IXTOC I: CHEMICAL CHARACTERIZATION AND ACUTE BIOLOGICAL EFFECTS P. L. Parker (Editor) Final Report submitted _to Environmental Protection Agency , in fulfillment of Department of Transportation Contract DOT-CG08-8174 by The University of Texas at Austin Marine Science Institute Port Aransas Marine Laboratory Port Aransas, Texas 73837 August 29, 1979 . .. ' TABLE OF CONTENTS Pages Section A. Chemical Characterization of Ixtoc I Samples and Oil-Seawater Mixtures· Used for Toxicity Studies (P.L. Parker, R.S. Scalan, J.K. Winters, D.C. Boatwright, and D.L. Scalan) •••••••.•••••••••Al-19 Section B. Effect of Mexican Oil on Phytoplankton and Seagrass Photosynthetic Activity after Short Time Exposure (W. M. Pulich) •••••••••••••••• Bl-9 Section C. Effects of Mexican Crude Oil on Redfish (Sciaenops ocellata) Eggs and Larvae (C. R. Arnold, S. C. ·Rabalais, N. S. Wohlschlag) ••••••••••• Cl-7 Section D. A Toxicity Study of Ixtoc I Oil for Zooplankton (W. Y. Lee, A. Mor:_ris) •..•••.••.•••••.••••••.•.•••••Dl-9 Section E. Mexican Oil Spill: Invertebrate Toxicity Tests (R.W. Flint, N.N. Rabalais, M.J. Poff, R.D. Kalke and J.A. Younk) .••••••••..••.••••••.•••••••. El-14 Section F. Effects of Ixtoc I Gulf of Mexico Oil Spill Materials on the Behavior and Respiratory Metabolism of the Spotted Seatrout, Cynoscion nebulosus· (D. E. Wohlschlag, F. R. Parker, Jr.) •••••Fl-27 For Reference Quote: Parker, P.L. (Ed.·), 1979. "Ixtoc I: Chemical. Characterization and Acute Biological Effects." -Final Report submitted to the Environmental Protection Agency in fulfillment of Department of Transportation Contract DOT-CG08-8174, 29 August 1979. The University of Texas, Marine · science Institute, Port Aransas Marine Laboratory, Port Aransas, Tx. 78373; 85 pp. • TABLE OF CONTENTS Section A. Chemical Characterization of IXTOC I Samples and Oil Seawater Mixtures Used for Toxicity Studies. Section B. Effect of Mexican Oil on Phytoplankton and Seagrass Photosynthetic Activity after Short-Time Exposure Section C. Effects of Mexican Crude ·oil on Redfish (Sciaenops ocellata) Eggs and Larvae Section D. A Toxicity Study of IXTOC I Oil for Zooplankton Section E. Mexican Oil Spill: Invertebrate Toxicity Tests Section F. Effects of IXTOC I Gulf of Mexico Oil Spill Materials on the Behavior and Respiratory Metabolism of the Spotted Seatrout (Cynoscion nebulosus) SECTION A CHEMICAL CHARACTERIZATION OF IXTOC I SAMPLES AND OIL-SEAWATER MIXTURES USED FOR TOXICITY STUDIES Report Submitted by: P. L. Parker R. s. Scalan J. K. Winters D. c. Boatwright D. L. Scalan University of Texas Marine Science Institute Port Aransas Marine Laboratory Port Aransas, Texas 78373 August 29, 1979 A-1 ' . INTRODUCTION The chemical component of these studies had two major objectives: 1) to characterize the composition of various IXTOC I oil samples representative of different stages of weathering and 2) to characterize oil-seawater mixtures prepared from the . specific mousse sample tested for acute toxicity. MATERIALS AND METHODS Samples of IXTOC I oil, mousse and beach tar were obtained from several government and private sources. About 25 2 of mousse were collected by personnel aboard the U. S. Coast Guard ship Pt. Baker at 22°50'N and 96°26'W. The Pt. Baker mousse was used for all biological t-C:>xicity studies. Five samples of oil collected from a beach in Mexico were also obtained from the Coast Guard. A sample of one of the first IXTOC I tarballs to come ashore on South Padre Island, Texas was supplied by Mr. Craig Hooper of NOAA. A sample of mousse collected near the well site 40 hrs after the blowout was received from Dr. Alfonso Botello of Mexico. Another sample of ''fresh" mousse was obtained from PEMEX through Dr. Carl Oppenheimer. Dr. John Robinson, NOAA, furnished samples of IXTOC I oil which had been separated from relatively unweathered mousse. Oil was accommodated in seawater for acute toxicity tests by shaking a 1% mixture of Pt. Baker mousse in seawater one hour on an Eberbach shaker. The mixture was allowed to settle for one hour after shaking before the aqueous phase was siphoned off for chemical and biological studies. For fish studies a different method of preparation was used due to the large volumes of seawater needed. A concentrated mixture of oil in seawater was prepared by blending oil and seawater in a Waring blender for about 30 seconds. The concentrated mixture was then diluted with seawater to a con A-2 .. centration which was similar to that prepared on the shaker. A "water soluble" fraction for chemical and biological studies was prepared from the oil-seawater mixture produced on a shaker as previously described. The mixture was filtered twice through glass fiber filters (Whatman GF/C) by gentle suction. Accommodated or "soluble" oil was extracted from seawater in separatory funnels with three extracts of dichloromethane. The crude oil was separated from water in mousse samples by addition of an equal volume of n-hexane followed by centrifugation at 10,000 RPM for 20 minutes. This separation indicated that the Pt. Baker mousse contained about 60% water by volume. Asphaltenes were removed from all samples by precipitation in n-pentane or n-hexane. A known quantity of each sample was fractionated by adsorptiqn on silica gel by usual column chromatograpic techniques. The sample was placed on the column in a small volume of hexane and saturate, aromatic and NSO fractions were eluted with hexane, hexane:benzene (1:1), and methanol respectively. Two column volumes each of hexane and the hex ane-benzene mixture were collected and methanol was collected until the eluate was colorless. Gas chromatography was carried out on a Perkin-Elmer 910 gas chromato graph equipped with a flame ionization detector. Electronic integr~tion of peak area was performed by a Hewlett-Packard 3352B Data System. The glass capillary column utilized for most analyses was OV-101, 11 m x .25 IIml ID. Oven temperature was progrannned from 70 to 255 Cat 3°/minute. Some analyses were performed on a 27 m OV-101 column progrannned from 100 to 260 Cat 2°/minute. Combined gas chromatography-mass spectrometry of selected samples was carried out on a Dupont 21-491 mass spectrometer with a Dupont 21-094 B data system. A-3 ' t Fluorescence spectra were determined with a Perkin-Elmer 204-A spectrophotometer. Samples were dissolved in hexane and excited at 265 run. The emission -spectra were recorded from 250 to 600 nm. RESULTS The total concentration of oil acconnnodated in seawater during the various preparations for biological and chemical studies varied from about 25-35 mg/£ (PPM). Specific concentrations for each individual preparation were measured and are reported in the appropriate biological section of this report. Approximately 90% of the oil acconnnodated into seawater was in the form of particles which were sufficiently large to be removed by filtration through glass fiber filters (Whatman GF/C). The "water soluble" portion therefore contained only about 10% or 2-4 PPM. Results of silica gel chromatographic separations are given in Table 1. The data indicate IXTOC I oil originally contained 52-53% saturates, 34-35% aromatics and 7-8% NSO compounds. The Pt. ~aker mousse used for toxicity tests was found to contain a similar percentage of saturates (51%} but a considerably lower percentage of aromatics (26%). The NSO fraction increased to 18% in the Pt. Baker mousse. The Mexican beach samples stud ied by the Coast Guard and the 1st IXTOC I tarball collected on South Padre