An LC-MS-based designated comparison method with similar performance to the Lp(a) reference measurement procedure to guide molar Lp(a) standardization.

BACKGROUND: The 2022 consensus statement of the European Atherosclerosis Society (EAS) on lipoprotein(a) (Lp(a)) recognizes the role of Lp(a) as a relevant genetically determined risk factor and recommends its measurement at least once in an individual's lifetime. It also strongly urges that Lp(a) test results are expressed as apolipoprotein (a) (apo(a)) amount of substance in molar units and no longer in confounded Lp(a) mass units (mg/dL or mg/L). Therefore, IVD manufacturers should transition to molar units. A prerequisite for this transition is the availability of an Lp(a) Reference Measurement Procedure (RMP) that allows unequivocal molecular detection and quantification of apo(a) in Lp(a). To that end an ISO 17511:2020 compliant LC-MS based and IFCC-endorsed RMP has been established that targets proteotypic peptides of apolipoprotein(a) (apo(a)) in Lp(a). The RMP is laborious and requires highly skilled operators. To guide IVD-manufacturers of immunoassay-based Lp(a) test kits in the transition from mass to molar units, a Designated Comparison Method (DCM) has been developed and evaluated. METHODS: To assess whether the DCM provides equivalent results compared to the RMP, the procedural designs were compared and the analytical performance of DCM and RMP were first evaluated in a head-to-head comparison. Subsequently, apo(a) was quantified in 153 human clinical serum samples. Both DCM and RMP were calibrated using external native calibrators that produce results traceable to SRM2B. Measurement uncertainty (MU) was checked against predefined allowable MU. RESULTS: The major difference in the design of the DCM for apo(a) is the use of only one enzymatic digestion step. The analytical performance of the DCM and RMP for apo(a) is highly similar. In a direct method comparison, equivalent results were obtained with a median regression slope 0.997 of and a median bias of - 0.2 nmol/L (- 0.2%); the intermediate imprecision of the test results was within total allowable error (TEa) (CVa of 10.2% at 90 nmol/L). CONCLUSIONS: The semi-automated, higher throughput, LC-MS-based method for Lp(a) meets the predefined analytical performance specifications and allowable MU and is hence applicable as a higher order Designated Comparison Method, which is ideally suited to guide IVD manufacturers in the transition from Lp(a) mass to molar units.

Quantitative protein mass-spectrometry requires a standardized pre-analytical phase.

OBJECTIVES: Quantitative protein mass-spectrometry (QPMS) in blood depends on tryptic digestion of proteins and subsequent measurement of representing peptides. Whether serum and plasma can be used interchangeably and whether in-vitro anticoagulants affect the recovery is unknown. In our laboratory serum samples are the preferred matrix for QPMS measurement of multiple apolipoproteins. In this study, we investigated the effect of different matrices on apolipoprotein quantification by mass spectrometry. METHODS: Blood samples were collected from 44 healthy donors in Beckton Dickinson blood tubes simultaneously for serum (with/without gel) and plasma (heparin, citrate or EDTA). Nine apolipoproteins were quantified according to standard operating procedure using value-assigned native serum calibrators for quantitation. Tryptic digestion kinetics were investigated in the different matrices by following formation of peptides for each apolipoprotein in time, up to 22 h. RESULTS: In citrate plasma recovery of apolipoproteins showed an overall reduction with a bias of -14.6%. For heparin plasma only -0.3% bias was found compared to serum, whereas for EDTA-plasma reduction was more pronounced (-5.3% bias) and variable with >14% reduction for peptides of apoA-I, A-II and C-III. Digestion kinetics revealed that especially slow forming peptides showed reduced formation in EDTA-plasma. CONCLUSIONS: Plasma anticoagulants affect QPMS test results. Heparin plasma showed comparable results to serum. Reduced concentrations in citrate plasma can be explained by dilution, whereas reduced recovery in EDTA-plasma is dependent on altered proteolytic digestion efficiency. The results highlight the importance of a standardized pre-analytical phase for accurate QPMS applications in clinical chemistry.

Novel insights into the genetics, morphology, distribution and hosts of the global fish parasitic digenean Proctoeces maculatus (Looss, 1901) (Digenea: Fellodistomidae).

