• • • • • • • • • • + • BIOLOGICAL PROCESS EFFECTS OF ORGANIC WASTES AT PUERTO RICAN OCEAN DUMPING SITES --Continuation Studies DRAFT FINAL REPORT 6 June 1980 Supported by Grant No. 04-8-MOl-54 1 March 1979 to 30 April 1980 from The Department of Commerce National Oceanic & Atmospheric Administration Ocean Dumping Program Rockville, Maryland to The University of Texas Marine Science Institute Port Aransas Marine Laboratory Port Aransas, Texas 78373 D. E. Wohlschlag, Principal Investigator • THE UNIVERSITY OF TEXAS • MARINE SCIENCE INSTITUTE Port Aransas Marine Laboratory June 6, 1980 Port Aransas, Texas 78373 Phone512 749-6711 • • Ocean Dumping Program National Ocean Survey NOAA, U.S. Department of Commerce Rockville, Maryland 20852 Attention: Dr. Thomas P. O'Connor • Dear Torn: Enclosed are three copies of a draft final report on the continuation of Grant No. 04-8-MOl-54 "BIOLOGICAL PROCESS EFFECTS OF ORGANIC WASTES AT PUERTO RICAN OCEAN • DUMPING SITES--CONTINUATION STUDIES" with additional copies available at your request. Please accept my apologies for the delay in getting this report to you. • Note that the report is in three sections: rnicroalgae, invertebrates, and fishes. I should like to call your attention to the :possibilities that continuous ocean dumping introduces a series of small pulse mortality situatiomto fish populations in • addition to the possibilities of general low level, sublethal chronic stresses. Some of the people interested in population dynamics models might be able to explain with simulations the relative differences in outcomes of subacute toxicities versus chronis sublethal situations. • I shall be interested in your comments and general critique. We send our very best regards, Cordia~,-; • Donald~hlag Professor and Principal Investigator • Encl: 3 report copies • • CONTINUATION STUDIES • --MICROALGAE C. VAN BAALEN • • • • • • • • .~ Final Report: NOAA Puerto Rico Pharmaceutical Wastes, • C. Van Baalen, January 11, 1980 . Individual pharmaceutical wastes from operations in Puerto Rico were examined for toxicity towards representative types of • microalgae. Three algal test species: the blue-green alga, Agmenellum quadruplicatum, strain PR-6 (our isolate); the green alga, Chlorella autotrophica, strain 580 (obtained from R.R.L • • Guillard); and the diatom, Cylindrotheca sp. strain N-1 (our isolate) were grown on medium ASP-2 (Provosoli, et al., 1957; Van Baalen, 1962) at 30°C, see Figure 1 for details. The wastes • supplied to us were upon receipt dispensed into clean screw cap glass tubes and frozen until tested. The Schering sample was received January 3, 1980 and will be tested shortly. The • untreated wastes were added directly to the growth medium, v/v, just before inoculation and incubation. Growth rates and lag times in initiation of growth were the • experimental endpoints. A representative set of growth curves is given in Figure 2. From such data the generation times recorded in Tables 1 and 2 were calculated. Lag times greater • than one generation time are significant. Lags could represent a lag in growth by all cells in the population or a fractional kill of the inoculum. The turbidimetric measure of grow~h as • used here will not distinguish between these two alternatives. The results with the blue-greenalga, PR-6; the diatom N-1; and the green alga, 580; demonstrate the highly tox~c nature of • the Bristol sample. The Upjohn sample was only inhibitcry to • 2 • • PR-6 (Table 1) . It should be noted that in algal assays such as these that the cell concentrations (in terms of chlorophyll a) • are roughly 1000 times that of natural populations. Hence in nature the ratio toxicant/cells will be higher and samples like Bristol or Upjohn could be effective at considerably lower concentrations. The Upjohn sample gave the same results with organism PR-6 • whether it was filtered or not filtered (0.4 µm Nucleopore polycarbonate type filter) . The biphasic nature of the Bristol sample precluded filtering it. The Bristol sample was examined • further using organism 'PR-6. It showed evident toxicity as low as 25 ppm, and at 50 ppm was completely inhibitory to the inoculum (Table 2 . The nature of the toxic material(s) in the • Bristol sample are unknown. Preliminary-experiments on the effect were of exposure to sunlight on the toxicity of Bristol sample conducted in quartz tubes containing 250 ppm Bristol sample in • filtered sea water. It appears that there is a lessening of toxicity after 24 hours followed by an increase in toxicity upon further exposure. This rather curious effect of sunlight on the • Bristol sample needs to be confirmed. In summary, of the six pharmaceutical wastes tested, four {Pfizer, Capri, Squibb, Merck) were not toxic as judged by their • effect on growth rate of three algal types. The Upjohn sample showed what has come to be a common observation, selective toxicity towards one kind of alga (Winters et al., 1977; Batterton • et al., 1978). The Bristol sample was not only toxic at rather low levels but was also toxic to all three test species. Presumably • 3 this indicates that the toxic material in it is a general metabolic • poison active in both prokaryotic and eucaryotic organisms . References • Batterton, J. C., K. Winters and C. Van Baalen. 1978. Anilines: • selective toxicity. to blue-green algae. Science 199, 1068-1070. Provasoli, L., J. J. A. McLaughlin and M. R. Droop. 1957. The development of artificial media for marine algae. Arch . Mikrobiol. 25, 392-428. Van Baalen, C. 1962. Studies on marine blue-green algae. Bot. mar. 4, 129-139. , I . Winters, K., J. c. Batterton and C. Van Baalen. 1977. Phenalen-1one: occurrence in a fuel oil and toxicity to microalgae. Environ. Sci. Technol •. 11, 270-272. I• • • • • • Table 1. Algal growth rates, as generations per day, in the presence of pharmaceutical wastes . • Waste Material Concentration Organisms Identification ppm PR-6 N-1 580 • Controls 0 5.3+.3 4.7+.3 2.9+.3 Bristol 250 NGa 2.4 NG • 500 NG NG NG 2500 NG NG NG 5000 NG NG NG • Upjohn 25.0 NG 4.5 3.0 500 NG 4.5 3.0 2500 NG 4.0 b 3.0 5000 NG 2.5(26) 3.1 Merck 250 4.9 4.7 3.0 500 4.9 4.5 3.0 2500 4.3 4.6 2.6 5000 2.5 4.6 2.6 • Squibb .250 · S.3 4.5 2.9 500 5.3 4.5 2.9 2500 4.5 3.5(17) 3.l 5000 4.1 3.5(24) 3.1 • Capri 250 5.4 4.7 2.9 500 5.4 4.7 2.9 2500 5.4 4.6 2.8 5000 5.0 4.6 2.8 • Pfizer 250 5.2 4.7 2.8 500 5.1 4.6 2.8 2500 5.1 4.7 2.8 5000 5.1 4.5 2.8 a NG means no growth for up to 5 days incubation. b Number in parenthesis is lag time in hours as compared to a control• • • Table 2. The effect of Bristol Waste on growth rate of organism PR-6 • • • • • • • • • • • Concentration Generations Bristol Waste Per Day ppm 0 {control) 5.0+.3 7 4.8 14 4.4 25 4. 4 (72) 50 NGa 125 NG 250 NG a NG means no gr_owth for up to 5 days incubation. Figure 1 . • • • • • • • • Protocol for testing waste material samples MAIN SAMPLE I so ML SAMPLES STORED AT -10°C I THAWED AND WELL-MIXED I SAMPLE ADDED TO STERILE GROWTH MEDIUM (V/V), FINAL VOLUME 20 ML IN 22xl75nun PYREX TUBES I INOCULATE, APPROX .. lOS CELLS/ML I INCUBATE -. AT . 30+0.1°C, WITH CONTINUOUS ILLUMINATION FROM TWO F40CWX FLUORESCENT LAMPS ON EACH SIDE OF GROWTH BATH POSITIONED 9 CM FROM CULTURE TUBES. CULTURE TUBES CONTINUOUSLY BUBBLED WITH l+0.1% co2-IN-AIR MEASURE GROWTH TURBIDIMETRICALLY (660 NM), LUMETRON COLORIMETER MODEL 402E • • • 2.0...----------------------...-----...-----.....-..-.---..-........------~..-------------..--. l.5 ~! ~ ~ -C> 0 .. )( 0 0 0 ~ 0 _J o/ / ~Q 1.0 ~~:y:~ VD//D 0.5 .