Toxicological evaluation of hydrocarbon removal from soils by an extraction and solubilization remediation process

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Spill Science & Technology Bulletin, Vol. 4, No. 3, pp. 147-154, 1997 © 1998 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1353-2561/98 $19.00+ 0.110

Pergamon

PII: S1353-2561 (98)00011-5

Toxicological Evaluation of Hydrocarbon Removal from Soils by an Extraction and Solubilization Remediation Process SEAN P. O'CONNELL*, R. MICHAEL LEHMAN & GREGORY A. BALA Idaho National Engineering and Environmental Laboratory, PO Box 1625, Idaho Falls, 1D 83415-2203, USA

Toxicity analyses were performed on manufactured frac sands and construction fill material to assess the efficacy of a pilot scale treatment system designed to remediate environments impacted with organic contaminants. Oiled proppants and backfill were collected from the North Slope in Alaska while unadulterated fill material from the Idaho National Engineering Laboratory (INEL) was collected and then contaminated with crude oil. Two commercial bioassays (Microtox, Polytox) were used to provide toxicological data for process waters, and treated and untreated solids from the soils treatment system. Total extractable hydrocarbon analyses were performed on treated and untreated solids, and indicated removal efficiencies greater than 95% for all samples tested. Microtox testing using sonicated extracts from solids and whole water samples provided the most sensitive and useful toxicological data, showing the soil treatment system to significantly reduce the toxicity of treated solids. © 1998 Elsevier Science Ltd. All rights reserved. Keywords: toxicity, hydrocarbons, Microtox, soil, remediation

Introduction Crude oil exploration, production, refining and transportation can result in negative environmental impact. Many Superfund, Department of Energy (DOE), Department of Defense (DoD), and energy exploration and recovery lands are contaminated with residual petroleum products. Ultimately, hydrocarbon contaminants may be degraded by microorganisms, weathered and evaporated in a complex ecological process that generally spans a long period. There exists a need for remediating such sites to capture lost oil, while returning the impacted area to a natural state by hastening the removal of pollutants (Raghaven et al., 1990). In remediation of soils, particular attention must be paid to the kind of system that will work best for a *Author to whom correspondence should be addressed. (Tel: (208)526-5587: Fax: 001 208 526 0828; E-mail: spo~ inel.gov)

certain sample type. Soils can be bioremediated (composting, landfarming) by naturally occurring bacteria in soil, which can aid in the removal of a desired pollutant (Atlas, 1977; Fredrickson et al., 1993; Lapinskas, 1989). Climate may be a chief factor, as dry or cold environments can make in situ bioremediation difficult because of the task of maintaining microbial activity high enough over an extended time period. Ex situ methodologies, such as thermal treatment, soil washing and solvent extraction, are best suited for handling smaller volumes of material; and in climates where rates of bioremediation are limited, these techniques may be the best alternative at the present. Since clays and silts are inherently more difficult to treat than sands and gravels because of their small size, greater surface-to-volume ratio and charged surfaces, ex situ treatment should accommodate the variety in grain size. 147

s. P. O'CONNELLet al. The purpose of this study was to test the ability of a new soil treatment technology to reduce toxicity of petroleum hydrocarbon contaminated soils (U.S. Patents #5,454,878 and 5,490,531). The remediation scheme is based on biochemical solvation (terpenes), materials classification (size fractionation), liquid/ liquid and liquid/solid extractions, mechanical and hydraulic scrubbing, phase separation of organic and aqueous fluids, isolation of the contaminants from the organic phase and recycling of all fluids. Toxicological analyses of water and sediment samples are usually quantified by measurements of individual contaminants or by their effects on commercially grown organisms (Microtox, Polytox). Wastewater effluent and polluted waterways and soils are generally tested with standard toxicity protocols. In this study, the efficacy of a soil treatment scheme in reducing toxicity of cleansed materials was tested using Microtox, a single organism test that relies on lumincscence, and Polytox, which tests for community respiration. Microbial tests were chosen because of

their ease of application and the reproducibility of results. Toxicity data sets compared with residual total extractable hydrocarbons (TEH) verified that hydrocarbon reduction resulted in toxicity reduction.