Island show similar percentages of silica gel fractions. These percentages are lower in aromatics and higher in NSO compounds than most tarballs collected ~n a study conduct_ed on St. Joseph and Padre Islands during the past years (Scalan, 1979). Gas chromatography indicated the saturate fraction of the Pt. Baker mousse (Figure lA) contained n-alkanes from C13 to greater than C36 with highest concentrations in the C1a to C20 range. The mousse sample collected 40 hrs after the blowout (Figure lB) contained n-alkanes lower than C10 A-4 TABLE 1 RESULTS OF SILICA GEL COLUMN CHROMATOGRAPHY OF IXTOC I SAMPLES Sample Description Source % Saturate % Aromatic % NSO Mousse, collected 40 hrs after blowout Dr. Botello, Mexico 52.0 34.0 N.A. Oil, unweathered Dr. John Robinson NOAA 51.4 31.8 6.9 Mousse, unweathered Dr. Oppenheimer PEMEX 53.0 34.7 7.5 Mousse, Pt. Baker 22°50'N 96°26'W u.s.c.G. 51.0 26.4 18.3 Pt. Baker mousse, accorrrrnodated particles UTMSI/PAML 48.0 18.7 19.4 Beached oil, lower beach_;) .Mexico U.S.C.G. 34.7 28.5 34.1 Beached oil, upper beach, Mexico U.S.C.G. 38.0 18.0 38.3 Beached oil, mid-beach, Mexico u.s.c.G. 37.3 14.0 40.5 Tarballs in surf Mexico u.s.c.G. 29.4 17.3 40.2 Tar on beach, Mexico U.S.C.G. 36.7 15.8 43.3 "First" IXTOC tarball-South Padre NOAA 34.4 15.4 39.4 Avg. of non-IXTOC I beach tars, 1978_..1979 St. Joseph Island,Tex Dr. Scalan UTMST/PAML 46.8 (8-77) 32.3 (14-55) 20.8 (8-41) A-5 I 1.. , 1 4 1 6_ -A 1 8 20 26 1 6 24 B 32 Figure i.- Gas chromatograms of the saturate fraction of a collected 40 hrs after the IXTOC I blowout (A), Baker mousse (B). mousse sample and the Pt. I • N MN DMN TMN DBT P MP MDBT DMP DMDBT A-6 TABLE 2 GAS CHROMATOGRAPHIC PEAK IDENTIFICATION Naphthalene Methylnaphthalenes Dimethylnaphthalenes (includes ethylnaphthalenes) Trimethylnaphthalenes (includes all methylethyl-and propyl naphatha.lenes) Dibenzothiophene Phenanthrene Methylphananthrenes Methyldibenzothiophenes Dimethylphenanthrenes (see DMN) Dimethyldibenzothiophenes (see DMN) A-7 I I . ' with the maximum at C13. Analysis of the aromatic fraction from the Pt. Baker mousse (Figure 2A) revealed the presence of alkyl naphthalenes, primarily C2-(dimethyl+ethyl) and C3 homologs. C3-naphthalenes were present at a concentration which was about one-third of that present in the 40 hr sample (Figure 2B). Three ring aromatic compounds such as phenanthrenes and dibenzothiophenes are present in both the Pt. Baker mousse and the 40 hr sample in similar relative ' concentrations. The IXTOC I oil contains a relatively high concentration of alkyl dibenzothiophenes with concentrations similar to those of alkyl phenanthrenes. Ana.lyses of the oil acconnnodated into seawater for biological testing indicated that the particulate oil which was removed by filtration had a saturate and aromatic compos~tion which was quite similar to the Pt. Baker mousse. The "water soluble" material from the acconnnodated oil-seawater mixture was not fractionated on silica gel. A typical chromatogram of the "water solubles" is shown in Figure 3. The increased concentration of more soluble aromatic compounds relative to the Pt. Baker mousse was evident. The presence of n-alkanes in the uwater soluble" fraction probably indicates that some oil particles were sufficiently small to pass through the glass fiber filters. The n-alkane distribution in the soluble fraction was from about C19 to greater than C37 with a maximum at about C31. This distribution was significantly different from the Pt. Baker mousse which had a maximum at about C1 9. The samples of oil from a Mexican beach gave similar results with some samples showing slightly more weathering. Typical chromatograms of saturate and aromatic fractions are given in Figure 4. Normal alkanes began at C1s with a maximum at C21. The aromatic fraction indicated that alkyl naphtha A~B I I . ' N NN DMN I 0 A TifN I MP DMP + MDBT + IDMDBT -1 I DBT ' p B DBT TMN \ . DMN ~ Figure 2. Gas chromatograms of the aromatic fraction of a mousse sample collected 40 hrs after the IXTOC I blowout (A), and the Pt~ Baker mousse (B). (See Table 2 for peak identifications.) MP + MDBT r-71 N DMN II TMN fl DBT p. 2 1 1'" \.0 Figure 3. Gas chromatogram of _the "water soluble" fraction prepared from Pt. Baker mousse. (See Table 2 for peak identification.) the 24 A 1 8 !I I i 3 2 .. l6 i JL__~ DMP + DMDBT MP + :t-IDBT ~! If) f I -i Figure 4. Typical chromatograms of the saturate (A), and aromatic (B), fractions of IXTOC I oil from a Mexican beach. (See Table 2 lat peak identification.) A-11 ' I lenes had been almost completely lost. Phenanthrene, dibenzothiophene and methyl homologs were also significantly lower in concentration relative to the C2 and C3 homologs. A chromatogram of the saturate fraction of the tarball from South Padre Island is shown in Figure SA. Maximum n-alkane concentration occurred at C20· The aromatic fraction (Figure SB) was found to contain a large con centration of alkyl benzenes and naphthalenes relative to all other aromatic fractions analyzed other than samples of very fresh mousse. A chromatogram of the aromatic fraction of Pt. Baker mousse has been included in Figure SC for comparison. Combined gas chromatography-mass spectrometry was used to verify iden tification of major components in saturate and aromatic fractions of repre sentative samples. Mass chromatograms as shown in Figure 6 and 7 for alkyl naphthalanes and phenanthrenes,respectively,were used routinely. Fluorescence emission spectra of several samples are presented in Fig ure 8. DISCUSSION Chemical analyses indicated the Pt. Baker mousse used for biological studies had been sufficiently weathered at sea to significantly alter the composition of the saturate and aromatic fractions. Virtually all alkyl benzenes and a large percentage of the two ring aromatics, primarily naph thalenes, had beeh removed. -Three ring compounds such as phenanthrenes and dibenzothiophenes, however, were quite similar in concentration to that present in samples of unweathered IXTOC I oil. Unlike the Pt. Baker mousse the tarball collected on South Padre Island contained a high percentage of low molecular weight aromatics including alkyl benzenes and naphthalenes. Analyses of a suite of tarballs collected A-.1-2 A 1 8 24 J\l__-----. DMP MN + DMDBT B r i DMN ~ MP + MDBT ,--, c DBT \ Figure 5. Gas chromatogram of the saturate (A), and aromatic (B), fractions of an IXTOC tarball from South Padre Island, Texas. Pt. Baker mousse aromatic fraction (C), shows much lower volatile aromatics. (See Table 2 for peak identification.) TAF?.BALL - 3820/1355~ BENZ. FXX B ·50 100 150 250 300 Figure 6. Mass chromatograms of the aromatic fraction from the South Padre Island tarball sample. Total ion chromatogram (A); m/e=l28 naphthalene (B); m/e=l42, methylnaphthalenes (C); m/e=l56, C2-naphthalenes (D); m/e=l70, C3-naphthalenes (E). ·A.:-14 TARBALL -SPL#l, PG39~ 3820/1355# BENZ. FX>C c ,;Tl r1' 11l ' i,!1!qe11ft J1li l•I ' 11 frl ; I i ·f uI1I=JaI 50 -100 .1s0 a00 as0 300 Figure 7. Mass chromatograms of the aromatic fraction from South Padre Island tarball sample. Total ion chromatogram (A); m/e=l78, phenanthrene (B); m/e=l92, methylphenanthrenes (C); m/e=206, C2-phenanthrenes (D); m/e=220, C3-phenanthrenes (E). A B·, c :-;i> .: 1 '-t-' V'l 550 250 nm 550 250 nm 550 ·250 nm 550 250nm Figure 8. Fluorescence ennnission spectra of IXTOC I oil samples A, B, C and a non-IXTOC tarball (D). Exitation.was at 265 nm. Lower trace represents spectrum obtained at one-half concentration. A-16 