Environ Sci Pollut Res DOI 10.1007/s11356-014-3403-9

BIOAVAILABILITY - THE UNDERLYING BASIS FOR RISK BASED LAND MANAGEMENT

Toxicity and oxidative stress induced by used and unused motor oil on freshwater microalga, Pseudokirchneriella subcapitata Kavitha Ramadass & Mallavarapu Megharaj & Kadiyala Venkateswarlu & Ravi Naidu

Received: 25 December 2013 / Accepted: 29 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Although used motor oil from automobiles is one of the major pollutants through storm water in urban environments leading to contamination of water bodies, very little information is available on its toxicity towards growth of microalgae. Also, to our knowledge, there are no data on the used motor oil-induced oxidative stress in microalgae. We therefore investigated the toxicity of used and fresh motor oil on growth and antioxidant enzymes of a microalga, Pseudokirchneriella subcapitata. In general, used oil was more toxic to the alga than fresh oil. Used oil at 0.20 % inhibited algal growth, measured in terms of chlorophyll a, by 44 % while fresh oil was nontoxic up to 2.8 %. Wateraccommodated fraction (WAF) of the used oil at >50 % concentration exhibited significant toxicity while WAF from fresh oil was nontoxic even up to 100 %. Used oil and its WAF, even at lower concentrations, increased the levels of antioxidant enzymes indicating algal response to the toxicity stress. When the alga was exposed to WAF from fresh motor oil, no alterations in the antioxidant enzyme levels were evident. The present investigation suggests that contamination of aquatic systems with used oil could potentially affect the

Responsible editor: Philippe Garrigues K. Ramadass : M. Megharaj : R. Naidu Centre for Environmental Risk Assessment and Remediation (CERAR), University of South Australia, Mawson Lakes, SA 5095, Australia K. Ramadass : M. Megharaj (*) : R. Naidu Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), Mawson Lakes, SA 5095, Australia e-mail: [email protected] K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India

ecosystem health via disruption of primary producers that are located at the base of the food chain. Keywords Used motor oil . Water-accommodated fraction (WAF) . Pseudokirchneriella subcapitata . Superoxide dismutase . Catalase . Peroxidase

Introduction Lubricants in the form of motor oil or engine oil are used for various internal combustion engines, and they consist of 80– 95 % of petroleum fractions and 5–20 % of additives (Pirro and Wessol 2001). The global annual production of used engine oil was estimated as 25–28 million tonnes excluding 12–15 million tonnes lost during engine operation (VazquezDuhalt 1989). A typical lubricant is a mixture of a wide variety of compounds such as hydrocarbons (C15–C50) including polycyclic aromatic hydrocarbons (PAHs); heavy metals like zinc (Zn), barium and lead (Pb); and other organic and inorganic additives such as amines and phenols. Toxic hydrocarbons and heavy metals (Va, Pb, Al, Ni, Fe and Cr and Zn) are present in higher quantities in used oil that comes out of automobile and generator engines’ leakages, services and disposals. Higher quantities of PAHs are also present in used motor oils due to the alteration that occurs during its engine use (Singh et al. 2006). Motor oil run-off from parking areas, roadways, accidental spillages, natural or man-made disasters and pipeline leakages are the direct sources of contamination that are detrimental to aquatic ecosystems. About 23–30 % of waste motor oil produced in USA is directly disposed of into the environment (Vazquez-Duhalt 1989). It seems a common practice in several underdeveloped nations for motor mechanics to carelessly dispose of the used motor oil into any available space in the environment as reported by Ogali et al. (2007) in case of

