ORIGINAL PAPER

John R. Haines Æ Eric J. Kleiner Æ Kim A. McClellan

Karen M. Koran Æ Edith L. Holder Æ Dennis W. King

Albert D. Venosa

Laboratory evaluation of oil spill bioremediation productsin salt and freshwater systems

Received: 19 July 2004 / Accepted: 2 March 2005 / Published online: 3 May 2005� Society for Industrial Microbiology 2005

Abstract Ten oil spill bioremediation products weretested in the laboratory for their ability to enhancebiodegradation of weathered Alaskan North Slopecrude oil in both freshwater and saltwater media. Theproducts included nutrients to stimulate inoculatedmicroorganisms, nutrients plus an oil-degrading inocu-lum, nutrients plus compounds intended to stimulate oil-degrading activity, or other compounds intended toenhance microbial activity. The product tests wereundertaken to evaluate significant modifications in theexisting official United States Environmental ProtectionAgency (EPA) protocol used for qualifying commercialbioremediation agents for use in oil spills. The EPAprotocol was modified to include defined formulas forthe exposure waters (freshwater, saltwater), a positivecontrol using a known inoculum and nutrients, twonegative controls (one sterile, the other inoculated butnutrient-limited), and simplified oil chemical analysis.Three analysts conducted the product test independentlyin each type of exposure water in round-robin fashion.Statistical tests were performed on analyst variability,reproducibility, and repeatability, and the performanceof the various products was quantified in both exposuremedia. Analysis of variance showed that the analysterror at each time-point was highly significant (P valuesranged from 0.0001 to 0.008, depending on water typeand oil fraction). In the saltwater tests, six productsdemonstrated various degrees of biodegradative activityagainst the alkane fraction of the crude oil and three

degraded the aromatic hydrocarbons by >10%. In thefreshwater tests, eight products caused >20% loss ofalkane hydrocarbons, of which five degraded the alkanesby >50%. Only four products were able to degradepolycyclic aromatic hydrocarbons (PAHs) by >20%,one of which caused 88% removal. However, when thevariability of the analysts was taken into consideration,only one of the ten products was found to yield signifi-cant percent removals of the PAH fraction and only infreshwater. Viable microorganism population analysis(most-probable-number method) was also performed onevery sample by each operator to measure the changes inaromatic and alkane hydrocarbon-degrading organismnumbers. In general, little evidence of significant growthof either alkane- or PAH-degraders occurred among anyof the ten products in either the saltwater or freshwatertesting.

Keywords Bioremediation Æ Biodegradation Æ Oil spill ÆProduct test protocol Æ Freshwater Æ Saltwater

Introduction

Following the Exxon Valdez oil spill in March 1989, theUnited States Environmental Protection Agency (EPA)made the decision to conduct a full-scale research pro-ject to determine whether bioremediation is a viabletechnology for the cleanup of such a catastrophic spill. Alarge team of scientists was put together to design andconduct this field study [16]. At that time, productvendors began pressuring EPA and other governmentalagencies to use their products for treating the spill. As aresult of this mounting pressure, the EPA Office of Re-search and Development (ORD) was mandated in thefall of 1989 to develop and validate a protocol to test theclaims of these product vendors on the effectiveness oftheir products in biodegrading crude oil spilled intothe environment. In November 1989, EPA requestedthe National Environmental Technology Applications

J. R. Haines (&) Æ E. J. Kleiner Æ K. A. McClellan Æ A. D. VenosaUnited States Environmental Protection Agency,26 W.M.L. King Drive, Cincinnati, OH 45268, USAE-mail: haines.john@epa.govTel.: +1-513-5697446Fax: +1-513-5697105

K. M. Koran Æ E. L. HolderDepartment of Civil and Environmental Engineering,University of Cincinnati, Cincinnati, OH 45268, USA

D. W. KingStatking Consulting, 759 Wessel Drive,Suite 6, Fairfield, Ohio, USA

J Ind Microbiol Biotechnol (2005) 32: 171–185DOI 10.1007/s10295-005-0218-1

Corporation (NETAC) to assemble a panel of scientificexperts to develop screening criteria that would comparethe efficacy and environmental safety of these products.In February 1990, EPA issued a public solicitation forproposals from the bioremediation industry to provideproducts for testing the feasibility of commercial bio-remediation agents for enhancing the degradation ofweathered North Slope crude oil. Meanwhile, ORD hadproposed a laboratory screening protocol based onmeasuring the disappearance of crude oil in shake-flaskscontaining seawater, weathered North Slope crude oil,and commercial products [17]. The results of the firstgeneration testing were presented in 1993 [18]. Sub-sequent work refined and adjusted the testing protocol[20].

