Toxicity of metal oxide nanoparticles: mechanisms, characterization, and avoiding experimental artefacts.

Metal oxide nanomaterials are widely used in practical applications and represent a class of nanomaterials with the highest global annual production. Many of those, such as TiO2 and ZnO, are generally considered non-toxic due to the lack of toxicity of the bulk material. However, these materials typically exhibit toxicity to bacteria and fungi, and there have been emerging concerns about their ecotoxicity effects. The understanding of the toxicity mechanisms is incomplete, with different studies often reporting contradictory results. The relationship between the material properties and toxicity appears to be complex and diifficult to understand, which is partly due to incomplete characterization of the nanomaterial, and possibly due to experimental artefacts in the characterization of the nanomaterial and/or its interactions with living organisms. This review discusses the comprehensive characterization of metal oxide nanomaterials and the mechanisms of their toxicity.

Application of artificial mussels (AMs) under South African marine conditions: a validation study.

Over the last three decades there has been a significant decline in the number of marine pollution monitoring-related studies in South Africa. Thus, the current study was conducted to assess the current state of metal contamination within the South African marine environment through the validation of the artificial mussel (AM). Indigenous reference mussels (Perna perna) were deployed alongside the passive device within the South African marine environment for a 6 week exposure period. Analysis of metal uptake (Cd, Cr, Cu, Pb and Zn) was determined by filtration and elution of the AM chelex resins, microwave digestion of the transplanted mussels, and determination of their metal concentrations by ICP-MS and ICP-OES analysis. Uptake patterns between the AM and transplanted mussels showed significant temporal and spatial correlation for the majority of the elements analysed. While the AM provided relevant and complementary information on the dissolved metal concentrations, limitations were also observed.

Brown mussels (Perna perna) and semi-permeable membrane devices (SPMDs) as indicators of organic pollutants in the South African marine environment.

A distinct lack of historical and current data on the status of organic pollutant contaminants within the South African marine environment is evident. This has highlighted the need for more current organic pollutant assessments. Reference mussels and SPMDs were transplanted at five South African harbour sites to assess organic bioaccumulation in brown mussels (Perna perna) and semi-permeable membrane devices (SPMDs). Spatial patterns of PAH and PCB contaminants were determined by GC-MS and GC-ECD after appropriate sample preparation. Significant (p<0.05) spatial differences were observed between the sites. Results indicate no correlations between the passive device and the transplanted mussels; however the SPMDs provided complementary information on the presence of dioxin-like PCBs within the environment not detected by the mussel. The results indicate that information provided by both the mussels and SPMDs allow for a more in depth scrutiny of environmental conditions as a result of anthropogenic influence.

A whole life cycle assessment on effects of waterborne PBDEs on gene expression profile along the brain-pituitary-gonad axis and in the liver of zebrafish.

Polybrominated diphenyl ethers (PBDEs) are now found ubiquitously in the aquatic environment and biota, and there is a growing concern that PBDEs may disrupt endocrine systems, leading to reproductive impairments of aquatic animals. In our study, zebrafish (Danio rerio) were exposed to the 5 ng/L, 1 µg/L and 50 µg/L of DE-71 for the duration of the whole life cycle (120 days, from eggs to adults). The expression of selected genes along the brain-pituitary-gonadal (BPG) axis and liver, and the levels of plasma sex hormones were examined. In male fish, up-regulation of GnRH in brain, FSHß and LHß in pituitary, FSH-receptor, LH-receptor, and CYP19a in testis was clearly evident, while down-regulation of CYP11a and 3ß-HSD was found in testis. In female fish, a 2.4-fold up-regulation of 3ß-HSD was found in ovary upon exposure to 50 µg/L of DE-71. GnRH in brain, FSHß and LHß in pituitary were also up-regulated, while ERß, TH and TPH in brain and GnRH-receptor in pituitary were significantly down-regulated. Hepatic ERα, AR and VTG in males were all down-regulated, while hepatic ERα and AR in female were up-regulated. Serum estradiol (E2) was reduced in both male and female upon exposure to DE-71, while significant increases in serum testosterone (T) and 11-keto-testosterone (11-KT) were only found in male but not female fish. The ratio of T/E2 as well as the ratio of 11-KT/E2 in male fish increased in a dose-dependent manner upon exposure to DE-71. Our overall results showed that whole life exposure of DE-71 altered the expression of regulatory genes and receptors at all three levels of the BPG axis in zebrafish, and the responses are sex dependent. The observed disruption of GnRH and GtHs can be further related to the subsequent disruption in both levels and balance sex steroid hormones.

Power analysis for biomarkers in mussels for use in coastal pollution monitoring.