Methods and materials Treatment technology An extractive counter current solids treatment process that utilizes a combination of aqueous treatment and solvent extraction in a materials classification system was used to ~clean hydrocarboncontaminated soils. Solvation was accomplished with controlled, defined terpene chemistry (Limonene DL in this study). An engineering scale treatment plant that was designed to accommodate 10 kg/min of soil was used (Fig. 1). Contaminated soils and terpenes were mixed and placed into a feed hopper and then fed from the hopper into a trommel-based 'revolving screen deck',

Fig. 1 Remediation device used to treat hydrocarboncontaminated solids through a process that utilizes terpenes as an extraction and solubilization agent. 148

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T O X I C I T Y T E S T I N G O F C R U D E O I L R E M E D I A T I O N IN SOILS

by a rotating screw that mechanically agitated the sample. Hydraulic, mechanical and chemical energies increased contaminant removal in the trommel. Solid, wetted material was moved through the trommel to maximize contact time with solvents. Lifters were incorporated in the design to agitate and turn' the solids in the trommel for even and complete contact with fluids and separatory screens. The screens were designed as removable elements to allow for replacement with screens of larger or smaller diameters, so that size classification of materials could be controlled, and to provide adequate liquid drainage. Treated coarse oversize materials exited the end of the trommel. Treated fine particles and fluids were diverted to a series of other systems to accomplish liquid/solid and liquid/liquid separations. Pretreatmerit and post-treatment water and solid samples (including treated, fine solids from a press filter) were collected for analyses (Polytox, Microtox, TEH). Soils r u n in the treatment system

The materials treated included artificial materials as well as natural clay, silt, sand and gravel mixtures that were contaminated with crude oil (Table 1). Frac sands (proppants), homogeneously-shaped and -sized

1:1,'~

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spherical particles that were used to maintain fracture-flow capacity after completion of a hydraulic fracturing treatment, were run through the system in two separate tests (Proppants 1 and 2). Fill materials that originated from the North Slope of Alaska (Reserve Pit) consisted of fine, sandy particles, pebbles, coarse-sized rocks and some artificial proppants. IRC Fill consisted of quarry clays, sands and pebbles, but included no frac sands. Propparlts were fed at a full design capacity of 10kg/min. Initial contaminant loading of the proppants was 50,000 ppm TEH. Total extractable hydrocarbon analyses were performed using EPA 3540 for extraction and EPA method 8000 for quantification (US EPA 1994). Reserve Pit material was fed at a rate of 1.6 kg/min. Solids that entered the process had a hydrocarbon residual level of up to 15,000 ppm. Contaminated and uncontaminated IRC fill materials were fed al a rate of 4.0 kg/min (Table 2). Process waters were analyzed on-line with a Grant YSI 3800 Water Quality Sonde. Unlike the other samples tested, the IRC solids had not been exposed to crude oil upon collection. IRC Fill Experimental, was mixed with crude oil to achieve a final oil concentration of 2.6%, and a further sample was not treated with oil (IRC Fill

Table I Particle size fractionation for Proppant, Reserve Pit, and IRC Fill materials run in soil treatment system (as determined by sieve screen analysis) Proppants

Reserve Pit

IRC Fill

Size (llm)

%Total

Size (/tm)

%Total

Size (llm)

%Total

10110-2000 0-50

- 95.00 - 5.00

6350.00 1191.26 419.1(I 210.82 149.86 73.66 38.111 (1

34.43 26.80 9.91 14.94 4.1t0 4.88 3.25 0.95

2360.00 850.00 300.00 212.00 150.00 106.00 90.00 53.00 0

76.38 10.60 4.84 2.(11 2.89 1.45 5.88 11.91 11.34

Table 2

Soil treatment parameters including beginning and ending total extractable hydrocarbons (TEH) in ppm, soil sizes in lira, soil feed rate in kg/min, mass of solids treated in kg, and overall T E H reduction expressed as percentages Sample

Beginning T E H

Sample size

Ending T E H

Feed rate

Soil mass treated

T E H reduction

Proppant 1

50,000

10.(1

50J)110

Reserve Pit

15.0011

144.8 NT ~ 172.0 2113.7 18.6 293.5 11.7 1.1 155.1 45.9

1(1.0

Proppant 2

>500 < 5011 >500 <500 >500 < 500 >500 < 500 > 5011 < 500

99.71 NAh 99.65 95.77 99.87 98.04 NA NA 99.38 99.81

IRC Fill Control IRC Fill Experimental

0 25,000

10.11

7.76

1.6

8.98

4.0

1.11

4.(I

3.56

~'No test: hnot applicable.

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S. P. O'CONNELL et al.

