Measuring lysosomal stability as an effective tool for marine coastal environmental monitoring

The use of lysosomal stability in the mussel, Mytilus galloprovincialis, as a potential biomarker of environmental contamination has been evaluated along the Portuguese coast. To this end, the neutral red retention (NRR) time was measured in mussel haemocytes gathered from nine different locations reflecting different degrees of anthropogenic contamination. Mussels collected in the vicinity of industrial and urban areas showed the lowest lysosomal stability. Additionally, no significant seasonal variability (winter-spring/summer) for NRR time was observed. In order to further support the usefulness of this method as an integrated tool for monitoring marine coastal environments, we compared the levels of xenobiotics in mussel tissues with the obtained NRR values. The results highlighted a consistent pattern, with the lowest lysosomal stability intimately correlated with the higher contaminant concentrations. In summary, this integrated approach further demonstrated that the NRR assay can provide useful and objective indications of the real health status of organisms subjected to different stress agents, being a valid option for environmental monitoring.     

Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in nearshore demersal fishes

Three biomarkers of hydrocarbon exposure, CYP1A in liver vascular endothelium, liver ethoxyresorufin O-deethylase (EROD), and biliary fluorescent aromatic compounds (FACs), were examined in the nearshore fishes, masked greenling (Hexagrammos octogrammus) and crescent gunnel (Pholis laeta), collected in Prince William Sound, Alaska, 7-10 years after the Exxon Valdez oil spill (EVOS). All biomarkers were elevated in fish collected from sites originally oiled, in comparison to fish from unoiled sites. In 1998, endothelial CYP1A in masked greenling from sites that were heavily oiled in 1989 was significantly higher than in fish collected outside the spill trajectory. In 1999, fishes collected from sites adjacent to intertidal mussel beds containing lingering Exxon Valdez oil had elevated endothelial CYP1A and EROD, and high concentrations of biliary FACs. Fishes from sites near unoiled mussel beds, but within the original spill trajectory, also showed evidence of hydrocarbon exposure, although there were no correlations between sediment petroleum hydrocarbon and any of the biomarkers. Our data show that 10 years after the spill, nearshore fishes within the original spill zone were still exposed to residual EVOS hydrocarbons.     

Mussels document loss of bioavailable polycyclic aromatic hydrocarbons and the return to baseline conditions for oiled shorelines in Prince William Sound, Alaska

Polycyclic aromatic hydrocarbons (PAH) were measured in mussels (Mytilus trossulus) collected between 1990 and 2002 from 11 sites on the shores of Prince William Sound (PWS), Alaska, that were heavily oiled by the 1989 Exxon Valdez oil spill (EVOS). This study, utilizing the methods of the NOAA Status and Trends Mussel Watch Program, found that concentrations of PAH released from spill remnants have decreased dramatically with time and by 2002 were at or near the range of total PAH (TPAH) of 3-355 ng/g dry weight obtained for mussels from unoiled reference sites in PWS. Time-series TPAH data indicate a mean TPAH half-life in mussel tissues of 2.4 years with a range from 1.4 to 5.3, yielding an annual mean loss of bioaccumulated TPAH of 25%. The petroleum-derived TPAH fraction in mussel tissues has decreased with time, reflecting the decreasing release of EVOS residues in shoreline sediments. These results show that PAH from EVOS residues that remain buried in shoreline sediments after the early 1990s are in a form and at locations that have a low accessibility to mussels living in the intertidal zone.     

Evaluation of the impacts of land-based contaminants on the benthic faunas of Jakarta Bay,Indonesia

