Sediment-associated contaminants and liver diseases in bottom-dwelling fish

High concentrations of chemicals have been found in sediments from urban areas of Puget Sound. Hundreds, of organic chemicals (including certain aromatic hydrocarbons [AHs] and various chlorinated compounds) were identified. Statistical methods were used to evaluate possible relationships between the chemistry data and fish diseases. Positive correlations were found between the frequencies of liver neoplasms (e.g., hepatocellular carcinoma) and other liver lesions in English sole (Parophrys vetulus) and concentrations of AHs in sediment; such correlations were not found with chlorinated hydrocarbons. Strong evidence was also obtained to show that many organic chemicals in sediment are bioavailable to bottom-dwelling fish. Stomach contents (consisting mainly of benthic invertebrates) from English sole had concentrations of a number of AHs similar to those in the sediment from which the fish were taken. In these same fish, metabolites of many aromatic compounds were found in bile using a procedure combining HPLC with fluorescence detection. Further, the concentrations of certain xenobiotic metabolites in bile were correlated positively with the occurrence of liver neoplasms in English sole.     

Exposure of juvenile chinook and chum salmon to chemical contaminants in the Hylebos Waterway of Commencement Bay, Tacoma, Washington

The Hylebos Waterway is an industrialized waterway ofCommencement Bay, Tacoma, Washington, that is severelycontaminated with aromatic and chlorinatedhydrocarbons in the sediment. Juvenile chinook (Oncorhynchus keta) and chum salmon (O.tshawytscha) inhabit this waterway for a few days orweeks during their outmigration from freshwaterstreams to saltwater. The purpose of thisinvestigation was to determine to what degree juvenilechum and chinook salmon captured from the HylebosWaterway might bioaccumulate organic contaminants. These levels of exposure will be compared to previousstudies where such exposures have been linked tobiological dysfunction in juvenile salmon. Theresults showed that juvenile chum and chinook salmonfrom the Hylebos Waterway take up a wide range ofchemical contaminants, compared to fish fromhatcheries or reference estuaries. These contaminantsinclude high and low molecular weight polycyclicaromatic hydrocarbons (PAHs), polychlorinatedbiphenyls (PCBs, including the toxic congeners 105 and118), hexachlorobutadiene (HCBD), hexachlorobenzene(HCB), DDTs, heptachlor, and several pesticides. Immunohistochemical examination of the gill and gut injuvenile chum salmon from the Hylebos Waterway showedthe induction of the P450 metabolizing enzyme. Moreover, concentrations of contaminants in juvenilechinook and chum salmon from the Hylebos Waterway arecomparable to levels previously shown to be associatedwith biological injury in juvenile chinook salmon,such as impaired growth, suppression of immunefunction as demonstrated by reduced B cell function,and increased mortality following pathogen exposure.     

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.     

DNA damage and biliary PAH metabolites in flatfish from Southern California bays and harbors, and the Channel Islands

Southern California bays and harbors have been shown to contain high concentrations of a variety of contaminants, including polycyclic aromatic hydrocarbons (PAHs), metals, and pesticides. Conventional monitoring tools do not assess exposure to PAHs in fish, or sublethal effects, which can be more sensitive indicators of stress than traditional methods. This study was conducted to evaluate DNA damage (a sublethal effect) and PAH exposure in flatfish from southern California bays and harbors, and the Channel Islands. California halibut (Paralichthys californicus) were collected from eight bays and harbors (Ventura Harbor, Channel Islands Harbor, Marina del Rey, King Harbor, Alamitos Bay, Long Beach Harbor, Newport Bay, San Diego Bay), and a reference site off Camp Pendleton. Pacific sanddab (Citharichthys sordidus) were collected near four of the Channel Islands (San Miguel Island, Santa Cruz Island, Anacapa Island, Santa Barbara Island). Metabolites of high molecular weight PAHs in fish bile were characterized using a semi-quantitative technique that measures fluorescent aromatic compounds (FACs) in fish bile. DNA damage in fish blood cells was assessed by measuring the amount of single-strand breaks in stained DNA using the Comet assay. The concentration of FACs in fish from all bays and harbors was elevated, with average concentrations ranging from three to ten times greater than FACs in reference fish. FAC levels were elevated in Pacific sanddabs from a Channel Islands station located between Santa Cruz Island and Anacapa Island. DNA damage varied by a factor of five among California halibut from bays and harbors, with significant damage occurring in fish from Alamitos Bay. There was a significant association between FAC concentrations and DNA damage in California halibut at Marina del Rey and Ventura Harbor, but not at other locations, presumably due to contaminants other than PAHs.     