Control). The experimental material was contaminated with a moderate weight of unweathered crude oil (25.4 ° API), which had been previously determined to contain 62.68% aliphatics, 22.53% aromatics, 6.38% pentane soluble resins and 6.4% non-pentane soluble resins (asphaltenes). Elemental analysis showed 82.3% carbon, 11.6% hydrogen, 0.35% nitrogen and 3.24% sulfur. All samples were treated in the soil treatment system with a few changes in sample processing between the tests, as described below. System parameters were adjusted after each run to increase the effectiveness of organic recovery and the cleaning of fine particles. The final two runs (IRC Fill) had the cumulative effect of improving process efficacy, produced paired contaminated and uncontaminated samples, and used fresh, domestic quality non-toxic water to start the process. No controls were used in the other three samples tested because pristine forms of the samples were not available.

Toxicity testing Various start and end points of solids and liquids were sampled from the process (Fig. 2). These

samples included initial water samples taken from a storage tank before they were introduced to the system and water samples taken at the end of the process. The end-samples were collected from the effluent of the system - - except for the Proppant 1 run where additional samples were taken from the end of the trommel at the same time as the coarse particles exited the system. Solid samples included untreated solids - - samples taken before addition of terpenes with no particle size differentiation - - and treated coarse and treated fine solids. Treated coarse solids were samples taken from the end of the trommel as they exited the system. Treated fine samples were collected from a fine filter trap. For some runs, both treated sample types were allowed to air dry and then tested using Microtox and Polytox. Triplicate samples were collected for all analyses (except Proppant 1 trommel water for which there were two samples) using I-CHEM certified glass containers (Fisher Scientific). For analysis of samples, either the Microtox Basic or 100% assay was used depending upon the toxicity of the sample (Microbics, 1992). The 100% test was used only when the toxicity of a sample was very low.

Fine Solids

Contaminated 8ample

~

System for Materials I Trommel HandlingandClassification

I I l

Aqueous Phase

Organic Phase

T I DistillationorDestructionor Fuel Blending Prooess

/

ConcentratedOrganics Fig. 2 Schematic diagram of process and sampling points where water and solids were removed for Microtox analysis. 'Solids' represents the point of coarse sample collection, 'fine solids' represents the point of fine solids collection, while water was removed from the 'water recycle tank' after being treated in the system for terpene and hydrocarbon removal. Untreated solids and beginning water samples were collected before introduction to the system. 150

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TOXICITY TESTING OF CRUDE OIL REMEDIATION IN SOILS Process waters were tested for toxicity by exposing the Microtox organism and Polytox consortium to undiluted waters. For Microtox soil toxicity testing, 0.1 g untreated and treated solids were sonicated in 10 ml 2% NaCI diluent for 15rain in a Branson 3200 sonicator (Branson, Inc.). Because of the use of freshly contaminated soils, the IRC Fill samples were sonicated in 10% dimethyl sulfoxide (DMSO) to solubilize the oil. This solvent sonication process was necessary because of the unweathered state of the oil. The extraction agent, DMSO, solubilizes hydrocarbons and is less toxic to the Microtox organism than alternative solvents (Campbell et al., 1992; Schiewe et al., 1985; K. Dowe, Microbics, personal communication). The resulting 1% sonicated solid extractants were used to gauge toxicity to the Microtox bacteria. The Microtox solid-phase protocol was initiated for some solids (treated fine Proppant-2 solids). It was incompatible with the samples from the treatment process because of particle sizes that were too large to be evenly suspended in solution, and that made a dilution scheme impossible. A Microtox toxicity analyzer model 2055 (Brinkmann Instruments, Inc.) was used to quantify light output by the Microtox cultures, and data were reduced using Microtox Reduction Software 7.11 (Microbics, Inc.). The data were expressed as ECs0: the effective concentration of a toxic substance at which light output was diminished in the Photobacterium phosphoreum organism by 50%. The lower the ECs0, the higher the toxicity of the substance.

