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BIOASSAY PROCEDURES for OIL and OIL DISPERSANT TOXICITY EVALUATION Gilles Laroche Quality Laboratory, U

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BIOASSAY PROCEDURES for OIL and OIL DISPERSANT TOXICITY EVALUATION Gilles Laroche Quality Laboratory, U University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Environmental Protection Agency Papers U.S. Environmental Protection Agency 11-1970 BIOASSAY PROCEDURES FOR OIL AND OIL DISPERSANT TOXICITY EVALUATION Gilles LaRoche Quality Laboratory, U. S. Bept. of the Interior, Federal Water Quality Administration Ronald Eisler Quality Laboratory, U. S. Bept. of the Interior, Federal Water Quality Administration Clarence M. Tarzwell Quality Laboratory, U. S. Bept. of the Interior, Federal Water Quality Administration Follow this and additional works at: http://digitalcommons.unl.edu/usepapapers Part of the Earth Sciences Commons, Environmental Health and Protection Commons, Environmental Monitoring Commons, and the Other Environmental Sciences Commons LaRoche, Gilles; Eisler, Ronald; and Tarzwell, Clarence M., "BIOASSAY PROCEDURES FOR OIL AND OIL DISPERSANT TOXICITY EVALUATION" (1970). U.S. Environmental Protection Agency Papers. 281. http://digitalcommons.unl.edu/usepapapers/281 This Article is brought to you for free and open access by the U.S. Environmental Protection Agency at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Environmental Protection Agency Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. BIOASSAY PROCEDURES FOR OIL AND OIL DISPERSANT TOXICITY EVALUATION Gilles La Roche, Ronald Eisler, and Clarence M. Tarzwell Hazards to marine and estuarine lid, and the grass shrimp Palaemonetes fauna associated with offshore drilling vulgaris, a decapod crustacean. of oil and with transport of large The authors emphasize that proce quantities of oils via tankers are nu dures presented herein are interim merous and understandable. At pres pending confirmation by independent ent, there is a growing body of evi testing laboratories and are not to be dence on adverse effects to these construed as the official FWQA method organisms of crude oil (1) (2) (3) (4) for evaluation of toxicity of oil-dis (5) (6) (7) (8) (9) and chemical oil persant mixtures to aquatic organisms. counteracting agents or dispersants Procedures (5) (6) (9) (10) (11) (12) (13) (14) (15) (16) (17). These effects are well In general, these methods represent summarized by Smith (18) and by modifications of bioassay procedures Carthy and Arthur (19). contained in the current edition of Comparatively few workers have ' ' Standard Methods " ( 21 ). Because of studied the influence of oil-dispersant special problems arising in the bioassay mixtures of marine life. Studies by of oils and oil-dispersant mixtures, it Rosenthal and Gunkel (20) and Kuhl was found necessary to supplement and Mann (5) have led them to con and adapt the procedures described in clude that crude oils are less toxic than "Standard Methods." These include oil-dispersant mixtures. Spooner (9) assay methods suitable for organisms states that work with dispersants and other than fish and the standardization oil together is important, but the vary of test conditions so that results may ing toxicities of the oils and their be compared. Among these conditions mechanical awkwardness in biological are: (a) test species; (o) a standard synthetic seawater formulation; (c) experiments makes it difficult to stan dardize such work and to compare it temperature, pH, salinity, and dis solved oxygen (DO) content of the test with that of others. medium; (d) a standardized agitation In this account standardized bioas procedure for mixing of oils and oil say procedures are presented, devised, dispersant combinations; and (e) use and developed for the evaluation of the of a reference toxicant. relative toxicities of oils, dispersants, and oil-dispersant mixtures to the Assay Species mummichog Fundulus heteroclitus, a Before testing, each species is ac eyprinodontiform teleost, the sand climatized at 20 ?C under subdued worm Nereis virens, a polychaete anne light in separate aquaria for 10 to 14 days to the synthetic seawater formu Gilles LaBoche, Bonald Eisler, and Clar lation described later. Mummichogs ence M. Tarzwell are, respectively, Program heavier than 1.5 g are not recom Coordinator, Biologist, and Direotor, Na mended and should be discarded be tional Marine Water Quality Laboratory, U. S. Bept. of the Interior, Federal Water cause of their comparatively high oxy Quality Administration, West Kingston, R. I. gen demand. All sizes of adults of 1982 Vol. 42, No. 11 BIOASSAYS 1983 sand worms and grass shrimp are satis 10. Add 15.65 kg NaCl. At this factory. Groups of test organisms ex point the pH is approximately periencing more than 20 percent mor 6.5. tality during the first 48 hr or more 11. Addl3gNa2SiO-9H20. than 5 percent after 48 hr and prior to 12. Add 0.4 g ethylene diamine tetra bioassay should be discarded. acetate (EDTA) tetrasodium salt. During acclimation all species may 13. Add 133 g NaHC03. The pH be fed fresh or live food or a prepared should be 8.0 ? 