Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons

To investigate the genotoxic effect of marine sediments on aquatic organism, sediment samples were collected from 13 sites along the coast of Gwangyang Bay (Korea). Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments were determined and the relationship between exposure of flounder blood cells to sediment extracts and DNA single-strand breakage in the blood cells was examined using the comet assay. Levels of DNA damage were proportionally increased by exposure concentration and the highest sediment-associated DNA damage was observed at the station showing the highest PAHs contamination. DNA damage in blood cells exposed to five types of PAHs (benzo[a]pyrene, fluoranthene, anthracene, pyrene and phenanthrene) and in flounder (Paralichthys olivaceus) exposed to benzo[a]pyrene (BaP) for 0, 2 and 4 days were assessed by measuring comet tail length. The tail lengths of five PAHs-exposed groups at 50 and 100 ppb were significantly different from the non-exposed group, and the genotoxic effect of BaP correlated with both concentration and duration of exposure. Throughout the study, significant differences in DNA breakage were recorded between cells exposed to sediment extracts or PAHs and non-exposed control. This study demonstrated the comet assay as a successful tool in monitoring contamination of marine sediments and assessing genotoxicity of PAHs in marine organisms, either in vitro or in vivo.     

Benzo[a]pyrene modulates the biotransformation,DNA damage and cortisol level of red sea bream challenged with lipopolysaccharide

In animals, biotransformation and the immune system interact with each other, however, knowledge of the toxic mechanism of benzo[a]pyrene (BaP) on these two systems is not well known. The present study investigated the toxic effects of BaP on the biotransformation system, cortisol level and DNA integrity of red sea bream (Pagrus major). The results showed that cortisol level was induced under the challenge of lipopolysaccharide (LPS). Short-term exposure (96 h) of BaP at environmental concentration significantly increased the cortisol level, hepatic EROD activity and CYP1A1 mRNA expression. When P. major was exposed to BaP for 14 d followed by LPS challenge this increased the cortisol level, EROD activity and hepatic DNA damage except CYP1A1 mRNA expression. Combined with our previous data, which showed that BaP exposure can modulate the immunologic response in P. major challenged with LPS, a hypothetical adverse outcome pathway of BaP on fish was suggested.     

Ecological Risk Assessment of Benzo[a]pyrene in Yellow River Delta

Ecological risk assessment is of great significance to promoting the rational management effectively for the oil‐polluted areas. A comprehensive evaluation method of ecological risk, including probabilistic risk assessment and regional ecological risk assessment, is developed through employing the contaminant benzo[a]pyrene as an indicator to assess ecological risk of five oil mining plots, respectively in Yellow River Delta. In this study, firstly we evaluate the ecological risk probability of five oil mining plots using overlapping area of probabilistic curves, and the results show that local ecological risk varies between the maximum 0.4 and the minimum 0.01. Then we overlay boundaries of five administrative divisions in Yellow River Delta and the spatial distribution patterns of ecosystems to generate new risk receptor plaques, and calculate the integrated value of 30 specifically classified plaques for comprehensive evaluation of ecological risk. The results, fluctuating within the range of 0.00005 and 0.25, indicate that local government should be vigilant to ecological risk of benzo[a]pyrene to some extent, although the current situation is not severe in whole.     

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.     

Role of a moderately halophilic bacterial consortium in the biodegradation of polyaromatic hydrocarbons

Polycyclic aromatic hydrocarbons are ubiquitous pollutants in the environment, and most high molecular weight PAHs cause mutagenic, teratogenic and potentially carcinogenic effects. While several strains have been identified that degrade PAHs, the present study is focused on the degradation of PAHs in a marine environment by a moderately halophilic bacterial consortium. The bacterial consortium was isolated from a mixture of marine water samples collected from seven different sites in Chennai, India. The low molecular weight (LMW) PAHs phenanthrene and fluorine, and the high molecular weight (HMW) PAHs pyrene and benzo(e)pyrene were selected for the degradation study. The consortium metabolized both LMW and HMW PAHs. The consortium was also able to degrade PAHs present in crude oil-contaminated saline wastewater. The bacterial consortium was able to degrade 80% of HMW PAHs and 100% of LMW PAHs in the saline wastewater. The strains present in the consortium were identified as Ochrobactrum sp., Enterobacter cloacae and Stenotrophomonas maltophilia. This study reveals that these bacteria have the potential to degrade different PAHs in saline wastewater.     

