Biodegradation of oil-contaminated aqueous ecosystem using an immobilized fungi biomass and kinetic study.

Remediation of environmental oil pollution with the usage of fungal organisms has proven to be a successful cleanup bioremediation method for organic contaminants. To investigate the breakdown of oil pollutants in water environments, biosurfactant-producing fungi have been isolated from oil-polluted soil samples. 16s rRNA sequencing technique was performed to identify the fungal organism and phylogenetic tree has been constructed. A variety of biosurfactant screening tests have demonstrated the better biosurfactant producing ability of fungi. The emulsion's stability, which is essential for the biodegradation process, was indicated by the emulsification index of 68.48% and emulsification activity of 1.3. In the isolated biosurfactant, important functional groups such as amino groups, lipids, and sugars were found according to thin layer chromatography analysis with a maximum retention value of 0.85. A maximum oil degradation of around 64% was observed with immobilized beads within 12 days. The half-life, and degradation removal rate constant of 20.21 days and 0.03 day-1, respectively, have been determined by the degradation kinetic analysis. GCMS analysis confirmed the highly degraded hydrocarbons such as nonanoic acid and pyrrolidine. The immobilized fungi exhibit better oil biodegradability in aqueous solutions.

Implications of microbial enhanced oil recovery and waterflooding for geochemical interpretation of recovered oils.

Biosurfactants and waterflooding have been widely reported thus far for enhancing oil production. Nevertheless, there is a lack of literature to explore enhanced oil recovered methods effects on its chemical composition. The aim of this work is to investigate the effects of a biosurfactant produced by Bacillus safensis and brine injection on the recovered petroleum composition, and their implications for geochemical interpretation. Original and oils recovered from displacement tests were analyzed by gas chromatography and ultra-high-resolution mass spectrometry, emphasizing saturated and aromatic biomarkers and basic and acidic polar compounds. Geochemical parameters based on some saturated compounds were subtly affected by the recovery methods, showing their reliable applicability in geochemical studies. Contrarily, parameters based on some aromatic compounds were more affected by biosurfactant flooding, mostly the low molecular weight compounds. Thus, these aromatic parameters should be applied with caution after such methods. The distribution of basic and acidic polar compounds can also be modified affecting the geochemical interpretation. In the case of the basic ones, the biosurfactant greatly influenced the N class species with favorable loss of lower aromaticity compounds. In addition to water solubilization, the compositional changes described in this study can be related to fractionation due to adsorption on reservoir rocks.

Utilization of surface-active compounds derived from biosolids to remediate polycyclic aromatic hydrocarbons contaminated sediment soil.

In the present study, surface-active compounds (SAC) were extracted from biosolids using an alkaline treatment process. They were tested for their remediation efficiency of crude oil-contaminated sediment soil and was compared with Triton x-100. The SAC exhibited a similar soil washing efficiency to that of the commercial Triton x-100, and under the optimized soil washing parameters, SAC exhibited a maximum of 91% total polycyclic aromatic hydrocarbons removal. Further, on analysing the toxicity of the soil residue after washing, it was observed that SAC from biosolids washed soil exhibited an average of 1.5-fold lesser toxicity compared to that of Triton x-100 on different test models-earthworm, a monocot, and dicot plants. The analysis of the key soil parameters revealed that the commercial surfactant reduced the soil organic matter and porosity by an average of 1.3-fold compared to SAC. Further, the ability of surfactants to induce toxicity was confirmed by the adsorption of the surfactants on the surface of the soil particles which was in the order of Triton x-100 > SAC. Thus, this study suggests that SAC can be applied as an effective bioremediation approach for contaminated soil for a greener and sustainable ecosystem.

Physico-chemical and thermal characteristics of sandy loam soils contaminated by single and mixed pollutants (mineral and vegetable oils).

