating hydrogen peroxide (H2O2). In response to rotenone, both GSH and catalase depletion were observed in the RTgill-W1 cells. In contrast, no changes were found in the GSH levels in ASG-10, while the catalase activity was increased. In summary, the two salmonid gill cell lines have different tolerance towards ROS, probably caused by differences in mitochondrial status as well as in GSH and catalase activities. This should be taken into consideration with the selection of experimental model and interpretation of results. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00560-0.

Alpha-chloralose poisoning in cats in three Nordic countries - the importance of secondary poisoning.

BACKGROUND: Alpha-chloralose (AC) is a compound known to be toxic to various animal species and humans. In 2018 and 2019 an increase in suspected cases of AC poisoning in cats related to the use of AC as a rodenticide was reported to national veterinary and chemical authorities in Finland, Norway and Sweden by veterinarians working in clinical practices in respective country. The aims of this study were to prospectively investigate AC poisoning in cats, including possible secondary poisoning by consuming poisoned mice, and to study metabolism and excretion of AC in cats through analysis of feline urine. METHODS: Data on signalment, history and clinical findings were prospectively collected in Finland, Norway and Sweden from July 2020 until March of 2021 using a questionnaire which the attending veterinarian completed and submitted together with a serum sample collected from suspected feline cases of AC-poisoning. The diagnosis was confirmed by quantification of AC in serum samples. Content of AC was studied in four feline urine samples, including screening for AC metabolites by UHPLC-HRMS/MS. Bait intake and amount of AC consumed by mice was observed in wild mice during an extermination of a rodent infestation. RESULTS: In total, 59 of 70 collected questionnaires and accompanying serum samples were included, with 127 to 70 100 ng/mL AC detected in the serum. Several tentative AC-metabolites were detected in the analysed feline urine samples, including dechlorinated and oxidated AC, several sulfate conjugates, and one glucuronic acid conjugate of AC. The calculated amount of AC ingested by each mouse was 33 to 106 mg with a mean of 61 mg. CONCLUSIONS: Clinical recognition of symptoms of AC poisoning in otherwise healthy cats roaming free outdoors and known to be rodent hunters strongly correlated with confirmation of the diagnosis through toxicological analyses of serum samples. The collected feline exposure data regarding AC show together with the calculation of the intake of bait and subsequent AC concentrations in mice that secondary poisoning from ingestion of mice is possible. The results of the screening for AC metabolites in feline urine confirm that cats excrete AC both unchanged and metabolized through dechlorination, oxidation, glucuronidation and sulfatation pathways.

Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids.

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC-MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26-C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC-MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90-92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

An Official Outbreak Investigation of Acute Haemorrhagic Diarrhoea in Dogs in Norway Points to Providencia alcalifaciens as a Likely Cause.

An outbreak investigation was initiated in September 2019, following a notification to the Norwegian Food Safety Authority (NFSA) of an unusually high number of dogs with acute haemorrhagic diarrhoea (AHD) in Oslo. Diagnostic testing by reporting veterinarians had not detected a cause. The official investigation sought to identify a possible common cause, the extent of the outbreak and prevent spread. Epidemiological data were collected through a survey to veterinarians and interviews with dog owners. Diagnostic investigations included necropsies and microbiological, parasitological and toxicological analysis of faecal samples and food. In total, 511 dogs with acute haemorrhagic diarrhoea were registered between 1 August and 1 October. Results indicated a common point source for affected dogs, but were inconclusive with regard to common exposures. A notable finding was that 134 of 325 faecal samples (41%) cultured positive for Providencia alcalifaciens. Whole genome sequencing (WGS) of 75 P. alcalifaciens isolates from 73 dogs revealed that strains from 51 dogs belonged to the same WGS clone. Findings point to P. alcalifaciens as implicated in the outbreak, but investigations are needed to reveal the pathogenic potential of P. alcalifaciens in dogs and its epidemiology.

In Vitro Metabolism of Azaspiracids 1-3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis).

