Synchronous fluorescence spectrometry of 1-hydroxypyrene: a rapid screening method for identification of PAH exposure in tissue from marine polychaetes

The uptake of polycyclic aromatic hydrocarbons (PAHs) by marine deposit-feeding invertebrates can be determined by screening for PAH-derived metabolites. We identified 1-hydroxypyrene as the only intermediate metabolite in tissue of four species of deposit-feeding polychaetes, Nereis diversicolor, Nereis virens, Arenicola marina, and Capitella sp. I exposed to pyrene spiked sediment. Synchronous fluorescence spectroscopy (SFS) provides a fast and simple method for both qualitative and quantitative analysis of 1-hydroxypyrene in all four species. The SFS assay was validated using HPLC with ultraviolet detection. A good correlation between 1-hydroxypyrene concentrations determined by the two methods was observed. We used HPLC with fluorescence detection combined with enzymatic hydrolysis of conjugated metabolites to investigate species specific metabolite patterns. A tentative aqueous metabolite identification scheme indicates that Nereid polychaetes predominately make use of glucuronide conjugation whereas Capitella sp. I. and Arenicola marina appear to utilize predominantly sulfate and/or glucoside conjugation. The usefulness of 1-hydroxypyrene as a biomarker for PAH exposure in deposit-feeding invertebrates is discussed.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

固相萃取-高效液相色谱-荧光检测法测定海水中的多环芳烃

建立了用固相萃取-高效液相色谱-荧光检测法测定海水中多环芳烃的方法,优化了色谱条件和萃取条件。除苊不能用荧光检测器检出外,其余15种多环芳烃的空白加标回收率在64.5%(苯并[g,h,i],茚并[1,2,3-cd]芘)～88.7%(苯并[a]蒽)之间,相对标准偏差(n=5)为4.9%(荧蒽,苯并[b]荧蒽)～11.1%(苯并[g,h,i],茚并[1,2,3-cd]芘),方法的检出限在0.72(蒽)～14.10 ng/L(荧蒽)之间,基本上达到了痕量分析的要求。利用该方法测得青岛湾表层海水中多环芳烃的浓度在0.125(苯并[k]荧蒽)～25.996 ng/L(萘)之间,但苯并[a]芘未检出。     

海水中多环芳烃的暴露浓度对鲈鱼体内脂质过氧化程度的影响

本文以鲈鱼为实验材料，以鲈鱼体内的丙二醛含量为指标研究了在水样中16种多环芳烃组分和外加芘为实验试剂的条件下，鲈鱼体内不同器官、组织的脂质过氧化程度的变化．实验结果表明：（1）在鲈鱼鳃、全血中的丙二醛浓度较高，而肝脏的丙二醛浓度较低。（2）水样中多环芳烃及外加芘的暴露能导致鲈鱼肝的丙二醛浓度升高，这表明多环芳烃以及芘的暴露能引起鲈鱼肝内脂质过氧化程度的提高。（3）水体中多环芳烃及外加芘浓度的变化对鲈鱼肝的丙二醛浓度的影响具有可逆性，且鲈鱼肝的丙二醛浓度与水环境的多环芳烃（包括芘）暴露浓度呈良好的正相关关系。这说明：鲈鱼肝的丙二醛浓度可作为指示水环境的多环芳烃污染程度的指标。由于生物体内的丙二醛浓度受多种因素的影响，在使用丙二醛浓度指标来指示海洋环境的多环芳烃污染时。最好与其他指标配合使用，以更准确地指示海洋环境多环芳烃污染。     

Evaluation of fixed wavelength fluorescence and synchronous fluorescence spectrophotometry as a biomonitoring tool of environmental contamination