on the recent MOUSSE I cruise of the R/V LONGHORN indicated that indeed most floating tarballs contained alkyl benzenes and naphthalenes in pro portions similar to the South Padre Island sample. The differences in chemical composition between the Pt. Baker mousse and the tarballs raise two important questions. The first question is whether or not the Pt. Baker mousse is representative of the large amount of oil which is impacting the various biological communities and therefore the validity of biolog~cal results based on this sample. A second question relates to the processes by which tarballs are formed from the IXTOC I oil. If the mousse formed at the well site has drifted north and slowly-disintegrated into smaller patches (pancakes) and ultimately into tarballs, as many people have speculated, how can such a high proportion of alkyl benzenes and naphthalenes be present in tarballs and not in an intermediate mousse (Pt. Baker)? Two additional subsamples of the Pt. Baker mousse were analyzed with special care to avoid loss of volatile components. The results of both subsamples indicated an absence of alkyl benzenes and low concentrations of naphthalenes similar to the previous analysis. Chromatograms of aromatic fractions of the Pt~ Baker mousse and South Padre Island tarball analyzed on a 27 m OV-101 column are given in Figures 9 and 10, respectively. Greater resolution on the longer columns should be useful for further more detailed studies of changes in composition during weathering. The large amount of alkyl benzenes and naphthalenes present in the tarball (Figure 10) was even more evident on the longer column. The fluorescence analyses were undertaken to develop a quick screening technique which would indicate with some certainty that a given oil, mousse, ' ; MP + MDBTI I DMP + DNDBT II I TMN f I I DBt p :t;t I . ~"'-J ' DMN 1.1 Figure 9. Gas.· chromatogram of the aromatic fraction from Pt. Baker column. (See Table 2 for peak identification.) mousse on a 27 m OV-101 N MN r.I >I H ~ DMN I.I Figure 10. Gas chromatogram of the aromatic fraction from South Padre Island tarball on 27 m. OV-101 column. (See Table 2 for peak identification.) A-19 or tarball was in fact from IXTOC I. The method can also be used to give a semi-quantitative estimate of the amount of oil present in a sample such as a sediment. The rate at which various weathering processes will alter the emission spectra and reduce the effectiveness of this procedure is currently being investigated. LITERATURE CITED Scalan, R. S. 1979. Quantitation and organic geochemical characterization of petroleum-like material found on undisturbed beaches of Padre Island National Seashore and St. Joseph Island. Unpublished results. SECTION B EFFECT OF MEXICAN OIL ON PHYTOPLANKTON AND SEAGRASS PHOTOSYNTHETIC ACTIVITY AFTER SHORT-TIME EXPOSURE Report Submitted by: Warren M. Pulich University of Texas Marine Science Institute Port Aransas l1arine Laboratory Port Aransas, Texas 78373 August 27, 1979 B-1 INTRODUCTION An attempt was made to assess acute toxicity of Mexican Oil to mixed phytoplankton populations and seagrasses by comparing photosynthetic rates in the presence and absence of a seawater-soluble fraction or oil-accomodated seawater preparation. This type of measurement is by nature a shortterm bioassay, which can only indicate whether the material being tested has an imaiediate effect on photosynthesis of the cells involved. Several studies have shown that seawater equilibrated with No. 2 fuel oil (Pulich et al., 1974; Gordon and Prouse, 1973} and some crude oils I ti z .L 2 Q..._____________________________..____________.____ 0.1°/o l.O°lo 10. 0°/o CONTROL OILED OILED OILED SEDIMENT SEDIMENT SEDIMENT SEDIMENT Results of sediment selectivity tests with Emerita sp. as the test organism. Two tests were run, one of 24 hr duration and the second of 72 hr duration. E-8 as tested by one-way ANOVA for these 24 hour tests. Thirty-five (35) percent of the crabs were observed in the control sediments, 28% in the 0.1% oiled sediments, 25% in the 1.0% oiled sediments, and 13% in the 10.0% oiled sediments. Less than one percent mortality was observed in all treatments over the experiment duration and no treatment showed significantly greater mortality than the controls. The 72 hour sediment selectivity tests showed much different results. For these tests there were no significant differences in selecting any of the treated sediments by the mole crabs at P < 0.05 (Vigure 1). This experiment did show a slightly different result concerning crab mortality. No dead crabs were observed in the control sediment. Mortality was 1% in the 0.1% oiled sediments, and 2% in the 1.0% and 10.0% oiled sediments. Toxicity Bioassays Mole Crab Percent mortality, percent survival (ps described in methods), and percent activity (number of mole crabs exposed above the sediment follow ing food stimulation) are recorded below: % Activity % Survival ff Visible after Food Stimulation Treatment % Mortality 96-hr 24-hr 48-hr 72-hr 96-hr 24-hr 48-hr 72-hr 96-hr 18 Control 0. 0 0 0 95 35 63 40 0 98 60 80 50 33 5% OAW 0 0 0 15% OAW 0 0 0 0 98 68 70 50 33 30% OAW 0 0 0 0 75 63 58 60 25 .60% OAW 0 0 0 0 100 78 70 53 38 No mortalities of mole crabs were recorded for any of the OAW treatments. Not all of the tests organisms were retrieved from the experiments. These unretrieved animals as well as pieces of mole crabs and molts may represent E-9 mortalities which could not be verified with an in-hand dead specimen. Acute subletha1 effects as determined by mole crab activity measurements were not noticeably different for the treatments. Percent activity decreased throughout the 96-hr period. Surf Clam Percent mortality and percent activity (number of surf clams with siphons extended and working .. following food stimulation)l are recorded below: % Mortality % Activity Treatment 24-hr 48-hr 72-hr 96-hr If w/siEhons ·extended & working 24-hr 48-hr 72-hr 96-hr Control 0 0 0 8 40 33 30 5 5% OAW 0 0 0 0 53 38 28 25 15% OAW 0 0 0 0 68 48 40 28 30% OAW 0 0 0 0 58 23 40 20 60% OAW 0 0 0 0 38 28 35 18 No mortalities were recorded for any of the surf clams in the OAW treat ments (8% in the control). All test animals were retrieved from the experiments. Sublethal effects as determined by surf clam activity measurements were not noticeably different for the treatments. Percent activity decreased throughout the 96-hr period. Polychaete Percent mortality and survival (as described in methods) are recorded below: %Mortality Treatment 24~hr 28-hr 72-hr 96-hr % Survival 96-hr Control 0 3 5 10 85 5% OAW 3 5 5 8 80 15% OAW 3 10 10 10 75 30% OAW 5 5 8 8 92 60% OAW 10 17 17 23 77 Not all the test organisms were retrieved from the experiments. These E-10 unretrieved animals as well as dead and decomposing body sections may represent mortalities which could not be verified with an in-hand dead specimen. The highest mortality (23% at 96 hours) occurred in the 60% OAW treatment. Sublethal effects as determined by polychaete activity could not be determined for any of the treatments. The number of polychaetes which were extended from their burrows after 24 hours was minimal for all treatments. Burrow openings and fecal pellets around openings were recorded, but artifacts of the previous day's activity could not be distinguished from the current day's·. In general, activity increased in all treatments after 48 hours then decreased through 96' hrs below the initial level. Qualitative observation of polychaetes