Results After treatment, large proppant particles ( > 500/zm) had total extractable hydrocarbon residues of 172 ppm and 144.8 ppm (runs one and two, respectively); large Reserve Pit particles ( > 5 0 0 / ~ m ) had a total extractable hydrocarbon residue of 18.6 ppm; and fine particles ( < 5 0 0 # m ) had a residue of 293.5 ppm (Table 2). Water T E H analysis values were no greater than those observed for the proppants. IRC Fill uncontaminated control material showed a hydrocarbon residue of 11.7 ppm for the large material ( > 500/~m) and 1.1 ppm for the small material. Treatment of contaminated fill resulted in a residual of 155.1 ppm for the large particles and 45.9 ppm for the small particles. Water analysis at the beginning of the first proppant run indicated a pI-I of 8.06, a temperature of 21.4°C, a conductivity of 0.418 mS/cm 2, salinity of 0.2%, redox potential of 247 mV and a turbidity of 1 NTU. Water analysis at the end of the run showed a pH of 8.28, a temperature of 22.7°C, a conductivity Spill Science & Technology Bulletin 4(3)

of 0.426 mS/cm 2, salinity of 0.2%, redox potential of 194mV and a turbidity of 12 NTU. Turbidity was determined to be a function of sonde placement and was believed to be a result of air bubbles within the system. Water analysis from subsequent treatments had similar values to those observed for the proppants. Toxicity of post-treatment water for all runs was generally greater than that in the initial water (Fig. 3a). In only two cases did the end water samples not increase in toxicity compared with the initial water samples. In the first proppant run, water collected from the end of the trommel was of equal toxicity to the pre-treatment water. The IRC Fill Control run also had start and end water measured at a 90% ECs0 (values at 90% reflect the upper detection limit and indicate very low toxicity). The Reserve Pit water samples produced the most toxic results of all the Microtox test runs. In general, the water tested from the Proppant 2 and Reserve Pit runs exhibited greater toxicity than that from the Proppant 1 and the IRC Fill trials. Treated coarse samples were reduced in toxicity compared with untreated soils from all samples (except Reserve Pit, where only one sample represents the datum point); and treated fine solids from the IRC control and experimental runs were less toxic than the initial solids (Fig. 3b). In tests performed on the proppant and Reserve Pit runs, drying the treated solids resulted in toxicity being reduced even further. The treated IRC fine particles were less toxic than other fine particles. High toxicity values were evident for Proppant 1 and 2 untreated solids, Proppant 2 treated fines and Reserve Pit treated fine, wet samples. Total extractable hydrocarbon reduction was greater than 95% for all solids tested in the system, with the Proppant 2 fine samples exhibiting the lowest reduction at 95.77%, and the Reserve Pit coarse samples showing the greatest reduction in T E H at 99.87%. Results from Polytox testing were not technically defensible and are not reported here.

Discussion The Microtox bioassay is a versatile, rapid toxicity test that uses decreased bioluminescence of the marine microorganism Photobacterium phosphoreum to indicate toxicity of a substance. Ribo and Kaiser (1987) and Brouwer et al. (1990) summarized the multiple tests designed for Microtox and described different studies that used these tests. Polytox is not well documented in published reports, but similar to Microtox, it is a common toxicity test that is performed on wastewater effluents (US EPA 1987). 151

Based on the results obtained for Microtox testing, in most cases the treatment process was found to be effective in decreasing toxicity of solids contaminated with crude oil. Ribo and Kaiser (1987) in their review of the literature dealing with the Microtox test found

good corroborative evidence for the versatility, accuracy, reproducibility and statistical significance of the procedure. Each dilution of a toxicant tested in the Microtox system is exposed to I(Y' cells of the Microtox organism rather than a few dozen

Proppant 1

IRC Fill

9O

80

70

6O

5O

o Ill

4O

I.l.I

CN v m

I.IJ

IJJ

I.kl

LI.I

Proppant 1

Proppant 2

Reserve Pit

IRC Fill

90

.o

|

!

70

60

! °

!

O

¢o

ILl

II 2O

'°|

,m, I--

,m, I--

I-

Fig. 3 (a) Results of Microtox toxicity testing for beginning and ending water samples from the soil treatment system where EC,, is equal to the effective concentration at which 50% of the light output has been reduced in the organism Photobacterium pho~sphoreum. The ECf~ values are expressed as the percentage of 1% toxicant extractant from each sample where 90% = very low toxicity and 0% = high toxicity. B = beginning; E = ending; Tr = trommel; CE = control ending; EE = experimental ending ( - 2 = second set of samples). Asterisks mark treated samples where EC~, was significantly different from untreated materials (Tukey's test after one-way A N O V A test for main effects; P < 0.05, n = 3 except materials followed by number of samples in parentheses). (b) Results of Microtox toxicity testing for untreated and treated solid extractants from the soil treatment system where ECs<~is equal to the effective concentration at which 50% of the light output has been reduced in the organism Photobacterium phosphoreum. The EC~<,values are expressed as the percentage of 1% toxicant extractant from each sample where 90% = very low toxicity and 0% = high toxicity. U = untreated; T = treated; W = wet; D = dry; C = coarse; F = fine (particle sizes); Ct = control; E = experimental. Asterisks mark treated samples where EC,<, was significantly different from untreated materials (Tukey's test after one-way A N O V A test for main effects; P<0.05, n = 3 except materials followed by number of samples in parentheses). 152