Establishing the Causal Relationship between Polycyclic Aromatic Hydrocarbon (PAH) Exposure and Hepatic Neoplasms and Neoplasia-Related Liver Lesions in English Sole (Pleuronectes vetulus)

For almost 25 years our laboratory has studied the impact of PAHs and related industrial contaminants on benthic fish, following an interdisciplinary approach involving chemical exposure assessment linked to synoptic detection of various effects at several levels of biological organization. These data demonstrate a cause-and-effect relationship between neoplastic and neoplasia-related liver lesions in English sole, and exposure to PAHs, and to a lesser degree, chlorinated hydrocarbons such as PCBs. In statistical analyses of data from multiple field studies conducted since 1978, exposure to PAHs measured in various compartments has consistently been identified as a highly significant, major risk factor for neoplasms and related lesions in this species, with PCB exposure shown to be a significant, but less consistent and less strong risk factor for these lesions. A cause-and-effect relationship between PAHs and toxicopathic liver lesions in this species is further supported by the experimental induction of toxicopathic lesions identical to those observed in field-collected fish, in sole exposed in the laboratory to model carcinogenic PAHs such as BaP or to PAH-rich extracts of sediments from Eagle Harbor, a severely PAH-contaminated site in Puget Sound. More recent field studies have identified significant associations between hepatic cytochrome P4501A (CYP1A) induction and xenobiotic-DNA adduct formation, and hepatic lesion prevalences in wild subadult English sole. Field studies in Eagle Harbor subsequent to capping of the most PAH-contaminated region of this harbor with clean dredge spoils have shown a decline in exposure to PAHs as assessed by biliary fluorescent aromatic compounds (FACs) and hepatic xenobiotic-DNA adducts. This decline in PAH exposure has been accompanied by a dramatic decline in risk of occurrence of toxicopathic hepatic lesions in English sole from Eagle Harbor. Further, laboratory studies have induced lesions in English sole by injections of extracts from PAHcontaminated sediments. Overall, these findings relating to exposure to PAHs and chlorinated hydrocarbons and the occurrence of hepatic neoplasms and neoplasiarelated lesions in English sole fulfill the classic criteria for causality in epizootiological or ecological risk assessment studies, including: (1) strength of association, (2) consistency of association, (3) specificity of association, (4) toxicological and biological plausibility, (5) temporal sequence/timing (i.e., exposure precedes disease, effect decreases when the cause is decreased or removed), (6) dose-response or biological gradient, and (7) supportive experimental evidence.     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Characterization of PAH Sources in Sediments of the Thea Foss/Wheeler Osgood Waterways,Tacoma, Washington

The character of polycyclic aromatic hydrocarbons (PAH) in sediments of the Thea Foss and Wheeler-Osgood Waterways in Tacoma, Washington, were investigated with the objective of determining the general source(s) of these compounds to the waterways. In this study, 42 near-surface sediment samples from the Waterways were collected and analyzed for their (1) concentration of 43 individual or groups of PAH, (2) total extractable hydrocarbon “fingerprint” and concentration, (3) grain size and (4) total organic carbon content. Analysis of the sediment data, including comparisons to standard reference materials, indicates that all but two samples contained PAH derived from a pyrogenic source(s), i.e., a non-petroleum source(s). The high concentrations and characteristic distributions of PAH in some sediment samples were consistent with the occurrence of manufactured gas plant (MGP) derived tar(s) or tar distillate(s), particularly in some sediments proximal to a historic MGP and tar distillate storage operation near the head of the Thea Foss Waterway. Most other sediment samples throughout the Waterways contained PAH distributions and concentration indicating (at least) a greater proportion of PAH are derived from urban runoff/fallout.     