Polycyclic aromatic hydrocarbons in coastal sediments of southwest Taiwan: An appraisal of diagnostic ratios in source recognition

Fifty-seven surface sediment samples were collected from the coast of southwest Taiwan and analyzed for polycyclic aromatic hydrocarbons (PAHs). Concentrations of total PAHs (28 PAH compounds) ranged from 15 to 907 ng g⁻¹ dry weight. Diagnostic ratios showed that PAHs in the sediments of the Gaoping estuary were predominantly of petroleum origin, whereas sediments from the Kaohsiung coast contained principally combustion-derived PAHs. Principal component analysis indicated that emissions from automobiles and coal burning were the main sources of combustion-derived PAHs. The relatively high ratios of perylene/penta-aromatic PAH isomers in sediments from the Tainan coast and some off-shore stations on the Kaohsiung coast suggest a significant diagenetic PAH contribution. The study shows that certain diagnostic ratios are useful and sensitive in delineating the distribution of PAHs from specific sources in southwest Taiwan. The phenanthrene/anthracene ratio is a better indicator than the methylphenanthrenes/phenanthrene ratio for tracing petrogenic PAHs, and the benzo(a)anthracene/chrysene and indeno(1,2,3-c,d)pyrene/benzo(g,h,i)perylene ratios are more specific than the benzo(a)pyrene/benzo(e)pyrene and benzo(b)fluoranthcene/benzo(k)fluoranthcene ratios in distinguishing PAHs from various pyrogenic sources.     

Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE

A microbial consortium was obtained by enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of a mixture of polycyclic aromatic hydrocarbons enrichment strategy. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial composition and community dynamic changes based on PCR amplification of 16S rRNA genes during batch culture enrichment. Using the spray-plate method, three bacteria, designated as BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequence analysis showed that BL01, BL02 and BL03 were phylogenetically close to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens, respectively. The degradation of benzo(a)pyrene (BaP), a model high-molecular-weight polycyclic aromatic hydrocarbon (HMW PAH) compound was investigated using individual isolates, a mixture of the three isolates, and the microbial consortium (BL) originally isolated from the oil port sea water. Results showed that the order of degradative ability was BL > the mixture of the three isolates > individual isolates. BL degraded 44.07% of the 10 ppm BaP after 14 days incubation, which showed the highest capability for HMW PAH compound degradation.Our results revealed that this high selective pressure strategy was feasible and effective in enriching the HMW PAH-degraders from the original sea water samples.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels “integrate” biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (α-HCH, aldrin, dieldrin, p,p′-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

The relationship between the concentrations and distribution of organic pollutants and black carbon content in benthic sediments in the Gulf of Gdańsk, Baltic Sea

The influence of the sediment’s physico-chemical properties and black carbon content, on the distribution of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in benthic sediments of the Gulf of Gdansk (Baltic Sea) was determined. Sediments from port, marine dump site of dredged spoils, the Vistula river mouth, Gdansk Deep were selected.The concentrations of ∑PAHs (fluoranthene, pyrene, benzo(a)anthracene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(ah)anthracene, benzo(ghi)perylene) were 294-2200 ng/g d.w. and for ∑PCBs (28, 52, 101, 118, 138, 153, 180) were 2.4-11.3 ng/g d.w.The sediments content of loss on ignition was 1.13-16.15%, total organic carbon was 0.89-7.15%, black carbon was 0.20-1.15%. The highest correlation coefficient values (r = 0.76-0.92, p < 0.05) for a relationship between the concentrations of organic pollutants, and organic matter, organic and black carbon contents were obtained in harbor sediments with low content of organic matter (<5%) and high share of black carbon in total carbon (up to 40%).     