Azaspiracids (AZAs) are a group of biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. Of the 60 AZAs identified, levels of AZA1, AZA2, and AZA3 are regulated in shellfish as a food safety measure based on occurrence and toxicity. Information about the metabolism of AZAs in shellfish is limited. Therefore, a fraction of blue mussel hepatopancreas was made to study the metabolism of AZA1-3 in vitro. A range of AZA metabolites were detected by liquid chromatography-high-resolution tandem mass spectrometry analysis, most notably the novel 22α-hydroxymethylAZAs AZA65 and AZA66, which were also detected in naturally contaminated mussels. These appear to be the first intermediates in the metabolic conversion of AZA1 and AZA2 to their corresponding 22α-carboxyAZAs (AZA17 and AZA19). α-Hydroxylation at C-23 was also a prominent metabolic pathway, producing AZA8, AZA12, and AZA5 as major metabolites of AZA1-3, respectively, and AZA67 and AZA68 as minor metabolites via double-hydroxylation of AZA1 and AZA2, but only low levels of 3ß-hydroxylation were observed in this study. In vitro generation of algal toxin metabolites, such as AZA3, AZA5, AZA6, AZA8, AZA12, AZA17, AZA19, AZA65, and AZA66 that would otherwise have to be laboriously purified from shellfish, has the potential to be used for the production of standards for analytical and toxicological studies.

Urinary deoxynivalenol as a biomarker of exposure in different age, life stage and dietary practice population groups.

The Fusarium mycotoxin deoxynivalenol (DON) and its modified forms are present in most samples of grain and grain-based products. Due to the widespread presence of DON in these highly consumed food commodities, nearly all individuals are exposed to DON. Previous estimates of the dietary DON intake in Norway indicated that children's dietary intake is close to or exceed the TDI of 1 µg/kg bw/day for the sum of DON and three modified forms. One aim of the current study was to determine whether the concentrations of DON in morning urine differ between population groups like men, women, children, vegetarians, and pregnant women. An additional aim was to compare a set of models for estimating the dietary intake of DON based on urinary DON concentrations and also compare these models with DON-intakes estimated using food consumption data. DON and metabolites were detected in the morning urine from 256 out of 257 individuals and with concentrations in similar range as reported from other countries. Children have higher urinary DON-concentration than adults and elderly. The urinary DON-concentration in pregnant women and vegetarians did not differ from other adults. The estimated intake of DON was higher for children than for other age groups on a body weight basis. The correlations between different models for estimating DON-intake based on urinary concentration as well as based on individual food consumption were good (0.79-0.99), but with some outliers. We conclude that Norwegians are exposed to DON in the same range as reported from other countries and that children have a higher exposure than adults. Furthermore, we conclude that intake estimates based on urinary DON concentration is a useful tool for evaluation of the exposure at population level, but due to outliers, the estimates for individuals are uncertain. There are also uncertainties in intake estimates both from food consumption and from urinary DON concentration, and we could not conclude on which approach provides the most accurate exposure estimate.

Identification of 24-O-ß-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Dinophysis Blooms, Dinophysis and Prorocentrum Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-ß-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1-5, the 24-O-ß-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4-6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

In vitro hepatic biotransformation of the algal toxin pectenotoxin-2.

We have investigated the in vitro metabolism of pectenotoxin-2 (PTX-2) using primary hepatocytes from Wistar rats in suspension. Purified PTX-2 was rapidly metabolized. Two major and several minor oxidized PTX-2 metabolites were formed, none of which had retention times corresponding to PTX-1, -11, or -13. Hydrolysis products, such as PTX-2 seco acid, were not observed. Preliminary multi-stage LC-MS analyses indicated that the major hepatic PTX-2 metabolites resulted from the insertion of an oxygen atom at the positions C-19 to C-24, or at C-44. The rapid oxidative metabolism may explain the low oral toxicity of PTXs observed in vivo studies.

Comparison of anticoagulant rodenticide concentrations in liver and feces from apparently healthy red foxes.