Influence of aromatic hydrocarbons on growth,plant growth promoting activities and survival in soil of Azotobacter chroococcum strain JL104

This study was undertaken to determine the effect of aromatic hydrocarbons on growth and plant growth promoting activities of Azotobacter chroococcum strain JL104. The organism was grown on Jensen’s media without sucrose, supplemented with different concentrations of aromatic hydrocarbons. Azotobacter chroococcum strain JL104 was able to grow in the presence of benzene, toluene, aniline and benzoic acid and was able to utilize these as sole carbon source as well. The culture showed the highest growth in presence of 0.5% concentrations of aniline and benzoic acid and 0.01% concentrations of benzene and toluene. Maximum indole acetic acid (IAA) production and acetylene reduction activity (ARA) were recorded with benzene and benzoic acid, respectively. Among other substituted benzene derivatives such as xylene, p-hydroxybenzoic acid, di-nitrophenol and di-chlorophenol, xylene was observed to be the least toxic and di-nitrophenol the most toxic hydrocarbon. The highest soil survival was found in soil amended with 1% sucrose however, the population of A. chroococccum strain JL104 declined continuously in unamended soil. Amongst various hydrocarbons, 0.1% toluene amended soil supported the maximum survival, indicating it to be least toxic aromatic hydrocarbon carbon in soil.     

Chemical analyses of water, sediment and biota in two small streams in southwest Germany

As a component of the VALIMAR project a comprehensive chemicalanalysis study was conducted in two small streams, the Körsch andthe Krähenbach, in southern Germany. The investigated pollutantswere heavy metals (HM), polycyclic aromatic hydrocarbons (PAHs),polychlorinated biphenyl's (PCBs) and a set of 33 pesticides.Samples from stream water, sewage treatment plant effluents,stream sediment and fish were analyzed. In the stream water, HMand PAHs showed slightly higher concentrations in the Körsch thanin the Krähenbach, whereas the pesticide concentrations (mainlyplant protective agents) were significantly higher in the Körschthan in the Krähenbach. Because of their similar occurrence andconcentrations in the Körsch water and in the sewage treatmentplant effluent, it appears that most of the pesticides wereintroduced into the river via the sewage treatment plant. Only inthe cases of terbuthylazine, triallate, chlorfenvinfos,metazachlor and mecoprop were the occurrence and concentrationsin the Körsch higher suggesting that run-off was the mostimportant route for these five compounds in this stream.The sediments of the two streams clearly differed in terms ofpollutant concentration. Polycyclic aromatic hydrocarbons, PCBsand HM were found in slightly higher concentrations in the Körschthan in the Krähenbach whereas pesticide concentrations weresignificantly higher in the Körsch. Results of each samplingdemonstrated extreme variations, due to frequent high-watersituations that led to high turnover and wash out of thesediment. In the sediment samples, most of the detected compoundswere more lipophilic than were those detected in water.Brown trout (Salmo trutta f. fario) and loach (Barbatulabarbatula) were sampled directly from the natural streams, from astream water fed bypass exposure system, and from a controlsystem in the laboratory. In the case of PAHs, PCBs andpesticides, concentrations were higher in fish exposed to theKörsch water than to the Krähenbach water. Heavy metals did notdifferentiate between the two streams. Polychlorinated biphenylsand pesticides showed higher concentrations in fish from thefield, which can be explained by longer exposure periods. Incontrast, PAHs showed the lowest concentrations under fieldconditions. Polycyclic aromatic hydrocarbons were possiblydisplaced by other compounds of high concentration. The pesticidepatterns in trout were quite similar in the different exposuretypes with primarily lindane, PCP and trifluraline contributingto these pattern changes. In loach, larger variations in allcontaminant concentrations were found but the same threecompounds also contributed most to the observed pattern changes.The analytical results were compared with various internationalobjective values for a good water quality as well as with valuesfrom other studies concerning the pollutant concentrations ofsediment and fish. All collected chemical data form a basis forthe biomarker investigations conducted by the other participantsof the VALIMAR project in order to find correlations betweenbiomarker responses and chemical pollution data of the twostreams.     