Data from literature on neutral red retention time (NRRT) in lysosomes, micronucleus (MN) frequency and condition index (CI) in mussel Mytilus, especially Mytilus edulis and Mytilus galloprovincialis, were re-analyzed to ascertain their statistical power in detecting a minimum 20% spatial/temporal change in field studies. Results showed that CI largely displayed higher statistical power (>90%) than lysosomal NRRT and MN frequency (<50%), suggesting that data from the latter two biomarkers may lead to erroneous conclusions if sample size is inadequate. Samples of green-lipped mussel Perna viridis were also analyzed in Hong Kong. To achieve statistically valid power, the optimal sample sizes for monitoring lysosomal NRRT, MN frequency, CI and gonosomatic index (GSI) were determined as >or=34, >or=90, >or=16 and >or=29, respectively. Natural variability of lysosomal NRRT and MN frequency was significantly greater than CI and/or GSI in mussels, rejecting the general belief in the greater variability of higher-tiered hierarchical biomarkers.

In situ hybridization to detect spatial gene expression in medaka.

A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka (Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with a 7d exposure to the model aromatase inhibitor, fadrozole. Fadrozole did not affect the magnitude of gene expression in testes, but significantly up-regulated CYP19a gene expression in ovaries. These results were confirmed with quantitative real-time-polymerase chain reaction (RT-PCR). Histological evaluation revealed that females exposed to 100microg/L fadrozole lacked mature oocytes. Male gonadal morphology was normal in all treatments. The ISH method developed in this study allowed tissue-specific resolution of gene expression in a whole animal model, as well as the ability to analyze cellular morphological detail in the same organism.

Influences of ammonia-nitrogen and dissolved oxygen on lysosomal integrity in green-lipped mussel Perna viridis: laboratory evaluation and field validation in Victoria Harbour, Hong Kong.

Lysosomal integrity in mussels has been applied as a biomarker to detect the pollution of trace organics and metals in the natural environments. However, few studies have examined the effects of water quality on the response of lysosomal integrity, in particular total ammonia-nitrogen (TAN) and dissolved oxygen (DO). This study demonstrated that high level of TAN (2.0mg/l) and low DO (2.5mg O(2)/l) could significantly reduce the lysosomal integrity in green-lipped mussel Perna viridis, respectively by 33% and 38%, whereas the mussel lysosomal integrity decreased by 70% in the combined treatment of TAN and low DO under laboratory conditions after one week. The mussel lysosomal integrity of all treatment groups could return to the control level after a three week recovery period. In the field validation in Victoria Harbour, Hong Kong during an one-year study period, lysosomal integrity in P. viridis identified the cleanest site east to the harbour, where the lowest TAN and highest DO concentrations were found. While lysosomal integrity in mussels seemed not affected by seasonal changes, approximately 40% of the variation of this biomarker could be attributable to the changes in TAN and DO in seawater. In conclusion, the response of the mussel lysosomal integrity can be confounded by both TAN and DO prevailing in the natural environments and thus caution must be exercised in relating the observed changes in lysosomal integrity to any specific pollutant in coastal water quality monitoring studies.

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis.

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

Spatio-temporal changes of marine macrobenthic community in sub-tropical waters upon recovery from eutrophication. II. Life-history traits and feeding guilds of polychaete community.

A two-year study was conducted in the vicinity of a harbour in sub-tropical Hong Kong, to examine the progress of recovery of macrobenthic community, based on analyses of both life-history traits and trophic guilds of polychaetes, upon cessation of organic pollution caused by sewage discharge. Seventy seven out of 83 species collected were classified under four ecological groups based on the life-history traits and sensitivity to organic gradients. The mean ATZI marine biotic index (AMBI) derived from these ecological groups showed spatial difference among the five sampling locations. In particular, the presence of different percentages of polychaete species from Groups III (unbalanced community) and IV (polluted community) suggested the presence of pollution stress in certain degree at all sampling locations. However, no significant temporal changes were noted over the study period. From all polychaete species identified, they were classified into 13 feeding guilds. The mean diversity of these feeding guilds at most of the sampling locations was significantly higher than that at one of the inside-harbour locations. The composition of feeding guilds was also significantly different spatially. At one of the inside-harbour locations, the dominant feeding guilds were motile/discretely motile surface deposit feeders with tentaculates or unarmed pharynx, and motile omnivores with jawed pharynx in the first year of study, but were replaced by motile, jawed carnivores in the second year of study. The increased proportion of carnivores over the study period can be seen as a sign of recovery in the community structure since abundance of predators is commonly higher in habitats with better environmental quality. The implications of using life-history traits and feeding guild analyses for benthic community are discussed.

Spatio-temporal changes of marine macrobenthic community in sub-tropical waters upon recovery from eutrophication. I. Sediment quality and community structure.