A tiered approach was developed from this effort [13],consisting of: (1) a base tier used to identify the presenceof chemical or biological agents that might be consid-ered unacceptable (pathogens, carcinogens, hazardoussubstances), (2) tier 1, in which a vendor provides adescription of the use of the product, its mode of action,and basic information on its toxicity, (3) tier 2, a labo-ratory batch-screening test that provides empirical evi-dence that a product is efficacious, (4) tier 3, a bench-topcontinuous-flow microcosm to simulate more closelyenvironmental conditions, and (5) tier 4, consisting ofactual field monitoring. Eventually, the final protocolconsisted of the first three tiers; and tiers 3 and 4 werenever adopted due to cost.

As more product tests were conducted throughoutthe 1990s, it became evident that refinements in the tier 2test were needed. The analytical chemistry procedure,which was based on gas chromatography/mass spec-trometry (GC/MS), was somewhat cumbersome; and thegravimetric and microbiological analyses became anunnecessary and burdensome step. Also, since the tier 2test was written exclusively for saline environments,there was no equivalent test for freshwater environ-ments. Because the test used natural seawater, it wasfound to give variable results due to the variability ofseawater, both in composition and in oil-degradernumbers. Finally, a positive control was needed as anindication that the test was performing properly.Therefore, modifications were made in the tier 2 proto-col. Such modifications included: (1) use of syntheticseawater and freshwater in separate tests, (2) incorpo-ration of a standard microbial inoculum and nutrients ina positive control, (3) incorporation of two negativecontrols (sterile control, nutrient-limited control withinoculum), and (4) streamlining of the GC/MS proce-dure. The inoculum is used in the protocol not only as apositive control but also as an inoculum to test abioticproducts such as fertilizers, enzymes, and other chemicalenhancements.

The objective of the investigation reported in thispaper was to perform the modified protocol in bothsynthetic saltwater and synthetic freshwater to deter-mine whether it screens commercial products for efficacyin the biodegradation of weathered crude oil so that they

may be listed on the National Contingency Plan (NCP)product schedule. Ten products were acquired fromvarious product vendors who were listed on the NCPproduct schedule (http://www.epa.gov/oilspill/ncp/ba-gents.htm). Three analysts conducted the tier 2 testing inround-robin fashion independently of each other, first insynthetic seawater and then in synthetic freshwater.Results were analyzed statistically for reproducibilityand repeatability and are presented in this paper. Adecision rule was established for qualifying productsthat pass the protocol.

Materials and methods

Source of inocula

Mixed cultures of microorganisms capable of degradingweathered crude oil were derived from several sources.Our laboratory collected sediment samples from marinebeaches and terrestrial locations contaminated withpetroleum products. The marine beach samples origi-nated from Alaska, Maine, Delaware, and Texas. Theorganisms for the freshwater tests were derived fromriverbank sediments, junkyard soils, or soils from man-ufactured-gas plant sites. Samples of the sediments orsoils were suspended in appropriate media with weath-ered crude oil and incubated on a shaker at 20�C for28 days. After three successive transfers, the activity ofeach mixed culture against crude oil was assessed usingthe same measurement protocol as described below.Cultures demonstrating good activity were then grownin 10-L batches for preparation of stock cultures. The10-L batch cultures were harvested by concentrating theorganisms by centrifugation, washing twice with salinesolution, and mixing with 10% glycerol as a cryopro-tectant. The concentrated cultures were dispensed into 5-mL plastic tubes and placed into a –80�C freezer forstorage. Periodically, frozen cultures were removed fromstorage and tested for oil-degrading ability. Culturesshowing good activity from terrestrial and marinesources were chosen as the positive control cultures forthis research.

Preparation of exposure water

A modified artificial seawater and freshwater were usedas the exposure media for the bioremediation producttest. Natural waters vary from location to location andseason to season. Therefore, consistency among productvendors and regulatory agencies is very difficult toachieve using natural waters. The seawater used in thisprotocol is derived from a marine aquarium formula(GP2) [15] used in zoos. The GP2 contained (per liter):21.03 g NaCl, 9.5 g MgCl2Æ6H2O, 3.52 g Na2SO4, 1.32 gCaCl2Æ2H2O, 0.61 g KCl, 0.088 g KBr, 0.034 g Na-B4O7Æ10H2O, 0.02 g SrCl2Æ6H2O, 0.17 g NaHCO3,0.05 g FeCl3Æ6H2O, 0.297 g Na5P3O10, 2.89 g KNO3.