Samples of contaminated top soil (0-30 cm) and uncontaminated soil (control) from two locations in Precambrian basement complex area were analyzed to assess the effects of single and mixed oil contaminants on the physico-chemical and thermal properties of soils. Pearson's correlation and analysis of variance (ANOVA) were used to study the interrelationships of the studied parameters as well as variation of studied soil characteristics under the different oil contaminants, respectively. Results showed insignificant impact of pollutant(s) on the textural class of contaminated soils. The highest and lowest mean soil resistivity (SR) values were found in petrol-contaminated and mixed surfactants (shampoo + conditioner) -contaminated soils, respectively. The least values of mean specific heat capacity (SHC), heat capacity (HC), and soil water diffusivity (SWD) were found in soils contaminated by mixed surfactants-contaminants. However, mixed mineral oils (petrol + diesel + engine oil) and mixed vegetable oils (palm oil + groundnut oil) had mean SHC values lower than those of control soils at the two sampling locations. The mixed surfactants-polluted soil is characterized by lowest mean bulk density (BD) and highest mean gravimetric water content (GWC) while lowest mean GWC and highest mean SWD characterized engine oil-contaminated soils. The analysis of variance (ANOVA) result revealed significant variation in % sand at 5% level (p < 0.05) for petroleum-derived contaminants but no significant differences in mean values of all analyzed soil properties under vegetable oil contaminants.

Surface-assisted Laser Desorption/ionization Mass Spectrometry Analysis of the Glycolipid Biosurfactants, Mannosylerythritol Lipids, Using an Ionization-assisting Substrate.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for the screening of glycolipid-type biosurfactants (BSs) from a crude extract of microbial products. However, it is unsuitable for the detection of lower molecular weight products because the observed ions are overlapped with matrix-derived ions at lower mass range. In this study, we applied a "matrix-free" surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis using a through-hole alumina membrane as an ionization-assisting substrate. Using this method, we could detect a variety of lower molecular weight products in an extract of a glycolipid BS producer with good sensitivity. In addition, the culture solution could be analyzed directly by this method.

Chemotaxis Toward Crude Oil by an Oil-Degrading Pseudomonas aeruginosa 6-1B Strain.

The chemotactic properties of an oil-degrading Pseudomonas aeruginosa strain 6-1B, isolated from Daqing Oilfield, China, have been investigated. The strain 6-1B could grow well in crude oil with a specific rhamnolipid biosurfactant production. Furthermore, it exhibits chemotaxis toward various substrates, including glycine, glycerol, glucose, and sucrose. Compared with another oil-degrading strain, T7-2, the strain 6-1B presented a better chemotactic response towards crude oil and its vital component, n-alkenes. Based on the observed distribution of the strain 6-1B cells around the oil droplet in the chemotactic assays, the potential chemotaxis process of bacteria toward crude oil could be summarized in the following steps: searching, moving and consuming.

Separation and determination of alkyl sulfate surfactants in wastewater by capillary electrophoresis coupled with contactless conductivity detection after preconcentration by simultaneous adsorption using alumina beads.

The aim of the present study is to determine four anionic alkyl sulfate (AS) surfactants with different alkyl chains, namely, C8, C10, C12, and C14, in wastewater by CE with capacitively coupled contactless conductivity detection (CE-C4 D). The conditions effective for the separation of the four AS surfactants were systematically optimized and found to be in a Tris-His (50 mM/20 mM) BGE solution at a pH of 8.95, using a separation voltage of +15 kV, hydrodynamic injection by siphoning using a 20 cm injection height and an injection time of 20 s. The LODs for C8, C10, C12, and C14 were 2.58, 2.30, 2.08, and 3.16 mg/L, respectively. The conditions used to achieve the simultaneous adsorption and preconcentration of the AS surfactants using Al2 O3 beads were pH of 3 and 0.1 mM NaCl. The adsorption efficiencies were found to be 45.6, 50.8, 81.7, and 99.9%, while the desorption efficiencies reached 66.1, 70.4, 83.9, and 100.0% for C8, C10, C12, and C14, respectively. The concentrations of the AS surfactants in wastewater samples were quantified by CE-C4 D after preconcentration by simultaneous adsorption using Al2 O3 beads. The results obtained from the proposed method were consistent with those obtained by HPLC-MS/MS, with a deviation of less than 15%. Our results indicate that the CE-C4 D performed after preconcentration by an adsorption technique using Al2 O3 beads is a new, inexpensive, and suitable method for quantifying AS surfactants in wastewater samples.