---------~-------------~--~~-----"----------------------------------- 22 27 12 37 42 TIM~J~~urs after inoculation) Figure 2. Growth of organism PR-6 in the presence of Squibb waste; 0 and I are controls,X plus 250ppm waste, 6 2500ppm, o 5000ppm. OD (optical density) is proportional to cell nunwer to OD = 1.0, at OD = 1.0 for organism PR-6 the cell dry weight is 0.25 mg m1-l. • • CONTINUATION STUDIES • --INVERTEBRATES J.A.C. Nicol :1w. Y. ·Lee A. Morris • • • • • • • • Toxicity of Ocean-Dumped Pharmaceutical Wastes to Marine Copepods and Ichthyoplankton by • J. A. C. Nicol, D. Phil., Co-Principal Investigator W. Y. Lee, • A. Morris, • • • • • • • Ph.D., Research Scientist Associate B.Sc., Research Scientist Assistant • Introduction • Previous research has shown that the composite organic wastes from Puerto Rico pharmaceutical companies, which are periodically dumped offshore, have detrimental effects on • marine organisms, including fish, invertebrates, and microalgae (Lee and Nicol, in press; Van Baalen and Batterton, 1978; Wohlschlag and Parker, in press). In this study, we continued our efforts to determine the individual toxicity of effluents to marine zooplankton. The materials tested were from, the six pharmaceutical • companies, Bristol, Capri, Merck, Pfizer, Squibb and Upjohn. Test animals included marine copepods and ichthyoplankton. Marine copepods were collected from the Gulf Stream off east • central Florida while the latter were from laboratory stock of the redfish (Sciaenops ocellata) . They were chosen as test animals because they live in the water column and are likely • to be exposed to the effluents which were released at the sea surface. Two experiments were designed to test the toxicity of • individual ~aste. One involved marine copepods, which were treated with a series of dilution of effluents at various exposure intervals and then transferred to clean sea water . • This was carried out to simulate a possible dilution process occurring at the dumpsite, and to determine the potential acute effects during early dilution and the delayed short-term effects • thereafter. The other experiment dealt with the comparative • • 2 toxicities of 3 effluents (Bristol, Merck, and Squibb) to theredfish larvae and eggs. A standard laboratory toxicity test • was performed to determine the values of 48h-LC50 . Behavioraland morphological aberrations. of larval fish observed during • the experiments were also described. Materials and Methods Experiments with. marine copepods • • Six industrial wastes used in this bioassay were fromcompanies of Bristol, Capri, Merck, Pfizer, Squibb, and Upjohn. • Each company supplied us with two bottles of samples, which. were different in both their color and in.the quantity of sediments.In order to have a representative sample for each company, we • drew 300 ml from each bottle of the same company and mixed them together. Sea water used in this part of the study came from offshoreFlorida, it was prefiltered through 1 µm glass fiber filter, and le bubbled with air for 20 minutes. Owing to a short duration ofthe experiment, no antibiotics were added to sea water. Zooplankton samples were collected at a station approximately • 17 miles east of central Florida off Fort Pierce. Each pollutantwas diluted to the following concentrations; 10, 1, 0.1, and 0.01%. • There were triplicates for each concentration, and for convenience they were designated as A, B, and C. A control was also maintained • during this study and used as a check for the experimental groups .. Zooplankton were sampled with a 0.5 m net (153 µm)at a depth of about 2 m below the surface. A total of 10 plankton hauls were • • 3 made on May 10, · 1979 between 10 AM and 2 PM. Each tow lasted • for 15 minutes. On board ship, the zooplankton was roughly divided into 21 parts and each part was transferred to a plexiglass cylinder (12.0 cm (L) x 6.8 cm (D)), with a 93 µm • netting attached to one end. The cylinder was submersed in a 32 oz. wide-mouth bottle, containing 500 ml of filtered sea water. At the sampling station, surface temperature of sea • water was 25.0 C and salinity was 36.6 o/oo,being 0.6 o/oo higher than the prefiltered sea water. To roughly simulate the dilution process of the early stage • at the dumpsite, zooplankton was exposed to the media for various time intervals. At the highest concentration (10%), exposure lasted for 2 minutes only, while at lower concentrations, it ·• lasted longer; 5 minutes for 1% mixture, 1 h for 0.1% and 2 h for 0.01%. At the end of exposure, sample B at all concentrations, including the controls, was stained with neutral red and then • fixed by a method described by Crippen and Perrier (1974). The remaining samples (A and C) were transferred to clean sea water and fixed 24 h later. The observed mortality in sample B • indicates the acute toxicity of each pollutant, while the difference between the mortality of sample B and the average mortality of samples A and C, represents the delayed toxicity • of pollutants • The field collection of the zooplankton and most of the experiments were carried out on R/V Johnson. The remaining work • after the vessel returned to the port was done at the Johnson • • 4 Science Laboratory of the Harbor Branch Foundation, in Link Port, • Florida. • The zooplankton of sample B was not fed because the experiment lasted only about 3 h. The zooplankton in samples A and C were starved for the first 6 h ~nd then fed with a mixture of Platymonas sp. and Dunaliella salina 4 times at an interval of 5 to 6 h. Experiments with redfish eggs and larvae • • The organic wastes that were tested were from 3 pharmaceutical plants in Puerto Rico: Merck, Squibb and Bristol. The sample from Merck differed from the others in being extremely black. Even at • 5% concentration, only a minimal amount of light was able to pass through the medium when held next to a fluorescent tube. Two experiments were carried out on each sample to determine • their relative toxicities to redfish eggs and larvae. The initial test solutions for both experiments (Part I and Part II) were in the following concentrations: 0% (control), 0.1%, 0.5%, 1% and 5% . • Effluents were diluted with glass fiber (pore sizes 0.2 to 10 µm) filtered sea water with a salinity. of 26%0. The stock sea water had been treated with the antibiotics, penicillin G and streptomycin • sulfate, and aerated for 30 minutes before the experiments. Two replicates were made for each concentration. • Part I Redfish larvae Approximately 700 fertilized eggs were obtained from the University of Texas Marine Fisheries Laboratory in Port Aransas, • • • 5 • Texas. The naturally spawned eggs were already in late gastrula. They were incubated for an additional 18 h until hatching occurred . I• Twenty-five larvae were then transferred by a large bore pipette to each of the diluted effluents. Survival counts were made at 6 h intervals from the time of exposure. Dead larvae were removed • from the bowls at the time of the counts. · A larva was considered dead when it had no detectable heart beat. The experiment lasted throughout the 48 h yolk sac phase of the larvae. It was therefore unnecessary to feed them. The survival data were plotted on graphs with probability x 2 log cycles. The Lcwas then calculated by the method described le 50 by Litchfield and Wilcoxon (1949). Part II Redfish eggs • The majority of the eggs were in late gastrula when obtained. Batches of 30 to 50 eggs were transferred to a 25 ml petri dish for observation under a stereoscope. Eggs that were deformed, • unfertilized, or otherwise not in late gastrula were discarded. The remaining eggs were transferred to the test media. Each bowl contained 25 eggs • • The first count was made 24 h after exposure. The resultant eggs and larvae were recorded as hatched-living larvae, hatched- dead larvae, unhatched-living embryo, and unhatched-dead ewDryo . All dead specimens were discarded at the time of the count. The surviving larvae were then transferred from the test solution to I I • • clean sea water. Subsequently, observations on the survival of organisms were made at ~ 6 h interval until the end of yolk sac stage . • 6 • Results Experiments with marine copepods • The majority of the zooplankton in the sample were copepods (Table 1). The occurrence and abundance of several dominant species in the test samples are listed in Table II. The cyclopoids, Farranula carinata and Corycaeus sp. together with the calanoids Paracalanus spp. and copepod nauplii make up about 75% of the number • counted in the 18 test samples. Other species such as the harpacticoids, Macrosetella gracilis and Miracia efferata were