Arch. Environ. Contam. Toxicol.

16, 239-246 (1987)

E nvironrnenta! C Ontamination
9 1987Springer-VerlagNew YorkInc.

Archives

of

Toxicity of Five Shale Oils to Fish and Aquatic Invertebrates

Daniel E Woodward 1, Edward E. Little, and L a w r e n c e M. Smith
National Fisheries Contaminant Research Center, United States Fish and Wildlife Service, Route 1, Columbia, Missouri 65201

Abstract. The chemical composition and toxicity of three shale crude oils (Tosco, Paraho, and Geokinetics), a hydrotreated oil (Paraho HDT), and a refined shale oil (Paraho JP-4) were assessed to de-

termine the potential hazards to native fish species and food chain organisms posed by accidental spills of such n~Jaterials. Colorado squawfish (Ptychocheilus lucius), fathead minnow (Pimephales promelas), cutthroat trout (Salrno clarki), and colonies of aquatic invertebrates were exposed to the watersoluble fractions of the shale oils for 96 hr to determine concentrations lethal to 50% of the exposed organisms (LC-50). The behavior of surviving fish was also n~teasured to determine the sublethal influences of e.xposure. The composition of the five water-soluble fractions was similar to that of the crude and refined shale oils from which they were made. H y d r o t r e a t e d and refined oils c o n t a i n e d fewer aromatic compounds than the crude shale oils. The cutthroat trout, a species endemic to oil shale regions, was less tolerant of shale oil exposure than the other species tested; the LC-50 concentrations; were 1.8 mg/L Geokinetics, 2.1 mg/L Tosco, and 1.3 mg/L Paraho. Exposure to concentrations of one-half to one-eighth those causing m o r t a l i t y r e d u c e d the swimming c a p a c i t y of squawfish and significantly impaired their ability to capture prey. The number of invertebrate taxa, species, and organisms colonizing plate samplers declined with increasing oil concentration. The genera Baetis and Isoperla were most sensitive to shale oil exposure; significant mortality occurred at the lowest concentration (0.5-0.7 mg/L) tested for each shale oil.

The demand to develop domestic energy resources in the United States fluctuates with the world market prices for crude oil. Synthetic crude oil produced from oil shale has been a viable substitute for conventional petroleum since the 1940s. The most concentrated area of oil shale deposits are in the Green River Formation in Colorado, Utah, and Wyoming, and major production is most likely to occur in this area (USEPA 1981). In an area of 44,000 km 2, about 600 billion barrels (95 billion cubic meters) of oil are recoverable by currently k n o w n technology. This amount is equivalent to m o r e than 90% of the world's estimated proven conventional reserves (Rio Blanco Oil Shale Company, unpublished report). Development of this magnitude in a small geographical area will result in several serious challenges to the environment. The oil produced will be transported by numerous mechanisms and spills and accidental discharges may result, as they now do, in the transportation of petroleum products. However, the composition of shale oil differs from that of petroleum oil, and only limited information is available on acute and s u b a c u t e toxicity to aquatic organisms. Organic composition of the water-soluble fraction (WSF) has not been completely characterized, but recent studies have indicated that shale oils have much higher concentrations of olefins, aromatic substances, and nitrogen heterocyclics than do petroleum crudes (Ghassemi and Panahloo 1984). Studies with freshwater algae have indicated that the WSF of oil shale is intermediate in toxicity b e t w e e n oil and liquid coal (Giddings and Washington 1981). A broader data base on chemical composition and ecological effects of shale oil products is necessary to adequately protect the environment. Streams draining the development area are tribu-

LNational Fisheries Contaminant Research Center, Field Research Station--Jackson, RO. Box 1089, Jackson, Wyoming 83001

taries of the upper Colorado River system and sup-

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port important sport fisheries and natural resource areas for recreation. In addition, four native fishes of the area--Colorado s q u a w f i s h (Ptychocheilus lucius), h u m p b a c k c h u b (Gila cypha), b o n y t a i l (Gila elegans), a n d r a z o r b a c k s u c k e r (Xyrauchen texanus)--are i n d a n g e r o f e x t i n c t i o n , a n d t h e U . S . F i s h a n d W i l d l i f e S e r v i c e is c h a r g e d w i t h t h e i r p r o t e c t i o n ( W y d o s k i et al. 1980). A n e v a l u a t i o n o f t h e sensitivities of endangered species, as well as standard test organisms, to potential contaminants is critical in an assessment of risks associated with the transportation of shale oils. The objectives of this research were to initiate a data base that would aid in the development of s h a l e oil r e s o u r c e s b y p r o v i d i n g i n f o r m a t i o n n e e d e d in hazard assessment and protection of endangered fish and aquatic organisms in the impacted area. Chemical characterization, lethal toxicity concentrations, sublethal effect concentrations, and methods to relate laboratory findings with current environmental monitoring techniques are included in this data base for different oil shale products.