On‐site bioremediation treatment system design and on‐site analytical screening of hydrocarbon‐contaminated dredge sediments

In early 1991, a petroleum refining facility located on the Blair Waterway in Commencement Bay near Tacoma, Washington, wished to deepen its berthing facility. Sediments had accumulated in the berthing area adjacent to the facility's petroleum handling dock to the extent that tanker ships could go aground during low tides. A preliminary sediment characterization program had indicated that elevated polynuclear aromatic hydrocarbons such as anthracene and phenanthrene, and total petroleum hydrocarbons exceeded Puget Sound Dredge Disposal Analysis maximum level guidelines for unconfined, open‐water disposal. Enviros designated an on‐shore sediment treatment facility to receive the dredged sediments. Design criteria and construction details of a treatment area capable of accommodating 8000 yd³ of sediments are presented. Onsite, real‐time sediment analysis for total petroleum hydrocarbon (TPH) concentrations was conducted by an onsite mobile laboratory. Laboratory techniques to expedite sediment analysis for TPH using infrared spectrophotometry are described.     

专一营养与兼性甲烷氧化菌降解氯代烃的研究现状、动力学分析及展望

生物修复技术被认为是氯代烃类污染物处理处置的最有效途径之一,而甲烷氧化菌在该领域表现出较大的应用潜力。近期研究发现,突破了仅能利用单碳化合物的局限,兼性甲烷氧化菌能够利用多种底物降解氯代烃,这一独特的新陈代谢特性,使其在污染物生物处置领域逐渐受到关注。结合本课题组研究成果,对甲烷氧化菌降解氯代烃进行了全面总结,主要包括:分析了不同菌株(纯菌株和混合菌株)对不同氯代烃的降解效果;比较了不同类型甲烷单加氧酶在不同底物体系中的活性表达和催化特性;总结了模型菌株甲基弯菌Methylosinus trichosporium OB3b降解氯代烃的动力学特性;概述了兼性甲烷氧化菌株降解氯代烃的特性及其应用潜力;最后讨论了甲烷氧化菌降解氯代烃存在的问题及未来发展方向。     

Residues of Aliphatic and Polycyclic Aromatic Hydrocarbons in Some Fish Species of Lake Temsah,Ismailia, Egypt: An Analytical Search for Hydrocarbon Sources and Exposure Bioindicators

Residues of aliphatic and polycyclic aromatic hydrocarbons (PAHs) were monitored in some fish species collected from Temsah Lake, near Ismailia, Egypt. Fish were selected to represent different feeding habits and ecological niches in the lake's ecosystem. Fish samples were extracted using organic solvents, and residues of aliphatic and PAHs were separated using column chromatography and detected using gas liquid chromatography. Fish species were Clupea sirm, Mugil sehli, Mugil capito, Morone labrax, and Sciasna sp. Clupea sirm, a surface feeder fish had the highest concentration of aliphatic hydrocarbons, 320 ± 54 ng g^?1, while Morone labrax, a predatory fish that live in the water column, had the highest concentration of PAHs, 315.87 ± 46 ng g^?1. Even-number aliphatic hydrocarbons were more frequently detected in all fish species in comparison to odd-number aliphatic hydrocarbons, suggesting a petrogenic origin of these compounds. Meanwhile, the pattern of PAHs detected in the present study suggested that they originate from atmospheric deposition rather than land-based runoff. Morone labrax fish and Clupea sirm fish were the most suitable candidate bioindicators of exposure to aliphatic hydrocarbons and PAHs through fish consumption of the five fish species examined in this study.     