after retrieval from experiments and prior to and during respiration measurements showed the 60% OAW organisms to be in poor condition, alive but motionless upon stimulation, and covered with mucus and attached sand grains and debris. The polychaetes from the control dishes, however, were robust, active, in good condition, and free of mucus.and debris'! Shrimp Percent mortality for the shrimp at the various treatment levels are recorded below: %Mortality Treatment '24-hr 48-hr · 72-hr 96-hr Control 3 6 6 6 5% OAW 3 3 3 3 15% OAW 0 0 0 0 30% OAW 3 3 3 3 60% OAW 9 9 9 9 All test animals were retrieved from the experiments. The highest mortal ities (9% at 24 to 96-hr) occurred in the 60% OAW treatment. E-11 Sublethal effects as determined by shrimp burrowing activity for 30-min of light exposure were not quantifiable. Initially most shrimp would burrow. Then within the 30-min period, others would come out of the sediment and burrow back up or remain on the surface of the sediment. _ After 96-hrs the shrimp were exposed to light for 5-min. and percent burrowing was recorded. None of the burrowed shrimp resurfaced during this time. This method was a more accurate representation of activity for the shrimp and the results are recorded below: % Burrowed % Burrowing % Burrowing at Treatment Initially w/in 5 min. end of 5 mins. Control 41 42 75 5% OAW 31 59 59 15% OAW 47 59 69 30% OAW n 43 90 60% OAW 71 50 93 In most treatments, approximately 50% of those above the sediment would burrow within the 5-min. exposure to light. Number of burrowed shrimp was highest in the 30 to 60% OAW treatments, both before and after exposure to light (5 mins.). Oxygen Consumption 1 Oxygen consumption (ppm 02 hr-1 g-) for organisms taken from various oiled treatments after 96-hrs are recorded in Table 1. The only measurable difference~ that wer~ shown to be significant (P < 0.05) between the control and any oiled treatment were for the mole crab. There appeared to be an increase in oxygen uptake in the oiled vs. the control treatments for the surf clam and shrimp but these differences were not significant. Oxygen consumption was lower for the polychaetes treated with 60% OAW than for the control and may possibly be a refelction of the poor physical condition of these worms as noted in the toxicity results section. E-12 Table 1. Oxygen consumption for experimental animals removed from control, 15% and 60% OAW treatments after 96 hours. Data are reported in 1 ppm 02 hr-1 g-• Organism TREATMENT Control 15% OAW 60% OAW Mole Crab* 20.14 24.88 26.95 18.24 18.47 15.32 x 23.99 17.34 Surf Clam 6.44 10.12 11.39 9.80 13.36 15.18 x 9.32 12.78 Polychaete 90.00 142.86 85.37 90.90 x 116. 43 88.14 Shrimp 1. 62 1.05 1.35 1.67 1.84 1.32 1.29 ~ 1.39 1.68 x 1.34 1.61 1.45 *Significant difference at P < 0.05 E-13 DISCUSSION The complexity of assessing the impact of petroleum hydrocarbons on the marine biota investigated~~ here was well documented by the results of these tests. Few observable impacts were noted and a problem exists in extrapolating these results to the real ecosystem. We did not determine letha:L. doses of the IXTOC I oil accomodated water to the biota studied. This could be related to the physical nature of the oil tested. It is also possible that the solutions tested (i.e. 5 to 60% of 25 ppm) did not mimic actual concentrations that are presently occurring in the Gulf of Mexico and on Texas beaches. Although by no means significant, we did observe a greater amount of mortality in the polychaetes than in any other fauna. This may be related to their feeding methods. The polychaete was the only one of the three beach fauna tested that was a surface deposit feeder. The other two were both suspension feeders. Feeding from the sediment surface, the polychaete may have injested the numerous micro-tarballs that were suspended in the OAW at treatment initiation and later settled to the sediment surface. This may help to explain the poor health of the worms in higher treatment concentrations as noted in the results. These tests also did not evaluate any of the sublethal biological effects on reproductive success, long-term health of the organisms, accumulation of _toxic substances, growth and development, or histopathological conditions. Many of these biological aspects may have been affected from chronic exposure to the Mexican oil. All that can be concluded here is that at the concentrations tested, the fauna examined appeared to show very few acute effects from oil treatment. Although these tests were not conclusive in terms of any significant E-14 mortality effects from acute exposure to the Mexican oil tested, because of the numerous unknowns including some of the questions raised above, we urge that care be taken to protect the south Texas marine environment from potential unknown effects related to repeated exposure. SECTION F EFFECTS OF IXTOC I GULF OF MEXICO OIL SPILL MATERIALS ON THE BEHAVIOR AND RESPIRATORY METABOLISM OF THE SPOTTED SEATROUT (Cynoscion nebulosus) Report Submitted by: Donald E. Wohlschlag Faust R. Parker, Jr. University of Texas Marine Science Institute Port Aransas Marine Laboratory Port Aransas, Texas 78373 August 24, 1979 ABSTRACT Exploratory experiments with a seawater extract {30 mg 1-1 oil) of IXTOC I crude oil "mousse" were behaviorly deleterious to subadult spotted seatrout {Cynoscion nebulosus) at about 7.5 mg 1-1 or higher immediately and after 24 hrs at 0.3 mg i-1 or higher. A similar experiment with fingerlings indicated a 96-hr TLM level in the range of 1.3 -7.5 mg 1-1, with about 10 g or smaller fish the more sensitive. Blazka respirometer experiments at near ambient temperatures of 28°C and at 35 o/oo salinity to measure metabolic levels up to maximum sustained swimming velocities indicated lethality within about two days at 5% dilutions of the seawater extract. Similar Blazka respirometry indicated that 1% dilutions would be effective up to about 4 days without perceptible morbidity. At 1% average maximum sustained swimming velocities were reduced 85% to 2.8 lengths sec-1 from 3.3 L sec-1 for the controls. From 2 to 7 days the performance levels decreased, but not from 2 to 5 days for the controls. During these periods the usual stress symptoms of behavioral and physical deterioration appeared after about four days at the 1% concentration, but not for the controls. Multiple regressions relating oxygen consumption rate to the body weight-and swinuning velocity made comparisons possible between control and experimental fish. At the average weights of the fish in each of the regressions the oxygen consumption rates were calculated for the active rates at average maximum sustained swimming velocities and at standard, zero velocity, rates. The oxygen consumption rates as metabolic rates per kilogram yielded metabolic scope values as the difference between active and standard rates. The pollutant reduced the scope to at least 89% of the control values, even though the "standard" was reduced from that of the controls. For larger ~ish (500 g) the same calculations of active and standard rates from the regressions yielded a scope reduction to 67% of controls. Thus it appears that larger fish, along with very small fish, are progressively more sensitive to the toxic materials. The relationships of the regression constants and statistics indicate that metabolic responses of the spotted seatrout to the toxicants are generally more variable than to control conditions. These preliminary ·experiments suggest that