To evaluate the impact of land-based contaminants on benthic faunas, as part of the Land-Ocean Contamination Study (LOCS), a study transect was established in Jakarta Bay, Indonesia during the 1996 southeasterly monsoon. The transect extended 72 km between the city's main port, Tanjung Priok, and the Pilau Seribu Island chain in the Java Sea. The dissolved concentrations of Pb, Cu, Zn, Cr and Ni in seawater, in suspended particulate matter (SPM) and surficial sea bed sediments were measured along the transect at nine sites. In addition, metal concentrations were measured in tissues of the corals Goniopora lobata and Lobophyllia hemprichii at five sites, and of the green mussel, Perna viridis at six sites. An assessment of the impact of contaminants on the faunas was made using a biomarker approach, employing coral community analysis and lysosomal stability with Perna viridis. The results of the study showed two main trends. The distributions of metals dissolved in seawater, in SPM, and in the coral and mussel tissues were similar, and failed to show a consistent graded response from inshore to offshore sites. This suggests that the concentration of metals in waters is the primary route for metal uptake by the coral and mussel tissues. By way of contrast, a clear offshore increase in coral generic diversity, coral cover, coral colony numbers and neutral-red retention time (lysosomal stability) was observed, suggesting increasing nearshore stress. Whilst the coral community stucture may reflect the seasonally-averaged metal distributions in the bay (shown by the metal content of sea bed sediments which increase shorewards), it is more likely that the coral community structure and lysosomal biomarker are responding principally to other nearshore stresses, such as sediment and nutrient loading of water (sewage) or, more likely, organic contaminants such as oils and other hydrocarbons.     

Environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status

The potential prognostic use of lysosomal reactions to environmental pollutants is explored in relation to predicting animal health in marine mussels, based on diagnostic biomarker data. Cellular lysosomes are already known to accumulate many metals and organic xenobiotics and the lysosomal accumulation of the carcinogenic polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is demonstrated here in the hepatopancreatic digestive cells and ovarian oocytes of the blue mussel. Lysosomal membrane integrity or stability appears to be a generic indicator of cellular well-being in eukaryotes; and in bivalve molluscs it is correlated with total oxygen and nitrogen radical scavenging capacity (TOSC), protein synthesis, scope for growth and larval viability; and inversely correlated with DNA damage (micronuclei), as well as lysosomal swelling (volume density), lipidosis and lipofuscinosis, which are all characteristic of failed or incomplete autophagy. Integration of multiple biomarker data is achieved using multivariate statistics and then mapped onto "health status space" by using lysosomal membrane stability as a measure of cellular well-being. This is viewed as a crucial step towards the derivation of explanatory frameworks for prediction of pollutant impact on animal health; and has facilitated the development of a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells, tissues and the whole animal. This model has also complemented the creation and use of a cell-based bioenergetic computational model of molluscan hepatopancreatic cells that simulates lysosomal and cellular reactions to pollutants. More speculatively, the use of coupled empirical measurements of biomarker reactions and modelling is proposed as a practical approach to the development of an operational toolbox for predicting the health of the environment.     

Persistence of oiling in mussel beds after the Exxon Valdez oil spill

Persistence and weathering of Exxon Valdez oil in intertidal mussel (Mytilus trossulus) beds in Prince William Sound (PWS) and along the Gulf of Alaska was monitored from 1992 to 1995. Beds with significant contamination included most previously oiled areas in PWS, particularly within the Knight Island group and the Kenai Peninsula. In sediments, yearly mean concentrations of total petroleum hydrocarbons ranged from < 60 micrograms/g in reference beds to 62,258 micrograms/g wet wt., or approximately 0 to 523 micrograms/g dry wt. total polynuclear aromatic hydrocarbons (TPAHs). In mussels, mean TPAH concentrations ranged up to 8.1 micrograms/g dry wt. Hydrocarbon concentrations declined significantly with time in some, but not all mussels and sediments, and should reach background levels within three decades of the spill in most beds. In 1995, mean hydrocarbon concentration was greater than twice background concentration in sediments from 27 of 34 sites, and in mussels from 18 of 31 sites.     

Effects of the MV Rena oil spill on intertidal rocky reefs in the Bay of Plenty,New Zealand

In the weeks following the 2011 Rena oil spill, a series of surveys was initiated on eight rocky intertidal reefs to describe the distribution of oil and to assess the impacts of oil on ecological communities. Consistent but relatively low cover of oil occurred at two sites (Mt Maunganui and Moturiki). The area covered by oil had decreased by c. 90% after 5 months due to natural weathering processes. There were immediate effects of oil fouling on the mussel Limnoperna pulex and its associated fauna, with reductions in the number of mussels and infaunal taxonomic richness. However, no ecological effects on any of the communities were detectable after 1 month. Overall, the ecological effects of the Rena oil spill on rocky shore intertidal communities were small and not long-lasting, but we stress that this does not consider potential sublethal effects and their consequences on organisms.     