Larval stages of the widely distributed digenean species Proctoeces maculatus (Looss, 1901) were reported 40 years ago from South Africa in the common octopus, Octopus vulgaris Cuvier (Octopodidae). However, the absence of adult specimens and molecular data from this region has hindered a comprehensive understanding of its distribution. In this study, we collected three species of intertidal and near-shore marine fishes [Clinus superciliosus (L.) (Clinidae), Diplodus capensis (Smith) (Sparidae) and Sparodon durbanensis (Castelnau) (Sparidae)] along the South African coast and discovered adult specimens of P. maculatus at five localities. By employing a combination of morphological and molecular techniques, including 28S rDNA, 18S rDNA and COI mtDNA analyses, the first report of adult P. maculatus from South Africa is presented. The findings encompass a comprehensive morphological description and molecular data, illuminating the true distribution of this species in the region.

A stable isotope analysis of the dietary patterns of the aquatic apex predator, the African tigerfish (Hydrocynus vittatus).

Stable isotope analyses, specifically δ13 C and δ15 N, are useful tools increasingly used to understand ecosystem function, food web structures, and consumer diets. Although the iconic tigerfish Hydrocynus vittatus is regarded as an apex predator in southern African freshwater systems, little information is available regarding their feeding behavior and how this may change with growth or differ between ecosystems, with most information stemming from stomach content analyses (SCA). The aim of the present study was to address this lack of information through a baseline study of the diet of large and small tigerfish in various lentic and lotic ecosystems in South Africa using stable isotope methods. Fish and various food web components and food sources were collected from two river and two lake ecosystems in South Africa. The δ13 C and δ15 N values for all samples were determined and multivariate analyses and Bayesian analytical techniques applied to determine the feeding ecology of H. vittatus and how this may differ with size and habitat type. Analyses revealed a substantial difference in the type and abundance of food sources contributing to the diet of H. vittatus between ecosystems, most prominently between the lotic systems, where less dietary specialization was observed, and lentic systems where more specialization was observed. Furthermore, there was a distinct difference in diet between small and large tigerfish, especially in the lotic system, indicating an ontogenetic diet shift as tigerfish grow and further supporting previous SCA studies. This is the first study of its kind on the African continent for H. vittatus and the findings illustrate the value of stable isotope analysis in providing in-depth information into the feeding ecology of consumers and how this may differ between size classes and habitat types.

Morphological and molecular characterisation of Alella igillimpethu n. sp. (Copepoda: Siphonostomatoida: Lernaeopodidae) parasitising the southern African endemic intertidal klipfish, Clinus superciliosus.

Lernaeopodidae Milne Edwards, 1840, is an ecological and economically important fish parasite family of copepods (Crustacea: Copepoda), consisting of 48 genera with 334 valid species. To date, approximately 17 genera have been documented from both teleost and elasmobranch hosts from South African marine waters. As part of parasitological surveys targeting parasites of the endemic intertidal klipfish, Clinus superciliosus (Linnaeus) (Clinidae) along the South African coast, a species of Lernaeopodidae was discovered on the gills of this host. Alella igillimpethu n. sp. is described from Clinus superciliosus from Langebaan marina on the west coast of South Africa, with a prevalence of 42.1% and mean intensity of 2.9 (ranging from 1-13). Morphological identification was done using light and scanning electron microscopy and the species description was generated with the aid of DEscription Language for TAxonomy (DELTA) software. The mitochondrial cytochrome c oxidase subunit 1 (COI) region, as well as partial 18S and 28S ribosomal RNA genes confirmed the classification within the family Lernaeopodidae. This is the first report of Lernaeopodidae from C. superciliosus, or in fact any member of Clinidae, as well as the first molecular characterisation of any marine lernaeopodid infecting teleost fish from South Africa. This study contributes valuable genetic and morphological data for this copepod family, as well as new host and distribution records.

A new species of Phoreiobothrium Linton, 1889 (Cestoda: Onchoproteocephalidea) from the spinner shark, Carcharhinus brevipinna (Valenciennes) off the coast of KwaZulu-Natal, South Africa.