Test Oil and Exposure Apparatus

Three crude shale oils were evaluated: Geokinetics, Paraho, and Tosco; one hydrotreated crude, Paraho HDT; and one refined product used for jet fuel, Paraho JP-4. Paraho crude, Paraho HDT, and Paraho JP-4 were obtained from the Oak Ridge National Laboratory, Department of Energy, Oak Ridge, Tennessee; Geokinetics crude from Geokinetics, Inc. (Vernal, Utah); and Tosco crude from Tosco Corporation (Golden, Colorado). Laboratory flow-through tests were conducted on the WSF of these oils for 96 hr, using the apparatus and procedures of Woodward et al. (1981, 1983). A 100 mg/L mixing ratio of oil to water was selected to simulate a spill. The WSF in the final separation tank made up the oil concentration in the highest exposure, and was also diluted four successive times by 50% to provide four additional exposure solutions that were 50, 25, 12, and 6% of the first. Warm or cold spring water used in the dilution also served as the experimental control solution. The watersoluble fraction in the final separation tank ranged from 1.5 to 6.5 mg/L depending on the oil. Duplicate exposure tanks of 40-L volume were maintained under identical conditions for each of the six solutions. Each tank received 1 L of new exposure water at 5-min intervals. For the warmwater test, the two duplicate sets of t a n k s c o n t a i n e d C o l o r a d o s q u a w f i s h and fathead minnows. In the coldwater test, one set of tanks contained cutthroat trout and the other contained the plate samplers colonized with aquatic invertebrates.

Methods

Chemical Analyses
Water-soluble fractions were prepared from each of the five shale oils by combining 0.5 g of oil and 350 ml of tap water into each of two 500 ml Erlenmeyer flasks. Flasks were capped and shaken for 16 hr on an Eberbach shaker. After settling for 2 hr, 250 ml were siphoned from below the surface of each flask and combined in a 1,000-ml separatory funnel for extraction with three 25-ml portions of methylene chloride. Before analysis, an internal standard of predeuterated naphthalene, pyrene, phenanthrene, perylene, anthracene, and benz(a)anthracene was added, and the sample was reduced to 1 to 8 ml under a stream of nitrogen. Samples were analyzed directly by gas chromatography (GC) and mass spectrometry (MS) with computer data analysis (DS). A model 4000 Finnigan MS with INCOS data system was used with electron impact ionization at 55 electron volts. Samples of 1 jxl were injected onto a fused silica capillary tube (60 m x 0.25 mm DB-5, J&W Scientific) by the on-column technique. The temperature program follows: 50~ hold 5 min; 5~ to 270~ l~ to 300~ hold 10 min. The gas carrier was helium at 10 psi. The analytical procedure used in these determinations required the development of an extensive GCMS search library specifically for these samples which entailed analysis and interpretation of several hundred individual components of these mixtures. The interpretations were made primarily by using the National Bureau of Standards mass spectrum library of over 30,000 spectra. Quantitations were made by the internal standard method. Relative response factors were determined for a number of specific aromatic and aliphatic compounds, and these factors were used for other members of the compound classes. Quantitations of compounds not having appropriate standards were made by assigning a response factor based on inspection of the mass spectra of these compounds. Total oil in water was determined for each of the six test solu-