0.1. homogenate of ground clam (80 per 14. Fill tank to 1,000-1 mark. cent by weight), dog biscuits (8 per A wrater temperature of 19.5? ? 0.5? cent), water (10 percent), and gum C, pH 8.0 ?0.1, salinity of 20 ? 0.5 arabic crystals (2 percent). This pre ppt, and DO concentration > 4.0 mg/1 pared diet should be frozen after mix is suggested during acclimatization of ing and completely thawed to room assay species. temperature before feeding. Unused thawed portions are discarded daily. Animals are fed twice daily to satiation Assay Procedures but are not fed 48 hr prior to, or dur General ing, the assay. Only those animals Disposable glass jars t measuring that feed actively and appear to be approximately 22.5 cm high, 15 cm in healthy are used for assays. diam, and 11 cm in mouth diam (approximately 4-1 size) were utilized Synthetic Seawater Formulation as the assay containers. These jars Instructions for preparing 1,000 1 were provided with screw-cap lids of 20 ppt salinity of synthetic seawater lined with a clear plastic nontoxic film. formulation (22) using technical grade Two liters of freshly prepared syn chemicals * and tap water t are pre thetic seawater were placed in each sented below. It is important that the test container by means of an auto listed order of ingredient addition be matic dispensing pipette of 2-1 capacity respected and that each ingredient be (Figure 1). These pipettes were satis dissolved before another is added ; this factory to expedite repetitive volumes. is best accomplished by constant mix Measured amounts of the compounds ing in the course of preparation. to be tested [viz., crude oil, chemical oil dispersants, oil-dispersant mixtures, 1. Add 890 1 of tap water f to a or dodecyl sodium sulfate (DSS)] suitable container, i.e., fiberglass. were then added. For each toxicant 2. Add 1.9 g NaF. tested, a minimum of six concentrations 3. Addl3gSrCl2-6H20. with five replicates per concentration 4. Add 20 g H3B03. is recommended. 5. Add 67 g KBr. Immediately after the addition of 6. Add 466 g KC1. the material to be tested, the jars are 7. Add733gCaCl2-2H20. tightly capped and shaken for 5 min 8. Add 2.66 kg Na2S04. at approximately 315 to 335 0.75-in. 9. Add 3.33 kg MgCl2-6H20. strokes/min on a reciprocal shaker. The shaker platform should be adapted * Exceptions : Reagent grades of EDTA to hold firmly six of the bottles de and SrCl2*6H20 are used because of unavail scribed above. Shaking and exposure ability of technical grades. t Containing 0.1 mg/1 nitrogen organics of the test organisms are carried out (N + NH3), <0.02 mg/1 Zn; < 0.05 mg/1 at 20??1?C. Cu ; < 0.1 mg/1 B ; and nondeteetable levels of Cd, As, P, Fe, Mo, Mn, Al, Be, Ag, Ni, 1131-oz flint wide-mouth screw-capped Co, Pb, Cr, and V. mayonnaise jars. 1984 JOURNAL WPCF November 1970 r?ft Dead animals are recorded and re moved daily. At 96 hr, the assay is terminated, and the TL50 values deter mined as described in "Standard Methods" (21). Values for TL100 500 (highest concentration tested with 100 GALIONS percent survival) and TL0 at 24, 48, and 96 hr are presented in tabular form along with estimated TL25, TL50, and TL75 values for these three time intervals. Sample data are presented later in this report. Test Toxicants Oil:?For bioassay of crude oils, it is recommended that all samples ar rive in sealed 100-ml containers. A minimum of 40 containers of each grade of crude should be available. Disposable serological pipettes are used to add various amounts of crude directly to the test containers. The unused oil from each opened container is discarded. As an example, the fol lowing amounts of crude were used in one series of tests: 36 ml (= 18 ml/1), 18 ml (= 9 ml/1), 12 ml, 8 ml, 4 ml, 2 ml, 1 ml (= 0.5 m/1). At least one control is set up for each series. Re sults of a preliminary assay on mum -I GALLON (US.) michogs are presented in Table I. Dispersant:?There are many com 2 LITERS mercially available oil dispersants. Studies of these products (23) indi FIGURE 1.?Schematic diagram of cate that most fall into one of six ma automatic dispensing pipette system used jor categories based on surface active in bioassay procedure. A = inflow from large holding reservoir; B = overflow from agents and solvent types. Some of other units in series; C = inflow to other these are primarily soluble in sea units. water and others have a greater af finity for petroleum derivatives. Re After agitation, the lids are removed gardless of this primary affinity, it and two test organisms, i.e., two mum TABLE^I ?Acute Toxicity of Crude Oil A to michogs, two sandworms, or two Mummichog at Three Time Intervals shrimp are added to each jar.
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