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Nigeria. Contamination with lubricating oils due to their largescale discharge into the inland and coastal waterways has often been neglected (Tam et al. 2005). Used motor oil is the single largest storm water pollutant in the urban environment. As a result, used and fresh lubricating oil pollution is now turning into a larger problem than the crude oil pollution, and therefore requires serious attention. Many studies on crude oil pollution and its impacts represent a popular area of environmental research, but data are scarce on lubricating oil toxicity. With their ubiquity in the natural environment, microalgae are the primary producers located at the base of the food chain. It is therefore important to understand how a subsystem of the ecosystem like microalgae reacts to the environmental pollutants. Bioassays serve as reputable pollution indicators, and microalgae are more sensitive to a variety of organic and inorganic toxicants through their rapid physiological response (Ramakrishnan et al. 2010). Only a few studies described toxicity of lubricating oil (Vazquez-Duhalt 1989; Tam et al. 2005), and investigations that compared the toxicity of fresh and used lubricant oil in microalgae are limited. Information on production of reactive oxygen species like superoxide, hydrogen peroxide and hydroxyl free radicals induced by lubricant stress in microalgae is also lacking. The present study describes the toxicity of used and unused motor oil on growth and induction of antioxidative enzymes like superoxide dismutase, catalase and peroxidase in Pseudokirchneriella subcapitata, a sensitive species of unicellular green algae that has been widely used in aquatic toxicity assays (Ramakrishnan et al. 2010).

Materials and methods Organisms and culture conditions A microalga, P. subcapitata (nonmotile, unicellular, crescentshaped (40 to 60 μm3), commonly found in most freshwaters), obtained from CSIRO Collection of Living Microalgae (Hobart, Australia), was used in this study. Axenic culture of the alga was maintained in Bold’s basal medium (BBM) at 25 ±2 °C in a growth chamber under continuous illumination (200 μE m − 2 s − 1 PPFD) (Megharaj et al. 1986; Subashchandrabose et al. 2013). Motor oils used in the study Used motor oil was obtained from a car that had its oil changed (after running 5,133 km) in an automobile service station at South Australia. The four-cylinder engine car was operated on regular unleaded gasoline for a total distance of 198,230 km. Fresh (unused) motor oil with similar specification to that of the used (SAE 15 W-50) was purchased under the brand name of Shell Helix plus.

Determination of hydrocarbons in motor oils Volatile and semivolatile hydrocarbons including benzene, toluene, ethylene and xylene (BTEX), total petroleum hydrocarbons (TPH) and PAHs were estimated by direct injection or using water-accommodated fractions (WAFs) or alkali extracts of both fresh and used motor oils. Direct injection Both the motor oils were injected directly, and TPH and PAH concentrations were determined. Aliquots (0.1 mL each) of motor oils, dissolved separately in 10 mL of hexane, were injected into gas chromatograph fitted with a flame ionization detector (GC-FID Agilent model 6890). Chromatography was performed on a fused silica capillary column BPX-5 from SGE (15-m×0.32-mm internal diameter) coated with HP-5 (0.10-μm film thickness). Helium was used as the carrier gas at 2.5 mL min−1, and the FID detector temperature kept at 300 °C. Splitless injection with a sample volume of 1 μL was applied. The oven temperature was increased from 50 to 300 °C at a gradient of 25 °C min−1 and held at this temperature for 5 min. The total run time was 19.6 min. Hydrocarbons were quantified using Agilent Chemstation Software by integration and calibration of peaks of a known concentration of external calibration standard—Hydrocarbon Window Defining Standard (C8–C40) from AccuStandard® (Risdon et al. 2008). Five concentrations of external calibration standard in the range expected in samples were analyzed to obtain a linear curve fit with a R2 value of 0.997. The Continuing Calibration Verification (CCV) was analyzed at the start and end of every ten samples, and CCV recovery was 95–110 % of true value. Hexane was run as blank with every ten samples to demonstrate that the system is free from contamination. The minimum concentration of TPH detected (MDL) following the analytical method described was 1.0 mg L−1. PAHs in motor oils were analyzed by gas chromatography/ mass spectrometry (GC-MS) following fractionation of hydrocarbon components by US EPA method 3630C with slight modification. Briefly, 0.5 mL of oil that was brought to 5 mL hexane and introduced into a column was packed with silica gel (100–200 mesh; 10 mm ×30 cm; topped with 2 cm Na2SO4; pre-eluted with 40 mL hexane). Elution of PAHs was carried out by the addition of 25 mL hexane followed by 40 mL methylene chloride/hexane (2:3, v/v). The eluent collected was concentrated to 1.0 mL and analyzed for 16 US EPA priority PAH compounds using a gas chromatograph fitted with mass selective detector (Hewlett-Packard 5890 Series II, Agilent Technologies, DE, USA) as per standard US EPA 8270C method. The PAHs were separated using a 30m-high resolution capillary column DB-5 (i.d. 0.25 mm, 0.25-μm film) coated with a 0.25-μm film (JW Scientific,