Test Organisms
The oil shale development area contains both a coldwater and warmwater fishery. Therefore, separate coldwater and warmwater tests were performed. The warmwater test species were the Colorado squawfish, selected because of its endangered and protected status, and the fathead minnow (Pimephales promelas), selected because of its general use as a standard test species (ASTM 1980). The cutthroat trout (Salmo clarki) was selected as a test species in the coldwater tests because it is a native to Rocky Mountain streams and lakes and is easily obtained (Lee et al. 1980). Colonized multiple-plate artificial substrate samplers (Merritt and Cummings 1978) were used in the coldwater test to evaluate effects on structure of aquatic invertebrate communities. Baseline surveys on macroinvertebrates were conducted before oil shale exploitation began in Colorado (Erman 1981). Therefore, identifying taxonomic groups sensitive to shale oil contaminants and common to the development area would be useful in assessing impact after a spill. Colorado squawfish were obtained from the Dexter (New Mexico) National Fish Hatchery, fathead minnows from our laboratory culture, and cutthroat trout from the Jackson (Wyoming) National Fish Hatchery. Before coldwater tests, multiple plate samplers were allowed to colonize for 4 to 6 weeks in Flat Creek on the National Elk Refuge, Jackson, Wyoming. Each artificial substrate sampler was made up of eight 7.6-cm square tiles spaced 0.64 cm apart. Warmwater tests were conducted in spring water at 20 -+ I~ pH was 8.4, alkalinity 196 mg/L (as CaCO3), and hardness 175 mg/L (as CaCO3). Coldwater tests were conducted at 10 - I~ pH was 7.5, alkalinity 149 mg/L, and hardness 158 mg/L. All fish were acclimated to the respective test waters for at least 10 days before tests were started.

Toxicity of Shale Oil to Fish and Food Chain Organisms tions at three different times during all warmwater and coldwater studies. During the 96-hr tests, 500-ml samples were pumped from below the surface of each of the six tanks at 1, 20, and 90 hr. Each 50C,-ml sample was extracted three times with 30-ml portions of t r i c h l o r o t r i f l u o r o e t h a n e in 1,000-ml separatory funnels. Extract volume was reduced to 10 ml under a stream of nitrogen and analyzed by infrared spectroscopy (Simard et al. 1952) to determine the total oil content. Actual concentration reported was a mean of these three determinations.

241 varied among experiments, depending on availability. The test was begun by transferring three randomly selected squawfish from one of the three exposure concentrations or the control to a "predator success" aquarium. This procedure was repeated 3 times per test solution (total of 12 tests per experiment)~ The experiment was terminated when 40 to 60% of the prey fish in the control tests had been captured. Predator success was measured by percentage of prey captured, and comparisons were made between exposure groups and controls by using a binomial chi-square test (Snedecor and Cochran 1967). "Prey success" experiments examined the ability of exposed fathead minnows to escape capture by unexposed squawfish and were conducted in three 27-L aquaria (45 x 30 x 20 cm) containing 24 L of warmwater and receiving 2 L of new water at 5-min intervals. Other conditions were similar to those for the " p r e d a t o r success" aquaria, except that each prey success aquaria c o n t a i n e d t h r e e u n e x p o s e d squawfish p r e d a t o r s (average weight, 10.5 g) maintained on a diet of fathead minnows. Predators were not fed for 4 days before the test to insure their being hungry. At 36 hr before the oil exposures, fathead minnows to be used were anesthetized with 2% tricaine methanosulfate, and the left base of the dorsal fin was injected with a dot-sized (1 mm) mark of red, orange, yellow, or blue latex dye to allow differentiation among individuals from different test solutions. The dye marks were randomized among solutions and did not affect the sensitivity of the fish to the oils or their vulnerability to predation. After the 96-hr exposure, 10 fatbead minnows from each of the four selected test solutions were added to each of the three prey success aquaria for a total of 40 prey fish per aquarium. Prey were initially separated from the predator squawfish by a screen, which was removed after 2 hr. Prey were counted at hourly intervals until about 50% had been eaten; all fish were then removed and the surviving fathead minnows in each test solution were identified and counted. Prey success was based on percentage of prey that escaped, and a binomial chi-square test (Snedecor and Cochran 1967) was used to compare exposure groups and controls.