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The synthetic freshwater formula (SFW) is a modifica-tion of Bushnell–Haas (BH) medium (Difco Laborato-ries, Ann Arbor, Mich.) and contained (per liter): 0.2 gMgSO4, 0.02 g CaCl2Æ2H2O, 0.5 g NaCl, 2.89 g KNO3,1.32 g K2HPO4, 1.00 g KH2PO4, 0.05 g FeCl3Æ6H2O.Trace minerals were not added as they have been foundto be unnecessary for microbial growth. Batches ofmedia sufficient for each product test were prepared anddispensed (100 mL) into 250-mL flasks prior to sterili-zation. The bicarbonate, phosphates, and minor com-ponents were prepared as concentrated solutions, filter-sterilized, and added to the flasks after the media hadbeen steam-sterilized.

Most probable number analysis

The populations of oil-degrading microorganisms ineach flask were estimated using a differential most-probable-number (MPN) method [20]. Microtiter 96-wellplates were used in the analysis, resulting in an 8-tube,11-dilutionMPN. One row was filled with sterile mediumas a sterility check control. Substrates used in the pro-cedure were hexadecane for alkane degraders (0.7% w/v)and a combination of phenanthrene, anthracene, fluo-rene, and dibenzothiophene (10:1:1:1) for polycyclicaromatic hydrocarbon (PAH)-degraders (0.5% w/v, finalconcentration). Positive wells for alkane-degraders werescored by observing the pink/red color change from theaddition of iodonitrotetrazolium violet (INTV) after a 2-week incubation period at 20�C. The PAH-degraderswere scored after a 3-week incubation period byrecording the brown/yellow color that develops naturallyduring PAH metabolism [5, 8, 9]. This color formation isthought to be due to the accumulation of products ofmeta-cleavage of aromatic rings [9, 14].

In thiswork, theMPNprocedurewas used as described[20] for saltwater and modified for fresh water by usingSFWmedium rather than GP2 [10]. For both alkane- andPAH-degrader enumeration, the MPN was calculatedusing a computerized enumeration program [11].

Residual hydrocarbon analysis

Following removal of 5 mL of sample from each flask forMPN analysis, a recovery surrogate solution consistingof D36-heptadecane, D50-tetracosane, D64-dotriacontane,D10-1-methylnaphthalene, D10-phenanthrene, D10-pyrene,and 5a-cholestane (androstane) in dichloromethane(DCM) was added to each reactor flask to obtain a finalsurrogate concentration of 4 ng lL-1 in the final extract.After addition of 50 mL of DCM to each shake-flask, theflasks were stirred for 10–15 min on a magnetic stirringplate. The DCM phase was passed through Na2SO4 toremove water. The DCM extracts were then exchangedinto hexane under a stream of dry nitrogen. The solventexchange was performed three times. The hexane extractswere prepared for GC/MS analysis [19] by adding an

internal standard mixture of deuterated alkane andaromatic hydrocarbons to yield a final concentration ofeach standard of 10 ng lL-1. Concentrations of hydro-carbons in the extract were quantified using a HewlettPackard 6890 series GC with a 5973 MSD-MS asdetector, operating in selected ion-monitoring (SIM)mode. The analytes included 28 alkanes ranging in car-bon number from nC10 to nC35, plus pristane, phytane,hopane, and 32 PAHs, consisting of 2-ring, 3-ring, and 4-ring PAHs (naphthalenes, phenanthrenes, dibenzothi-ophenes, fluorenes, naphthobenzothiophenes, pyrenes,chrysenes) and their alkylated homologues. The columnwas a DB5 column (30 m long, 0.25 mm ID, 0.2 lm li-quid phase; Supelco, Bellefonte, Pa.). Alkane and PAHconcentrations were summed to obtain the total alkaneand total PAH concentrations in each flask. The percentremaining at each sampling event was determined rela-tive to the concentrations of alkanes and PAHs in flaskssacrificed at time 0. The chemical analysis procedure gi-ven in the Federal Register [6] is more complex with morepossibilities for error. The latter procedure differs fromthe one used in this research in that sample transfer to aseparatory funnel is required as opposed to extraction inthe incubation flask, a Kuderna-Danish concentrator isused to concentrate the analytes to quantifiable amounts,and the analytes listed include pyrogenic hydrocarbons,which were excluded from this research. In our experi-ence, the extraction can be performed directly in theculture flask, solvent exchange can be performed in thesample storage vial, and the analytes can be changedwithout sacrificing the reliability of the method. Naph-thobenzothiophenes and C3 and C4 chrysenes were ad-ded to the analysis and the 5-ring and 6-ring pyrogenicPAH compounds were eliminated. The pyrogenic PAHcompounds, found in low concentrations in crude oil areof little importance in quantifying large changes in oilconcentration. Hopane as a biomarker is useful in esti-mating physical losses of oil in open systems, but nor-malization to hopane is not necessary for closed systemswhere abiotic loss mechanisms are minimal.