Enhanced marine monitoring and toxicity study of oil spill dispersants including Corexit EC9500A in the presence of diluted bitumen.

Observations made for the analysis of the oil spill dispersant tracer dioctyl sulfosuccinate (DOSS) during LC50 toxicity testing, highlighted a stability issue for this tracer compound in seawater. A liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (LC/QToF) was used to confirm monooctyl sulfosuccinate (MOSS) as the only significant DOSS breakdown product, and not the related isomer, 4-(2-ethylhexyl) 2-sulfobutanedioate. Combined analysis of DOSS and MOSS was shown to be applicable to monitoring of spill dispersants Corexit® EC9500A, Finasol OSR52, Slickgone NS, and Slickgone EW. The unassisted conversion of DOSS to MOSS occurred in all four oil spill dispersants solubilized in seawater, although differences were noted in the rate of MOSS formation. A marine microcosm study of Corexit EC9500A, the formulation most rapid to form MOSS, provided further evidence of the stoichiometric conversion of DOSS to MOSS under conditions relevant to real world dilbit spill. Results supported combined DOSS and MOSS analysis for the monitoring of spill dispersant in a marine environment, with a significant extension of sample collection time by 10 days or longer in cooler conditions. Implications of the unassisted formation of MOSS and combined DOSS:MOSS analysis are discussed in relation to improving dispersant LC50 toxicity studies.

Effects of physical parameters and chemical dispersant on the formation of oil-particle aggregates (OPAs) in marine environments.

Floating oil and sediments can interact to form oil-particle aggregates (OPAs) in marine environments. Laboratory batch experiments were conducted to investigate the effects of the concentration and size of sediment, temperature, oil types and chemical dispersant on the formation of OPAs. The results showed that the mass of OPAs and oil-particle aggregation rate are mainly related to the sediment concentration. Under the same mass concentration, more oil droplets can be trapped by smaller particles. Nevertheless, larger particles tend to interact more quickly with oil droplets. The effect of temperature on the formation of OPAs is substantially attributed to its influence on oil viscosity, and there is a threshold for oil viscosity which will bring about the maximum OPAs. Spilled oil with a high asphaltene can interact more effectively with the sediments. Appropriate addition of chemical dispersant is favorable for the formation of OPAs while excess addition will inhibit it.

Development of Palm Fatty Acid Distillate-Containing Medium for Biosurfactant Production by Pseudomonas sp. LM19.

High production costs of biosurfactants are mainly caused by the usage of the expensive substrate and long fermentation period which undermines their potential in bioremediation processes, food, and cosmetic industries even though they, owing to the biodegradability, lower toxicity, and raise specificity traits. One way to circumvent this is to improvise the formulation of biosurfactant-production medium by using cheaper substrate. A culture medium utilizing palm fatty acid distillate (PFAD), a palm oil refinery by-product, was first developed through one-factor-at-a-time (OFAT) technique and further refined by means of the statistical design method of factorial and response surface modeling to enhance the biosurfactant production from Pseudomonas sp. LM19. The results shows that, the optimized culture medium containing: 1.148% (v/v) PFAD; 4.054 g/L KH2PO4; 1.30 g/L yeast extract; 0.023 g/L sodium-EDTA; 1.057 g/L MgSO4·7H2O; 0.75 g/L K2HPO4; 0.20 g/L CaCl2·2H2O; 0.080 g/L FeCl3·6H2O gave the maximum biosurfactant productivity. This study demonstrated that the cell concentration and biosurfactant productivity could reach up to 8.5 × 109 CFU/mL and 0.346 g/L/day, respectively after seven days of growth, which were comparable to the values predicted by an RSM regression model, i.e., 8.4 × 109 CFU/mL and 0.347 g/L/day, respectively. Eleven rhamnolipid congeners were detected, in which dirhamnolipid accounted for 58% and monorhamnolipid was 42%. All in all, manipulation of palm oil by-products proved to be a feasible substrate for increasing the biosurfactant production about 3.55-fold as shown in this study.

In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing.