Exposure of wildlife and domestic animals to anticoagulant rodenticides (ARs) is a worldwide concern, but few methods exist to determine residue levels in live animals. Traditional liver detection methods preclude determining exposure in live wildlife. To determine the value of assessing AR exposure by fecal analysis, we compared fecal and liver residues of ARs in the same animals. We collected liver and fecal samples from 40 apparently healthy red foxes (Vulpes vulpes) potentially exposed to ARs, and quantified brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen residues by liquid chromatography-tandem mass spectrometry. Residues of ARs were detected in 53% of the fecal samples and 83% of the liver samples. We found good concordance between AR residues in feces and liver for coumatetralyl, difenacoum, and difethialone. Bromadiolone occurred in significantly greater frequency in livers compared to feces, but no significant difference in concentration between feces and liver in individual foxes could be detected. Brodifacoum displayed a significant difference in concentration and occurrence of positive samples between liver and feces. Our findings demonstrate that fecal analysis of ARs provides a feasible and valuable non-lethal means of determine AR exposure in live wildlife.

Isolation of the Tephrosia vogelii extract and rotenoids and their toxicity in the RTgill-W1 trout cell line and in zebrafish embryos.

This study focused on identifying the rotenoids from the Tephrosia vogelli plant (fish-poison-bean), investigating the toxic potency of a crude T. vogelii extract and individual rotenoids (tephrosin, deguelin and rotenone) in vitro and in vivo and assessing the mode of action. A trout (Onychorynhis mykiss) gill epithelial cell line (RTgill-W1) was used to determine the cytotoxicity of rotenoids and effects on cell metabolism. Zebrafish (Danio rerio) aged from 3 h post fertilization (hpf) to 72 hpf were used for testing the developmental toxicity. The crude T. vogelii plant extract significantly decreased the cellular metabolic activity and was cytotoxic at lower concentrations (5 and 10 nM, respectively), while tephrosin, deguelin and rotenone showed these effects at concentrations ≥ 50 nM. The crude T. Vogelli extract had the highest toxic potency and induced adverse health effects in zebrafish including deformities and mortality at the lowest concentration (5 nM) compared to rotenone (10 nM) and deguelin and tephrosin (50 nM). These results indicate that the crude T. Vogelii extracts are highly potent and the bioactivity of these extracts warrant further investigation for their potential use to treat parasites in human and veterinary medicine and as a natural alternative to pesticides.

Anabolic-androgenic steroid use and correlates in Norwegian adolescents.

This paper surveys the prevalence and correlates of anabolic androgenic steroids (AAS) use among Norwegian adolescents, and examines the degree to which sports participation is a mediating or moderating factor to well-known correlations between AAS use and problem behaviour. The data come from the "Ungdata" study, a cross-national youth survey system offered to all municipalities in Norway (response rate: 74%, N = 77,572). The study demonstrates a lifetime prevalence of AAS use of 1.27% and a higher prevalence among boys (1.81%) than girls (0.76%). The analyses show that AAS use is clearly related to problem behaviour such as violence and other substance use. When controlling for problem behaviour, there are no correlations between AAS use and exercising in a sports club or on one's own, whilst there is a weak positive correlation between AAS use and exercising in a gym or engaging in other forms of physical exercise such as dancing or martial arts. These patterns are more or less the same for boys and for girls. We conclude that adolescent AAS use is a low-prevalence phenomenon that primarily takes place in smaller subgroups of individuals who engage in other forms of problem behaviour as well.

Occurrence of cyclic imines in European commercial seafood and consumers risk assessment.

Cyclic imines constitute a quite recently discovered group of marine biotoxins that act on neural receptors and that bioaccumulate in seafood. They are grouped together due to the imino group functioning as their common pharmacore, responsible for acute neurotoxicity in mice. Cyclic imines (CIs) have not been linked yet to human poisoning and are not regulated in the European Union (EU), although the European Food Safety Authority (EFSA) requires more data to perform conclusive risk assessment for consumers. Several commercial samples of bivalves including raw and processed samples from eight countries (Italy, Portugal, Slovenia, Spain, Ireland, Norway, The Netherlands and Denmark) were obtained over 2 years. Emerging cyclic imine concentrations in all the samples were analysed on a LC-3200QTRAP and LC-HRMS QExactive mass spectrometer. In shellfish, two CIs, pinnatoxin G (PnTX-G) and 13-desmethylspirolide C (SPX-1) were found at low concentrations (0.1-12µg/kg PnTX-G and 26-66µg/kg SPX-1), while gymnodimines and pteriatoxins were not detected in commercial (raw and processed) samples. In summary, SPX-1 (n: 47) and PnTX-G (n: 96) were detected in 9.4% and 4.2% of the samples, respectively, at concentrations higher than the limit of quantification (LOQ), and in 7.3% and 31.2% of the samples at concentrations lower than the LOQ (25µg/kg for SPX-1 and 3µg/kg for PnTX-G), respectively. For the detected cyclic imines, the average exposure and the 95th percentile were calculated. The results obtained indicate that it is unlikely that a potential health risk exists through the seafood diet for CIs in the EU. However, further information about CIs is necessary in order to perform a conclusive risk assessment.