The extreme sparsity of collection efforts in many parts of the word, including southern Africa, leaves a vast hidden diversity of marine cestode species, such as species of Phoreiobothrium. The examination of a common South African coastal-pelagic shark species, Carcharhinus brevipinna (Valenciennes), resulted in the discovery of a new species of Phoreiobothrium Linton, 1889. Phoreiobothrium martini sp. n. is undoubtedly different from all other congeners in several morphological characteristics, however, it is most easily distinguished from other species by its total length, the size of its hooks, and the number of post vaginal testes. The addition of P. martini sp. n. increases the total number of valid species of Phoreiobothrium to 20 worldwide. Furthermore, P. martini sp. n. marks the description of representatives of only the second onchoproteocephalidean genus from southern Africa, therefore expanding the generic biogeographical representation and introducing new host associations. Apart from the description of this new species of cestode, the discovery of additional species of Phoreiobothrium will be beneficial regarding future ecological investigations. With the high degree of host-specificity found amongst species of Phoreiobothrium, species such as P. martini sp. n. could be used as sentinel species for the accurate identification, separation and diagnosis of commonly misidentified shark species.

A new species of Siphoderina Manter, 1934 (Digenea: Cryptogonimidae) infecting the Dory Snapper Lutjanus fulviflamma (Teleostei: Lutjanidae) from the east coast of South Africa.

Parasitological assessment of marine fishes at Sodwana Bay in the iSimangaliso Marine Protected Area on the east coast of South Africa revealed a new species of cryptogonimid trematode infecting the pyloric caeca of the Dory Snapper, Lutjanus fulviflamma (Forsskål) (Lutjanidae). The new species is morphologically consistent with the concept of the large genus Siphoderina Manter, 1934; its phylogenetic position within this genus was validated through molecular sequencing of the ITS2 and partial 28S ribosomal DNA sub-regions. We name this species Siphoderina nana n. sp. and comment on the current state of understanding for this genus of cryptogonimids.

Using Confocal Microscopy and Pigment Analyses to Detect Adverse Insecticide Effects in non-target Freshwater Diatom species - a proof-of-concept Using Nitzschia palea.

The persistence of insecticides in aquatic environments is a cause of concern and to date hardly any studies have focused on the effects that DDT and deltamethrin have on non-target freshwater diatom communities. The application of diatoms in ecotoxicological studies is well acknowledged and therefore this study used laboratory bioassays to determine the effects that DDT and deltamethrin have on a monoculture of a diatom indicator species, Nitzschia palea. The insecticides affected the morphology of chloroplasts at all exposure concentrations. These effects were a maximum reduction in chlorophyll concentrations (4.8% and 2.3%), cell viability (51% and 42%), and increases in cell deformities (3.6% and 1.6%) following exposure to DDT and deltamethrin respectively. Based on the results we propose that methods, such as confocal microscopy, chlorophyll-α analysis and cell deformities are useful tools in assessing the effects of insecticides on diatoms.

Development and Provisional Validation of a Multiplex LC-MRM-MS Test for Timely Kidney Injury Detection in Urine.

Kidney injury is a complication frequently encountered in hospitalized patients. Early detection of kidney injury prior to loss of renal function is an unmet clinical need that should be targeted by a protein-based biomarker panel. In this study, we aim to quantitate urinary kidney injury biomarkers at the picomolar to nanomolar level by liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples, denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS analysis. Stable-isotope-labeled peptides functioned as internal standards, and biomarker concentrations were attained by an external calibration strategy. The method was evaluated for selectivity, carryover, matrix effects, linearity, and imprecision. The LC-MRM-MS method enabled the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and SLC22A2 and the detection of TGF-ß1, cubilin, and uromodulin. Two to three peptides were included per protein, and three transitions were monitored per peptide for analytical selectivity. The analytical carryover was <1%, and minimal urine matrix effects were observed by combining immunocapture and targeted LC-MRM-MS analysis. The average total CV of all quantifier peptides was 26%. The linear measurement range was determined per measurand and found to be 0.05-30 nmol/L. The targeted MS-based method enables the multiplex quantitation of low-abundance urinary kidney injury biomarkers for future clinical evaluation.

A first for Southern Africa: description of a new Heterobothrium (Monogenea: Diclidophoridae) parasitizing the evileye pufferfish Amblyrhynchotes honckenii (Tetraodontiformes: Tetraodontidae).