大连湾石油污染沉积物中细菌群落结构分析

大连新港"7.16"输油管道爆炸溢油事故发生后,为探究石油污染与细菌群落结构变化之间的关系及在石油生物降解过程中起重要作用的细菌菌群,本研究对大连湾表层沉积物中石油烃含量和细菌宏基因组16SrDNA V3区进行分析。结果表明:溢油初期2010年8月DLW01站位表层沉积物石油烃含量高达1 492mg/kg,符合第三类沉积物质量标准,随着时间推移,2011年4月、2011年7月、2011年12月航次各站位沉积物中石油烃含量基本呈下降趋势,且均符合第一类沉积物质量标准;16S rDNA PCR-DGGE方法分析表明,石油烃含量高的区域优势细菌种类少,反之则较丰富;海洋环境中同一地点的细菌群落能保持一定稳定性;大连湾石油污染沉积物中变形菌门γ-变形菌纲和拟杆菌门一直保持较高的优势度,是在石油生物降解过程中起重要作用的细菌菌群,而厚壁菌门只在石油烃含量低的区域出现;此外,出现的对污染物敏感的嗜冷杆菌可作为石油污染指示生物进行深入研究。     

多环芳烃油指纹应用于船舶溢油鉴别研究

溢油种类主要包括船舶燃料油和原油,二者性质的差异决定了鉴别方法也相应不同,寻求适合于船舶溢油的鉴别方法具有重要意义。在使用柱色谱层析方法对样品进行分离前处理的基础上,以气相色谱/质谱方法(GC-MS)为主要分析手段,对溢油样品和可疑船舶溢油源样品的多环芳烃油指纹特征进行对比,并在多环芳烃油指纹参数的基础上进一步进行多环芳烃内组成三角图分布特征与聚类分析研究,成功为珠江口水域某船舶溢油事故追踪到肇事溢油源。结果表明:取自丁船的油样和现场溢油样芳烃油指纹特征最为相近,是此次溢油事故的溢油源。受风化作用后的船舶燃料油中饱和烃类化合物数量稀少,且含量极低,不适合用于溢油鉴别,而多环芳烃类化合物较饱和烃类化合物而言具有含量高、种类丰富的特点,是该类溢油鉴别的主要油指纹依据。使用油指纹参数进行可疑溢油源识别时,充分考虑油品中有机分子所受风化影响程度的不同是风化条件下溢油鉴定的关键。因此,多环芳烃油指纹可以有效应用于船舶燃料油溢油的鉴别。     

Long-term changes in rocky intertidal macrobenthos during the five years after the <Emphasis Type="Italic">Hebei Spirit</Emphasis> oil spill,Taean, Korea

The Hebei Spirit oil spill reached the west coast of Taean County, Korea, on December 7, 2007. At that time, this was the largest oil spill that had occurred in the country. The incident devastated local marine ecosystems. In this study, we examined changes in these ecosystems and the recovery pattern on impacted rocky shores. Macrobenthos monitoring of polluted and control sites was performed seasonally from January 2008 through October 2013. At polluted sites, ecological indices increased gradually from 2009, thereby providing evidence of recovery. However, the mean density of macrobenthos was still less than 50% of the level at the control site, indicating that the ecological status of the polluted sites has still not recovered to that which prevailed before the oil spill. Although densities of the most dominant species were higher in the control site than in polluted sites, the densities of Lottia spp. and Odostomia aomori were higher in polluted sites. These two taxa were found in lower numbers at the control site, where they were out-competed by Chthamalus challengeri. Nonmetric multidimensional scaling ordination divided the macrobenthic community structure into three clusters according to differences in the abundance of dominant species over the 6 years after the oil spill. An ANOSIM test detected significant differences between the polluted and control sites. Consistent long-term differences indicate that complete recovery had not occurred during the duration of our study.     

Relation between responses in the neutral red retention test and the comet assay and life history parameters of Daphnia magna.