the Blazka respiratory metabolism technique of determining metabolic scope and its reduction under sublethal stresses is highly sensitive and quantitative. It can be used when there is no a pr·iori chemical information on the nature and effects of toxic materials. F-3 INTRODUCTION This s.tudy was designed to contribute preliminary information on toxicity levels and sublethal respiratory metabolic effects of Bay of Campeche crude oil on Gulf of Mexico fish species. The spotted seatrout (Cynoscion nebulosus) was the fish of choice due to its occurrence in both nearshore and estuarine environments. It is a sensitive, euryplastic species on which a considerable amount of baseline data have been acquired in studies by Wohlschlag and Wakeman (1978) and Wohlschlag and Parker (in progress). Initial toxicity tests were conducteq on adult c. nebulosus at various concentrations of oil accommodated in water (OAW) prepared from samples of "mousse" collected by the U.S. Coast Guard A second set of toxicity tests were made Cutter Point Baker. utilizing fingerling or underyearling trout to ascertain size-related Rationale for these experiments included determination of effects. toxic concentrations of the spill material and establishment of appropriate concentration levels for investigation of sublethal effects on the respiratory metabolism and swimming performance of C. nebulosus. Respiratory metabolic responses of marine fish have been shown sufficiently sensitive to sublethal pollutant levels to be useful in biological monitoring (Wohlschlag et al. 1978; Wohlschlag and Parker 1978). The use of respiratory metabolism is based on the fact that it is often measurably influenced by toxic substances in dilutions far below usually perceived lethal concentrations. The use of respiratory scope --the difference between oxygen consumption at the maximal sustained aerobic activity and at the F-4 minimal maintenance, or standard, level --for the assessment of environmental quality was suggested by Fry (1947, 1957, 1971}. Theoretical and empirical studies indicate that metabolic scope tends to be reduced by stresses when standard rates may be increased, active rates reduced, or both. Wohlschlag et al. (1978} and Wohlschlag and Parker (1978) have demonstrated scope reductiorls in the red snapper ·(Lutjanus campechanus) exposed to sublethal concentrations of ocean dumped industrial wastes. Ecologically, most fishes generally operate at a routine rate that lies-between the standard and maximum. This rate is operationally minimal and is around twice the standard level to account for about 1 length sec-1 swimming (foraging} , specific dynamic action (assimilation} and other functions, excluding growth, spawning, extended migrations, etc. (Fry, 1971; Kerr, 1971: Mann, 1969; Winberg, 1956; Wohlschlag and Wakeman, 1978}. Stresses also can depress routine metabolic rates (Beamish, 1964; Wohlschlag and Cameron, 1967; Kloth and Wohlschlag, 1972; Cech and Wohlschlag, 1975}, although a depressed routine rate appears to be less definite than scope for maximal sustained activity for species that may have maximal swimming metabolic activity levels 4 -8 times standard levels (Randall, 1970). The specific aim~ of these studies were to use the spotted seatrout, a well known commercial and recreational species, as a test organism to make preliminary determinations: 1. On acutely toxic levels of oil spill materials on adults and fingerlings or underyearlings; 2. On metabolic results at active and standard (or resting) levels for detection of scope diminution even though the chemical composition of the spill material could be considered unknown; F-5 3. On what levels of the spill material produce observable metabolic depression; 4. For additional information of basic energetics data on a species of general importance in fishery and ecological considerations. MATERIALS AND METHODS Spotted seatrout were used throughout the study and were captured near Port Aransas, Texas by hook-and-line or seine. Capture temperature (30C) and salinity (32 o/oo) were near test levels for respiration experiments but toxicity test fish required additional acclimation to the lower (23C) . room temperature used. Fish were transported to the laboratory in insulated boxes and placed in indoor holding tanks for acclimation to the appropriate test condition. Any fish that behaved abnormally or appeared unhealthy were discarded. I The preparation of the oil spill material to produce a stock "100% . solution" was carried out as follows: Point Baker "mousse" was blended with seawater from laboratory settling tanks at 35 o/oo. No measured amounts of "mousse" were used; the procedure involved high speed blending of the oil in water for 45 seconds, after which the water was allowed to clear for a few seconds, then the water was siphoned into a holding container. This water fraction will be referred to as oil accommodated in water (OAW) . After a sufficient quantity of this solution was collected it was diluted with two parts seawater to produce what we will call a 100% solution. Prior to the onset of each test and at the termination point, samples of each appropriate dilution were taken for later chemical F-6 analysis. Initially the concentration of the stock was 27 mg i-1 oil for the adult fish toxicity tests and 30.1 mg 1-1 for the fingerlings and underyearlings. Analyses have been made by Dr. Kenneth Winters and further detailed chemical analyses are pending. Toxicity tests on adult C. nebulosus were conducted in 20gallon glass aqua~ia equipped with aerators. Six dilutions were used (100%, 50%, 25%, 12.5%, -5% and 1%); control fish were maintained in a larger holding tank. Observations were recorded for a 96-h period on each of the 6 tanks which contained 2 fish of 100 -200 g weight. No temperature control was available for toxicity tests, so that room conditions dictated 23C and a salinity of 35 o/oo was used throughout all tests. Fingerling or underyearling fish tests were carried out in 6 20-gallon aquaria at 5 dilutions (100%, 50%, 25%, 12.5%, and 5%) and one control tank. Again observations on behavior and any At the termination fatalities were recorded for a 96-h period. of all experiments, fish were weighed and lengths measured. Specimens were then frozen for later inspection. From the behavioral observations on the adult fish it was determined that respiration experiments should be attempted at 5% of the 100%.dilution. However this dilution produced 100% mortality in 63.5 hours at 28C and 35 o/oo during a 2-day+ acclimation period. The test dilution was then lowered to 1%. Fish were held in a temperature controlled circular tank at 1% OAW dilution for 2 days prior to testing. Fish were fasted during this period and a slow current within the circular tank provided some acclimation to swimming before respiration measurements. The holding tank and respirometer tank were well aerated throughout experiments. Active and resting metabolism rates were made in a 207-1 Blazka chamber (Blazka et al., 1960; Fry, 1971) as utilized by Wohlschlag and Wakeman (1978) • The entire chamber was immersed in a 3,024-1 temperature-salinity controlled system, which was a contiguous part of the circular holding tank. No filtration system was utilized for these tests. Fish were maintained for 2 days swimming at low velocities (about 1 L sec-1) prior to active measurements. After swimming in the chamber at an intermediate speed for a period of time sufficient to calm the fish, the velocity was