much less abundant and their numbers were always less than 30 in • -' a sample of about 250 individuals. Except for the cyclopoid, F . • carinata, the other species or groups varied much in the order of dominance from sample to sample. For example, copepod nauplii were the most abundant taxa in sample 5, but were one of the • least taxa in sample 18. Percentage similarities calculated among species ranged from 16.4 to 90%, with a mean and a standard deviation of 59.5 and 16.4, respectively (Table III). This contrasts to what we obtained for the test samples collected off the ship channel of Port Aransas. In the latter case, percentage similarity between samples was always greater than 70% a.nd the overall mean was 88.7% (Lee et al., unpublished manuscript). Zooplankton in Bristol were found to be covered with oily • brown materials which not only prevented the specimens from being adequately stained by the neutral red but also added much difficulty in copepod identification. We therefore decided to discard all • specimens in Bristol samples. Revised techniques are required for further studies on the toxicity of Bristol waste to small marine crustaceans . • • Table I • Composition of marine zooplankton collected from the Gulf Stream, 17 miles east of central Florida. • Calanoids: • Paracalanus spp. (probably parvus, quasimodo and indicus) Ternora stylifera Clausocalanus sp. Clausocalanus furcatus Candacia longimana Candacia pachydactyla • Candacia varicans Labidocera sp. Calocalanus pavo Euchaeta marina Neocalanus copepoqites Undinula vulgaris Acartia spinata Unknown species • Cyclopoids: • Farranula carinata oithona simplex Oithona spp. Oncaea mediterranea Oncaea sp. Corycaeus amazonicus Corycaeus speciosus Corycaeus sp. Harpacticoids: • Macrosetella gracilis Miracia efferata Chaetognaths: • Sagitta spp . Salps Siphonophores • Ostracods Crab zoea Fish eggs and larvae • Table II Abundance and occurrence of common copepods in the 18 test samples. The last column represents the percentage of each as shown below this table. taxon in total counts (5133) . Each sample was assigned with an Arabic numeral Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total % 125 187 82 95 111 26 102 107 106 208 2373 46.2 Farranula carinata 175 162 192 50 51 176 203 215 54 102 26 74 44 25 621 12.1 Paracalanus spp. 6 14 3 29 30 3 7 25 2 47 62 68 6 96 0 527 10.3 - Copepod nauplii 2 19 14 25 145 1 6 9 16 7 83 e 0 88 2 334 6.5 Corycaeus sp. 19 19 25 25 9 26 2 18 8 7 36 9 26 20 34 23 11 17 4 36 3 10 246 4.8 Oncaea spp. 21 39 6 21 5 26 3 5 6 17 1 35 7 1 2 11 10 2 22 13 4 12 212 4.1 Macrosetella gracilis 17 9 11 19 9 15 18 9 16 13 Corycaeus amazonicus 9 23 29 29 6 27 15 16 3 23 1 ~5 6 0 5 2 5 5 200 3.9 7 55 1.1 Corycaeus s12eciosus 14 0 4 3 1 4 3 2 2 0 1 5 1 0 5 3 0 Control A=l Control B=2 Control C=3 Pfizer A=4 Pfizer B=5 Pfizer C=6 Capri A=7 Capri B=8 Capri C=9 Upjohn A=lO Upjohn B=ll Upjohn C=l2 Squibb A=l3 Squibb B=l4 Squibb C=l5 Merck A=l6 Merck B=17 Merck C=18 CX> ~ ~ ........ • 9 • The acute and delayed toxicity of a sample varied (Table IV) . Based on the survival data at the end of 3 h exposure, the organic waste from Merck was the most toxic; mortality being about 50% of the test population. For the remaining wastes, the toxicities in • order were Pfizer>Capri>Squibb>Upjohn. Survival of zooplankton in Upjohn was 94.8% compared to 96% in the control. The last column in Table IV shows the difference between • survival of organisms at the end of 3 h exposure and at the end • of 24 h after being transferred to clean sea water. Although there were high mortalities in the controls (25%), delayed mortalities in all wastes were still significant. The highest delayed mortality was observed in the Upjohn sample and the lowest in the Merck sample . The order of toxicity for these two samples were just • the reverse recorded at the end of 3 h exposure. Nevertheless, the overall toxicity of each test waste ended up with a very similar result. The survival of marine zooplankton at the end of • the experiment was low and in most ·wastes the percentage of survival was less than 10% (see column 4 in Table IV) . • Experiments with redfish larvae A. Squibb The mortality of redfish larvae increased with both increasing concentration of the Squibb effluent and length of exposure (Fig. 1) • The estimated 48h-Lcwas 0.42% (v/v) with the confidence limits • 50 between 0.34 and 0.52%. • Dead fish larvae were observed after 6 h exposure. All of the larvae in the 5% solutions and more than half of those in the 1% solutions had expired at that time. The dead larvae in the 1% • - ~ ,. ! Table III Percentage similarities among samples. The index was calculated by the following equation suggested by Whittaker anQ n Fairl;>anks (1958). p = 100 (1 -0.5 E IPij -pikl ), where P~ . and P.k are the observed proportions of species i in 1) 1 i=l samples j and k, and n is the total number of species in the paired samples. < ~ u < ~ u < ~ u < ~ u rl rl rl < ~ u 0 0 0 1-1 1-1 1-1 i:: i:: < ~ u i:: ..Q ..Q ..Q 1-1 1-1 1-1 Q) Q) Q) ·rf ·rf .µ .µ ·rf .c: .c: .c: ..Q ..Q ..Q ~ ~ ~ .µ N N N 1-1 1-1 1-1 0 0 0 ·rf ·rf ·rf 0 0 0 i:: i:: i:: ·rf ·rf ·rf 0. 0.. 0.. ·n ·n ·n ::s ::s ::s 1-1 1-1 1-1 0 0 0 4-l 'H 'H ni ni ni 0.. 0.. 0.. 01 01 01 Q) Q) Q) u u u Al Al Al u u u p p p Cl) Cl) Cl) ~ ~ ~ Control A 100 79.5 83.2 54.7 31.0 89.0 81.0 64.0 82.5 76.8 39.6 56.1 67.0 21.8 54.7 62.3 48.6 84.4 Control B 100 73.7 62.3 41. 4 82.7 70.8 74.2 66.5 79.9 49.2 67.0 71. 3 32.0 51. 7 67.6 58.3 75.0 Control C 100 62.0 42.2 89.7 82.6 67.6 79.4 79.7 45.1 54.3 68.5 25.0 79.1 34.5 50.5 82.5 Pfizer A 100 57.5 59.7 44.5 .62. 7 38.9 60.5 63.0 73.9 59.7 50.5 61.8 70.7 61.4 49.0 Pfizer B 100 30. 2 31.2 64.7 29.9 44.0 64.6 46.4 41.0 61.8 40.8 44.6 74.4 37.8 Pfizer C 100 79.0 . 62.0 76.0 79.0 40.0 60.0 71.0 20.0 53.l 60.7 46.3 81.2 Capri A 100 60.0 90.0 76.0 36.0 50.6 63.0 17.4 48.7 49.9 47.0 83.6 Capri B 100 57.0 69.2 70.7 60.3 68.6 53.5 55.3 62.5 80.0 67.2 Capri C 100 71.0 34.6 45.1 62.4 16.4 46.4 50.2 45.2 83.3 Upjohn A 100 52.0 71.1 79.0 34.2 56.0 67.0 63.8 80.5 Upjohn B 100 60 .1' 65.0 74.0 53.5 63.7 75.1 45.7 Upjohn C 100 70.6 4 3 .1 62.8 86.8 63.l 55.8 Squibb A 100 44.4 61.0 77 .9 59.5 75.4 Squibb B 100 34.8 53.6 68.3 26.3 Squibb C 100 65.7 54.6 57.4 Merck A 100 66.4 61. 7 Merck B 100 54.6 Merck C 100 ...... 0 Table IV Survival (%) of marine zooplankton in pharmaceutical effluents and in the controls. Live and dead determination of marine zooplankton was made at the end of 3 h exposure to effluent for sample B, and at 24 h after transferred to clean sea water for samples A and C. B A c Average of 2&3 (1) ( 2) (3) (4) (1)-(4) Control 96.7 75.2 24.8 68.6 71.9 Bristol -* Capri 71. 8 5.7 61.0 15.8 10.8 Merck 51.6 4.5 2.0 3.3 48.3 Pfizer 63.2 0.3 16~2 8.3 54.9 Squibb 72.6 o.o 0.6 0.3 72.3 Upjohn 94.8 4.2 4.6 4.4 90.4 *Samples were discarded, because zooplankton were covered with oily brown materials, which made live-dead determination impossible. ....., ....., • • • • 0% • a.a MEAN SURVIVAL • 0 6 12 18 24 30 36 . 