Toxicity
Where possible, the standard practices for performing acute toxicity tests were followed (ASTM 1980). At the start of each warmwater test, 20 Colorado squawfish and either 30 or 50 fathead minnows were placed in each test solution. For coldwater tests, :20 cutthroat trout and 3 colonized plate samplers were placed in each test solution. Dead fish were removed daily, and the concentration lethal to 50% of the organisms (LC50) and 95% confidence interval were calculated for each species at 48 and 96 hr (Litchfield and Wilcoxon 1949). After 96 hr of exposure, live and attached invertebrates were removed from the plate samplers and stored in 70% ethyl alcohol. Organisms from each plate sampler were identified and enumerated by a private laboratory (Susswasser, RO. Box 1255, Paso Robles, CA 93446). The three colonized plate samplers from each test solution were combined to give one value for total number of organisms, total number of taxa, and the Shannon-Weaver Index (Pielou 1975). The Shannon-Weaver or diversity index was based on the lowest taxonomic classification used (either genus or species) and would be properly referred to as diversity of taxa. At the end of each 96-hr warmwater test, the influence of sublethal exposure was measured during swimming and predatorprey tests of survivors from the three highest exposure concentrations that resulted in no mortalities, and from the control. Swimming ability of Colorado squawfish was measured in a stamina chamber modified from Howard (1975). Individual fish were placed in the chamber, acclimated for 5 rain to a water velocity of 9.2 cm/s, and then subjected to incremental increases of 6.7 cm/s in water velocity every 3 rain until they failed to swim. The fish were weighed and measured for total body length. Swimming capacity was measured for each fish by duration of activity or total swimming time in seconds and by body lengths per seconds (BL/S) as calculated by using the equation: BL/S = A + (B/180 * 6.2) TL

Results
The aromatic fraction represented 70% or more of the total compounds identified in the WSF of the crude shale oils; Geokinetics, Tosco, and Paraho ( T a b l e 1). I n c o n t r a s t , t h e a r o m a t i c f r a c t i o n c o m posed only 30-40% of the total WSF of the upgraded and refined products, Paraho HDT and Paraho JP-4. The components in the WSF of these two products were dominated by aliphatics. Almost all o f t h e a r o m a t i c s i n P a r a h o H D T a n d J P - 4 w e r e monoaromatics, whereas heterocyclics (ketones, pyridines, quinolines, etc) dominated in the crude s h a l e o i l W S F s . H e t e r o c y c l i c s w e r e n o t d e t e c t e d in the refined and upgraded products. Actual average oil concentrations were 5.6 mg/L in the highest exposure solution of the three crude oils, as compared with 2.8 mg/L in the high exposure solutions of the hydrotreated and refined oils. The higher test concentrations achieved with the

where A is the water velocity before the failure interval, B is the swimming time during the failure interval, and TL is the total length of the fish. Both time in seconds and BL/S were analyzed by a multiple means comparison test and least significant difference (Snedecor and Cochran 1967). Experiments on the predatory success of squawfish were conducted in 12 30-L aquaria (40 x 29 x 25 cm) with a gravel substrate and clusters of artificial Cabomba planted at each end. Each aquarium contained 20 L of warmwater and received new water at the rate of 500 mI/min. Illumination was provided by fluorescent lights positioned 90 cm over each aquarium. Unexposed fathead minnows were the prey species and either 17, 23, or 25 were put in each aquarium 24 hr before the experiment was started. The number added was constant within experiments but

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Table 1. Concentration (mg/L) of different classes of organic compounds in the water-soluble fraction (WSF) of five shale oil products Shale oil products Crude Compound Geokinetics Tosco Paraho Upgraded Paraho HDT Refined Paraho JP-4

Aliphatic, cycloaliphatic, and olefinic hydrocarbons 5.2 Aromatic and polar compounds Monoaromatic 3.7 Diaromatic 0.98 3-Ring aromatic 0.02 Aliphatic ketones <0.003 Phenols 4.6 Pyridines 2.8 Indoles 0.92 Quinolines <0.002 Alkyl thiophenes 0.06 Total 18.3

0.33 2.9 0.30 <0.01 0.74 2.0 1.8 0.42 0.32 0.35 9.2

1.6 0.38 0.08 <0.01 <0.003 1.9 1.0 0.32 <0.002 0.19 5.5

18.5 8.8 0.05 0.07 <0.003 <0.002 <0.002 <0.003 <0.002 <0.001 27.4

14.5 8.5 0.66 <0.01 <0.003 <0.002 <0.002 <0.003 <0.002 <0.001 23.7

Table 2. Toxicity of the water-soluble fraction of five shale oils to Colorado squawfish (CS), fathead minnows (FM), and cutthroat trout (CT) Mean size Oil and fish species Geokinetics CS FM CT Tosco CS FM CT Paraho CS FM CT Paraho HDT CS FM CT Paraho JP-4 CS FM CT
a