increased gradually until the fish "broke" pace. At this instant the velocity was lowered (usually quite slightly) to the highest possible velocity at which normal swimming persisted without breaking. With this "training" regimen, the maximum sustained velocity could be reproducible for each fish. The Umax (total lengths sec-1) swimming velocity was determined, after which the fish was tested for at least 1 h for a consistent Umax· Following the 1 h or longer runs, the fish were left in the chamber at intermediate and/or zero velocities with oxygen rate measurements to detect any respiratory irregularities that could have resulted had the Umax been associated with undesirable anaerobic metabolism. Oxygen consumption rates were measured by withdrawal of small samples for use in a Radiometer model E-5046 with a PHM 71 electrode equipped with acid-base analyzer. Following completion of a set of experimental oxygen consumption measurements, the fish were removed and lengths and weights recorded. Along with lengths, weights, oxygen consumption rates, and swimming velocities (total lengths sec-1), salinities and temperatures were recorded to 0.1 ppt and O.lC. From this, a simple multiple regression was calculated at control and experimental conditions in the form: Y= a +bwXw + bvXv where: Y= expected 02 consumption rate in log10 mg02h-l, a = constant, Xw = log10 weight in grams, Xv = 1 sec-1. The various b values are the respective partial regression coefficients. Simi~ar procedures have been used by Wohlschlag and Juliano (1959), Wohlschlag and Cameron (1967), Wohlschlag and Cech (1970), and others. Temperature and salinity values remained near 28C and 35 ppt respectively and were not included in the regression calculations. Control data were acquired from a study on ocean dumped pharmaceutical wastes by Wohlschlag and Parker (in progress) . Standard metabolic rate determinations were made from the appropriate active regression equation utilizing the Brett (1964) technique. This involves drawing a line parallel to the active regression line through the lowest Umax value and using the Y intercept value as a realistic estimate of the standard rate. F-9 RESULTS The preliminary experiment to assess the toxicity of the oil to subadult fish in 20-gallon aquaria accommodated water (OAW) indicated that 100%, 50% and 25% dilutions were definitely At dilutions deleterious --at least behaviorly over 96 hours. of 12.5%, 5% and 1% adverse effects usually appeared about 24 h later. The descriptive details are in Appendix Table 1. The second preliminary experiment to assess 96 h toxicity to fingerlings with similar but more definitely lethal results .. From the crude results is described in Appendix Tables2 and 2a. a 96 h TLM level would be a range from 5-25% dilution (1.3 -7.5 mg 1-1). The smallest of the fish generally died first; that would indicate a critically susceptible size smaller than about 10 g, although there may have been uncontrolled variables from one aquarium to the next. The first Blazka respiratory measurement attempts under controlled conditions failed during acclimation (habituation) to a 5% OAW level when some deaths occurred. In the appendix (following Table 2a) are appropriate notes that indicate severity of the stress at 5% OAW. Accordingly the remainder of the results deal with respiratory metabolism measured under control or 1% OAW conditions in the Blazka apparatus. The more definitive results from the Blazka chamber experiments yield the following equation for control data: y = 0.06995 + 0.71242 Xw + 0.11192 Xv, (Eq. 1) where the average weight was 114 g and the average of 14 determinations at Umax was 3.3 L sec-1. Total N = 34. The data at 1% OAW yield: ~ = 0.87662 + 0.36468 Xw + 0.11381 Xv, (Eq. 2) where average weight was 164 g, average of 10 Umax measurements was 2.8 L sec-1. Total N = 27. The following schedule of statistics for these equations are also useful: Eq. N R p p 1 34 0.92 0.07901 0.12416 <0.001 0.0092 <0.001 2 27 0.77 0.13624 0.02123 <0.001 0.0190 <0.001 Fig. 1 is the plot of oxygen consumption rate of all control fish (calculated from Eq. 1 and adjusted to the average weight of 114 g) against observed swimming velocities. A Y = 1.53533 + 0.11192 Xv· The equation is The Brett (1964) extrapolated standard metabo lism rate is 29.17 mg o2h-l or 256 mg o2kg-lh-l. Fig. 2 is the plot of the oxygen consumption of the experimental fish (calculated from Eq. 2 and adjusted to the average weight of 164 g) against observed swimming rates. The equation is A y = 1.68433 + 0.11381 xv. By the Brett (1964) method, the extrapolated standard rate is 35.08 mg o2h-l or 214 mg kg-lh-1. Some pertinent notes on the condition of the fish in the Blazka chamber experiments are most revealing. For 2 days initial acclimation and 7 more days for making the metabolic performance 200 2.2 . 100~ 6 ~ '..c ~ • @ @ ~2.Q C\J 50 ~~- --------- (!) I ___ -------• -- 1-8 C\J O • ------• 00 ttj i'k_____ • Eor ~ E 251 0 Ol ~ l4 l2 10 lO 0 1 2 3 . -1 VELOCITY ( Lsec ) Fig. 1. Calculated oxygen consumption rates for control spotted seatrout at 114 g average weight plotted against observed swimming rates. Estimated standard rate shown b¥ arrow (see text) • Encircled points are for maximum sustained (Umax) swimming performances. 200 2.2 0 ------100 • 0 • • (;r@ ~2-o 0 ----------- •I 0 • • 0 ~ I II ' ---0 ~ £ ..c 50~ ----l 8 (\J (\J • --• 0 O •I --_..---• a a> ik----. E ~ I E '1 lG 0 ~ 25l a0 ~ l4 l2 10 lO 0 1 2 3 VELOCITY ( L sec -1 ) Fig. 2. Calculated oxygen consumption rates for spotted seatrout in 1% crude oil extract at 164 g average weight plotted against observed swimming rates. Standard rate estimate shown by arrow (see text) . Encircled points are for maximum sustained (Umax) swimming performances. runs, there was no perceptible deterioration with Umax values showing no trend downward. The fish in the 1% solution showed no deterioration for the first 4 days, during which no abnormal effects were observed during the 2-day acclimation period and the first 2 days of experiments. By the fifth day, fish in the Blazka chamber swam less well and appeared to have equilibrium problems. Fish were definitely sluggish in the sixth day with obvious prevalence of tail rot. Of three fish remaining on day 7 one died, one failed to swim faster than 1.6 L sec-1 and was afflicted with tail rot, gill lesions and-. coughing spasms, and the last fish with the same afflictions swam weakly at only 1.5 L sec-1. DISCUSSION General Remarks The initial trials which indicated a 1% OAW extract was adequately toxic, were apparently misleading, inasmuch as cumulative effects did not become evident until after 4 days. Just what protocol should be used for evaluating delay reactions to unknown toxins at unknown concentrations is not clear at this time when relative concentrations, exposure time and effects in the open ocean are unknown. Some crude oil spill pertinence to fishes is available from the literature, however, even though marine sublethal effects on fishes may be unknown in quantitative terms. Also very little laboratory information is available for larger fish. McKeown and March (1978) have observed severe damage to gills in rainbow trout (Salmo gairdneri) exposed to Bunker C oil. Minchew and Yarbrough (1977) found fin erosion in Mugil cephalus exposed to 4-5 mg 1-1 crude oil in estuarine pond ecosystems. Sindermann (1978) reviewed the recent literature on the generalized nature of fin rot occurrences in degraded estuarine and coastal