42 48 54 60 HOURS • • Figure 1. Mean survival of Sciaenops ocellata larvae in four concentrations of a pharmaceutical effluent (Squibb) • • • • 13 were corkscrew-shaped from the yolk sac to the caudal region. • Only one larva with this configuration showed a heartbeat. The corkscrew-shaped larvae were not found in the 5% solutions. It is possible that these larvae died very soon after exposure. • After 12 exposure, some behavioral abnormalities were noted among the larvae in the 0.5% mixtures. The majority of the larvae drifted about, but displayed very little or no escape response • when approached with the tip of a pipette. Several larvae were also found to be motionless having only a slight and irregular heartbeat. However, death did not occur in this concentration • ~. . . for an additional 18 h. At the time of death, the larval develop • ment had progressed up to the appearance of pectoral fin folds, which was also observed in the controls at that hour. A significant difference in yolk size was observed in the larvae of 0.5% solution as compared to the controls. The dead larvae had little or no materials in the yolk sac; it was drawn • up tightly around the oil globule. These larvae, which lay motionless on their sides, had approximately 1/4 of the original material in the yolk sacs, while in the controls the larvae had • about 1/2 of the original material . • All larvae in the 0.1% survived throughout the yolk sac phase. Even at the end of 62 h, the mortality of the larvae did not differ significantly from that in the controls. Also, no behavioral or morphological abnormalities were observed throughout the experiment. B. Merck e Due to the dark color of this waste, it is impossible to recover any larvae from the 5% solutions for observation. All • • 14 • these larvae were presumably dead. Three crippled larvae were found in the 1% solution. Each was bent approximately 90 • laterally. These larvae were dead at 12 h. The survivors in the 1% solutions lay motionless on the bottom of the culture dish at the end of 18 h exposure. Repeated • prodding with water outflow from a pipette resulted in no escape response. For the remaining 30 h the only detectable sign of life for these larvae was the presence of heartbeat. Most of the :• larvae survived in this condition until 36 h. Less severe effects were observed for the larvae in the 0.5% solutions. When approc;tched with the tip of a pipette, th~_ larvae • were able to react and swim 1-2 cm before resettling to the bottom of the bowl. An occasional quivering was observed when viewed under the stereoscope. The heartbeats of these larvae did not • differ much from the controls; each was measured at 2 to 3 beats per second. The mortality of larvae ·in the 0.1% was low; only 4 of 25 • were lost at 48 h. Mean survival of S. ocellata larvae in all test concentrations during the 48 h exposure is shown in Fig. 2. The estimated Lc50 was 0.49%, with confidence limits from 0.39 to 0.61. C. Bristol • e A preliminary test on the Bristol effluent showed it to be much more toxic than the other samples. Therefore, concentrations lower than those tested for the Squibb and Merck wastes were used for the Bristol samples. For the latter, it ranged from 0.01 to 0.5% . • • 15 • • • (0%) ( .1%) • 2 MEAN SURVIVAL (. 5%) II • 1% 0 6 12 18 24 30 36 42 48 HOURS • Figure 2. Mean survival of Sciaenops ocellata larvae in four concentrations of pharmaceutical .effluent (Merck) • • • • • I' • 16 • Significant mortalities were recorded at concentrations> 0.02% (Fig. 3). At concentrations >0.5% 100% mortality occurred within 6 h. The estimated 48h-LCwas 0.045% (confidence limits, 0.039 50 0.050), one order of magnitude more toxic than the other two • samples tested. • Morphological abnormalities were noted in the 0.1% solution at 6 h.. All of the larvae were twisted and knotted from the yolk sac to the caudal region. No muscle contraction was noted, except for the heartbeats, which were faint and irregular. These larvae were dead within 12 h of exposure. • Behavioral abnormalities were observed in the 0.02% group . • At the end of 6 h of exposure, the larvae would swim only 0.5-1 cm as compared to 5-10 cm in the control when approached with the tip of a pipette. The larvae became rigid and no movement other than heartbeat was detected from 18 h to the end of the experiment. • Another test was conducted using concentrations of 0%, 10 ppm • and 50 ppm (0.005%). The exposed larvae, in both concentrations, showed virtually no escape response compared to the controls. However, the larvae did not become rigid as those in the 0.02% solutions or the previous test. At concentrations < 50 ppm, survival was not much different from that of the controls during • the 48 h exposure. Lack of availability of the redfish eggs prevented further toxicity testing at concentrations lower than 10 ppm. • • • • • • le • • • • • • 17 MEAN SURVIVAL (0%) (.01 & .02%) 0 6 12 18 24 HOURS 30 36 42 48 (.04%) (.06%) Figure 3. Mean survival of Sciaenops ocellata larvae in seven concentrations of pharmaceutical effluent (Bristol-Alpha) • .'-\ • 18 Experiments with redfish eggs • A. Squibb • Egg and larval mortality increased with increasing concentrations of effluent, as indicated in Table v. More than 85% of the eggs in the 0.1% solutions were hatched and survival ·of these larvae was also high after they were transferred from the test solutions to clean seawater (Fig. 4). At higher concentrations (0.5 and 1%), • most larvae were hatched but apparently died shortly after hatching • • The two survivors in the 0.5% solutions were motionless on the bottom of culture bowl and could not be induced to move. In the SS!,0, none of the eggs were hatched. B. Merck As in the Squibb waste, no hatching occurred in the 5% solutions (Table VI). At the end of the 24 h exposure, all of the embryos in this concentration were dead and none showed signs of development after the initial exposure. • Extensive morphological and behavioral aberrations were observed in the 1% solution. Of the 42% that hatched, about 5% of the larvae were deformed. In each case, the yolk sac was • highly distended, while the posterior half of the body was atrophied. Movement was limited to an occasional twitching of the body~ These larvae also had highly irregular heartbeats. • Many of the non-crippled larvae had abnormal swimming patterns . When touched with a pipette, the larvae would swim in small circles rather than darting away from the pipette as in the • controls . • I• 1 Table V ~ i ·s.~ i Hatching success of redfish eggs after 24 h of exposure to a pharmaceutical effluent (Squibb). j ; i ! Hatched Unhatched live larvae dead larvae live embryo dead embryo A B Mean A B Mean A B Mean A B Mean Control 25 25 25 0 0 0 0 0 0 0 0 0 .1% 23 21 22 0 0 0 0 0 0 2 4 3 .5% 0 2 1 22 20 21 0 0 0 3 3 3 1% 0 0 0 18 12 15 0 0 0 13 7 10 5% 0 0 0 0 0 0 0 0 0 25 25 25 I '-' • 20 • • i• 25 • (0\) MEAN SURVIVAL ·C.1%) • 5 c.s\) 0 6 12 18 24 30 36 42 48 HOURS FIG. 4 MEAN SURVIVAL OF LARVAE AFTER TRANSFER TO STOCK SEA WATER -(SQUIBB). • • • • Ii ~ Table VI Hatching success of redfish eggs after 24 h of exposure to a pharmaceutical effluent (Merck) . Hatched Unhatched live larvae dead larvae live embryo dead embryo -A B Mean A B Mean A B Mean A B Mean 0 0 0 0 Control 25 25 25 0 0 0 0 0 1 • 19'0 24 23 23.5 0 1 .5 0 0 0 1 1 .5% 24 25 24.5 0 0 0 0 0 0 1 0 .5 1% 8 13 10.5 0 0 0 0 0 0 17 12 14 •. 5 5% 0 0 0 0 0 0 0 0 0 25 25 25 N t-' • 22 After the transfer of hatched larvae in the 1% solution to • clean sea water, the progress of the behavioral aberrations was • arrested. In fact, the larvae gradually became more active and the abnormal circular swimming patterns were no longer observed after 18 h in fresh sea water. However, the exposed larvae never • became as active as the controls. Their distance of travel in the escape response was 1-2 cm compared to 8-12 cm in the controls. The routine activity of the larvae from the 1% exposed group was • largely drifting or laying on the bottom, whereas the larvae in the controls drifted and darted about intermittently. No behavioral aberrations in the 0.5% and 0.1% solutions were evident. Survival • of all larvae which hatched from exposed eggs were high in clean sea water (Fig. 5), as was also observed in the experiment on the Squibb sample . c. Bristol Percentages of egg hatching in all test concentrations were > 85%. At the highest concentration (0.08%) tested, an average of 90% of the exposed eggs hatched. However, all of these larvae died soon after hatching, while there are more than 80% survival • in the groups, controls, 0.02, 0.04 and 0.06% (Table VII) . • Morphological and behavioral aberrations occurred in all concentrations tested. In the 0.02% solution, all larvae lay motionless on their sides, with movement limited to an occasional quivering of the muscle along the spine. About 80% of the larvae in this concentration was severely malformed, having the corkscrew • shaped configuration from the yolk to the caudal region. The remaining 92% had a distinct S-shaped spine compared to the relative straight spine observed in the specimens of the controls . • • • • 25i=====================---- 2 • MEAN SURVIVAL .15 io.r------------- • 5 (0.1%) (0.5%) (0%) (1%) 0 6 12 18 24 30 36 42 48 HOURS • • Figure 5. Survival of redfish larvae(from exposed eggs) after transfer to stock sea water (Merck) . • • • • Table VII Hatching success of redfish eggs after 24 h of exposure to a pharmaceutical effluent (Bristol} . .Unhatched Hatched live larvae dead larvae live embryo dead embryo A B Mean A B Mean A B Mean A B Mean 0 0 0 Control 25 25 25 0 0 0 0 0 0 0 .02% 24 25 24.5 1 0 .5 0 0 0 0 0 2 .04% 21 22 21.5 0 1 .5 0 2 1 3 0 .06% 24 19 21.5 1 4 2.5 0 1 .5 0 1 .5 .08% 0 0 0 24 21 22.5 0 0 0 1 4 2.5 N ~ • 25 In the 0.04 and 0.08% solutions, the hatched larvae had • more severe damages than were noted in the 0.02%. A quarter of the larvae were corkscrew-shaped and the remaining 75% had a pronounced S-shaped spine. In addition, all larvae in these • two concentrations had faint and irregular heartbeats . Mortality of redfish larvae after transfer to clean sea water was low for the first 36 h and then began increasing. At • the end of 48 h only about 1/3 of the hatched larvae at 0.06% and 2/3 at 0.04% were still alive. However, for the entire 48 h post-hatching period, there was no evidence that the larvae • recovered from the morphological or behavioral aberrations that were present at the time of hatching. • Discussion Experiments with zooplankton Of the five pharmaceutical wastes, samples from Merck and • Pfizer were acutely toxic to marine copepods, survival in these two samples was < 65% at the end of 3 h exposure. Animals in other 3 samples had low initial mortalities but high delayed • mortality. Twenty-four hours after zooplankton were transferred to clean sea water, the highest percentage of survival was recorded in sample of Capri (10.8%). These data suggest that • samples varied in both the acute and delayed toxicity. Any toxicity study, therefore, should be designed to investigate these two factors in order to obtain meaningful results as • suggested by Wright (1976) . • • 26 • • • MEAN • SURVIVAL • • Figure 6 • Survival of redfish larvae(from exposed eggs) -after transfer to stock sea water (Bristol-Alpha) • • • • , . 27 I• Initial mortality of the controls was low. At the end of 27 h, the percentage of survival dropped to about 72%, a 25% • • loss during a 24 h period. This loss could be caused either by the inadequate food added or by the changing temperature encountered during the experiment. On the ship, the study was carried out on a bench outside the room. There the temperature changed from time to time depending on the position of the place relative to the sun. • If the factor of temperature was taken into consideration, the high delayed mortalities observed in all samples were probably not caused by the toxicity of pollutants alone, but by the combined • stress of pollutants arid varying temperature. Hence, the laboratory results, when applied to field effects, should be cautiously • interpreted. The biological consequences of waste dumping at the dump site depend on many factors, such as the toxicity of dumped material, the rate of dilution, and the stability of the in situ biological • community. In turn, the latter two factors are more or less • governed by the oceanographic conditions of the dumping ground. Since the conditions of the sea vary daily_and seasonally, it is impossible to select one model to describe all the possible waste • plume behaviors or the structure of the plankton community. However, field observations on the plume growth and the chemical analysis of N,N-dimethylaniline in sea water suggested that one of the three equations discussed by Simpson et al. (1979) was very close to the actual plume behavior in the ocean (O'Connor, 1979). • 115 This equation has a diffusion coefficient of K = 0.01 1· 2 cm /sec, and predicts a maximum concentration of 4 ppm, 24 h • 28 after the dumping. According to the same equation, the initial minimum dilution factors estimated for the first 3 h ranged from 3 4 • 5 x 10 to about 2 x 10. For the same interval, the test 5 2 concentrations in our biological study varied from 10ppm to 10 ppm. Compared to the theoretical predict1ons (from Fig. 6, • O'Connor, 1979), the concentrations employed in our study for the first hour of exposure were too high, while the concentration for the period of the next two hours was reasonable. The results • obtained from this laboratory study may not represent the outcome of the large dumping area, but of a small region near the center of the waste plume. There., at least.- half of the zooplankton • populations would be killed at the end of 24 h, assuming that the in situ zooplankton responds the same way to the dumped wastes as did the Gulf stream populations . • Experiments with redfish larvae and eggs The toxicities of the three effluents to larval fish are • ranked as follows: Bristol>Squibb>Merck. The values of 48h-Lc 50 for Squibb and Merck were relatively close (0.42 and 0.49%, respectively), while the 48h-Lcfor Bristol was 0.045%, 50 • approximately 10 times lower than either of the other two samples . _Our previous study (Lee et al., 1978) found the composite waste samples to be toxic to anemones at a concentration of 0.49% • (96h-Lc50), juvenile amphipods at 0.25% (96h-Lc50), isopods at 1.4% (96h-Lc50), jellyfish at 1% (96h-Lc50), and mixed coastal zooplankton at 1% (24h-Lc). These values cannot be directly • 50 compared to that of the present study because the two experiments • • 29 differed in two factors, namely, test animals and test pollutants • (individual vs. composite). If we assume that the mixed zooplankton, • amphipods, isopods, anemones, or jellyfish were comparable to the redfish larvae, it could then be concluded that an individual waste was more nocuous than the composite samples. Indeed, it was true especially when the toxicity of Bristol sample was considered. Although the chemical composition of the three samples varied • considerably (Hatcher and Harvy, 1978; Atlas et al., 1980), the symptoms of the eggs and larvae induced by the toxic effects of the wastes were not much different from each other. The severity • of the symptoms and the frequency of occurrence depends on both the concentration of pollutant and its toxicity. Generally, a short-term exposure of redfish larvae or eggs to a concentration • > 0.5% of any company's effluent, would result in reduced swimming activity, deformity, irregular heartbeat, and also some deaths. Those larvae which hatched from the eggs exposed to the Merck • sample appeared to be partially revived after several hours in clean sea water, while in samples of Squibb and Bristol, the damage done to larvae was irreversible. • At the dumpsite, the maximum concentration of waste is about 2 _10 ppm and this concentration covered about 35 kmover the mixed layer (O'Connor, 1979). It appeared that concentrations of composite wastes at the station were well below the level of toxicity for the redfish eggs and larvae. However, the higher toxicity of Bristol sample compared with the samples from the other companies is of concern and interest. Atlas et al. (1980) have reported that N,N-dimethylaniline (DMA) was the major component • • 30 of the Bristol sample. It is therefore very possible that the • higher toxicity of Bristol waste is attributable to the compound DMA. Futhermore, the analyses of DMA at and near the sumpsite indicated that DMA was very persistent in the • environment. Owing to these characteristics, we suggest that the acute toxicity of DMA and its sublethal effects on marine animals is studied in the future program. • Acknowledgments For the redfish larvae and eggs, we are indebted to Dr. C. Arnold, who always generously provided more eggs than • we asked for. For the Gulf Stream zooplankton, we wish to thank Dr. R. S. Jones, the Director of the Johnson Science • Laboratory, Harbor Branch Foundation in Fort Pierce, Florida, for providing the shiptime and much assistance. We are also grateful to many of his colleagues, especially Drs. Youngbluth, • J. Miller, Jeff Prentice and