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Agilent Technologies, DE, USA). The oven temperature was held initially at 40 °C for 4 min, and then ramped up to 270 °C at 10 °C min−1. The calibration was done for each PAH by external calibration using a certified mixture (TCL Polynuclear Aromatic Hydrocarbons Mix-Ref 4-8905, Supelco, Bellefonte, PA, USA). Deuterated surrogate standards (1-methylnaphthalene-d8, fluorene-d10, anthracene-d10, pyrene-d 10 , p-terphenyl-d 14 , BaP-d 12 and benzo(ghi)perylene-d12) were used to monitor PAH losses during extraction and clean-up. Consistent recovery (>90 %) was observed during analysis. The reliability of the calibration was checked periodically by injecting known standards and solvent blanks into the column (Ssempebwa et al. 2004; Thavamani et al. 2012) WAF extract preparation and analysis for hydrocarbons Since petroleum hydrocarbons occur in two forms, dissolved or dispersed petroleum hydrocarbons (DDPHs) and adsorbed or absorbed petroleum hydrocarbons (AAPH), WAF is the better mode to know the water-soluble hydrocarbon concentration of motor oils (Singer et al. 2000; Faksness and Brandvik 2008). WAFs were prepared by slight modification in the procedure followed by Bejarano et al. (2006). Briefly, 720 mL of 0.22-μm filtered milliQ water and a Teflon-coated stirring bar (2 cm) were placed into a 1,000-mL glass bottle. Then, motor oil (80 mL) was layered on the top of the water surface by means of a glass tight syringe. The flask was sealed tight, headspace air-purged through the Teflon septa with a stainless steel needle attached to a gas tight syringe, and the 200-mL headspace filled with nitrogen (>99 % purity) to prevent oil degradation/oxidation. The bottle was placed in a refrigerated incubator (20±1.5 °C) on a magnetic stirrer plate, with stirring speed adjusted to avoid a large vortex and formation of oil droplets. After 24 h of shaking, the water extract portion was drained out from the bottom and was considered as 100 % WAF and analyzed for BTEX, TPH and PAHs. BTEX and other volatile compounds were directly estimated in WAFs of the motor oil using EPA methods 5030 and 8260B (GC-MS with purge and trap extraction for volatile organics). Standard volatile organic compound mix (VOC Mix 502 Alltech VOC-2JM-A) was used to quantify BTEX and other organic compounds. Preparation of standards and WAF samples and their injection into the purge and trap system were similar to those described above. MSD productivity software installed in the GC-MS instrument 5975 VL MSD (Agilent Technologies, DE, USA) was used for the calibration and quantification of BTEX and other compounds. 4-Bromofluorobenzene was added as surrogate at the concentration of 20 μg/mL. The recovery (>90 %) for all the compounds was consistent. TPH in the WAF sample was extracted according to standard extraction procedure (US EPA 3510C) with few modifications. Briefly, each WAF sample (100 mL) along with