Measurement of metabolites of polycyclic aromatic hydrocarbons in fish bile for comparative assessment of coastal water pollution

In the laboratory experiment and in fish caught in conditionally clean and polluted areas, the presence of metabolites of polycyclic aromatic hydrocarbons in bile by methods of conventional and synchronous spectrofluorimetry was determined. The study showed that this approach is well suited for the detection of metabolites of polycyclic aromatic hydrocarbons with 2–3, 4, and 5–6 aromatic rings. If the content of metabolites of polycyclic aromatic hydrocarbons with 4 and 5–6 rings in bile is low, the method of synchronous spectrofluorimetry is less sensitive than conventional spectrofluorimetry. Both methods of monitoring are simple and fast and reflect the real picture of water pollution.     

Inhibition of lipolysis by hydrocarbons and fatty alcohols

The hydrolysis of long-chain triglyceride by pancreatic lipase (EC 3.1.1.3) is inhibited by hydrophobic solutes that are dissolved in the fat. Solutes tested included n-alkanes (C10-C16), aromatic and chlorinated aromatic hydrocarbons (including a PCB and DDT), n-alcohols (C10-C16), and cholesterol. Except for cholesterol, which stimulated lipolysis at low concentrations, all compounds produced roughly similar inhibition curves that followed the pattern of a typical Langmuir adsorption isotherm (Mattson, F. H., R. A. Volpenhein, and L. Benjamin, 1970. J. Biol. Chem. 245: 5335-5340). According to this interpretation, hydrophobic solutes dissolved within fat droplets partition between the interior oil phase and the surface monolayer where lipolysis occurs. Although the aromatic and chlorinated aromatic hydrocarbons were approximately 25% more inhibitory than the long-chain aliphatic hydrocarbons, as a single class, hydrocarbons were 7-10 times weaker inhibitors of lipolysis than fatty alcohols. In contrast to the alcohols whose inhibitory action may involve several mechanisms, the hydrocarbons behaved like simple dilution inhibitors; i.e., at 50% inhibition the mass ratio of hexadecane to triglyceride was 0.42. The lack of a chain length effect indicates that the hydrocarbons are not adsorbed at the interface but interdigitate the triglyceride molecules and align parallel to the lipid acyl chains. Inhibition by hydrophobic solutes was not reversed by the presence of 4 mM taurodeoxycholate and pancreatic procolipase or colipase.     

Compounds in urban air compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the receptor protein

Acetone extracts of filter-collected urban airborne particulate matter contain compounds which can competitively inhibit 2,3,7,8-[1,6-3H]tetrachlorodibenzo-p-dioxin (TCDD) binding to the rat liver TCDD-receptor protein. The concentration of conventional polycyclic aromatic hydrocarbons (PAHs) or chlorinated dioxins and dibenzofurans cannot account for more than 1-30% of the observed competition for [3H]TCDD binding to the receptor protein. The difference in potency between samples collected in urban areas during different periods of the year and a background sample is 25-400-fold. Collecting samples in the presence of increased concentrations of nitrogen dioxide, nitrous acid, nitric acid or ozone did not increase the amount of compounds with receptor affinity. However, with nitrogen dioxide and especially with nitric acid, a substantial increase of the mutagenic effects in the Ames Salmonella assay in the absence of mammalian activation as well as a degradation of several PAHs were noted. Affinity for the TCDD-receptor protein, mutagenicity in the absence of mammalian metabolic activation in the Ames Salmonella assay and PAH-content are characteristics of urban particulate matter showing the presence of compounds, that represent potential health risks. The compounds with affinity for the receptor may constitute a group of substances different from both conventional PAHs and direct-acting mutagens.     