Ms. Lindy Eyster and Ross Neville, • who helped us in various aspects either during our stay in the Johnson Science Laboratory or on board the R/V Johnson. Finally, we wish to especially thank our colleague, S. Anderson, • who not only shared with me the long drive from Texas to Florida but also assisted in the field and laboratory. Without his help, this zooplankton project would have been impossible to complete . • • • 31 References • ATLAS E., G. MARTINEZ and C.S. GIAM (1980) Chemical character ization of ocean-dumped waste materials. In: Ocean dumping of industrial wastes, B. H. Ketchum et al., • editors, in press • CRIPPEN R.W. and J.L. PERRIER (1974) The use of neutral red and Evans blue for live-dead determinations of marine • plankton. Stain Technology, !2_, 97-104 . HATCHER P.G. and G.R. HARVEY (1978) Trace organic studies at the Puerto Rico pharmaceutical waste dumpsite. Final • report to NOAA/ODMP, 17 pp • LEE W.Y. and J.A.C. NICOL (1980) Toxicity of biosludge and pharmaceutical wastes to marine invertebrates. In: Ocean • dumping _of industrial -wastes, B.H Ketchum et al., editors, in press. LEE W.Y., N. HANNEBAUM and J.A.C. NICOL (1978) Toxicity of • Puerto Rican organic waste materials to marine invertebrates. University of Texas Marine Science Institute, Final report to NOAA/ODMP, 37 pp • • LEE W.Y., A.MORRIS and D. BOATWRIGHT (1980) A toxicity study of oil accommodated in seawater on marine invertebrates. Marine Pollution Bulletin, in press . • LITCHFIELD, J.T. JR. and F. WILCOXON (1949) A simplified method of evaluating dose-effect experiments. Journal of_Pharmacology and Experimental Therapeutics, 2..§_, 99-113 . • O'CONNOR T.P. (1979) Ocean dumping research and monitoring at Puert_o Rico dumpsite. NOAA/ODMP, 35 pp. (Unpublished manuscript.) • • 32 • SIMPSON D.C., T.P. O'CONNOR and P.K. PARK (1979) Deep ocean dumping of industrial wastes. In: Marine pollution research, R.S. Geyer, editor, in press. • VAN BAALEN C. and J.C. BATTERTON (1978) Effects of pharmaceutical wastes on growth of microalgae. University of Texas Marine • Science Institute, Final report to NOAA/ODMP, 9 pp. WOHLSCHLAG D.E. and F.R. PARKER, JR. (1980) Metabolic sensitivity of fish to ocean dumping of industrial wastes. In: Ocean • dumping of industrial wastes, B.H. Ketchum et al., editors, in press. WRIGHT A. (1976) The u~e df recovery as a criterion for toxicity . Bulletin of Environmental Contamination and Toxicology, 15, 747-749 . • • • • • • • • • • • • • • • • • BIOLOGICAL PROCESS EFFECTS OF ORGANIC WASTES AT PUERTO RICAN OCEAN DUMPING SITES --CONTINUATION STUDIES --FISHES Draft Final Report by Donald E. Wohlschlag and Faust R. Parker, Jr . • ABSTRACT • The respiratory metabolic rates and swimming performances of the spotted seatrout, Cynoscion nebulosus, at 28°C and 35 • o/oo salinity were used to assess the possible degree of a • sublethal 0.125% concentration of a composite ocean-dumped pharmaceutical waste. Three successive groups of fish were added at about 2-day intervals to the aerated waste-contaminated aquaria and their metabolic and swimming rates monitored. Swimming rates were much depressed initially and started to • improve after the first day, while the active metabolism declined for the first group of f·ish ·in the first 2 days. The second group had depressed, but highly variable, metabolism • and improving swimming rate.. The third group (after aquaria had been aerated for about 4 days} still showed severely and uniformly depressed active metabolism, but good swimming • performance. These results suggest that the maximum effects • of toxicity developed over more than one day of exposure and that toxicity tended to persist after 4 days at an expected level of about 0.06% waste concentration, from which future recovery in -nontoxic waters might be likely. However, extended exposure to the relatively slowly attenuating toxicities would • most likely result in excessive metabolic stresses had the • experiments been continued. Exposure-recovery experiments at 28°C and 35 o/oo salinity with the same species indicated that exposures of 2 hrs or less to the composite waste or 6 separate industrial waste sources • • at concentrations from 0.5% to 0.00629%, depending on each • source, were subacute and just sufficient to cause definite visible stress symptoms. Following the appearance of stress, fish were immediately transferred to clean waters for • subsequent determinations of respiratory metabolic rates from resting to maximum sustained active levels over periods of from 2 to 4 days. The metabolic scope, the difference between • • the active and standard (maintenance) metabolism, over this period compared to controls was generally diminished because the standard rate tended to remain at high levels and the active level was usually depressed, except for the stimulatory effects of the Upjohn and possible the Merck, Bristol and Pfizer wastes. However in all the exposure-recoveryexperiments, • the swimming performance always declined. The scope remained markedly reduced for the composite waste recovery experiment, possibly due to the interactions among the various wastes. All • other scopes decreased, except for the Upjohn experiment, in which there was some indication that the larger fish tended to be the more stressed. All fish in all experimental treatments showed at l~ast some signs of fin rot and incipient morbidity after 2-4 days in clean water which is interpreted to mean that toxic levels of wastes high enough to produce visible stress effects within 2 hrs or less are ultimately lethal. The effects on fishes of vµrious open ocean toxic levels and short-term pulse exposures followed by various intervals for recovery or delayed lethality need to be investigated in terms • • of both real world data and population models of fishes that • live permanently in or migrate through areas of dumping. Such effects also need to be compared also with both direct acute toxicities and indirect chronic sublethal toxicities. • Both of these types of experimental techniques on scope diminution could be used for early warning and general biological monitoring of fishes either directly or as ~hecks • on other types of biological manifestations useful for monitoring • • • ! • • ·• • • INTRODUCTION • The purpose of this study is to expand a preliminary study, "Sensitivity of Marine Fish to Organic Pollutants," • which was reported to NOS in December 1978. The result of this study was that the samples of composite wastes from the Puerto Rican pharmaceutical industries had an inhibitory effect • on metabolism and swimming performance of the red snapper. The wastes in leaky plastic containers had been excessively exposed to both heat and light in delayed transit, but they still • markedly affected the metabolic scope at sublethal concentrations of 0.0625% with a 48-hr exposure. Because metabolic scope was depressed still more for fish exposed to the same concentration • of wastes transported in glass, the experiments were repeated for wastes shipped only in glass without unreasonable delays or detoxification• • The concept of metabolic scope--the difference between aerobic respiratory metabolism at maximum sustained active rates and metabolism at minimal standard rates--has proved to • be a useful concept (Fry 1947, 1957, 1971). When there is a sublethal stress on an organism, the scope tends to be reduced because the metabolic costs for maximum sustained activity tend • to be reduced, or the standard maintenance costs are increased, or both. Examples are illustrated for natural stresses (Wohlschlag and Wakeman 1978) or for pollutant stresses • {Wohlschlag and Parker in press), both cases of which also show that the maximum sustained swimming rates also are reduced • 2 at less than optimal conditions. The scope also represents the total amount of energy that would be available for all functions, • such as foraging, assimilation, growth, etc. as partitioned by various authors, e.g., Webb (1978). Several useful features of assessing and evaluating • stresses by the utilization of scope diminution measurements are clearly evident with respect to fishes. Firstly, the effects of sublethal stresses on scope diminution can be • ascertained without any ~ priori knowledge of the stressor, or stressors, in terms of chemical or physical properties. This feature is especially useful in cases~ of effects by mixed • pollutants, possibly fro~ multiple sources, when the individual toxic compounds and their modes of action on organisms may be unknown or only partially understood. • Secondly, the system of measuring the scope for activity has good possibilities for being used directly in continuous monitoring procedures for detecting any decline in water quality • • (Wohlschlag and Parker in press). Indirectly, the measurement of maximum sustained swimming performance by itself could also be used with fish as biological monitors, inasmuch as the maximum sustained swimming velocity and the metabolic scope closely parallel each other in relation to stress (Wohlschlag and Wakeman 1978). Swimming velocity measurements without • oxygen consumption rate measurements would thus be relatively much easier to make than both oand swimming rate measurements. 