Test procedure

Ten commercial bioremediation products were selectedfor this work. Commercial product manufacturers wereadvised that products were to be tested in both fresh-water and saltwater systems. Table 1 lists the types ofproducts and their assigned letter designations. Productsare listed by letter to preserve confidentiality. Manu-facturers of each product provided the product sampleand instructions for use. The manufacturers specifiedwhether the product required additional nutrients orinoculum. The appropriate additions were made to test-flasks at the beginning of each test. The manufacturerswere: Acorn Biotechnical Corp., B&S Research, Bio-NutraTech, Elf Aquitaine, Enviro Zyme, Land and SeaRestoration LLC, Marine Systems, Oppenheimer Bio-technology, PetroRem, and Waste Microbes. Products

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identified by letter in the results are not in alphabeticalorder by manufacturer. Tests were run independently bythree different analysts to simulate a round-robin orinter-laboratory comparison type of process. All pro-ducts and controls were run concurrently in a singlemedium by a single operator. One set was completedbefore another was started.

The experimental design for this study included onepositive and two negative controls and ten producttreatments, all in triplicate. Products that were powdersor dry materials were weighed into small sterile test-tubesto provide the proper concentration for each flask. Li-quid products were dispensed on the day the test wasstarted. If a product supplied nutrients, the exposurewater formula was modified by removing nitrogen.Products were supplemented with either nutrients orinoculum only at the specification of the manufacturer.Microbial products could have nutrients added. Fertil-izer products could have inoculum added. Some requiredneither nutrients nor inoculum. Erlenmeyer flasks(250 mL) were filled with 100 mL of SFW or GP2, 0.5 gof weathered Alaska North Slope crude oil (ANS 521,previously heated to 272�C in vacuo to remove the light-end hydrocarbons), and product per the manufacturers’recommended concentrations. Each flask was inoculatedwith 1.0 mL of enriched culture (marine or freshwater)unless the product was a microbial product. Positivecontrols received 1.0 mL of culture, 0.5 g of ANS 521,and nutrients in 100 mL of exposure water. Negativecontrol 1 (the sterile control) consisted of flasks withexposure water containing the nutrients specified and0.5 g of ANS 521 but no inoculum. Negative control 2consisted of 1 mL of inoculum, 0.5 g of ANS 521, andexposure water lacking nitrogen. Flasks were incubatedat 20�C on an orbital shaker table rotating at 200 rpm.Triplicate shake flasks for each treatment were sacrificedon days 0, 7, 14, 21, and 28 of incubation and analyzedfor microbial number by MPN and residual oil, as de-scribed above.

Statistical analysis

An overall analysis of variance (ANOVA) was con-ducted in each time-period. The ANOVA model in-

cluded product, analyst, and product-by-analystinteraction. Then the least squares product from theANOVA (adjusted for analyst and interaction) werecompared with the negative control without inoculum.The seawater and freshwater analyses were conductedseparately. The purpose of a round-robin multipleanalyst evaluation of a method is to enable a regula-tory group to formulate a precision statement about amethod. The precision of a method is a function oftwo types of variability: the variances in reproducibil-ity (R1) and repeatability (R2). The repeatability vari-ance represents the within-laboratory variability, i.e.,the variability of the method when carried out onidentical samples in the same laboratory by the sameanalyst. The reproducibility variance represents thesum of within-laboratory and among-laboratory vari-ability, i.e., the variability of the method when con-ducted on identical samples in two or morelaboratories by two or more analysts. One goal of thisstudy was to estimate the repeatability and reproduc-ibility standard deviations and the correspondingrepeatability and reproducibility coefficients for thelaboratory method to be used to obtain biodegrad-ability measurements from various commercial prod-ucts by various analysts. To obtain the repeatabilityand reproducibility parameters, ASTM method E691[2] was used and SAS served as the software programfor making the calculations.

Results

Residual oil analysis

Figures 1, 2 summarize the alkane and aromatichydrocarbon degradation in both SFW and GP2; andFig. 3 shows the three controls. The data from all threeanalysts were compiled and averaged for the figures. Thealkane data are plotted against the left axis and thearomatic data are plotted against the right axis. Of theten products tested in SFW, nine demonstrated statisti-cally significant degradation of the total alkane hydro-carbons analyzed at day 28, with P values ranging from0.044 for product H to P<0.0002 for all other productsand the positive control. Fewer products demonstratedstatistically significant degradation of aromatic hydro-carbons in SFW. Products C, E, H, I, and the positivecontrol caused statistically significant degradation ofaromatics (P<0.0001) at day 28. Table 2 shows thepercent loss of aliphatic and aromatic hydrocarbons inSFW and GP2.