A field study was carried out to investigate whether polyaromatic hydrocarbon (PAH) contamination in shore crabs (Carcinus maenas) could be measured using fluorescence spectrometry. Crabs were collected as part of an integrated study to assess PAH contamination in Southampton Water, UK. Urine was collected from crabs via a non-destructive technique and analysed using fixed wavelength fluorescence (FF) and synchronous fluorescence spectrophotometry (SFS) in order to determine the presence of PAH metabolites, indicative of PAH exposure. By scanning at wavelength pairs specific to a variety of PAH groups the fluorescence of metabolites was analysed and attributed to the following PAH groups; naphthalenes, pyrenes and benzo[a]pyrene (BAP). Naphthalene-type, pyrene-type and BAP-type metabolites were detected using FF and SFS techniques and two distinct parameters were measured; Peak Intensity and Peak Area. Results showed that crabs obtained near a petroleum refinery, a point source of PAH discharge, exhibited significantly higher PAH metabolites for all PAH groups, than at sites sampled along the estuary (ANOVA, p<0.05). This study highlights the effectiveness of using FF and SFS as a rapid and cost-effective biomonitoring tool for detecting PAH contamination.     

夏季珠江口水体中多环芳烃的分布、组成及来源

利用1999年7月对珠江口海域的调查资料,对该区表层海水中优控多环芳烃的分布、组成及来源进行了分析和讨论,结果表明:(1)夏季珠江口海域表层海水中14种溶解态多环芳烃[苊、芴、菲、蒽、荧蒽、芘、苯并(a)蒽、艹屈、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、二苯并(a,h)蒽、苯并(g,h,i)艹北、茚并(1,2,3-cd)艹比]的质量浓度为63.8~171.7 ng/L,且沿着冲淡水流向呈降低趋势;(2)颗粒态中15种多环芳烃[萘、苊、芴、菲、蒽、荧蒽、芘、苯并(a)蒽、艹屈、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、二苯并(a,h)蒽、苯并(g,h,i)艹北、茚并(1,2,3-cd)艹比]的质量浓度为60.7~186.7 ng/L,其分布与水体载沙量及悬浮颗粒物的性质、粒径有关,具有从河口内向外海降低的分布特征;(3)多环芳烃组成和特征参数比值的分析表明,珠江口海域高温裂解来源的多环芳烃在伶仃洋海区输入最多,且主要为人类活动中煤燃烧排放的,而在香港岛周围海区的输入则相对较少,且主要为油燃烧排放的;(4)与法国塞纳河及长江口等河口相比,珠江三角洲海域水体中存在高菲含量排放源。     

Phototoxicity of pyrene and benzo[a]pyrene to embryo-larval stages of the Pacific oyster Crassostrea gigas

There is a growing body of evidence to suggest that certain polycyclic aromatic hydrocarbons (PAHs) pose a greater hazard to aquatic organisms than previously demonstrated, due to their potential to cause photo-induced toxicity when exposed to ultraviolet (UV) radiation. The consequences of photo-induced toxicity are reported here for embryo-larval stages of the pacific oyster Crassostrea gigas, following exposure to pyrene and benzo[a]pyrene. During laboratory investigations, significant increases in toxicity were observed in the presence of environmentally attainable levels of UV-radiation, compared with embryos exposed to PAH alone, at levels previously deemed to have little acute biological effect. The phototoxicity of pyrene and benzo[a]pyrene completely inhibited the development to the D-shell larval stage when embryos were simultaneously exposed to 5 microg l(-1) PAH and ultraviolet light (UVB = 6.3 +/- 0.1 microW/cm2 and UVA = 456.2 +/- 55 microW/cm2). A linear relationship was also demonstrated for benzo[a]pyrene phototoxicity with decreasing UV light intensity.     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

Inhibition by polycyclic aromatic hydrocarbons of ATPase activities in Sebastiscus marmoratus larvae: relationship with the development of early life stages

Sebastiscus marmoratus larvae were exposed to waterborne polycyclic aromatic hydrocarbons (PAHs) containing 3-5 rings, benzo[a]pyrene (BaP), pyrene (Py) or phenanthrene (Phe), respectively at 0.01, 0.1 and 1 μg L?1. Cumulative mortality, frequency of dorsal curvature and rate of pericardial and yolk sac edema in larvae treated for 8 days were significantly increased in a dose-dependent manner. All three PAHs resulted in reduction of the lower jaw, and the extent of reduction increased with increasing concentrations of the PAHs. Na+/K+-ATPase and Ca2+-ATPase activity in larvae exposed to the three PAHs were all significantly inhibited in a dose-dependent manner. Analysis using the Pearson correlation coefficient indicated a significant correlation between the rate of the dorsal curvature and edema and the inhibition of Na+/K+-ATPase and Ca2+-ATPase activity, suggesting that the developmental defects caused by PAHs were related to their inhibition of Na+/K+-ATPase and Ca2+-ATPase activity.     