2 It should be noted that monitoring systems already in use • (Cairns 1980) that depend on behavioral characteristics like • • 3 • gill movement rates or coughing frequencies could be coupled to a non-invasive technique for measuring metabolic scope and swimming rates as a "check" or "calibration" procedure. Physiological rate measurements coupled with behavioral • movement measurement should provide very useful and powerful monitoring tools. Thirdly, metabolic scope measurements for detection and • monitoring of environmental quality perturbations are additionally useful because they can provide data on a number • of subjects of direct interest in environmental management, fish physiology, ecology, hydomechanics of swimming, bioenergetics, and related .fields. An important, but often • overlooked interest, is the cumulative effect of sublethal, chronic environmental stresses on relatively long-lived • ecosystem components like fishes compared to the effects on short-lived components. The length of life cycles and the relative efficiencies of energy conversion at different • ecotrophic levels, both of which are adversely affected by stresses (e.g. culminating in higher mortality rates, metabolic costs, etc.), are directly related to the survival and well • being of the higher ecotrophic levels to which fish belong (Steele 1974). Thus not only are organisms like fish directly sensitive to cumulative sublethal stresses, they also are • sensitive to the cumulative effects these stresses may have on other supportive organisms in lower ecotrophic levels. Steele (1974) notes that the lower ecotrophic levels may be more definitively and adversely affected by stresses than are • • 4 • fishes, but that definitive data and theory on this point need better verification• • Fourthly, the use of metabolic scope diminution techniques to detect sublethal stress provide the rationale for the two classes of experiments in this report, which is for the purpose of exploring: • a. The possible effects of attenuation of toxicity to fishes after an initial addition of toxic pharmaceutical • wastes followed by the usual aeration over a period of several days. These experiments with a composite of individual wastes at sublethal concentrations were • set up to measure the metabolic scope reduction at successive intervals of about 2 days to determine the degree to which toxicity disappears and the degree to which more refractory, but still toxic, materials may remain. • b. The effects of a short-term "shock" exposure at a subacute • level of pharmaceutical wastes on fishes that are then removed to clean waters. These exposure-recovery experiments with the composite and 6 individual wastes • were set up for the purpose of detecting any suspected occurrences of delayed effects on metabolic scope diminution over and above the effects on swimming • performance. Both these types of experiments were for the purpose of extending the concept of sublethal effects at a given low concentration to the more realistic situations in the natural environment where • • 5 • ocean dumping at intervals allows fishes to be exposed to only a limited area of lethal toxicity, a larger area of acute toxicity and for a longer time, and a still larger area of sublethal toxicity persisting for a still longer time and • possibly overlapping dumping time intervals• • • • • • • • • 6 • METHODS Experimental Fish Cynoscion nebulosu~, the spotted seatrout, were the fish of choice throughout all experiments. Capture was by hook and line, usually with shrimp as bait, in coastal waters adjacent to the • Port Aransas area. Transfer was in insulated boxes to the laboratory where fish were held for 2 days to allow for discarding of unfit specimens. Subsequent two-day acclimation was at I• ! 28°C and 35% under controlled conditions before experimental studies. Rather than maintain laboratory stocks of fish with the usual awkward and erratic feeding , regimes that characterize • • this species, conduct of all experiments was with freshly captured groups of wild specimens. Control Experiments In addition to the metabolic data at 35% salinity and 28°C • on this species in Wakeman (Ph.D. diss.) and Wohlschlag and Wakeman (1978), a preliminary series of 34 metabolic rate determinations at these conditions and from 0 to 3.6 body lengths (cm) per second (L sec-1 ) provided comparative "control" data. The techniques for determining metabolic rates are in the above • references or_in the earlier unpublished reports on effects of Gulf waste dumping. The analyses of the data for the controls and for the • subsequent series of experiments was by arithmetic computation -1 -1 of the mg o2 consumption rate usually as Ykg=log1 mg0 kg hr , • 2at the averages the that swam at their maximum for fish sustained swimming rates (Umax) in L sec-1. Because body sizes varied, • • 7 smaller fish tend to have higher lkax values in L sec-1 than • larger fish, and especially because the oxygen consumption rate and body weight are log-log linear, multiple regressions incor porated these relationships as: 1. Y A = a + bw Xw + bv Xv , where Y A = the expected log mgo 2 hr-1 consumption rate; Xw= log weight in grams; -1 X = swimming rate in body lengths L(cms) sec ; v • a = constant; b =partial correlation coefficient relating log weight to w • log consumption rate; ando2 b ~ swimming partial regression coefficient relating v increase in Xv to Y • • (In several cases below another te~m +ht Xt was used to consider the btrate and recovery time Xt hours on the oconsumption rate.) 2 The use of multiple regression in a similar study by • Wohlschlag and Wakeman (1978) follows the conventional statistical procedures, eg. in Sendecor and Cochran (1967) and similar manuals or pretested statistical software packages . • For both controls and other experiments, the estimated standard rates were obtained by utilizing regressions at resting A rates extrapolated down to Ykg by the Brett (1965) method . • Essentially this calculated standard rate was derived by a • 8 • regression of log10 mg02 hr-1 adjusted to a group average weight and plotted against Xv. At the lowest of the adjusted • Y at X = U a parallel line was then calculated and the v max new a intercept below the original a intercept atzero velocity (resting) was taken as the standard rate. Thus for a new • equation the standard rate A Y=a+b X .... • w w since the bvXv.... and btXt if originally calculated, are not • relevant. For any particular group of measurements, the oxygen consumption rates were calculated at a given mean weight, Xw and for both U = X and standard donditions. The rates, max v converted to Ykg = mg10 mg kg-1hr-l could then be used to o2 calculate the metabolic scope for activity by subtracting the the standard from the active rates. 