In the last decade, extensive application of hydraulic fracturing technologies to unconventional low-permeability hydrocarbon-rich formations has significantly increased natural-gas production in the United States and abroad. The injection of surface-sourced fluids to generate fractures in the deep subsurface introduces microbial cells and substrates to low-permeability rock. A subset of injected organic additives has been investigated for their ability to support biological growth in shale microbial community members; however, to date, little is known on how complex xenobiotic organic compounds undergo biotransformations in this deep rock ecosystem. Here, high-resolution chemical, metagenomic, and proteomic analyses reveal that widely-used surfactants are degraded by the shale-associated taxa Halanaerobium, both in situ and under laboratory conditions. These halotolerant bacteria exhibit surfactant substrate specificities, preferring polymeric propoxylated glycols (PPGs) and longer alkyl polyethoxylates (AEOs) over polyethylene glycols (PEGs) and shorter AEOs. Enzymatic transformation occurs through repeated terminal-end polyglycol chain shortening during co-metabolic growth through the methylglyoxal bypass. This work provides the first evidence that shale microorganisms can transform xenobiotic surfactants in fracture fluid formulations, potentially affecting the efficiency of hydrocarbon recovery, and demonstrating an important association between injected substrates and microbial growth in an engineered subsurface ecosystem.

Microbial Surfactants: Alternative to Vegetable Oil Surfactants.

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.

Biosurfactant-modified palygorskite clay as solid-stabilizers for effective oil spill dispersion.

An effective and conventional remediation technique in marine oil spills is to apply chemical dispersants to emulsify oil slicks into small oil droplets. Still, the potential hazards of chemical dispersants onto the marine ecosystem have motivated the research for environmentally friendly alternative while keeping exceptional dispersion ability. Here, we showed that the mixture of palygorskite (PAL) and rhamnolipid (Rha) formed a biocompatible alternative to synthetic surfactants used for oil spill dispersion. The oil droplets dispersed by R-PAL presented a small average size and long-term stability, which illustrated the synergistic interactions between Rha and PAL acting as an efficient dispersant in artificial sea water (ASW). Due to the strong flocculation caused by high salinity, PAL alone was not effective emulsifiers in ASW. A small amount of Rha could played a major role in modifying the surface characteristics of PAL and decreasing oil-water interfacial tension. Therefore, PAL particles irreversibly adsorbed onto the oil-ASW interface and formed a rigid interfacial film around oil droplets in the presence of Rha, which offered an efficient barrier to droplet coalescence. The synergistic interactions between PAL and Rha could enable the dispersion of tetradecane in ASW. Such a functionality was further tested in dispersing crude oil in ASW. The study presents a new strategy of using a mixture of PAL and Rha for oil dispersion, thus providing an ecofriendly alternative to conventional dispersants.

Field-collected crude oil, weathered oil and dispersants differentially affect the early life stages of freshwater and saltwater fishes.

The Deepwater Horizon (DWH) oil spill was the biggest in US history and released 3.19 million barrels of light crude oil into the Gulf of Mexico. In this study, we compared the toxicity of water accommodated fractions (WAFs) of naturally weathered crude oils, source oil, and source oil with dispersant mixtures and their effects on developing sheepshead minnow and zebrafish. Although a freshwater fish, zebrafish has been used as a model for marine oil spills owing to the molecular and genetic tools available and their amenability to lab care. Our study not only aimed to determine the effect of crude oil on early life stages of these two fish species, but also aimed to determine whether dissolved crude oil constituents were similar in fresh and saltwater, and if freshwater fish might be a suitable model to study marine spills. Weathering and dispersant had similar effects on WAF composition in both fresh and saltwater, except that the saltwater source oil + dispersant WAF had markedly higher PAH levels than the freshwater equivalent. WAF exposure differentially affected survival, as the LC50 values in %WAF for the zebrafish and sheepshead minnow exposures were 44.9% WAF (95% confidence interval (C.I.) 42.1-47.9) and 16.8% WAF (95% C.I. 13.7-20.5); respectively. Exposure increased heart rate of zebrafish embryos, whereas in sheepshead, source oil exposure had the opposite effect. WAF exposure altered mRNA expression of biotransformation makers, vitellogenin and neurodevelopment genes in both species. Muscle deformations were only found in oil-exposed zebrafish. This is one of the most comprehensive studies to date on crude oil toxicity, and highlights the species-specific differences in cardiotoxicity, estrogenic effects, biotransformation enzyme induction and potential neurotoxicity of crude oil exposure.