The monogenean genus Heterobothrium (Monogenea: Diclidophoridae) currently consists of 12 accepted species described globally from various pufferfishes (Tetraodontidae). This includes the economically important Heterobothrium okamotoi Ogawa 1991 that causes severe disease in the cultured tiger puffer Takifugu rubripes in Japan. During parasitological surveys targeting near shore marine fishes of South Africa, a new Heterobothrium was discovered on the gills of five evileye pufferfish Amblyrhynchotes honckenii with a prevalence of 100% and mean intensity of 23 (4-72). Specimens were morphologically studied using both light and scanning electron microscopy and molecularly characterized by sequencing the partial ribosomal gene 28S. Heterobothrium victorwepeneri n. sp. differs from its 12 congeners by a combination of morphological characters, such as fourth clamp pair (anteriormost, 180° inverted) as the smallest with some of the sclerites differing in shape from the other pairs, 8 to 9 genital hooks, number of testes 40-50, and absent isthmus. The 28S tree depicted two main branches, one clustering together species of mazocraeidean families while the other clustered together only species of the Diclidophoridae. Heterobothrium victorwepeneri n. sp. is the first species of its genus to be recorded and described from South Africa and from the tetraodontid A. honckenii. This study also provides for the first time 28S sequence for a species of this highly host-specific genus.

Pseudodactylogyrus anguillae (Yin & Sproston, 1948) from the giant mottled eel Anguilla marmorata Quoy & Gaimard, 1824, in the Phongolo River, South Africa: an invader on the African continent.

The global invasive anguillid gill parasite Pseudodactylogyrus anguillae (Yin and Sproston, 1948) has only recently been documented from eels in South Africa. As there is no known eel trade in South Africa, the source of introduction of this parasite has been debated, and its status as an alien parasite was rendered uncertain. We report on the first infection of Pseudodactylogyrus anguillae from the giant mottled eel Anguilla marmorata from the Phongolo River (South Africa) using classic morphological and molecular methodologies and clarify the introduction status category of this parasite as alien and invasive.

Regional Scale Risk to the Ecological Sustainability and Ecosystem Services of an African Floodplain System.

The Phongolo floodplain is one of southern Africa's most important systems. In this study, we carried out a regional scale ecological risk assessment to evaluate the risk of multiple stressors associated with the use of the aquatic resources in the floodplain to selected social and ecological endpoints representing its sustainability. The floodplain has undergone significant changes as a result of the impacts of multiple stressors. This includes high risk of impact and threatened sustainability between the Pongolapoort Dam and the Ndumo Game Reserve (NGR). This compares to relatively low risk to the maintenance of the endpoints within the NGR. The reserve provides a protection and refuge function for regional biodiversity maintenance and ecosystem sustainability processes. In the study a range of scenarios were considered and demonstrate that the system will respond to protection measures and or increased resource use options. Should flood reductions or water quality pollution drivers continue on increasing trajectories, the condition of the Phongolo River and floodplain will probably deteriorate into an unacceptable, unsustainable state. Removal of the protection services of the NGR would result in an unsustainable ecosystem and loss of ecosystem services for regional vulnerable African communities. Additional evidence should be obtained from monitoring and research to refine, validate, and update the assessment in an adaptive management context. The risk assessment framework approach implemented in the Phongolo floodplain can contribute to the management of other floodplains ecosystems and the sustainability management of social and ecological attributes and processes of these important ecosystems.

Sea lice (Copepoda: Caligidae) from South Africa, with descriptions of two new species of Caligus.