The fate of polyaromatic hydrocarbons in an intertidal sediment exposure system: Bioavailability to macoma inquinata (mollusca: Pelecypoda) and abarenicola pacifica (annelida: Polychaeta)

Macoma inquinata, a detritus feeding clam, was exposed for 60 days to coarse-grained, and Abarenicola pacifica, a burrowing polychaete, to fine-grained, sediment. Each sediment contained ¹⁴-C-labelled phenanthrene, chrysene or benzo(a)pyrene. Over 70% of the chrysene and benzo(a)pyrene, but only 8% of the phenanthrene, remained in the coarse sediment during the exposure. Essentially all of the chrysene and benzo(a)pyrene and 70% of the phenanthrene remained in the fine sediment. The concentrations of chrysene and benzo(a)pyrene in the clams rose steadily, reaching levels 11·6 and 5·2 times as high as those in the sediment. The tissue phenanthrene concentration rose for 3 days, then fell to one-eighth of the initial concentration. The concentrations of each of the aromatic hydrocarbons in Abarenicola tissue increased for 2 weeks to four to six times the sediment levels. The tissue concentration of chrysene remained constant thereafter, but the levels of phenanthrene and benzo(a)pyrene fell to three-quarters of their peak values. No intermediate degradation products of any of the hydrocarbons were identified in extracts of sediment from either exposure system, or in solvent extracts of tissue or of tissue digestates.Significant ¹⁴C-activity, associated with non-solvent extractable compounds, remained in tissue digestates of individuals of both species exposed to phenanthrene.     

High matrix interference affecting detection of PAH metabolites in bile of Atlantic hagfish (Myxine glutinosa) used for biomonitoring of deep-water oil production

The characteristic biology and wide distribution of hagfish species makes them relevant for use in pollution biomonitoring at great water depths, particularly in regions where deep-water oil production may take place. The exposure of fish to petrogenic contaminants can normally be detected from the level of polycyclic aromatic hydrocarbon (PAH) metabolites in bile fluid. Some of these metabolites are strong fluorophores, allowing analytical detection by means of simple fluorometric techniques such as fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS). In the present study bile from Atlantic hagfish (Myxine glutinosa) collected in pristine areas (Barents Sea and southwestern Norway) displayed strong bile fluorescence levels, suggesting the presence of PAH contaminants. However, gas-chromatography-mass spectrometry (GC-MS) analyses ruled out PAHs as the origin for this fluorescence signal. Rather, the bile of Myxine contains components resulting in unusually strong background fluorescence interfering at the wavelength pairs used for detection of PAH metabolites. Possible background for the observed matrix interference and implications for detection of PAH metabolites in hagfish is discussed.     

Evaluation of the relationships between biochemical endpoints of PAH exposure and physiological endpoints of reproduction in male California Halibut (Paralichthys californicus) exposed to sediments from a natural oil seep