In GP2 significant alkane degradation (P<0.033–0.0001) occurred with six of the ten products after28 days of incubation (A, C, E, G, H, I). Virtuallycomplete biodegradation occurred in the positive controlwithin 7 days. As shown in Figs. 1, 2, some of theproducts exhibited degradation earlier in the incubationperiod, but the most extensive degradation was observedat 28 days.

Table 1 Product type and letter designation

Letter designation Product type

A Bacteria/enzymeB Biological additiveC Oleophilic fertilizerD Microorganisms with nutrientsE Microorganisms with nutrientsF Biological additiveG Microorganisms with nutrientsH Sorbent with microorganismsI Oleophilic fertilizerJ Microorganisms

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Degradation of aromatic hydrocarbons in GP2 wasunimpressive. Only three products (C, E, I) caused sta-tistically significant reduction of PAHs (P<0.0032–0.0004), although in absolute terms these reductionswere only about 15–17%. In contrast, approximately86% reduction of PAHs occurred at day 28 in the po-sitive control (P=0.0001; Fig. 3). These results weresurprising, because the products tested were originallyintended for use in a saltwater environment. Yet, betterdegradation of PAHs occurred in SFW.

MPN analysis

Figures 4, 5, 6 summarize the alkane-degrader MPNdata for the ten products in both exposure waters, and

the three controls. The inoculum added to the abioticproducts (C, F, I) and the control flasks containedapproximately 104–106 cells mL-1 alkane-degradingorganisms at time 0 in freshwater and ca. 107 cells mL-1

in the saltwater medium. Flasks with products A, B, D,E, G, H, and J were not inoculated because they con-tained their own microbial source. The ability of thesecultures to grow on ANS521 in the two exposure waterswas highly variable. Some grew better in saltwater thanin freshwater, some did the opposite, and still othersgrew poorly or moderately in both. The positive controlin SFW (Fig. 6) grew by two orders of magnitude byday 7 and then remained steady for the remainder of theincubation. The positive control culture derived from amarine source did not show the same growth pattern.

Fig. 1 Alkane (solid lines) andaromatic (broken lines)hydrocarbon degradation inflask test. A–E designateproducts

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Figures 7, 8, 9 depict the MPN estimates for PAH-degrading organisms from both freshwater and seawatertests and the controls at each sampling event. Theinoculum added to the abiotic products (C, F, I) and thecontrol flasks contained approximately 103–104 cells mL-1 PAH-degrading organisms at time 0. Fewof the products tested demonstrated significant growthof aromatic degraders in either water type. The MPNs inproduct C increased approximately two orders of mag-nitude in SFW; and product H increased approximatelyfive orders of magnitude. Product F showed no changein MPN over the course of the test. Growth in GP2 wasmuch less.

Reproducibility and repeatability

Table 3 summarizes the average R1 and R2 standarddeviations derived from the ten-product test. The within-analyst error (R2) should always be lower than theamong-analyst error (R1). In compiled data the R2

standard deviation was slightly lower than the R1 stan-dard deviation, although R1 and R2 standard deviationswere of similar magnitude. For individual products, thiswas not always the case (data not shown). Also, thevariability at day 28 was higher than at day 0 whenviewed as a percentage of the corresponding means. Thisis not surprising, since more variation is expected as

Fig. 2 Degradation of alkane(solid lines) and aromatic(broken lines) hydrocarbons inthe flask test. F–J designateproducts

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concentrations of hydrocarbons decline with time. Incases where the R2 error was greater than or similar tothe R1 error, this signifies that it is somewhat difficult foran individual analyst to get precisely the same result forall three replicates. Biological variability is relativelyhigh, especially as biodegradation takes place. For thisreason, a pass/fail decision rule (discussed below) mustaccount for the inherent variability expected from theprotocol.

Discussion

This research was undertaken to determine whether themodified protocol was an adequate test of the ability ofcommercial products to demonstrate successful biodeg-radation of crude oil hydrocarbons. Two types ofexposure water were used for these tests, synthetic salt-water and freshwater, and results indicated large differ-

ences between the two inocula originating from eachsource. This suggests that commercial products shouldbe screened in both types of environments if the productvendor wishes his product to be used in both marine andfreshwater spill responses. This agrees with the findingsof Blenkinsopp et al. [4], who also used two differentexposure waters for testing bioremediation products.

In the modified procedures, the exposure waters werechemically defined, a positive control was added, aninoculum was used for testing abiotic products, andresidual oil analysis was simplified. Using synthetic wa-ters eliminates the need to rely on natural waters toprovide the oil degraders for testing abiotic productssuch as new fertilizers or other chemical stimulants. If anabiotic product is able to stimulate the inoculum todegrade the oil, this procedure should be able toaccommodate that activity reproducibly. Chemicalanalysis was simplified by reducing the number ofsample-transfers and the solvent-exchange procedure.