Thirteen species of sea lice (family Caligidae) are reported from a range of elasmobranch and actinopterygian fishes caught off South Africa or obtained from public aquaria in South Africa. Two new species of Caligus Müller, 1785 are described: C. linearis n. sp. from Pomatomus saltatrix (Linnaeus) and C. tumulus n. sp. from Chrysoblephus cristiceps (Valenciennes). A supplementary description is provided for both sexes of Caligus tetrodontis Barnard, 1948 taken from Amblyrhynchotes honckenii (Bloch) and previous records of this parasite from South African fishes are critically reviewed. It is concluded that Caligus material from Arothron hispidus Linnaeus was previously misidentified as C. tetrodontis and is in urgent need of re-examination. Morphological and molecular observations on Caligus furcisetifer Redkar, Rangnekar & Murti, 1949 indicate that this copepod is phenotypically and genetically identical to Lepeophtheirus natalensis Kensley & Grindley, 1973, and the latter becomes a junior subjective synonym of C. furcisetifer. We include new geographical distribution records for Caligus longipedis Bassett-Smith, 1898, C. rufimaculatus Wilson, 1905 and Lepeophtheirus spinifer Kirtisinghe, 1937, extending into South African waters, as well as both new distribution and host records for Alebion gracilis Wilson, 1905, Caligus dakari van Beneden, 1892 and Lepeophtheirus acutus Heegaard, 1943. The molecular analysis confirmed the monophyly of the genus Caligus. The South African species of Caligus did not cluster together, but the two included South African species of Lepeophtheirus were recovered as sister taxa.

Ultrastructure of the uterus, embryonic envelopes and the coracidium of the enigmatic tapeworm Tetracampos ciliotheca (Cestoda: Bothriocephalidea) from African sharptooth catfish (Clarias gariepinus).

Transmission electron microscopy (TEM) was used to study the ultrastructure of the uterus and egg morphology in the enigmatic bothriocephalidean tapeworm Tetracampos ciliotheca. The uterine wall, underlain by well-developed muscle bundles, consists of a syncytial epithelium which is characterized by the abundance of free ribosomes, mitochondria and cisternae of granular endoplasmic reticulum (GER). On the apical surface of the uterine epithelium, there is an abundant network of cytoplasmic microlamellae projecting into the uterine lumen. The lumen is filled with freely lying eggs which are located close to the uterine wall but do not contact with the microlamellae of the uterine epithelium. The developed eggs possess an oncosphere surrounded by four envelopes: (1) a thin egg shell; (2) an outer envelope; (3) a syncytial, ciliated inner envelope; and (4) the oncospheral membrane. The mature hexacanth is armed with three pairs of oncospheral hooks, as well as somatic and hook muscles and five types of cells (1) binucleated subtegumental cell, (2) somatic cells, (3) penetration gland cells, (4) nerve cells and (5) germinative cells. Considering the relative scarcity of descriptive and comparative studies on the ultrastructure of the uterus and egg morphology in the order Bothriocephalidea, we concluded that Tet. ciliotheca displays a unique type of egg development. Based on these results, we discuss plausible ideas relating to the function of these structures for consideration in future studies.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

Biomarker Responses in the Freshwater Shrimp Caridina nilotica as Indicators of Persistent Pollutant Exposure.

Biomarkers are applied as early warning indicators of organisms' exposure to pollutants. The aim of this study was to utilise a multi-biomarker approach in the freshwater shrimp Caridina nilotica (Decapoda: Atyidae) as indicators of persistent pollutant exposure. A suite of biomarkers was selected to cover oxidative stress and damage, and energetics of the organisms. Five sites, representing an agricultural and pesticide application gradient, were sampled during two flow related hydro-periods in rivers of the Phongolo floodplain, north-eastern South Africa. Cytochrome P450 (CYP) activity was significantly higher in shrimp at sites directly adjacent to regions of increased human activity. Increased oxidative responses, i.e. catalase (CAT; p < 0.01) and protein carbonyl (PC, p < 0.01) were also found at these sites. The energetics biomarker did not show any influence of increased contaminant exposure. We demonstrated that the biomarkers of exposure (CYP) and effect (CAT, PC) were suitable to detect effects of stressors, probably persistent pollutants.

Hooked on you: shape of attachment structures in cymothoid isopods reflects parasitic strategy.