Characterization of Deoxynivalenol-Glutathione Conjugates Using Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass Spectrometry.

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,ß-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,ß-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC-MS with thiol derivatization.

Microcystins are cyclic heptapeptides from cyanobacteria which are responsible for poisonings of livestock and humans. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analysed by LC-MS. Thiol derivatization of the α,ß-unsaturated amide present in most microcystins was recently shown to simplify analysis of LC-MS chromatograms of a Microcystis culture, making it easier to identify peaks corresponding to microcystins in complex mixtures. This method was applied to analysis of extracts taken from a natural cyanobacteria bloom in Mwanza Gulf, Lake Victoria, Tanzania, in 2010, revealing the presence of numerous putative microcystin analogues in the sample. Results were verified using LC-MS², LC-MS/MS with precursor-ion scanning, and LC-HRMS, leading to identification of 8 major and 17 minor microcystins in the sample, including analogues of microcystin-RY, -RL and -RA. Microcystin-YR (2), -RR (3), and -RY (9) were isolated from bloom material from Lake Victoria, and the structure of 9 was confirmed by NMR spectroscopic analysis and NMR spectral comparison with 2 and 3. Confirmation of the structure of MC-RY (9) facilitated detailed analysis of its MS² spectrum, thereby supporting the structures of related analogues tentatively established on the basis of MS analyses.

LC-MS analysis with thiol derivatization to differentiate [Dhb(7)]- from [Mdha(7)]-microcystins: analysis of cyanobacterial blooms, Planktothrix cultures and European crayfish from Lake Steinsfjorden, Norway.

Kinetic studies showed that [Asp(3), Dhb(7)]MC-RR reacted with mercaptoethanol hundreds of times more slowly than MC-RR and a range of other [Mdha(7)]-containing microcystin congeners. The difference in reaction rate was sufficiently large that derivatization of microcystin-containing samples with mercaptoethanol, followed by LC-MS analysis, clearly discriminated between microcystins containing the isobaric [Dhb(7)]- and [Mdha(7)]-groups. Application of this approach, using LC-MS with both-ion trap and triple-quadrupole mass spectrometers, to water samples and Planktothrix cultures from Lake Steinsfjorden, Norway, demonstrated the presence of [Asp(3), Dhb(7)]MC-RR (5), [Asp(3)]MC-RY (14), and [Asp(3)]MC-LY (16), as well as analogues tentatively identified as [Asp(3)]MC-RR (4), [Asp(3), DMAdda(5), Dhb(7)]MC-LR (6), [Asp(3), Dhb(7)]MC-HtyR (8), [Asp(3)]MC-HtyR (9), [Asp(3), Dhb(7)]MC-LR (10), [Asp(3)]MC-LR (11), [Asp(3), Dhb(7)]MC-RY (15), and [Asp(3), Dhb(7)]MC-LY (17), together with low levels of several other analogues. This is the first use of this thiol-based LC-MS approach to identify Dhb-containing microcystins, and allowed identification of LC-MS peaks in a mixture of [Mdha(7)]- and [Dhb(7)]-congeners of [Asp(3)]MC-RR (4, 5), -RY (14, 15), and -LY (16, 17) in the samples from L. Steinsfjorden. This is also the first report of MC-RY-congeners outside of Africa, or in Planktothrix spp. Analysis of European crayfish (Astacus astacus) taken from L. Steinsfjorden revealed the presence of only trace levels of microcystins in the edible parts.