Polycyclic aromatic hydrocarbons in commercial fish and lobsters from the coastal waters of Madagascar following an oil spill in August 2009

Concentrations of polycyclic aromatic hydrocarbons were determined in species of commercial fish and lobsters following an oil-spill just off the protected Madagascan coastline. Samples were collected along the coastline within and outside the affected area. Summed PAH concentrations ranged from 1.9 μg kg⁻¹ to 63 μg kg⁻¹ wet weight, but with no higher molecular weight PAHs (>202 Da) being detected. All concentrations of benzo[a]pyrene, benz[a]anthracene and dibenz[a,h]anthracene were <0.1 μg kg⁻¹ wet weight, well within the EU and UK set limits for the protection of human health. Additionally, samples were calculated as the benzo[a]pyrene toxic equivalency quotient (TEQ) and found to be well below the level of concern in relation to health of human consumers. Evaluation of the biota PAH data indicated the origin of PAH was predominantly petrogenic with >80% arising from oil sources. Profile studies indicate a low-level multisource petrogenic contamination probably representing a pre-spill background for the area.     

Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp. VA1

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants in the environment and are derived from both man-made and natural resources. The present study is focused on the degradation of PAHs by a halotolerant bacterial strain under saline conditions. The bacterial strain VA1 was isolated from a PAH-degrading consortium that was enriched from marine water samples that were collected from different sites at Chennai, India. In the present study, a clearing zone formed on PAH-amended mineral salt agar media confirmed the utilization of PAH by the bacterial strain VA1. The results show that the strain VA1 was able to degrade anthracene (88%), phenanthrene (98%), naphthalene (90%), fluorene (97%), pyrene (84%), benzo(k)fluoranthene (57%) and benzo(e)pyrene (50%) at a 30 g/L NaCl concentration. The present study reveals that the VA1 strain was able to degrade PAHs in petroleum wastewater under saline conditions. The promising PAH-degrading halotolerant bacterial strain, VA1, was identified as Ochrobactrum sp. using biochemical and molecular techniques.     

Concentrations of unsubstituted polycyclic aromatic hydrocarbons in softshell clams from Coos Bay,Oregon, USA

Concentrations of benzo[a]pyrene (BAP) were measured in subpopulations of softshell clams, Mya arenaria, from four intertidal sites in Coos Bay from June 1976 to June 1978. Subsequently, concentrations of 15 unsubstituted polynuclear aromatic hydrocarbons (PNAH) were determined in two subpopulations from September 1978 to August 1979. There were significant differences between BAP concentrations in clams from the four sites. For the two-year period, they were highest in clams inhabiting areas adjacent to the industrialized bayfront and lowest in clams inhabiting more remote areas. There were no significant seasonal variations in BAP concentrations during this period. During the 1978–1979 study, the average total PNAH concentration in clams from the bayfront area was 555.1 μg kg^?1 compared to 76.3 μg kg^?1 for clams from a more remote environment. In general, PNAH concentrations were lowest in the fall-winter and highest during the spring-summer.     

Heavy metals and polycyclic hydrocarbon compounds in benthic organisms of the Upper Gulf of Thailand

Concentrations of heavy metals and polycyclic aromatic hydrocarbons (PAHs) were measured in bivalves collected in the Upper Gulf of Thailand. PAHs detected included acenaphthene, acenaphthylene, benzo[a]pyrene, fluoranthene, methylphenanthrene, phenanthrene and triphenylene. Benzo[a]pyrene was detected in all species at concentrations varying from 1.0 to 8.2 ng·g^?1. Concentrations of cadmium, cobalt, copper, lead and nickel were significantly lower in bivalves from the Gulf than in green mussels collected from the mouth of the Chao Phraya River. No correlation was found between metal concentrations in animals and sediment, with the exception of copper. Copper concentrations in polychaetes and clams appeared to correlate with the copper:iron ratio of sediments. High rates of degradation were observed when radiolabelled chlorobenzene, phenanthrene and chrysene were added to water and sediment of the Chao Phraya River. Rates were lower in the waters and sediment of the Gulf of Thailand. The calculated half-lives of chlorobenzene in the Gulf of Thailand and the Chao Phraya River were about 130 and 68 days, respectively.     

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.     

Sediment hydrocarbon contamination at the site of the Baie Verte,Newfoundland, oil spill: Results of a Four Year Study

Sediments samples were taken at 3, 15, 27, and 39 months following a spill of diesel oil at Baie Verte, Newfoundland, to determine the effects of weathering on diesel oil trapped in the sediments and to attempt to develop a suitable method for extraction and analysis.Extraction was carried out using hexane, concentration was effected by a procedure modified from Junk et al. (1974) and analysis was completed within 18.0 min by high performance liquid chromatography (HPLC) using a bonded amino silane-(NH₂) column (5 μm, 5 mm × 25 cm) with hexane as mobile phase. A backflush cycle of 22.0 min using hexane and methl-t-butyl ether (90:10%) was performed to elute any polar compounds from the column.This class analysis by HPLC indicated a decrease in the amount of naphthalene, fluorene, phenanthrene and benzo[a]pyrene equivalents over time and at the conclusion of the study (39 months) no PAHs except for fluorene were found in the sediments from Baie Verte.     