systems. The coughing response has been shown to be directly related to concentrations of toxic substances (Barnett and Toews, 1978; Carlson and Drununond, 1978; among others). Any of these, or other similar deficiencies, would be expected to suppress metabolic scope. Choice of Experimental Concentration of Seawater Extract From the descriptions of the preliminary toxicity tests and the Blazka chamber respiratory experiments, there is no obvious biological clue as to a "good" concentration or a "good" exposure interval. Whatever the identifications of toxic materials are, it is apparent that a 5% extract is acutely toxic within about 2 days. In about 7 days the 1% dilution could be considered acutely toxic. Whether the acute toxicity depends upon bioaccumulation past a threshhold concentration, or the progressive breakdown of a biochemical system once a toxin or combination of toxin initiates a degradational process, should provide an important point of departure for future experiments. Such experiments should provide contrasting acute and sublethal chronic toxicity levels with the sublethal levels (1) low enough to allow for flow-through experiments conducted at a continuously added, constant pollutant level and (2) low enough to detect further degradation, if any, by a single initial addition of the pollutant. The experimental concentration of the pollutants in this study yield about what might be ordinarily expected of sublethal levels. The Blazka respirometer experiments as summarized by Equations 1 and 2 and the plotted processed data in Figures 1 and 2 show that the variability of the controls is considerably less with high R multiple correlation and low standard error of the regression, sv. The greater variability of the plotted data in Fig. 2 show up at Xv = 0 and at the maximum sustained (encircled) values as compared to the Fig. 1 control data. The greater variability of the standard error (sbw) fo~ the bw of the control (0.12 compared to 0.02) is not clear, although similar ranges of standard errors are common and may be associated with the relatively smaller average size Xw = 114 g and the smaller range of weights for the control fish. Relative Scope Depression at Observed Average Weights Average weights of 114 g for controls in Equation 1 and 164 g for 1% Ixtoc I samples in Equation 2 reveal somewhat emphatically the differences between control and experimental metabolic levels when the average maximum activity is decreased from 3.3 L sec-~ to 2.8 L sec-1, or to 85% of the maximum sustained value. At 114 grams, Eq. 1 yields 704 mg o2 kg-lh-1 at maximum activity, Xv = 3.3 L sec-1. From Fig. 1, extrapolated from the minimum active level of this zero (standard) level by the Brett (1964) method, the standard rate is 256 mg 02 kg-lh-1. The difference, or scope, is 448 mg 02 kg-lh-1 (704-256). At 164 g, Eq. 2 (1% Ixtoc I) yields at Xv = 2.8, 614 mg o2 kg-lh-1, which is considerably depressed• . A corresponding Brett type of extrapolation gives a standard rate of 214 mg o2 kg-lh-1, somewhat lower than the control as might be expected for continuously stressed fish. The difference, 614-214, 400 mg o2 kg-lh-1 is a scope that is about 89% of the 448 control values. The depression in the bw coefficient in Eq. 2 to 0.36 compared to 0.71 in Eq. 1 indicates that polluted waters adversely and selectively affect the larger fish. This situation has been repeatedly observed both in current studies in progress on effects of industrial wastes and in published studies by Wohlschlag and Cameron (1967), Kloth and Wohlschlag (1972), among others. For this study an extrapolation from Equations 1 and 2 to a larger size, say 500 g, is instructive. For control fish the scope is (460 -167) = 293 mg o2 kg-lh-1 with Xw = 500 g and Xv = 3.3 L sec-1 in Eq. 1. For the oil exposed fish, the scope is (302 -105) = 197 mg o2 kg-lh-1 at Xw = 500 g and Xv = 2.8 L sec-1. Thus for a 500 g fish the oil exposed fish have a scope value that is about 67% of the control fish. Clearly the implications are that even these low levels of chemical toxicants in the 1% solution of the mousse water phase can have a severe effect on the overall metabolism of organisms. In fisheries the disappearance of larger members with exploitive stresses is well known, but little work has been extended to show how natural or pollution stresses at very low, sublethal levels can have the same ultimate effect, i.e. the older and larger members tend to disappear from a population structure while the younger and smaller members survive --providing some recruitment is maintained, of course. LITERATURE CITED Barnett, J. and D. Toews. 1978. The effects of crude oil and the dispersant, Oilsperse 43, on respiration and coughing rates in Atlantic salmon (Salmo salar). Can. J. Zool. 56:307-310. Beamish, F. W. H. 1964. Respiration of fishes with special emphasis on standard oxygen consumption. Can. J. Zool. 42:177-188. Blazka, P., M. Volf, and M. Cepela. 1960. A new type of respirometer for the determination of metabolism of fish in the active state. Physiol. Bohemoslov 9:553-558. Brett, J. R. 1964. The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Bd. Can. 21:1183-1226. Carlson, R. W. and R. A. Drummond. 1978. Fish cough response -A method for evaluating quality of treated complex effluents. Water Res. 12:1-6. Cech, J. J., Jr. and D. E. Wohlschlag. 1975. Summer growth depression in the striped mullet, Mugil cephalus L. Contr. mar. Sci. 19:91-100. Fry, F. E. J. 1947. Effects of the environment on animal activity. Univ. Toronto Studies Biol., Ontario Fish. Res. Lab. 68:1-62. Fry, F. E. J. 1957. The aquatic respiration of fish. pp. 1-63. In: M.E. Brown (ed.) The Physiology of Fishes. Academic Press, New York. Fry, F. E. J. 1971. The effect of environmental factors on the physiology of fish. p. 1-98. In: W.S. Hoar and D.J. Randall (eds.} Fish Physiology, Vol. 6, Environmental Relations and Behavior. Academic Press, New York and London. Kerr, s. R. 1971. A simulation model of lake trout growth. J. Fish. Res. Bd. Can. 28:815-819. Kloth, T. c. and D. E. Wohlschlag. 1972. Size-related metabolic responses of the pinfish, Lagodon rhomboides to salinity variations and sublethal pollution. Contr. mar. Sci. 16: 125-137. Mann, K. H. 1969. The dynamics of aquatic ecosystems. Adv. Ecol. Res . 6 : 1-81 • McKeown, B. A. and G. L. March. 1978. The acute effect of Bunker C oil and an oil dispersant on: 1 serum glucose, serum sodium and gill morphology in both freshwater and seawater acclimated rainbow trout (Salmo gairdneri). Water Res. 12:157-163. Minchew, C. D. and J. D. Yarbrough. 1977. The occurrence of fin rot in mullet (Mugil cephalus} associated with crude oil contamination of an estuarine pond-ecosystem. J. Fish. Biol. 10:319-323. Randall, D. J. 1970. Gas exchange in fish. pp. 253-29 2. ·rn: W. s. Hoar and.D. J. R~ndall (eds.} Fish Physiology, Vol. 4, Academic Press, New York. Sindermann, C. J. 1979. Pollution-associated diseases and abnormalities of fish and shellfish: A review. Fish. Bull. 76 (4} :717-749. Winberg, G. G. 1956. Rate of metabolism and food requirements of fishes. Fisheries Research Board of Canada Translation Series 194:1-253. Wohlschlag, D. E. and J. N. Cameron. 1967. Assessment of a low level stress on the respiratory metabolism of the pinfish (Lagodon rhomboides). Contr. mar. Sci. 12:160-171. Wohlschlag, D. E. and R. o. Juliano. 1959. Seasonal changes in· bluegill metabolism. Limnol. Oceanogr. 