Thiol derivatization for LC-MS identification of microcystins in complex matrices.

Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,ß-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS(2) spectra was facilitated by addition of the mass associated with the thiol to the α,ß-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O'-methyl-hexa(ethylene glycol) (MEMHEG) (M(w)t. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.

Assessment of acetylcholinesterase activity in Clarias gariepinus as a biomarker of organophosphate and carbamate exposure.

The objective of this study was to investigate the response of acetylcholinesterase (AChE) activities in Clarias gariepinus in response to Organophosphates (Ops) and carbamate exposure. The AChE activities were determined in plasma, and eye and brain homogenates of unexposed and exposed fish using Ellman's method and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) chromophore. The baseline AChE activities in plasma, eyes and brain tissues in unexposed fish were comparable between males and females (P > 0.05). Concentrations of pesticides that inhibited 50% (IC(50)) of AChE activities in brain homogenates following in vitro exposures were 0.003, 0.03, 0.15, 190, 0.2, 0.003 and 0.002 microM for carbaryl, chlorfenvinphos, diazinon, dimethoate, fenitrothion, pirimiphosmethyl and profenofos, respectively. The in vivo dose-effect relationships were assessed using chlorfenvinphos and carbaryl at different concentrations that ranged from 0.0003 to 0.06 microM and 0.0005 to 0.05 microM, respectively. Acetylcholinesterase activities were comparable in plasma, and eye and brain homogenates from control and carbaryl-exposed fish. Following exposure of fish to chlorfenvinphos at concentrations above 0.03 microM, a significant inhibition of AChE activities in plasma (84%) and eye homogenate (50%) was observed. The AChE activities in brain homogenate were comparable between chlorfenvinphos-exposed fish and controls. Because carbaryl cause reversible inhibition of AChE activities was found to be more potent than chlorfenvinphos that cause irreversible inhibition following in vitro exposure. Contrary, carbaryl was less potent than chlorfenvinphos after in vivo exposure possibly due to more rapid biotransformation of carbaryl than chlorfenvinphos. Findings from this study have demonstrated that inhibition of AChE activity in C. gariepinus is a useful biomarker in assessing aquatic environment contaminated by anticholinesterases.

Assessment of pollution in sewage ponds using biomarker responses in wild African sharptooth catfish (Clarias gariepinus) in Tanzania.

The interactive effects of mixed pollutants in sewage wastewater on biomarker responses were investigated using wild male African sharptooth catfish (Clarias gariepinus) in Morogoro, Tanzania. A total of 58 fish were used, of which 21 were from Mindu dam (reference site) and 22, 9 and 10 from Mafisa, Mazimbu and Mzumbe sewage ponds, respectively. Liver somatic index (LSI) and gonadosomatic index (GSI) were significantly greater (two- to threefold) and (five- to sixfold), respectively, in fish from all sewage ponds. Haemoglobin concentration and gill filament 7-ethoxyresurufin O-deethylase (EROD) activities were significantly higher (1.2-fold and twofold, respectively) in fish from Mzumbe sewage ponds than in fish from Mindu dam, whereas liver EROD activity was significantly higher in fish from Mzumbe and Mafisa sewage ponds (5-fold). A HPLC method for determination of enzymatically formed p-nitrophenyl-glucuronide (PNPG) was developed and applied to measure UDP-glucuronosyl transferase (UGT) activities that was significantly higher in fish from all sewage ponds (2-2.5-fold) than in fish from Mindu dam. Kinetic characteristics and assay dependence of UGT were studied with microsomal preparations. Metallothionein (MT) content was significantly lower (three- to fourfold) in fish from sewage ponds than in fish from Mindu dam, and corresponded with cumulative levels of cadmium, lead and mercury. Condition factor, vitellogenin (Vtg), acetylcholinesterase (AChE) activities in plasma, eyes and brain, haematocrit, plasma protein and cytosolic glutathione S-transferase (GST) activities were comparable in fish from sewage ponds and Mindu dam. Although specific pollutants other than the metals were not identified by chemical analysis, application of a suite of biomarkers in C. gariepinus demonstrated that all sewage ponds were contaminated by pollutants of public health concern.