LC50 (mg/L total oil) a exposure time (hr) 96 4.5 (3.9-5.2) 2.9 (2.6-3.6) 1.8 (1.6-2.0) 2.3 (2.0-2.7) 2.3 (2.1-2.5) 2.1 (1.7-2.4) 4.2 (3.5-5.0) >1.7<3.3 1.3 (1.0-1.6) >2.4 >2.4 >5.0 >2.1 >2.1 >1.5

Length Weight (mm) (g) 48 4.5 (3.9-5.2) 2.9 (2.6-3.6) 1.8 (1.6-2.0) >3.7 3.0 (2.6-3.4) 4.4 (3.7-5.2) 4.2 (3.5-5.0) 3.5 (3.2-3.8) 1.7 (1.4-2.1) >2.4 >2.4 >5.0 >2.1 >2.1 >1.5

120 30 51 77 30 46 72 30 55 87 37 41 84 29 61

12 0.24 1.1 3.1 0.21 0.83 2.3 0.19 1.5 4.4 0.39 0.88 3.7 0.23 1.9

Values expressed as total oil concentration lethal to 50% of the test organisms (LC50); 95% confidence interval in parentheses. Test temperatures were 20~ for CS and FM and 10~ for CT

crude shale oils enabled us to measure 96-hr LC50s (Table 2). No mortality was observed in any of the high exposure solutions for Paraho HDT and Paraho JP-4. Comparisons of LC50s at 96 hr indicated that Colorado squawfish were slightly more tolerant than fathead minnows and cutthroat trout to the crude shale oils.

Sublethal effects were observed at concentrations of one-half to one-eighth of those causing acute toxicity (Table 3). Predator success was the most sensitive sublethal response measured. Swimming performance and prey success were reduced significantly (P ~< 0.05) in the highest concentration tested for Geokinetics, Tosco, and Paraho. As judged by sublethal effects, Paraho JP-4 was the least toxic oil, and Tosco, Paraho, and Paraho HDT were the most toxic (predator success was affected at less than 1.0 mg/L total oil). Stream colonization of the plate samplers produced a community represented by four major orders of aquatic insects--Ephemeroptera, Plecoptera, Tricoptera, and Diptera--plus representatives of Coleoptera and Mollusca. Because of the similarity of community response to some exposure concentrations within an oil, only the effects from two or three of the five exposure concentrations along with the control were presented (Table 4). After 96 hr in the control solution, invertebrate communities were composed of 18 to 34 taxa and 151 to 211 total organisms. As the exposure concentration increased, total number of organisms, and both number and diversity of taxa generally decreased. Response of the colonized community was a sensitive index of toxicity when assessed on a taxon basis by percentage composition of total organisms. The most common genera or family representing each of the major insect orders were Baetis, Isoperla, Brachycentrus, and Chironomidae; they composed more than 50% of the total organisms collected. The most sensitive of these genera were Baetis and Isoperla. One or both showed a significant (P ~< 0.05) reduction in percentage composition in the lowest concentration tested for all shale

Toxicity of Shale Oil to Fish and Food Chain Organisms

243

Table 3. Effects from 96-hr exposure of the water-soluble fraction of five shale oil products on swimming performance and predator success of Colorado squawfish, and prey success of fathead minnows Swimming performance Oil and concentration (mg/L) Geokinetics Control 0.8 1.4 2.2 Tosco Control 0.9 1.1 1.8 Paraho Control 0.5 0.9 1.7 Paraho HDT Control 0.8 1.3 2.4 Paraho JP-4 Control 0~8 1.3 2.1 Mean weight (g) 13 10 12 11 2.9 3.1 3.1 3.0 2.4 2.2 2.2 2.2 3.7 4.4 5.6 4.0 4.0 3.2 3.6 3.8 quration of activity (s) 1,533 1,350 1,286 1,521 1,295 1,104 1,134 1,006 1,264 1,119 1,144 1,084 1,239 1,103 992 1,169 1,466 1,513 1,435 1,556 (59) ~ (288) (264) (59) (265) (309) (195) (138)d (208) (161) (108) (65) d (327) (270) (114) (299) (114) (59) (185) (63) Swimming capacity (BL/S) b 4.0 3.7 3.5 4.1 5.3 4.4 4.5 4.0 5.3 4.9 4.9 4.8 4.7 3.8 3.3 4.3 5.3 6.1 5.6 5.9 (0.4) a (1.0) (0.7) (0.5) (1.3) (1.4) (0.8) (0.5) (1.3) (0.8) (0.6) (0.5) (1.5) (0.9) (0.4) (1.2) (0.9) (0.5) (1.0) (0.5) Predator success (% captured) 60 49 48 31 59 41 35 0 (75) a (75) (75) (75) r (75) (75) c (75) c (75) c Prey success (% escaped) 59 63 67 22 73 60 57 20 77 87 60 37 67 77 60 70 47 33 37 37 (27p (27) (27) (27) c (30) (30) (30) (30) c (30) (30) (30) (30F (30) (30) (30) (30) (30) (30) (30) (30)