Coal Oil Point (COP) is a natural oil seep off the coast of Santa Barbara, California. Although most studies examining the fate and effects of petroleum have focused upon urbanized or anthropogenic sources of inputs, few have examined the effects of polyaromatic hydrocarbons (PAHs) derived from natural seeps. In order to evaluate the effects of PAHs derived from COP on marine fish populations, hatchery-reared California Halibut (Platichthys californicus) were exposed for 30 days to seven dilutions of sediments collected from COP. Hepatic cytochrome P450 1A (CYP1A), biliary fluorescent aromatic compounds (FACs), gonadal somatic indices, and plasma steroid concentrations. Sixteen USEPA priority PAHs were targeted for analysis in each sediment dilution. In general, biochemical responses were somewhat recalcitrant to dose-response relationships and were less sensitive than the literature values established for the same indicators following exposure to urbanized PAHs. Trends toward reductions in plasma 17beta-estradiol concentrations were observed, but reductions in gonadal somatic indices were not observed. FAC values for naphthalene, benzo(a)pyrene, phenanthrene-related compounds reached maximums at 33-100% COP sediment. The resulting insensitivity may be unique for exposure to "natural" petroleum due to a higher concentration of lower molecular weight PAHs or uncharacterized inhibitors.     

苯并（a）芘诱导下岩虫CYP基因的表达特征

Rapid amplification of cDNA ends(RACE) and real-time polymerase chain reaction(RT-PCR) were carried out to analyze the CYP4 gene expression in polychaete Marphysa sanguinea exposed to benzo[a]pyrene(BaP) in this study. The full length of MsCYP4 cDNA was 2 470 bp, and it encoded 512 amino acids. The deduced amino acid sequence showed 47% identity with CYP4 F from frog Xenopus tropicalis and shared high homology with other known CYP4 sequences. To analyse the role of CYP4 in protecting M. sanguinea from BaP exposure, three BaP groups were established: 0.5, 5 and 50 μg/L. Polychaetes were sampled after 3, 7 and 12 d. At 0.5 μg/L, the effect of BaP on MsCYP4 gene expression increased with time prolonged. MsCYP4 gene expression curve showed Ushaped trend with time in 5 and 50 μg/L BaP groups. Therefore, MsCYP4 gene may play an important role in maintaining the balance of cellular metabolism and protecting M. sanguinea from BaP toxicity.     

苯并(a)芘、芘及其混合物暴露对梭鱼肝脏谷胱甘肽硫转移酶活性的影响

在实验生态条件下,观察苯并(a)芘、芘及其等浓度混合物暴露对梭鱼(Mugil so-iuy)肝脏谷胱甘肽硫转移酶(GST)活性的影响。结果显示,在7d的暴露中,苯并(a)芘、芘对肝脏GST活性的影响主要为诱导效应,芘对GST活性的诱导比苯并(a)芘强。混合物在15d的暴露中未观察到GST活性的诱导,而是在暴露的后期出现GST活性的抑制。实验表明,肝脏GST活性的诱导指示受到PAHs污染胁迫,而GST活性抑制则是受到较长时间或较严重的污染。     

In vivo formation of (+)-anti-benzo[a]pyrene diol-epoxide-plasma albumin adducts in fish.

Benzo[a]pyrene (BaP), a procarcinogenic polycyclic aromatic hydrocarbon (PAH), is bioactivated to BaP diol-epoxides (BPDEs) that can form adducts with DNA and blood proteins. We report here for the first time the in vivo formation of adducts between BPDE and plasma albumin (Alb) from two fish species experimentally exposed to BaP. Brook trout (Salvelinus fontinalis) received either a single i.p. dose (10 mg/kg) or two separate i.p. doses (25 mg/kg; 7 days apart) of BaP, and blood was collected 2 (single exposure) or 3 (multiple exposure) days post-treatment. Arctic charr (Salvelinus alpinus) received 10 i.p. doses (3 mg/kg; a single dose every 6 days), and blood was collected 2 days after the second, sixth, and 10th injections. BPDE-Alb adducts were measured by an improved HPLC/fluorescence method developed to detect and quantify BaP-tetrols released after acid hydrolysis of adducted Alb. HPLC/fluorescence chromatograms of Alb from BaP-treated fish revealed only BaP-tetrol I-1, thus indicating the formation of adducts exclusively via the (+)-anti-BPDE metabolite. Levels of (+)-anti-BPDE-Alb adduct ranged from 0.68 to 19.6 ng of tetrol I-1 per gram of Alb. Notably, adduct level was not related to BaP dose and there was no accumulation of adducts with repeated exposure, which may indicate a very short half-life (< 2 days) of plasma Alb in fish. The data suggest that BPDE-Alb adducts in fish could be useful as a non-destructive biomarker of recent exposure to bioactivated BaP.     