Production and characterization of a biosurfactant produced by Streptomyces sp. DPUA 1559 isolated from lichens of the Amazon region.

Surfactants are amphipathic compounds containing both hydrophilic and hydrophobic groups, capable to lower the surface or interfacial tension. Considering the advantages of the use of biosurfactants produced by microorganisms, the aim of this paper was to develop and characterize a biosurfactant produced by Streptomyces sp. DPUA1559 isolated from lichens of the Amazon region. The microorganism was cultured in a mineral medium containing 1% residual frying soybean oil as the carbon source. The kinetics of biosurfactant production was accompanied by reducing the surface tension of the culture medium from 60 to values around 27.14 mN/m, and by the emulsification index, which showed the efficiency of the biosurfactant as an emulsifier of hydrophobic compounds. The yield of the isolated biosurfactant was 1.74 g/L, in addition to the excellent capability of reducing the surface tension (25.34 mN/m), as observed from the central composite rotational design when the biosurfactant was produced at pH 8.5 at 28°C. The critical micelle concentration of the biosurfactant was determined as 0.01 g/mL. The biosurfactant showed thermal and pH stability regarding the surface tension reduction, and tolerance under high salt concentrations. The isolated biosurfactant showed no toxicity to the micro-crustacean Artemia salina, and to the seeds of lettuce (Lactuca sativa L.) and cabbage (Brassica oleracea L.). The biochemistry characterization of the biosurfactant showed a single protein band, an acid character and a molecular weight around 14.3 kDa, suggesting its glycoproteic nature. The results are promising for the industrial application of this new biosurfactant.

High-resolution Kendrick Mass Defect Analysis of Poly(ethylene oxide)-based Non-ionic Surfactants and Their Degradation Products.

Matrix assisted laser desorption ionization (MALDI) high-resolution mass spectrometry (HRMS) and the recently introduced high-resolution Kendrick mass defect (HRKMD) analysis are combined to thoroughly characterize non-ionic surfactants made of a poly(ethylene oxide) (PEO) core capped by esters of fatty acids. A PEO monostearate surfactant is first analyzed as a proof of principle of the HRKMD analysis conducted with a fraction of EO as the base unit (EO/X with X being an integer) in lieu of EO for a regular KMD analysis. Data visualization is greatly enhanced and the distributions detected in the MALDI mass spectrum are assigned to a pristine (H, OH)-PEO as well as mono- and di-esterified PEO chains with palmitate and stearate end-groups in HRKMD plots computed with EO/45. The MALDI-HRMS/HRKMD analysis is then successfully applied to the more complex case of ethoxylated hydrogenated castor oil (EHCO) found to contain a large number of hydrogenated ricinoleate moieties (up to 14) in its HRKMD plot computed with EO/43, departing from the expected triglyceride structure. The exhaustiveness of the MALDI-HRMS/HRKMD strategy is validated by comparing the so-obtained fingerprints with results from alternative techniques (electrospray ionization MS, size exclusion and liquid adsorption chromatography, ion mobility spectrometry). Finally, aged non-ionic surfactants formed upon hydrolytic degradation are analyzed by MALDI-HRMS/HRKMD to easily assign the degradation products and infer the associated degradation routes. In addition to the hydrolysis of the ester groups observed for EHCO, chain scissions and new polar end-groups are observed in the HRKMD plot of PEO monostearate arising from a competitive oxidative ageing.

Effects of salinity on oil dispersant toxicity in the eastern mud snail, Ilyanassa obsoleta.