1.0 mL of surrogate (o-terphenyl) was extracted with methylene chloride, reduced to 1.0 mL and analyzed by GC-flame ionization detection in HP6890 GC (Hewlett-Packard) to quantify TPH following EPA SW-846 Method 8015B. Freshly prepared WAFs were analyzed for PAHs following EPA SW-846 Method 3510C. WAF samples (100 mL each) were transferred to 250-mL separation funnels followed by additions of 50 μL of surrogate standard solutions (2fluorobiphenyl (96 %) and p-terphenyl-d14 (98 %), Aldrich®, St. Louis, MO, USA). PAHs were extracted thrice with methylene chloride (6 mL) by shaking vigorously for 2 min each time and collecting the organic phase into a 40-mL borosilicate glass vial. The combined extracts were filtered through a 60° Pyrex glass funnel equipped with filter paper loaded with 2 g anhydrous sodium sulphate. Each filtered extract was collected into 40-mL vials, brought down to 1.0 mL with a gentle stream of nitrogen and transferred to 2-mL amber vials. After the addition of 50-μL internal standard (i.e. phenathrene-d10), 16 PAH analytes (2–6 ring structures) were quantified based on the protocol as described above. Aqueous alkali extract preparation and analysis for hydrocarbons The methodology for extraction of hydrocarbons consisted of shaking a mixture of 9 g of oil and 30 g of saturated sodium bicarbonate in a separating funnel for 15 min, and the pH of this aqueous solution was adjusted to 3.0 by adding phosphoric acid. This acidified solution was then extracted with methylene chloride and analyzed for TPH content by GC-FID (Sepcic 2003). Heavy metal analysis in motor oils The determination of ‘total’ metal concentrations in the motor oil samples was carried out in triplicate by digesting 0.5 g of oil samples in 9 mL of HNO3 and 3 mL of HCl using a MARS 5 Microwave digester (HP 500, CEM) according to the US EPA method 3052. Included in all runs was a certified reference soil (Montana Soil SRM2711) and blanks. Digests were filtered through 0.45-μm Millipore filters and diluted appropriately for analysis via inductively coupled plasma-mass spectroscopy (ICP-MS) (Agilent 7500c). Algal toxicity of motor oils Algal growth inhibition test was conducted by exposing the microalga to different concentrations of oil (0–5 %, v/v) and different dilutions of WAF (0–100 %) for 2 weeks. Motor oils were tested for their toxicity as whole product through direct addition in various concentrations of 0–5 % for unused motor oil and 0–0.8 % for used oil on v/v basis. Whole oils were

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added into BBM inoculated with algal cells from a 7-dayold exponentially growing culture. The initial cell density of the culture was maintained at 5×105 cells mL−1, and the total volume of the test medium was 100 mL. WAFs were also tested for their toxicity and compared with the whole oil toxicity. Aliquots of freshly extracted WAF were combined with appropriate volumes of sterilized milliQ water and BBM in clean 250-mL conical flasks to provide 0, 10, 25, 50, 75 and 100 % WAF in 100 mL. BBM, instead of water, was used while preparing WAF in order to serve as 100 % WAF. Algal cells were inoculated into all the flasks. Cultures with no oil and 0 % WAF which contained only sterilized milliQ and BBM served as controls. The test cultures were maintained in a temperature-controlled (25 °C) orbital shaker set at 120 rpm under cool white fluorescent illumination of about 200 μE m−2 s−1 PPFD. At the end of 4, 8 and 12 days, algal growth was measured in terms of chlorophyll a, an indicator of algal biomass (Megharaj et al. 1999; Deasi et al. 2010). EC50 (median effective concentration that causes 50 % inhibition in growth) values for 96-h exposure (US EPA 1989) were calculated from the values of percent inhibition in growth relative to those of untreated controls by Probit analysis using Minitab 16 statistical software (Palma et al. 2008). The acute toxicity experiment was performed twice under reasonably constant test conditions, and the data showed that the precision (CV) of EC50 values in triplicate of each sample was 11.2 %. TPH extraction from algal cells exposed to motor oils To determine the extent of hydrocarbon accumulation in the microalga, cell suspension exposed to whole oil and WAF was centrifuged at 10,000×g for 10 min at 4 °C. TPHs in the aqueous supernatant were then extracted twice with equal volumes of hexane by liquid–liquid extraction, using oterphenyl as the internal standard surrogate (Lei et al. 2002). The extracts were combined, concentrated to