苯并(a)芘和芘对梭鱼肝脏DNA损伤的研究

用苯并(a)芘、芘以及它们的等量混和物,分别在浓度为0.1,1,10,20,50μg/dm3浓度下对梭鱼暴污,5d后取梭鱼肝脏和鳃用碱解旋法分别测定其DNA的损伤,结果随着污染物浓度的增加,肝脏DNA损伤程度增加;在相同浓度下,苯并(a)芘和芘的联合毒性大于苯并(a)芘和芘分别作用时的毒性之和。所以苯并(a)芘和芘对DNA损伤的联合作用应为加强作用。     

A laboratory and field comparison of sediment polycyclic aromatic hydrocarbon bioaccumulation by the cosmopolitan estuarine polychaete Streblospio benedicti (Webster)

Polycyclic aromatic hydrocarbon (PAH) bioaccumulation by the polychaete worm Streblospio benedicti (Webster) was measured under exposure to PAH-contaminated sediments in the field and for 28 d in the laboratory. Streblospio benedicti collected from field sediments contaminated at 2.94, 1.07, and l.52 μg g⁻¹ fluoranthene (FLU), benz[a]anthracene (BAA), and benzo[a]pyrene (BAP), bioaccumulated those PAHs at 1.53, 0.215, and 0.332 μg g⁻¹, while worms isolated from less contaminated field sediments (0.399, 0.228, 0.288 μg g⁻¹ FLU, BAA, and BAP) had FLU, BAA, and BAP body burdens of 0.543, 0.236, and 0.083 μg g⁻¹. Worms incubated for 28 d in PAH-spiked sediments (1.52, 0.991, 0.504 μg g⁻¹ FLU, BAA, and BAP) bioaccumulated those PAHs at 0.382, 0.966, and 0.602 μg g⁻¹, respectively. Data normalization to organism lipid and sediment organic carbon (biota-sediment accumulation factors [BSAFs] strongly suggest that Streblospio PAH bioaccumulation was directly related to percent sediment organic carbon, but BSAFs were substantially lower than predicted by equilibrium partitioning theory. BSAFs decreased with increasing PAH log K_ow, in worms collected from field sediments, but in spiked sediments BSAFs increased with increasing PAH hydrophobicity. This disparity may have been caused by insufficient spiked-sediment equilibration time (1.5h) in the case of the laboratory test sediments.     

Bioaccumulation and biomarkers in the sea star Asterias rubens (Echinodermata: Asteroidea): a North Sea field study

During two North Sea field trips in March 1995 and September 1996 sea stars, Asterias rubens, were collected at various stations along pollution gradients in order to study the relation between biochemical markers and levels of accumulated contaminants. Biomarkers measured were: cytochrome P450 level, benzo[a]pyrene hydroxylase (BPH) activity, acetylcholinesterase (AChE) activity and DNA integrity. Accumulation levels of heavy metals, polychlorinated biphenyls, organochlorine pesticides and polycyclic aromatic hydrocarbons (PAHs) in the pyloric caeca of sea stars indicate different pollution gradients, influenced by rivers in The Netherlands, UK and Germany. For some contaminants, especially PAHs, relatively high levels were found in the central part of the North Sea (Dogger Bank). On the basis of multivariate statistics, stations near the mouth of the Elbe and the Rhine/Meuse were shown to have different patterns of biomarker responses. Sea stars from stations in coastal zones showed relatively high levels of cytochrome P450 and 'P418', another haemoprotein that is present in most marine invertebrates. The station nearest to the Elbe Estuary showed the lowest BPH and AChE activity. DNA integrity was lower especially in stations near the Dutch coast and in a station near the Tees/Tyne estuaries. Using these biomarkers as early warning signals of exposure and/or adverse effects, this type of monitoring can be used also in the future to study the spatial and temporal trends in the quality of coastal waters.