Fig. 3 Alkane (solid lines) andaromatic (broken lines)hydrocarbon degradation inflask-test controls. I Innoculum,N nitrogen, Neg negative, Pospositive

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The number of analytes was changed to focus on themore prevalent components of crude oil. These modifi-cations, which were consistent with the Canadian pro-

tocol [4, 7], were developed to improve thereproducibility and repeatability of the protocol. Resultsdemonstrated that, of the three abiotic products tested in

Table 2 Loss of hydrocarboncomponents over 28 days Product or treatment Freshwater Seawater

Alkane loss (%) Aromatic loss (%) Alkane loss (%) Aromatic loss (%)

Positive control 97.6 39.5 99.3 86.3Negative control 1 4.0 5.4 2.9 7.5Negative control 2 2.1 4.0 9.7 9.2A 41.4 2.5 22.0 0.5 IncreaseB 9.1 0.2 7.4 3.0C 68.4 29.0 53.9 17.2D 57.4 3.3 7.7 8.2E 75.1 22.5 46.2 16.7F 0.4 2.9 5.9 3.5G 27.9 6.6 24.0 0.2H 21.3 23.0 21.6 3.5I 98.4 88.2 67.3 14.6J 71.3 4.4 17.4 0.03 Increase

Fig. 4 MPN of alkane-degrading organisms. A–Edesignate products

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both GP2 and SFW, two (products C, I) significantlydegraded both the aliphatics and the aromatics in bothexposure waters, whereas the other (product F) causedno biodegradation activity in either medium. Althoughstatistically significant differences from the negativecontrols were observed for products C and I, the abso-lute degradation compared with the positive control wasmuch lower in seawater. This was not true for product Iin freshwater, which was able to achieve much betterdegradation of the PAH fraction than the positive con-trol. These findings suggest that product C did notsupply enough nitrogen and phosphorus to even ap-proach the same level of degradation that the positivecontrol did. Product I, however, did supply sufficientnutrients, but this stimulation occurred only in fresh-water. Since the positive control cultures for freshwaterand saltwater were from different sources, it is unclear

why this product was unable to elicit the same stimula-tion for both cultures. One of the three abiotic products(product F) contained a substantial concentration ofalkane hydrocarbons present in the product itself; andno degradation of either oil fraction was observed withthat product.

An additional experiment with six of the ten products(A, B, C, D, H, I) was repeated later in GP2 with fivetimes the product manufacturers’ recommended dosage.Table 4 shows the results of the higher concentrationtests. Products A, B, and D did not show anyimprovement in the degradation of hydrocarbons at ahigher dose. The positive control elicited virtually iden-tical results to the earlier tests performed. Products C,H, and I showed improved PAH degradation. Theproducts were oleophilic fertilizers and one sorbent withorganisms. This suggests that the nutrient products were

Fig. 5 MPN of alkane-degrading organisms. F–Jdesignate products

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nitrogen-limited and/or phosphorus-limited in the ori-ginal tests. The manufacturers recommended a dose ratethat may be inadequate for a batch-type test with nocontinuous input from the environment, such as wouldhappen in an open beach setting.

With respect to the seven products containingmicrobial cultures, only product E was able to signifi-cantly biodegrade both oil fractions in both exposurewaters. The other biotic products partially degraded thealiphatic fraction in one or other exposure water, butnone of them degraded the PAH fraction significantly.This was despite the fact that they had the same nutrientconcentration as the positive control. This suggests thatthe cultures in the biotic products were not effective inmetabolizing the oil fractions within the 28-day periodof the test. The positive control in saline GP2 was muchmore effective in metabolizing the PAH fraction than theSFW positive control. Average degradation in the lattercase was 39.5%, whereas average degradation in GP2was 86.3%. These results contrast with those of Blen-kinsopp et al. [4], whose cultures showed that muchbetter biodegradation occurred in synthetic freshwaterthan in marine water.