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TOXICITY TESTING OF CRUDE OIL REMEDIATION IN SOILS individuals of a species, as in other tests (Ekundayo & Benka-Coker, 1994; Harkey et al., 1994; Tay et al., 1992). Furthermore, the Microtox test has bccn found to be more sensitive than many other toxicity tests, and gives results that may show a more accurate reading of ecosystem health, because microorganisms have biochemical functions that are similar to animal and plant life and will thus react similarly to physiological stress (Ribo & Kaiser, 1987). However, Ribo and Kaiser (1987) point out that the Microtox bioassay is best used in a battery of tests to gain a better understanding of the overall picture of an environmental sample. They suggest that other trophic levels should be incorporated into any pollutant study, and that the Microtox assay could also be used to screen samples before other more costly assays are performed. In data not shown, the Polytox results were found either unreliable or inconclusive. The Polytox system was incompatible with samples collected from the process, as residual hydrocarbons or, more likely, terpenes, rendered oxygen meter membranes useless. Also, solid-phase testing, a technique favored by many toxicologists, was not practical in this study (Brouwer et al., 1990; Harkey et al., 1994). In all runs in the treatment system, coarse solids contaminated by crude oil were reduced in toxicity after treatment (Fig. 3b). It was found that drying the solids after treatment decreased toxicity. This was probably because of volatilization of residual organics. The terpene formulation used in treating the soils was extremely toxic to the Microtox organism in its pure form (0.03% ECs0). However, the addition of tcrpcnc to sample processing - - although terpenes were added to the untreated solids at full strength before being run through the system - - showed no effect in increasing the toxicity of the samples. In the IRC Fill runs, the control began with uncontaminated material that was subjected to the same rigors as the crude oil-contaminated samples, including addition of terpenes. At the end of treatment, the solids were again not toxic to the Microtox organism, which suggests removal of terpenes from within the system. These results suggest that crude oil constituents (or perhaps some synergistic combination of terpcnes and hydrocarbons from the oil) arc most likely involved as toxicants, even after the soil has been treated (Fig. 3b). As seen in Fig. 3b, in runs prior to the IRC tests (Reserve Pit and Proppant 2), treated fine particles showed considerable toxicant concentrations before drying. Modifications to the system (repeated fine particle flushing and filtering, use of clean water at start of process) after the Reserve Pit run may have remedied this phenomenon, as it was not evident in the IRC Fill samples. Even in the experimental run, Spill Science & I'eclmoh)gy Bulletin

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I ~ ~'~lI ~1~'~I ~][I~, I the fine particle samples were non-toxic (90% ECs~) after treatment. However, the fines in the IRC Fill and the Proppant 2 and Reserve Pit wcrc different with respect to crude oil exposure, and this probably accounts for the difference in results. Improvements to the system also resulted in lower toxicity values for the IRC Fill water samples (Fig. 3a). For the first three runs, water from the Idaho National Engineering Laboratory (1NEL) facility at Test Area North (TAN), where the treatments took place, was used for processing samples. This water, designated by a site review as non-potable, contained elements toxic to the Microtox organism in the Proppant 2 and Reserve Pit runs. During the IRC Fill runs, water from the Idaho Falls municipal supply was used. As in the other tests for solids toxicity, the water at the end of the process had more of a toxic effect on the Microtox bacteria. This was probably because of residual hydrocarbons rather than terpenes. The IRC Fill control end water was non-toxic (90% ECs~) and showed no effect from terpene carryover (Fig. 3a). The higher water toxicity values for the second proppant and the Reserve Pit runs most likely were a result of the first water being toxic. As previously stated, T E H concentrations were greatly reduced upon treatment of the solids (Table 2). The least efficient run, Proppant 2 fines, exhibited T E H at 2113.7 ppm, which was by far the greatest concentration of hydrocarbons seen in treated materials. This probably explains why the same sample exhibited the highest toxicity of all the samples (Fig. 3b). The sample with the next highest toxicity value was the Reserve Pit fine, wet solids. Unlike the other samples tested, the Reserve Pit material had other detectable contaminants that may have contributed to its toxicity values. Additionally, the water used at the beginning of the process was toxic. Thus, these two fine-sized samples possibly accumulated other toxicants besides the hydrocarbons already present. The Microtox results show that, as expected, fine particles potentially harbor higher concentrations of contaminants (based on surface-to-volume ratio) and thus must be given extra treatment in the cleaning process. The system is effective at removing hydrocarbons from the solids that were treated here, and no additional contaminants were added. Raghaven et al. (199(I) discussed the need for continued development of soil treatment practices rather than containment of polluted soils. Because of the results that have been obtained from this study, the similar treatment of soils marred by crude oil could bc a costeffective means of returning an impacted site to a more natural condition. This technology could bc especially useful in the Arctic and other dry environ-