BACKGROUND: Parasite attachment structures are critical traits that influence effective host exploitation and survival. Morphology of attachment structures can reinforce host specificity and niche specialisation, or even enable host switching. Therefore, it is important to understand the determinants of variation in attachment structures. Cymothoid isopods are striking ectoparasites of fishes that include the infamous 'tongue-biters.' They are known to parasitise hosts in one of four qualitatively distinct anatomical regions. Here, we quantify variation in cymothoid attachment structures - hook-like appendages called dactyli - and test whether differences in dactylus shape are correlated with parasite mode (where they attach), allometry, or both, using multivariate ordinary least squares regression. We also assess the influence of shared ancestry on shape using a molecular phylogeny to weight our models using phylogenetic generalised least squares regression. RESULTS: We find clear differences in shape between externally-attaching and internally-attaching cymothoids but also between anterior and posterior dactyli across various species with the same attachment mode. Allometric effects are significant for anterior but not posterior dactyli. Mouth-attaching species show greater shape variability than gill- and mouth-attaching species. We find no evidence that there are clade-specific patterns of association between parasite mode and dactylus shape. CONCLUSIONS: Parasite mode appears to be the main driver of attachment morphology. This likely reflects several components of parasite ecology including feeding and functional demands of attachment in different microhabitats. Geometric morphometric approaches to the quantification of shape variation of simple structures is an effective tool that provides new insights into the evolvability of parasite attachment.

Monophyly of the species of Hepatozoon (Adeleorina: Hepatozoidae) parasitizing (African) anurans, with the description of three new species from hyperoliid frogs in South Africa.

Haemogregarines (Apicomplexa: Adeleiorina) are a diverse group of haemoparasites reported from almost all vertebrate classes. The most commonly recorded haemogregarines to parasitize anurans are species of Hepatozoon Miller, 1908. To date 16 Hepatozoon species have been described from anurans in Africa, with only a single species, Hepatozoon hyperolli (Hoare, 1932), infecting a member of the Hyperoliidae. Furthermore, only two Hepatozoon species are known from South African anurans, namely Hepatozoon theileri (Laveran, 1905) and Hepatozoon ixoxo Netherlands, Cook and Smit, 2014, from Amietia delalandii (syn. Amietia quecketti) and three Sclerophrys species, respectively. Blood samples were collected from a total of 225 individuals representing nine hyperoliid species from several localities throughout northern KwaZulu-Natal, South Africa. Twenty frogs from three species were found positive for haemogregarines, namely Afrixalus fornasinii (6/14), Hyperolius argus (2/39), and Hyperolius marmoratus (12/74). Based on morphological characteristics, morphometrics and molecular findings three new haemogregarine species, Hepatozoon involucrum Netherlands, Cook and Smit n. sp., Hepatozoon tenuis Netherlands, Cook and Smit n. sp. and Hepatozoon thori Netherlands, Cook and Smit n. sp., are described from hyperoliid hosts. Furthermore, molecular analyses show anuran Hepatozoon species to be a separate monophyletic group, with species isolated from African hosts forming a monophyletic clade within this cluster.

Polycyclic aromatic hydrocarbons (PAHs) in sediments from a typical urban impacted river: application of a comprehensive risk assessment.

Soweto and Lenasia, the most densely populated area of South Africa, is simultaneously a thriving metropolis, with a fair share of people still living in squalor conditions directly dependant on the natural resources. Because of industrialisation the populace and environment in this urban area are exposed to various pollutants. The aquatic environment was selected as a proxy to study the effect of industrial pollution in this area. The concentrations, source identification, and various environmental risks of polycyclic aromatic hydrocarbons (PAHs) were determined in sediments of the upper reaches of the Klip River. Composite sediment samples collected in low-flow conditions in 2013 and 2014 ranged from 270-5400 ng/g. The PAHs in this aquatic ecosystem were dominated by 4-ring congeners and could be attributed to combustion of organic fuels by chemical mass balance. Heavy traffic and industrial complexes in the northern part of the study area were responsible for the PAH fingerprints. Probable adverse effects such as toxicity to benthic biota were proven after comparison with international sediment quality guidelines (SQG) both survey years. Toxic equivalence quotients (TEQs) calculated for the sediments using fish potency factors (FPFs) were up to 30 times greater than the Canadian guideline for dioxin-like compounds, indicating high probability of carcinogenic effect to fish mediated through the aryl-hydrocarbon receptor. Finally, sediments in the area posed moderate to high ecological risk, which corroborates the other toxicity assessments. The advantage of investigating multiple risk endpoints, is the comprehensive results obtained that allows for a more realistic representation of the study area. Consequently more aspects are kept into account that results in better conclusions.