Energetics of interactions of aromatic hydrocarbons with water

The thermodynamics of transfer of aromatic (benzene, toluene) and aliphatic (ethane, propane, butane) hydrocarbons from the gas phase into water in the temperature range 5–125°C have been analyzed in order to determine the net hydration effect of these compounds. In the case of the aromatic hydrocarbons the enthalpic contribution predominates over the entropic contribution to the Gibbs energy of hydration. This results in a negative value of the hydration Gibbs energy of aromatic hydrocarbons, in contrast to the positive Gibbs energy of hydration of aliphatic hydrocarbons. The different sign of the hydration Gibbs energies indicates that the mechanism causing hydrophobicity of aromatic hydrocarbons has different nature than that causing the hydrophobicity of aliphatic hydrocarbons. The comparison of hydration of aliphatic and aromatic hydrocarbons leads to the following thermodynamic parameters for these additional interactions between the benzene ring and water at 25°C: enthalpy −5.4 kJ/mol, entropy 26.8 J/K mol and Gibbs energy −13.4 kJ/mol. The large enthalpic contribution to the Gibbs energy of hydration of aromatic hydrocarbons probably comes from the ability of the aromatic ring to accept hydrogens from water, forming hydrogen bonds.     

The influence of seasonality,fish size and reproductive status on EROD activity in Plagioscion squamosissimus: Implications for biomonitoring of tropical/subtropical reservoirs

Ethoxyresorufin-O-deethylase (EROD) activity is considered an important biomarker for aquatic environmental contamination. Although EROD activity has been widely used as a biomarker of exposure to polycyclic aromatic hydrocarbons in fish, this activity can be influenced in the field by spatial-, seasonal- or individual-related factors. We therefore performed a comparative study of hepatic EROD activity levels in the croaker Plagioscion squamosissimus to determine whether variations existed in enzyme activity levels, especially in relationship to reproductive status, fish size, age, and seasonality. For this purpose, we collected fish from three reservoirs with different pollution levels during the early-rainy (November 2012), rainy (March 2013), and dry (July–August 2013) seasons from the Tietê River, Brazil. We tested whether size, age and sex affected EROD activity among the localities and seasons. We found a marked effect of pollution during the dry season on variation among the localities in EROD activity in P. squamosissimus. An analysis of covariance indicated that sex had a significant negative effect on the seasonal variability of the EROD activity levels at the most polluted locality (near São Paulo). A possible explanation for the statistical association between EROD activity and sex is that the reproductive status of the females influenced the EROD activity levels. A possible explanation for the statistical association between EROD activity and sex is that the reproductive status of the females influenced the EROD activity levels, also largely reported in the literature. Our results suggest that reproductive status can be a significant confounding factor for determining EROD activity in female P. squamosissimus in freshwater ecosystems when compared to males. Moreover, our results suggest that the presence of phenanthrene during the dry season at Barra Bonita reservoir might explain the highest EROD activity responses in this period of study.     

Biodegradation of Aliphatic vs. Aromatic Hydrocarbons in Fertilized Arctic Soils

The objectives of this study were to (1) test a simple bioremediation treatment strategy in the Arctic and (2) examine the effect of fertilization on the degradation of aliphatic and aromatic hydrocarbons. The site is a coarse sand pad that once supported fuel storage tanks. Concentrations of diesel-range organics at the beginning of the study (July 1996) ranged from 250 to 860 mg/kg soil. Replicate field plots treated with fertilizer yielded final concentrations of 0, 50, 100, or 200 mg N/kg soil. Soil samples were collected three times during the thaw season and analyzed for physical and chemical properties, microbial populations and activities, and concentrations of semivolatile hydrocarbons. Soil pH and soil-water potentials declined as a result of fertilizer application. Addition of fertilizer significantly increased soil respiration potentials, but not the populations of microorganisms measured. Fertilizer addition also resulted in ∼50% loss of measured aliphatic and aromatic hydrocarbons in surface and subsurface soils. For fertilized plots, hydrocarbon loss was not related to the amount of fertilizer added. Losses of aliphatic hydrocarbons were attributed to biotic processes, whereas losses of aromatic hydrocarbons likely were a result of both biotic and abiotic processes.     

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (approximately 750 kb) linear plasmid.