153

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II

ments, and it has the added benefit that it results in ecologically functional soil. That is, the soil contains viable microorganisms that can begin the process of reestablishing the ecosystem from which the soil came (O'Connell et al., in review). Furthermore, the process was designed to recycle fluids, and so minimize waste streams and maximize economic benefit. Thus the resulting contaminant stream is concentrated and can be used for co-generation of plant power, recovery of oil that can be sold, destruction or disposal. Acknowledgements--We wish to acknowledge the supportive work of Charles P. Thomas, F. S. Colwell, Janice D. Jackson and Raelene A. McMillan. This work was conducted through the United States Department of Energy contract to EG and G Idaho, Ine. (DE-AC07-76ID01570), and sponsored by Laboratory Directed Research and Development funds.

References Atlas, R. M. (1977). Stimulated petroleum biodegradation. CRC Critical Reviews of Microbiology, 5, 371-386. Brouwer, H., Murphy, T. & McArdle, L. (1990). A sedimentcontact bioassay with Photobacterium pho,whoreum. Environmental Toxicology and Chemistry, 9, 1353-1358. Campbell, M., Bitton, G., Koopman, B. & Delfino, J. J. (1992). Preliminary comparison of sediment extraction procedures and exchange solvents for hydrophobic compounds based on inhibition of bioluminescence. Environmental, Toxicology and Water Quality, 7, 329-338.

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e, al

Ekundayo, J. A. & Benka-Coker, M. O. (1994). Effects of exposure of aquatic snails to sublethal concentrations of waste drilling fluid. Environmental Monitoring and Assessment, 30, 291-297. Fredrickson, J. K., Bolton, H. Jr. & Brockman, F. J. (1993). In situ and on-site bioreclamation. Environmental Science and Technology, 27, 1711-1716. Harkey, G. A., Landrum, P. F. & Klaine, S. J. (1994). Comparison of whole-sediment, elutriate and pore-water exposures for use in assessing sediment-associated organic contaminants in bioassays. Environmental Toxicology and Chemistry, 13, 1315-1329. Lapinskas, J. (1989). Bacterial degradation of hydrocarbon contamination in soil and groundwater. Chemistry and Industry 1989, 784-789. Microbics Corporation (1992). Microtox Manual." A Toxicity Testing Handbook, Vols 1-5. Carlsbad, CA, U.S.A. Raghaven, R., Coles, E. & Dietz, D. (1990). Cleaning excavated soil using extraction agents: a state-of-the-art review. Environmental Protection Agency Publication 600/$2-89/034. Ribo, J. M. & Kaiser, K. L. E. (1987). Photobacterium phosphoreum toxicity bioassay: test procedures and applications (1). Toxicity Assessment, 2, 305-323. Schiewe, M. H., Hawk, E. G., Actor, D. I. & Krahn, M. M. (1985). Use of a bacterial bioluminescence assay to assess toxicity of contaminated marine sediments. Canadian Journal of Fisheries and Aquatic Sciences, 42, 1244-1248. Tay, K.-L., Doe, K. G., Wade, S. J., Vaughan, D. A., Berrigan, R. E. & Moore, M. J. (1992). Sediment bioassessment in Halifax Harbour. Environmental Toxicology and Chemistry, 11, 1567-1581. United States Environmental Protection Agency (1987). Permit writer's guide to water quality-based permitting for toxic pollutants. EPA document 440/4-87-005. United States Environmental Protection Agency (1994). Test methods for evaluating solid waste: physical/chemical methods. EPA document SW-846.

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