Responses of the neutral red retention (NRR) assay as test for lysosomal stability and the comet assay as test for DNA integrity were measured in the water flea, Daphnia magna, and compared with mortality and effects on population growth rate during short- or long-term exposure to seven different toxicants. The NRR test and the comet assay were performed with fresh preparations of pieces of tissue from the digestive tract or with cell preparations from whole daphnias. Five toxicants caused responses of the NRR test or the comet assay after short-term exposure at concentrations below the acute toxicity level. Preliminary results of long-term exposure experiments suggest that these biomarker responses can be related to chronic effects on survival and/or reproduction of D. magna. This type of research should provide the basis for future use of the NRR test and the comet assay as early warning biomarkers for effects of toxicants on Daphnia populations.     

海上溢油极化特征及其探测研究

The SAR(Synthetic Aperture Radar) has the capabilities for all-weather day and night use. In the case of determining the effects of oil spill dumping, the oil spills areas are shown as dark spots in the SAR images.Therefore, using SAR data to detect oil spills is becoming progressively popular in operational monitoring, which is useful for oceanic environmental protection and hazard reduction. Research has been conducted on the polarization decomposition and scattering characteristics of oil spills from a scattering matrix using allpolarization of the SAR data, calculation of the polarization parameters, and utilization of the CPD(Co-polarized Phase Difference) of the oil and the sea, in order to extract the oil spill information. This method proves to be effective by combining polarization parameters with the characteristics of oil spill. The results show that when using Bragg, the oil spill backscattering machine with Enopy and a mean scatter α parameter. The oil spill can be successfully identified. However, the parameter mechanism of the oil spill remains unclear. The use of CPD can easily extract oil spill information from the ocean, and the polarization research provides a base for oil spill remote sensing detection.     

Restoration of oiled mussel beds in Prince William Sound, Alaska

Natural loss of hydrocarbons was often low from mussel (Mytilus trossulus) beds (which were typically not cleaned after the Exxon Valdez oil spill), thus this habitat remained a long-term source of oil. Consequently, experimental restoration of nine contaminated beds was attempted in 1994; mussels were removed, contaminated surface sediment was replaced (33 metric tons), and original mussels were returned. Hydrocarbon concentrations and mussel populations were monitored for 5 years thereafter. Post-restoration mussel population fluctuations were indistinguishable from regional changes. Increased short-term oil loss was apparent, but long-term (5 year) improvement was equivocal and difficult to distinguish from natural losses. By 1999, oil concentrations in mussels were typically at baseline levels in restored and oiled reference beds; concentrations in replaced sediment were elevated in one third of restored beds, indicating recontamination from underlying or surrounding sediment. Our results suggest mussel relocation is feasible but suggest oil might more effectively be removed from sediment mechanically or chemically than manually.     

Effects of polynuclear aromatic hydrocarbons on lysosomal membranes in molluscs

Lysosomal sequestration of polynuclear aromatic hydrocarbons (PNAHs), a major class of environmental contaminant, is a well-established phenomenon;¹ considerably less is known about their pathological effects on lysosomes. Marine molluscs contain a number of lysosome-rich tissues and PNAHs are known to induce deleterious alterations in lysosomal structure and latency of lysosomal enzymes.² The latter are presumed to involve destabilisation of the lysosomal membrane, resulting in increased permeability and reduced enzyme latency. If lysosomal injury involves derangement of membrane-lipid structure due to the interaction of PNAHs then it would be expected that membrane damage would be closely linked to the structural characteristics of the intruding molecule. Our results show that the effects of the isomeric PNAHs phenanthrene and anthracene on digestive cell lysosomal stability were markedly different in the marine mussel (Mytilus edulis) over the same range of tissue concentrations. Lysosomal membrane stability was determined using a cytochemical test for enzyme latency.³     

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-β-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.     

Diet restriction induced autophagy: a lysosomal protective system against oxidative- and pollutant-stress and cell injury

Nutrient deprivation or dietary restriction (DR) confers protection against ageing and stress in many animals and induced lysosomal autophagy is part of this mechanism. The effects of dietary restriction on the toxicity of copper and the polycyclic aromatic hydrocarbon phenanthrene have been investigated in the common marine mussel Mytilus edulis. The findings show that DR-induced autophagy facilitates the recovery of the digestive gland (i.e., molluscan liver analogue) from cell injury caused by both copper and phenanthrene. It is inferred that DR-induced autophagy and lysosomal proteolysis results in improved cellular "housekeeping" through the more efficient removal of oxidatively and pollutant damaged proteins (e.g., protein carbonyls, protein adducts, etc.) and that this contributes to stress resistance.     

Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy)

A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments.     

Effects of nanoparticles in Mytilus edulis gills and hepatopancreas - a new threat to marine life?

Every day new extraordinary properties of nanoparticles (a billionth of a meter) are discovered and worldwide millions are invested into nanotechnology and nanomaterials. Risks to marine organisms are still not fully understood and biomarkers to detect health effects are not implemented, yet. We used the filter feeding blue mussel as a model to analyse uptake and effects of nanoparticles from glass wool, a new absorbent material suggested for use in floating oil spill barriers. In both, gills and hepatopancreas we analysed uptake of nanomaterials by transmission electronmicroscopy (TEM) in unstained ultrathin sections over a period of up to 16 days. Lysosomal stability and lipofuscin content as general indicators of cellular pathology and oxidative stress were also measured. As portals of uptake, diffusion and endocytosis were identified resulting in nanoparticle accumulation in endocytotic vesicles, lysosomes, mitochondria and nuclei. Dramatic decrease of lysosomal membrane stability occurred after 12h of exposure. Lysosomal damage was followed by excessive lipofuscin accumulation indicative of severe oxidative stress. Increased phagocytosis by granulocytes, autophagy and finally apoptosis of epithelial cells of gills and primary and secondary digestive tubules epithelial cells indicated progressive cell death. These pathological responses are regarded as general indices of toxic cell injury and oxidative stress. By the combinational use of biomakers with the ultrastructural localisation of nanoparticle deposition, final evidence of cause-effect relationships is delivered.     

The occurrence of hydrocarbons and histopathological abnormalities in oysters for seven years following the wreck of the Amoco Cadiz in Brittany (France)

A study of the main oyster areas polluted by the Amoco Cadiz oil spill (Abers, Baie de Morlaix) was carried out between 1978 and 1985 by chemical analyses (mainly aromatic hydrocarbon determinations) and histopathological observations of lesions in the digestive tracts and gonads of oysters. The hydrocarbon content in oyster tissues was monitored and analyzed by UV spectrofluorimetry, and gas and liquid chromatographic techniques.Seven years after the Amoco Cadiz wreck, the oysters sampled in the AberBenoit and in the Baie de Morlaix (Carantec) are still polluted with residual aromatic hydrocarbons, corresponding, respectively, to about five and two times the values from an unpolluted site (Baie de Saint-Brieuc). Since petroleum residues are buried in anaerobic sediments, especially in the Abers areas, they are expected to persist and accordingly may still contaminate oysters for several more years.Depuration kinetics of contaminated flat and Pacific oysters from the heavily polluted zones (Abers, Carantec) were studied and compared with that of healthy oysters transplanted in the same areas. The decontamination rates of oysters after transfer to a clean environment were heavily dependent on the duration of oil exposure.Necrosis and inflammation were the most significant and noticeable changes occurring during the first months after the pollution. Atrophy of the gonadal cells was detected in the oysters from the Abers only during the first six months of the study. No neoplastic and parasitic diseases have been related to this oil spill.     

一种面向港口溢油监测的小型激光荧光遥测系统

目前,针对港口溢油巡航监测缺乏行之有效的技术手段。为探索激光诱导荧光光谱技术应用于港口溢油监测的可行性,以微型光纤光谱仪搭建小型激光荧光遥测实验系统,并对其性能进行测试分析。结果发现,在光谱仪曝光时间设为最小(1.11 ms)的情况下,环境光对荧光测量仍会产生影响,利用减背景法可以有效去除溢油荧光信号中环境光的影响;在相同条件下,系统测得不同油质溢油样品的荧光光谱表现出不同的光谱特征,轻质油较重质油的荧光峰蓝移、荧光更强;在上述环境光背景影响和光谱测量分析的基础上,进一步测试了系统的信号探测稳定性,结果显示,系统重复测量的相对标准偏差RSD=2.54%,表明该系统具有较好的信号探测稳定性。综上,基于微型光纤光谱仪的小型溢油激光荧光遥测系统可望在近岸港口溢油巡航监测中发挥重要作用。