4:195-209. Wohlschlag, D. E. and F. R. Parker Jr. Metabolic sensitivity of fish to ocean dumping of industrial wastes. First International Ocean Dumping Symposium, University' of Rhode Island, w. Alton Jones Campus, Kingston, Rhode Island, October 10-13, 1978. Wohlschlag, D. E., F. R. Parker, Jr., J. R. Burns and J. A. Kinney. 1978. Sensitivity of open Gulf fishes to ocean dumped wastes. In: Studies of chemical and biological process effects of industrial wastes released into the Gulf of Mexico by ocean dumping. Report to The Department of Commerce, National Oceanic and Atmospheric Administration, Ocean Dumping Program, Rockville, Maryland. pp. 84-143. Wohlschlag, D. E. and J. M. Wakeman. 1978. Salinity stresses, metabolic responses and distribution of the coastal spotted seatrout, Cynosc!on nebuiosus. Contr. mar. Sci. 21:171-185. APPENDIX TABLES AND NOTES ... Appendix Table 1 Observation& of Q. nebulosua placed in various concentratlona of Pcmex Gulf of Mexico apUl, otl accornodated in water. Two fish placed in each 20 gallon aquaria at 1730 houre on 8/7/79. . OBSERVATIONS at 23C and 35 o/oo Time Tank# l* 2• J* 4 5 6Cone. 1001. 507. 257. l2.5't Sl 17. 1750 ' head shaking alive equilibrium normal normal normal(8/7) convulsing poor vie. loss, headshaking 1830 Coughing Coughing Coughing normal nonnal normal(8/7) irregular irregular ventllatlon ventilation 1-rj 2010 Coughing Coughing Coughing normal labored normal I(8/7) irreg.-labore4 irregular N irregular ventllatlon very ~ ventllatlon ventilation ventilation como. to ll calm 2130 A• above increased As above normal one flah As above(8/7) coughing moving some,irrea. vent. seems stres1 ' 2230 Aa above As above as above As above Large fish A1 above(8/7) swlmningunnaturally 0035 Ae above 88 above a8 above A1 above As above Ae above(8/8) 0215 Irregular As above Labored Coughing Coughing As above(8/8) ventllatlon ventilation Venttlotlon Irregular Largo fish . Coughing not forced Ventilation Actlvc/labo 2-3 tlrnca/min red Vent. Appendix Table 1 (cont.) . OBSERVATIONS at 23C and 35 o/oo . Time Tank# t* 2• 3• 4 s Cone. 1001. 6 507. 251 12.5'1 Sl 11. 0425 . A• above Aa Above As Above Aa above (8/8) Aa above Aa above Water Clearln1 Water r.1 ..n..-fno 0630 Fish Active Fish Active Fish Active (8/8) Coughing Calm Fish Active Flsh Active Coughing ClearinR Cont, ClearlnR ClearinR Cont 0900 l"ij Host Active Rapid ventll. As above VentU. Slight. (8/8) Mouths Agape Calm Calm I high, but caln &nonnal &normal N Ra2id Ventll1 N 1030 Active Active Active Calm(8/8) Rauid Ventll. kapid Ventll. Ventll Norm. As Above A11 above Nonnal 1300 As Above As Above As Above As above (8/8) · As above Aa above 1400 As Above Aa Above Moderate Ventll. Above (8/8) Slightly Aa above Coughing Coughing Ventilation norm., Active active -·--· Cousd1lnR 1515 .. Ventilation and ~decreaalng to ..... Calm r (8/8) Activity highest -I I . *Fins erect at higher concentration quite frequentlyr rarely at lower concentrations. **These conditions persisted until fish were terminated on 8/11/79 at 1730 hours.96 hours exposure. Appendix Table 2 Observations of fingerling Q. nebulosua placed in various concentrations of Pemex Gulf of Mexico Oil Spill moterinl occomodated in water. Six fish placed in each concentration at 1615 hours on 8/14/79. Observations at 23C and 35 o/oo Time Tank# 1 2 l 4 s 6 Cone. 1001 507. 257. 12.51 51 Control. .. .... ---·· Sluggish Swinning in Rapid Vent., Alert and ln2035 Rapid Some Coughing (8/14) !Sluggish spurts, one Coughing good condition dying, coughin1 , Sluggishrapid vent. 2400 u u II ti ti ti (8/14) One Dead One Dead 0300 ti II l'%j " II II ti I(8/15) One Dead N w 0515 Pne dying, Coughing, Coughing and Coughing, Alert and (8/15) !Ventilation Active Active : Calmer than Active ti llopid and !;wimmln9 1,2,l Labored ti 0830 Pne Dead " .. Call'ft Cal111 (8/15) " sooo II (8/15) ti " " " " 1100 II .. II II (8/15) ti " 1400 -" " " " " (8/15) " 1600 u II II u II " {8/15) 2050(8/15) " " " " •• ti Tank Clearing Tank ClenrlnR Tank ClearinR ~ppendix Table 2 Time Tank# Cone• . 0800 (8/16) 0930 (8/16) I 1115 (8/16) 1300 (8/16) 1400 (8/16) 1500 (8/16) 2100 (8/16) 0815 (8/17) 0930 (8/17) 1100 (8/17) 1300 18/17) (cont.) 1 1001. II Two Near Surfa~e Bad Shaoe One near Surfate' One Dead Others 11 One Dead " One small fish looks bad, rest " " ti Raoid Vent. II SWflllllin~ hard " " Observations at 23C and 35 o/oo 2 3 4 50'Z 257. 12.51 . II II " One Dead One Dead II II II II II " " II II .. " " II " " .. " " II II " Rapid Vent. Two Dead II II " Rauld vent. II II " II " " s 51 II Two Dead " ti " " " " " " " " 6 Control " " " " t'tj I N " .i:- " One dead Rest Healthy " II " " Appendix Table 2 (cont.) Observations at 23C and 35 o/oo Time Tank# 1 2 3 4 5 6 Cone. 1001. 501 251 12.51 51 Control II II II ti .. 1600 I " (8/17) One Dead . ti ... 0800 " " " " ' (8/18) One Dead II ti II 1030 " " " t'rj(8/18) I One Dead Five fish alive Three Alive, 'Ihree Alive, Twc 111ree Alive Five fish in N 1615 One Live fish all very sluggish Healthy and healthy, one and healthy excellent Ln (8/18) remains, alugg-Active. small -sluggisl shape TERMINATION lab, mod. tall rot. TOTAL 5 l* 3 J. 3 1 FATALITIES * Thia tank cleared very feat and dld not exhibit a toxicity effect ln line wltla the other concentrations. It was noticed that this system had a very high rate of aeration compared to the other tanks, which may have resulted in a much faster breakdown of toxic components of the oil mixtuee. Appendix Table 2a Weight, standard and total lenqths for subadult £· nebulosus utilized in toxic concentration studies on Mexican oil spill materials. Fi.sh Killed Cone. Weight (g) Std. Langi:h Total Length A.vg. Wt. (Cm) (QU) (g) LO~ l) a.s a.a 10.9 2) 4 • .5 6.8 8.6 3). 7 • .3 8.2 9.7 a.a 4) 3.0 6.0 7.6 5) 20.6 11.4 1.3.8 501. l) 4.2 6.7 8.4 4.2 251. l) 4.J 6 • .5 1.S 2) 6.2 7.l 8.7 5.4 3) s.a 7.0 8.5 12..5'?. l) 5.4 6.9 8 • .3 2) s.o 6.7 8.l 4.l 3) 2.0 5 • .3 6.5 l) 6.9 7.2. 8.62) 2S.9 12.2. 15.0 13.4 3) 1.3 7.7 9.3 01. (Conttol) l) 6.7 7.7 9.4 6.7 Fish Alive 1001. 1) 40.l 14.8 17.8 40.l 501. 1) 30.7 12.7 1.5•.5 2) 19.l ll.2. 13.7 3) 8.9 8.6 10.6 14.8 4) 9.8 8.7 10•.S 5) 5.6 7.4 9.l 251. 1) 34.7 13.0 l.S.8 2) 26.9 12.4 15.0 25.9 3) 16.0 10.3 12.4 12 • .51. l) 8.4 8.4 10•.3 2) 6.l 7.7 9.5 9.5 3) 14.0 9.8 12.2 51. l) l.S.O 10.0 12.S 2) 10.0 8.4 10.4 10.l 3) 5.2 6.9 8.8 ~ (Couttol) l) 22.l ll.4 14.0 2) 14.6 10.4 12.7 3) ll.O 9.2 11•.S 12.0 4) 7.6 8.1 10.l 5) 4.9 6.9 8.6 F-27 Appendix Note Blazka Acclimation Notes l. Nine fish exposed to 5' Ixtoc I oil accommodated in water. This 5' •mixture• w.. prepared by blendinq •mous~e· with water to obtain a concentrated sample .which was then diluted with two parts seawater to. obtain what we will rater to u a 100' sample. Analysis of the content of these samples are underway at this time. 2. Initial. •Xi>O•ur• at 1630 hours on 8/lJ/79 in the Blazka exercise tank. T~rature 28C; Salinity JS o/oo. 3. On 8/15/79 at 0830 hours four fish ware dead.. Three fish swimminq on surface ra~id ventilation, mouths aqape, sluqqish. 4. 8/lS/79 at· lSOO hours -TWo more fish dead. S. At 0800 on 8/16, finai t1fO fish dead. 6. Data on fish: Wuqht. (q) T.L-(cm) s .r.. (cm) i1s.a 28.0 24-.S 90.6 23.8 20.7 178.2 28.4 2S.8 166.4· 27.8 24.5 142.6 26.9 23.S 109.• 8 24.0 21.S lOQ.2 23.3 20.0 133.l 2S.7 22.2 l09 .2 24-.6 20.6 ~: l. All fish exhibited liqht to moderate tail rot. Oil and lesions on qill.s. 2. TotaJ. exposure time • 63.S hours. 3 •. Stat:ic taats at 23 •c -Blazka acclimation at 2s•c Sr. difference + swimminq stra•s pr.educed by currant apparently were too severe.