Chemical dispersants can be a beneficial method for breaking up oil slicks; however, their use in mitigation could pose potential toxic effects on the marine ecosystem. Dispersants may be transported to lower salinity habitats, where toxicity data for aquatic species have not been established. This study examined the effect of salinity on oil dispersant toxicity in the eastern mud snail, Ilyanassa obsoleta, using two dispersants authorized for oil spill response, Corexit® 9500A and Finasol® OSR 52. Median lethal toxicity values (LC50) and sublethal effects were examined at 10, 20, and 30 ppt salinity in adult and larval mud snails. Two biomarkers (lipid peroxidation and acetylcholinesterase) were used to measure sublethal effects. The 96-h static renewal LC50 values indicated significant differences in toxicity between dispersants and salinities. Larval snails were significantly more sensitive than adult snails to both dispersants, and both life stages were significantly more sensitive to Finasol than to Corexit. Larval snails were more sensitive to dispersants at lower salinity, but adult snails were more sensitive at higher salinities. Dispersants increased lipid peroxidation and decreased acetylcholinesterase activity. These results demonstrate that dispersant toxicity varies among compounds and organism life stages, and that physicochemical properties of the environment, such as salinity, can affect the potential toxicity to estuarine species.

Ophthalmic effects of petroleum dispersant exposure on common murres (Uria aalge): An experimental study.

The safety of chemical dispersants used during oil spill responses is largely unknown in birds. We captured common murres in Monterey Bay, CA and exposed them to Corexit EC9500a, crude oil, or a combination in artificial seawater. We performed ophthalmic examinations and measured intraocular pressures and tear production before and after exposure. Loglinear analysis found that exposure to oil or dispersant was related to the development of conjunctivitis and corneal ulcers. Odds ratios for birds exposed to oil or dispersant were positive and significant for the development of conjunctivitis, while odds ratios for the development of corneal ulcers were positive and significant only for birds exposed to a high concentration of oil. Ocular exposure to dispersants and petroleum in seabirds may cause conjunctivitis and may play a role in the development of corneal ulcers. These results have implications for policymakers who develop protocols for the use of dispersants during marine oil spills.

Determination of chloride in brazilian crude oils by ion chromatography after extraction induced by emulsion breaking.

The present paper reports on the development of a novel extraction induced by emulsion breaking (EIEB) method for the determination of chloride in crude oils. The proposed method was based on the formation and breaking of oil-in-water emulsions with the samples and the consequential transference of the highly water-soluble chloride to the aqueous phase during emulsion breaking, which was achieved by centrifugation. The determination of chloride in the extracts was performed by ion chromatography (IC) with conductivity detection. Several parameters (oil phase:aqueous phase ratio, crude oil:mineral oil ratio, shaking time and type and concentration of surfactant) that could affect the performance of the method were evaluated. Total extraction of chloride from samples could be achieved when 1.0g of oil phase (0.5g of sample+0.5g of mineral oil) was emulsified in 5mL of a 2.5% (m/v) solution of Triton X-114. The obtained emulsion was shaken for 60min and broken by centrifugation for 5min at 5000rpm. The separated aqueous phase was collected, filtered and diluted before analysis by IC. Under these conditions, the limit of detection was 0.5µgg(-1) NaCl and the limit of quantification was 1.6µgg(-1) NaCl. We applied the method to the determination of chloride in six Brazilian crude oils and the results did not differ statistically from those obtained by the ASTM D6470 method when the paired Student-t-test, at 95% confidence level, was applied.

Isolation, screening, and characterization of surface-active agent-producing, oil-degrading marine bacteria of Mumbai Harbor.

Diverse marine bacterial species predominantly found in oil-polluted seawater produce diverse surface-active agents. Surface-active agents produced by bacteria are classified into two groups based on their molecular weights, namely biosurfactants and bioemulsifiers. In this study, surface-active agent-producing, oil-degrading marine bacteria were isolated using a modified Bushnell-Haas medium with high-speed diesel as a carbon source from three oil-polluted sites of Mumbai Harbor. Surface-active agent-producing bacterial strains were screened using nine widely used methods. The nineteen bacterial strains showed positive results for more than four surface-active agent screening methods; further, these strains were characterized using biochemical and nucleic acid sequencing methods. Based on the results, the organisms belonged to the genera Acinetobacter, Alcanivorax, Bacillus, Comamonas, Chryseomicrobium, Halomonas, Marinobacter, Nesterenkonia, Pseudomonas, and Serratia. The present study confirmed the prevalence of surface-active agent-producing bacteria in the oil-polluted waters of Mumbai Harbor.