61 (75) 44 (75) c 15 (75) c 21 (75) ~ 41 8 4 0 64 65 65 65 (51) (5IF (51) ~ (51) c (69) (69) (69) (69)

Numbers in parentheses represent standard deviations for size and swimming performance and number of observations for predator/ prey success; rates b Mean body lengths per second c Significantly different from control (X 2, P ~< 0.05) a Significantl[y different from control (LSD, P ~ 0.05)

oil products. On the other hand, B r a c h y c e n t r u s was one of the more tolerant genera; its percentage composition of the total number increased as exposure concentration increased. The Chironomidae were intermediate in response.

Discussion The chemical compositions of the WSF of the crude and refined shale oils are highly variable, and the WSFs of the three crude oils differ significantly from the compositions of shale oils reported elsewhere (Ingram e t al. 1983, Ghassemi and Panahloo 1984; Griest e t al. 1981). In general, crude shale oils contain about 20% alkanes, 45% aromatics, and 35% olefins and cycloaliphatics. Nitrogen and sulfur hete, rocyclics are significantly higher in crude shale oils than in petroleum crudes. Crude shale oil is hydrotreated to produce a transportable and suitable refinery feedstock. Hydrotreatment results in

a reduction in nitrogen and sulfur heterocyclics, a reduction in aromatics, and an increase in aliphatics as hydrogen is incorporated during treatment (Cada and Kenna 1985). Similar differences in composition were observed in this study when the WSFs of Paraho HDT were compared with those of Geokinetics, Paraho, and Tosco crude shale oils. The refined product, Paraho JP-4, was similar in composition to Paraho HDT, and the concentration of JP-4 in the WSF was similar to that reported by Fisher et al. (1985). Aromatic components of these oils, including nitrogen heterocyclics and phenols, are more soluble and much more toxic than the aliphatic components (Coleman et al. 1984; Rice et al. 1977). The development of concentrations of HDT and JP-4 in water high enough to induce lethality or to determine LC50 in fish was not possible. On the other hand, lethal water concentrations of the three crude shale oils were attained.

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Table 4. Effects of exposing colonized artificial plate samplers for 96 hr to different concentrations of the water-soluble fraction of five shale oil products. Each value is based on the total n u m b e r of organisms collected from three replicate samplers for each concentration Product and concentration Geokinetics Control a 0.7 1.5 2.3 Tosco Control 0.6 1.4 3.1 Paraho Control 0.7 1.3 2.6 Paraho H D T Control 0.5 1.3 2.6 Paraho JP-4 Control 0.5 1.5 Percentage of total n u m b e r No. taxonomic groups Diversity of taxa

Baetis

Isoperla

Brachycentrus

Chironomidae

Total no.

20 1b 2b 0b 24 4b 0b 0b 5 0b 0b 0b 4 5 0 0 42 4b 38

16 7b 2b 0b 15 11 15 3b 17 19 12 0b 15 5b 0b 5 19 6b 3b

14 19 40 b 45 b 19 15 27 62 b 12 13 8 13 11 22 b 15 43 b 10 35 b 42 b

26 37 22 9 25 41 21 5b 43 31 24 b 29 28 2b 3b 0b 7 8 7

-155 85 22 211 168 94 39 166 104 122 31 151 67 40 21 173 52 31

-22 15 8 21 22 18 7 18 21 21 10 34 17 16 9 18 14 14

-3.9 3.4 2.7 3.4 3.5 3.5 1.8 3.6 3.6 3.3 2.7 4.5 3.6 3.7 2.8 2.8 3.2 3.0

a Sample not taken; values represent means from other control samples b Significantly different from control (X 2, P ~< 0.05)