Oxidation of Benzo(a) Pyrene Catalyzed by Phenol Oxidases of Alga Nitella sp. and Potato Tubers

The kinetics of oxidation of carcinogenic benzo(a)pyrene (0.1 nM solution in phosphate buffer) catalyzed by enzyme protein of soil and water plants (potato tubers and alga Nitella sp.) has been studied. The benzo(a)pyrene oxidation rate is determined by the phenoloxidase activity of the enzyme protein from both sources to an equal degree and expressed by the Michaelis-Menten equation. The values for k_cat and apparent K_m were (0.97 ± 0.14) Ms^?1 and 33 ± 6 μM, respectively. The presence of other compounds (including protein substances) in acetone preparations of potato tubers considerably suppresses its catalytic activity in relation to benzo(a)pyrene. Consequently, in the polluted biosphere some water plants, such as alga Nitella sp., and potato tuber can transform carcinogenic benzo(a)pyrene (BP). As oxidation products, all three BP-quinones, i.e. 1,6-, 3,6-and 6,12-diones, were identified.     

Cooxidation of Benzo(a)pyrene and Phenols on Activated Sludges

In the presence of the cancerogenic benzo(a)pyrene (4 × 10^?7 mole/1) the rate the of biochemical oxidation of phenols and a mixture of oil-shale alkylresorcinols (5 × 10^?5 mole/1) on activated sludges adapted to these phenols is reduced up to 30%. The oxidation of benzo(a)pyrene occurs at a high rate, the rate constant on both sludges being 6.8 × 10⁸ mole/1 · sec and exceeds the index of the processes of its selfpurification by two orders. The high efficiency of the biooxidation of benzo(a)pyrene is retained at a repeated use of the same biomass, thus no additional regeneration being needed. Some products of the, biooxidation of benzo(a)pyrene, such as 1,6- and 3,6-quinone, derivatives of chrysene and naphthalene, are identified.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels “integrate” biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (α-HCH, aldrin, dieldrin, p,p′-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

Cooxidation of Carcinogenic Benzo(a) Pyrene and Phenols by Horseradish Peroxidase (Armoracia lapathifolia)

The catalytic activity of horseradish (Armoracia lapathifolia) peroxidase in the oxidation reactions of benzo(a)pyrene (BP) and phenols, separately and in admixture, has been studied. Experiments were carried out with model phenols from industrial sewage (phenol, m-, p-cresols, catechol, hydroquinone, resorcinol, orcinol), keeping to the real concentration ratio of reagents, 0.5 mM for phenols, 0.1 nM for benzo(a)pyrene. The total oxidation process for all reagents tested is described by the second-order formal kinetic equation up to 60 … 90% of their conversion. Addition of peroxidase increases the rate of oxidation of benzo(a)pyrene and phenols 2 … 6 times as compared with the control test (with hydrogen peroxide), whereas a linear dependence of the rate of benzo(a)pyrene oxidation on the enzyme concentration is observed. By cooxidation a mutual inhibition of benzo(a)pyrene and phenols is established whose degree depends on the phenol-chemical structure and varies in the range of 23 … 58% for BP under the influence of phenols and 35 … 80% for phenols under the influence of benzo(a)pyrene. The results obtained permit a supposition that peroxidase containing plants are capable of simultaneously transforming carcinogenic benzo(a)pyrene and toxic phenols in water reservoirs and in the soil, thus promoting the detoxication of environment.     

Composition and sources of pollutant hydrocarbons in the Severn Estuary

Hydrocarbon distributions in sediment from the Severn Estuary can be explained mainly in terms of a pollutant origin. The aliphatic hydrocarbon distribution is similar to that found in biodegraded crude oils. The distribution of polycyclic aromatic hydrocarbons, including the carcinogen benzo(a)pyrene, is similar to that derived from the pyrolysis of fossil fuels, especially petroleum.