Laboratory-scale evaluations of bioremediationproducts have been performed in the past. However, thetesting protocols have not been standardized, leading to

an inability to compare results directly. Aldrett et al. [1]tested 13 products using a protocol similar to the EPA,and four of those products performed as well as or betterthan the nutrient controls, which achieved approxi-mately 80% removal of alkanes and 35% removal ofaromatic hydrocarbons. The four products reduced ali-phatics of chain length less than C31 by more than 90%.Removal of C31–C35 alkanes ranged from 31% to 90%.Pristane and phytane were completely removed by twoproducts and were 73% and 89% removed by the othertwo products. Removal of two-ring aromatics rangedfrom 30% to 93%, three-ring aromatics from 13% to77%, and four-ring aromatics from 11% to 28%. Theauthors observed unpredictable variability in the naturalinoculum, which was a limitation to accurate andreproducible assessment of commercial products. Nera-lla and Weaver [12] tested ten commercial products insalt marsh microcosms over a 90-day period at twotemperatures (10�C, 30�C). A 1-g amount of oil wasadded to a 1-cm layer of water covering salt marshsediment and biodegradation was monitored with andwithout nutrient addition. After 90 days, only oneproduct enhanced oil degradation without added nutri-ents at 10�C. With added nutrients, nine of ten productsenhanced degradation at 10�C, compared with the fer-tilized control. At 30�C, seven of ten products enhanced

Fig. 6 MPN of alkane-degrading organisms in controlflasks

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the biodegradation of total petroleum hydrocarbons(TPH) after 90 days without nutrients, whereas none ofthe products in the presence of nutrients was able todegrade TPH better than the nutrients alone. Fochtet al. [7] developed a defined bacterial consortium of sixbacteria for use as a standard inoculum for testingfreshwater oil-spill bioremediation agents. This consor-tium was able to degrade both the alkane and aromaticfractions of crude oil reproducibly and predictably.Similar to the inoculum used in our study, it was notintended to be a super-concoction of oil degradersagainst which all commercial products would be com-pared. Rather, it was used as a quality control bench-mark for product testing and as a reproducible inoculumfor testing non-living biostimulation products.

Bachoon et al. [3] examined microbial communitydynamics in salt marsh microcosms treated with oil andtwo bioremediation products. The products were abacterial culture plus nutrients plus an activator solutionand a dispersant. The treatments were oil plus sediment,oil plus nutrients plus sediment, and oil plus the twoproduct treatments at the manufacturer’s dosage. The

results showed that nutrient formulas were most effec-tive in stimulating population growth, as measured byDNA content and oil degradation. Oil degradation withthe nutrient treatment was greater than 90% removal ofalkanes and a significant reduction of aromatics, com-pared with the control or either product. The bacterialproduct with nutrients showed greater than 90%reduction of alkanes C19 and above. Pristane and phy-tane were reduced to about 76% of the control. Aro-matics were not significantly reduced. Plate counts andMPN estimates of hydrocarbon-degrading populationswere of limited utility.

In previous work conducted by our laboratory, Ha-ines et al. [10] proposed a freshwater protocol for testingbioremediation products to include performance targets,a positive control, and a defined nutrient formula. Fourproducts were tested and two were able to degrade>90% alkane and >60% aromatic hydrocarbonswithin 28 days. The Canadian freshwater protocol [4, 7]differs in the incubation period (14 days), inoculum size,and oil content. In our opinion, a longer incubationperiod, 28 days, allows more time for the growth of

Fig. 7 MPN of PAH-degraders. A–E designateproducts

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organisms that may be slow to metabolize some oilcompounds, especially PAHs. A smaller inoculum in theorder of 103–104 cells mL-1 rather than 106 cells mL-1 issomewhat more typical of what may be found in nature.In our research, the inoculum contained about 103–104 PAH-degraders mL-1 and about 106 alkane-degraders mL-1. The response of an abiotic productwithout its own inoculum should reflect actual useconditions. In regards to the method of analyzinghydrocarbon degradation in the protocol, the Canadianprotocol [4, 7] uses GC with flame ionization detection(FID) to measureme the aliphatic, aromatic, and totalpetroleum hydrocarbons and uses GC/MS-SIM for thetarget aliphatic and aromatic analytes. Our laboratoryproposes using only GC/MS, because GC/MS is able toresolve and quantify the alkylated PAHs that GC-FIDcannot. We also recommend only two sampling times,day 0 and day 28. The intermediate sampling events arenot necessary to establish efficacy. The initial oil contentin our protocol is 5 g L-1. This is high enough to providea good distinction between starting analyte concentra-tions and final analyte concentrations, especially withregard to aromatic hydrocarbons. The saltwater and

freshwater exposure media are defined formulas, to en-able interlaboratory comparison and to limit inoculumvariability that would occur if natural water were used.Positive and negative controls must be included to en-sure the procedures are working properly and thatmicrobial contamination does not occur.