A two-year study was conducted in the vicinity of a harbour in sub-tropical Hong Kong, to examine the progress of improvement in sediment quality and recovery of macrobenthic community upon cessation of organic pollution caused by sewage discharge. Median sediment particle diameter (Mdphi) and levels of total organic carbon (TOC), total Kjeldahl nitrogen (TKN), ammonia-nitrogen (NH(3)-N) and total phosphorus (TP), as well as macrobenthic species composition and abundance were determined bi-monthly at three inside-harbour and two outside-harbour locations. At the two inside-harbour locations, significantly higher levels of TOC, TKN, NH(3)-N and TP in sediments were observed than the outside-harbour locations. However, no significant temporal change of nutrient levels was detected at all sampling locations during the two-year study, except a significant decrease in TKN and NH(3)-N levels at one outside-harbour location and a decline in NH(3)-N content at another outside-harbour location. Spatially, the highest in mean total species number, individual number, species diversity H' and lowest evenness J was found at one outside-harbour location, whereas the other four locations had relatively similar values. H' was negatively correlated with TOC, TKN, NH(3)-N and TP content in sediments while J was positively correlated with MDphi. Across the study period, the patterns of macrobenthic community were significantly different among all samplings and that the spatial difference of benthic community was best correlated with MDphi, TOC, TKN and water depth. A weak sign of recovery in macrobenthic community structure was detected at inside-harbour locations, with replacement of opportunistic by ubiquitous species over the two-year study. The slow recovery of macrobenthic community was related to sediment characteristics. Results of a larval settlement bioassay using the polychaete Capitella sp. I also demonstrated that the inside-harbour sediments were still unfavourable for colonization and larval settlement of species sensitive to pollution. The slow biodegradation of the organic pollutants and continuous presence of heavy metals in the sediment may hinder settlement and colonization of benthic animals. However, increases of H' and J were observed in a longer time scale when comparing the present data with those obtained four years ago. This suggested that a detectable recovery of benthic community in the harbour may take at least three years and a complete recovery may even take longer duration.

A field experimental study on recolonization and succession of subtidal macrobenthic community in sediment contaminated with industrial wastes.

A field experiment was carried out in Hong Kong to study the patterns of recolonization and succession of subtidal macrobenthos in defaunated sediment contaminated with industrial wastes and to determine the time required for benthic recovery in the industrial-contaminated sediment. A total of 50 species was found with an average of 172 animals/tray and 24 species/tray recorded one month after deployment. Initial colonizers were predominantly polychaetes (96 animals/tray, accounting for 55.7%) and gastropods (47 animals/tray, accounting for 27.2%). Abundance of macrobenthos increased quickly to a peak (505 animals/tray) after four months, declined afterwards, and increased again till the end of the experiment. Species number peaked (57 species/tray) in the same month as abundance did, and gradually declined thereafter. Abundance, species number and diversity were significantly lower in the industrial-contaminated sediment as compared to the controls during the early successional stages, indicating the harmful effects of industrial wastes on recolonization and succession of macrobenthos. Although no significant differences in community parameters between the industrial-contaminated and the control sediments were found after eleven months, significant difference in species composition still existed after fourteen months, showing a relatively long-term impact of industrial wastes on macrobenthic community structure.

Ultraviolet damages sperm mitochondrial function and membrane integrity in the sea urchin Anthocidaris crassispina.

Effects of ultraviolet A (UVA) and ultraviolet B (UVB) on mitochondrial function and membrane integrity of sea urchin sperm were investigated using flow cytometry and fluorescent probes. Both UVA and UVB impaired sperm mitochondrial function in a dose-dependent manner. Covariance analysis further showed that the slopes of change in mitochondrial function in relation to UVA and UVB were significantly different, suggesting that the modes of action were different. UVA did not affect membrane integrity, while membrane integrity showed a linear reduction with increasing UVB doses. Sperm mitochondria function showed significant positive correlations with sperm motility and subsequent fertilization success. Overall, our results showed that both UVA and UVB could decrease sperm motility and fertilization success through impairment of mitochondrial function, whereas UVB alone could cause additional damage through impairing the functional integrity of sperm membrane. Mitochondrial function of sperm may also offer a reliable ecotoxicological biomarker for predicting fertilization success in urchins.

Application of the comet and micronucleus assays to the detection of B[a]P genotoxicity in haemocytes of the green-lipped mussel (Perna viridis).

Green-lipped mussels (Perna viridis) were exposed to water-borne benzo[a]pyrene (B[a]P) at nominal concentrations of 0, 0.3, 3 and 30 microg l(-1) for up to 12 days, and both the relative levels of DNA strand breaks (assessed using an alkaline comet assay) and the proportion of micronucleus (MN) formation were monitored in mussel haemocytes at days 0, 1, 3, 6 and 12. The results of the comet assay indicated that an increase in the proportion of strand breaks occurred generally with increasing B[a]P concentration, but a significant decrease in the levels of DNA damage was observed after exposure for 12 days at all concentrations tested, suggesting that the patterns of changes in the levels of DNA strand breakage can be explained by the threshold dependent DNA repair theory. Moreover, the relatively slow development and recovery of the DNA damage response in mussel haemocytes in comparison with previous findings utilizing P. viridis hepatopancreas suggests that the response of DNA alteration upon exposure to B[a]P may be tissue-specific in this species. Monitoring the frequency of micronucleus development in mussel haemocytes indicated both dose- and time-response relationships within the exposure period. Furthermore, the levels of DNA strand breakage correlated well with the levels of micronucleus induction, suggesting a possible cause and effect relationship between the two damage types. We suggest that DNA strand breakage and micronucleus formation in mussel haemocytes can potentially be used as convenient biomarkers of exposure to genotoxicants in the marine environment.

Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp.

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and pI of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.