Exposure to the WSF of Paraho HDT did not induce mortality in fish; however, sublethal effects on predator success were significant at the lowest exposure concentration of HDT. Although exposure to JP-4 produced neither mortality nor behavioral change in fish, this refined oil was as toxic as the crude shale oils to the invertebrates Isoperla and Baetis. The substantial difference in the effects of HDT and JP-4 is surprising, because of their similarities in composition. JP-4 was expected to be somewhat more toxic than HDT because of its higher concentration of diaromatics in the WSE In any event, the effects of Paraho HDT were fairly similar to those of the crude shale oils, even though the compositions of the WSF of the crude oils included high concentrations of aromatics containing nitrogen and oxygen. Consequently, no simple correlation between chemical composition and toxicological effects was apparent in this study. Comparative acute toxicity data are available for JP-4 (Fisher et al. 1985; Klein and Jenkins 1977). Investigations with fathead minnows and golden shiners (Notemiqonus crysoleucas) under static conditions resulted in 96-hr LC50s of 3.8 to 24

mg/L. The 96-hr LC50 to fathead minnows in the present study exceeded the maximum flow-through concentration of 2.1 mg/L. Swimming performance and predator/prey behavior are often sensitive indicators of sublethal stress (Little et al. 1985). Changes in these critical behavioral responses can deleteriously influence long-term survival. The behavioral impairments observed during the present study are consistent with previous studies which examined behavioral aberrations induced by sublethal exposure to petroleum hydrocarbons. Increased predation-induced mortality was associated with a loss of schooling behavior among fathead minnows exposed to Wyoming crude oil (E. E. Little, unpublished data). The aromatic hydrocarbon, fluorene, impaired feeding efficiency and inhibited prey consumption in bluegills, Lepomis macrochirus (Finger et al. 1985); and food consumption was inhibited in coho salmon, Oncorhynchus kisutch, following sublethal exposure to crude oil (Folmar et al. 1981). Gill damage and fin erosion induced in cutthroat trout following exposure to Wyoming crude oil (Woodward et al. 1983) would also impair swimming capacity. Aquatic insects tested in this study represented

Toxicity of Shale Oil to Fish and Food Chain Organisms m a n y o f t h e s a m e g e n e r a s a m p l e d in b a s e l i n e s t u d i e s o f t h e o i l - s h a l e r e g i o n s in C o l o r a d o ( E r m a n 1981; P o l l a r d a n d K i n n e y 1979). T h e t a x a c o m m o n to all studiies w e r e t h e C h i r o n o m i d a e a n d t h e g e n e r a

245 gasoline, kerosene, and No. 2 fuel oil that partition into the aqueous phase after mixing. Arch Environ Contam Toxicol 13:171-178 Erman DC (1981) Stream macroinvertebrate baseline surveys: A comparative analysis from the oil-shale regions of Colorado, USA. Environ Manage 5:531-536 Finger SE, Little EE, Henry MG, Fairchild JF, Boyle TP (1985) Comparison of laboratory and field assessment of fluorine. Part 1: Effects of fluorine on the survival, growth, reproduction, and behavior of aquatic organisms in laboratory tests. In: Boyle TP (ed) Validation and predictability of laboratory methods for assessing the fate and effects of contaminants in aquatic ecosystems. ASTM STP 865, American Society for Testing and Materials, Philadelphia, PA, pp 120-133 Fisher JW, Hunt TP, Putnam ME, Livingston MJ (1985) Toxic effects of petroleum and shale JP-4 and JP-8 aviation fuels on fathead minnows. Water Resour Bull 21:49-52 Folmar LC, Graddock DR, Blackwell JW, Joyce G, Hodgins HO (1981) Effects of petroleum exposure on predatory behavior of coho salmon (Oncorhynchas kisutch). Bull Environ Contam Toxicol 27:458-465 Ghassemi M, Panahloo A (1984) Comparison of physical and chemical characteristics of shale oil fuels and analogous petroleum products. Environ Toxicol Chem 3:5t 1-535 Giddings JM, Washington JN (1981) Coal liquefaction products, shale oil, and petroleum. Acute toxicity to freshwater algae. Environ Sci Technol 15:106-108 Griest WH, Guerin MR, Yeatts LB Jr, Clark BR (1981) Sample management and chemical characterization of the Paraho/ Sohio/U .S. Navy crude and refined shale oil suite. In: Griest WH, Guerin MR, Coffin DL (eds) Health Effects Investigation of Oil Shale Development, Ann Arbor Science Publishers, Ann Arbor, MI, pp 17-44 Howard TE (1975) Swimming performance of juvenile coho salmon (Oncorhynchus kisutch) exposed to bleached kraft pulpmill effluent. J Fish Res Board Can 32:789-793 Ingrain LL, Ellis J, Crisp PT, Cook AC (1983) Comparative study of oil shales and shale oils from the Mahogany Zone, Green River formation (USA) and Kerosene Creek seam, Rundle formation (Australia). Chem Geol 38:185-212 Klein SA, Jenkins D (1977) Environmental Quality Research: Fish and Aufwuchs Bioassay. Air Force Aerospace Medical Research Laboratory Report No. AMRL-TR-77-54. WrightPatterson Air Force Base, OH, 58 pp Lee DS, Gilbert CR, Hocutt CH, Jenkins RE, McAllister DE, Stauffer JR Jr (1980) Atlas of North American Freshwater Fishes. North Carolina State Museum of Natural History, Raleigh, NC, 854 pp Litchfield JT Jr, Wilcoxon F (1949) A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 96:99-113 Little EE, Flerov BA, Ruzhinskaya NN (1985) Behavioral approaches in aquatic toxicity: A review. In: Mehrle PM, Gray RH, Kendall RL (eds) Toxic substances in the aquatic environment: An international perspective. American Fisheries Society, Water Quality Section, Bethesda, MD, pp 92-98 Merritt RW, Cummings KW (1978) An Introduction to the Aquatic Insects of North Americm Kendall/Hunt, Dubuque, IA, 441 pp Pielou, EC (1975) Ecological Diversity. John Wiley & Sons, New York, 165 pp Pollard JE, Kinney WL (1979) Assessment of macroinvertebrate monitoring techniques in an energy development area. U.S.