One of the objectives of this investigation was toestablish quantitative decision rules that a product mustbe able to meet to qualify for use in an oil spill. Ourfreshwater positive control culture yielded 97.6% alkaneand 39.5% aromatic hydrocarbon degradation in28 days, while the saltwater culture yielded 99.3% al-kane and 86.3% aromatic hydrocarbon degradation.Since the entire purpose of listing products on the NCPproduct schedule is to have a readily available source ofeffective bioremediation agents that responders can useto clean up an oil spill, it is reasonable to set a minimumtarget aliphatic and aromatic concentration level that aproduct must be able to meet in order to pass the testand therefore qualify for NCP listing. It is recognizedthat the higher-molecular-weight compounds in both oilfractions are more difficult and therefore slower to de-grade than the lower-weight compounds. It is also rec-

Fig. 8 MPN of PAH-degraders. F–J designateproducts

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ognized that the 60+ target compounds measured bythe GC/MS-SIM method represent only a minor frac-tion of the total hydrocarbons present in crude oil.Nonetheless, quantification of those compounds is thebest we can do at the present time to infer that biodeg-radation is taking place. Most of the products tested inthis research did not degrade the PAH fraction much atall, even though most products degraded the alkanefraction by a significant amount. Since PAHs are themost toxic compounds in oil, a product must show someevidence that it is able to degrade PAHs within the 28-day time-frame of the protocol. Since our two positivecontrol cultures are known to degrade PAHs, and since

the poorer performing culture was the freshwater inoc-ulum, it is reasonable to set a minimum percent PAHreduction level at 39% after 28 days for both thefreshwater and saltwater tests.

Recognizing that the data from this protocol can behighly variable, it is important to incorporate anaccounting of the variability when establishing a targetdecision rule. We propose this be done as follows. First,calculate the mean and standard deviation of the totalaliphatic and total aromatic concentrations from thethree independent replicates at day 0 and day 28. Then,from those data, calculate the upper 90% confidencelevel (UCL90) at day 28 using the following formula:

Fig. 9 MPN of PAH-degradersin control flasks

Table 3 Reproducibility andrepeatability standarddeviations

Water type Oil fraction Day 0 Day 28

Mean R1 R2 Mean R1 R2

Freshwater Alkane 49,306 2,703 2,112 28,749 6,333 4,730PAH 18,147 1,505 796 14,934 1,605 900

Saltwater Alkane 59,266 4,371 2,343 42,560 5,188 4,893PAH 17,517 1,237 1,027 15,157 1,644 1,456

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UCL90 ¼ �xþ t0:90;2df � rffiffiffi

np

� �

ð1Þ

where �x is the total alkane or total PAH mean of threereplicates on day 28, t0.90,2df is the 90% 1-tailed t valuewith two degrees of freedom (1.886), r is the standarddeviation of the three replicates on day 28, and n is thenumber of replicates (three). Finally, calculate the per-centage reduction of each oil fraction from day 0 today 28, using the day 28 UCL90 value instead of theday 28 mean.

Using the above procedure, the freshwater positivecontrol percent-reduction values for the aliphatic andaromatic fractions were calculated to be 96.6% and23.6%, respectively, compared with 97.6% and 39.5%based simply on the means. For the saltwater tests, thepositive control values for the aliphatic and aromaticfractions were 98.9% and 79.8%, respectively, comparedwith values of 99.3% and 86.3%, respectively, based onthe means. Since the freshwater tests gave the poorerperformance of the two tests, setting a target where theUCL90 has to meet a percent reduction of >90% foraliphatics and >20% for aromatics would be a rea-sonable requirement for passing the test. Using thesecriteria, only one of the ten products in these tests wouldpass the protocol in freshwater (product I) and nonewould pass in saltwater.

The above data are similar to the requirementsestablished in Canada [4], which are 35% reduction forTPH, 30% for total aliphatics, and 10% for totalaromatics. The 30% reduction in total saturates(including all resolvable and unresolvable but GC-detectable aliphatics) is equivalent to roughly 80%reduction in total GC/MS-resolvable target n-alkanes,according to the Canadian data; and the 10% reductionin total aromatics is equivalent to an approximately50% reduction of the 5-PAH homologue group, con-sisting of naphthalene, fluorene, dibenzothiophene,phenanthrene, chrysene, and their alkylated homo-logues. Our PAH series includes two other four-ringPAHs in addition to those five PAH series consideredby Blenkinsopp et al. [4]. Thus, the United States andCanadian protocols are quite similar. Before the UnitedStates EPA protocol is adopted, it must go through

public comment and be approved by EPA headquar-ters. This will be pursued in 2005.

References

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Table 4 Hydrocarbon reduction with products at five-fold higherconcentrations in seawater

Product or treatment Alkane loss(%)

Aromaticloss (%)

Positive control 99.6 90.6Negative control(innoculum, without nitrogen)

18.3 11.1

A 6.5 9.2B 8.9 8.7C 97.1 35.0D 4.8 1.7H 29.3 11.8I 68.6 19.8

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