Ephemerella, Baetis, Rithrogena, Isoperla, Hydropsyche, a n d Hydroptila. A m o n g t h e s e , C h i r o n o m i d a e , Baetis, a n d Isoperla w e r e s e l e c t e d f o r s t u d y

b e c a u s e oF t h e i r u b i q u i t o u s o c c u r r e n c e in field p l a t e s a m p l e s a n d t h e i r s e n s i t i v i t y to l o w c o n c e n t r a t i o n s o f oil. I n a s m u c h as Baetis a n d Isoperla w e r e t h e most sensitive genera, their absence could provide a n e a r l y w a r n i n g in t h e f i e l d o f t h e o c c u r r e n c e o f l o w l e v e l s ( 0 . 5 - 1 . 0 rag/L) o f s h a l e oil c o n t a m i n a tion. A l t h o u g h fish m o r t a l i t y w o u l d b e u n l i k e l y at these concentrations, impaired swimming performance and decreased predator success might be e x p e c t e d . T h u s , t h e u s e o f a q u a t i c i n s e c t s a n d fish b e h a v i o r in this m a n n e r m a y p r o v i d e a m e a n i n g f u l a n d realislfic m e a n s o f r e l a t i n g l a b o r a t o r y r e s u l t s to e n v i r o n m e n t a l e f f e c t s m o n i t o r e d in t h e field. Actual concentrations of hydrocarbon contamin a n t s in a s t r e a m a f t e r a p i p e l i n e b r e a k o r a c c i d e n t a l spill d e p e n d o n m a n y f a c t o r s . U n d e r a simul a t e d o i l s p i l l o f 100 m g / L a n d t h e c o n d i t i o n s p r e s e n t e d in this s t u d y , a n i m p a c t on a q u a t i c org a n i s m s w a s o b s e r v e d . T h e d a t a b a s e n e e d s to b e e x p a n d e d , b u t c o m b i n e d field a n d l a b o r a t o r y app r o a c h e s u s e d in t h e p r e s e n t s t u d y p r o v i d e a t e c h n i q u e u s e f u l in t h e f i e l d a s s e s s m e n t o f b i o l o g i c a l d a m a g e f r o m s h a l e oil s p i l l s . B e n t h i c o r g a n i s m s avoid stress by drifting; and their relatively slow r a t e o f r e c o l o n i z a t i o n a f t e r s t r e s s , g i v e s this a s s e s s ment technique the advantage of being applicable days after contamination has occurred. A measure of the degree of contamination could be estimated from the insect species affected, and contaminant stress to higher organisms of more economic or aesthetic value could be predicted.

Acknowledgments. We thank Jim Johnson and Bob Knowlton

for their respective assistance in chemical analysis and toxicity studies of the shale oils.

References

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