1 I I• Composite Waste Toxicity Attenuation Experiment (1.0) The preliminary trials were conducted with a frozen composite waste sample that was about one year old and no doubt partially degraded. The concentration tested initially was 0.125 v/v% • for spotted _seatrout, Cynoscion nebulosus, double the concentration that indicated a minimal metabolic scope reduction for the red snapper, Lutjanus campechanus experiments (Wohlschlag, • et al. 1978 report). At this concentration there was for the trout no observable effect after 29 hours, but there was evidence of a metabolic scope reduction. • With fresh composite waste material, the initial concentration was 0.125 v/v% in the 28°, 35% salinity water in the entire • 9 • Blazkaapparatusand exercise acclimation tank, which was well aerated continuously. Six fish previously acclimated were placed I · in the exercise tank in the morning as soon as the composite wastes had been well mixed within the system. Two fish were then run through the swimming rate and oxygen consumption rate pro • cedures in the Blazka respirometer in the afternoon, two more the second morning after about 26 hours and two more the second afternoon after about 30 hours. One fish was "left over" in the • first group for another set of rate determinations starting I immediately in the a.m., two in the p.m., 2 in the a.m. next day and 2 in the p.m. the same day . • A second set of experiments was carried out with 3 succes sive groups of 6 fish each group every other day at the same pollutant concentration, temperature and salinity. • • • • • 10 The schedules for the two sets of data were: Set 1 5 IX 79 at 0845 Pollute Blazka apparatus; 6 fish in. • P.M. 2 fish tested. 6 IX A.M. 2 fish tested. • P.M. 2 fish tested. 7 IX A.M. 6 fish placed in exercise tank. • P.M. 2 fish tested. 8 IX A.M . 2 fish tested. P.M. 2 fish tested. ! Set 2 12 IX 79 at 0830 Pollute Blakza apparatus; 6 fish in. • P.M. 2 fish tested• 13 IX A.M. 2 . fish tested. P.M. 2 fish tested. • 14 IX A.M . 6 ·fish placed in exercise tank. P.M. 2 fish tested• . 15 IX A.M. 2 fish tested. • P.M• 2 ffsh tested. 16 IX A.M. 6 fish placed in exercise tank P.M. 2 fish tested. • 17 IX A.M . 2 fish tested. P.M. 2 fish tested. The maximum sustained metabolic rates and the maximum • swimming rates for each ·fish at each successive time interval were tabulated and plotted against elapsed time as indicated best by a review of Appendix Table 1.0 and Fig. 1, which also show rate averages at daily time intervals for e~ch of the fish • • 11 groups~ A multiple regression that related log oxygen consumption rates to log weights, swimming rates, and elapsed times was calculated. From this equat~on, at the average experimental weight, and the maximum observed x_ = 3.9 L sec-, the calculated v • 1 • maximum active metabolic rate was calculated. Also, for the same weight and activity, the Equation 8 of Wohlschlag and Wakeman (1978, p. 187, Table 2} was used to make a comparative calculation. • Composite Waste Exposure-Recovery Experiment Fish were secured as described above and acclimated (2.0} for two days at 28° C and 35% salinity. Initial trials were ~ith the • fresh composite waste at a concentration (v/v) of 0.5% at the acclimation conditions and for a period of 2 hr, during which it was observed that visible signs of stress had become established or immi!ent within one hr. One-hour exposure at 0.5% v/v was utilized for all the composite waste experiments. Respiratory rates were determined daily for two fish at maximum (U }, and • max intermediate swimming rates, and at resting conditions. Three sets of fish were examined in this manner so that data up to elapsed recovery times of about 4 days were available. A set of • regressions as described above, with and without time, were computed for active rates. A regression to estimate the ~stressed standard'' rate was also computed by th~ Brett (1965) method • For comparative purposes~ the latter standard was calculated • for a fish of average weight at ·resting conditions (really a calculated resting rate) for comparison to a calculated standard rate from the control regression. The maximum activity level of • • • • • • • • • • • • 12 the control observed at 3.6 L sec -1 was used with the average weight of 175 g for the experimental fish to calculate a metabolie level (2) at maximum activity. The maximum activity at 3.9 L sec-l for the fish in the aerated composite waste experiments above was used in the control regression equation with the average weight of 175 g to calculate a maximum metabolic level (1) at sustained activity_ Individual Waste Exposure~Recovery Experiments (3.1-3.6) Initial trials with fish in various concentrations of the wastes (starting at 0.5% v/v) at various time exposures were visually noted for init£al behavioral, stress manifestations for each of 3 separate industrial waste$. The principal stress manifestations were coughing, head shaking, rapid or erratic ventilation (gill pumping) rates~ rapid or erratic swimming or other non-locomotry movements and loss of equilibrium or supination. The preliminary observations resulted in the following arbitrary concentrations and times of exposure for each of the industrial wastes as follows ~ 3 ~1 Upjohri 3.2 Merck 3.3 Bristol 3.4 Capri 3.5 Squibb 3.6 Pfizer In each case the time interval 0.5% 1 hr 0 .. 5% 2 hr 0.00625% 2 hr 0 .. 5% 2 hr 0 ~ 5% 110 min 0.5% 2 hr was arbitrarily selected as the • 13 time when fish first exhibited acute stress, or when 2 hours had elapsed, after which the fish were placed in the exercise tank · of the Blazka rig with clean water at 28° C and 35% salinity. The respiratory rate determinations preceded as in the previous set of experiments at zero, intermediate and maximum swimming velocities. For each of the wastes, regressions at both active and ,. standard respiratory levels are in the same form as for the exposure-recovery experiment with the composite waste described above. For each of the separate wastes the observed metabolic rate • data were plotted at time. For comparison • but at control V ~ 1 i max I u and resting (X =O) rates against ~lapsed max · v ~ at control conditions of the same weight, X = 3.6, the act~ve control rate was plot v ted. Also at the same weight, the control equation was used to calculate a standard control rate for comparison. ,. For each of the wastes the maximum swimming plotted against the elapsed time . • • • • rates we~e • 14 • RESULTS Individual Respiration Measurements All the data used in making the various regression calculations are in the appendix tables, starting with the control data in Table A. l , the aerated composite waste experimental data • • in Table A. 2, the composite exposure-recovery data in Table A. 3, and the successive individual industry waste exposure-recovery data in Table A. 4 • Regression Data and Means The ranges of the variables for these respective data and the pertinent regressions are in Tabl~ 1. . , • In Table 2 are the regressions without the time (Xt) variable, except for the continuously aerated composite waste dilution experiment to which fish were added at successive intervals . Also included are the equations for the standard rates as determined by the Brett (1965) method. The appropriate statistics and probability levels for • occurence of given values are in Table 3 for the ~ctive respiratory rate equations of Table 2. The same data used in Tables 2 and 3 without time Xt are in Tables 4 and 5 with Xt retained, although only two equations have statistically significant bt values (Table 5). • Time Series-Composite Waste Toxicity Attenuation Experiment (1.0) As plotted in Fig. l~ the three groups of fish did not tend to show successively higher active metabolic rates with • continued aeration of the composite waste, which should have • Table 1. Averages and ranges of variables used in regression equations for respiration rate experiments on Cynoscion nebulosus (spotted seatrout) under control conditions and various concentrations • of pharmaceutical wastes at 28°C and 35 o/oo salinity, Equation No. Weight (g) Velocity (L sec-1) Umax Velocity (L sec-1)and N Range Average Range Average Range AverageDescription 0.0 Control 34 72 -174 114 o.o -3.6 1.7 2,9 -3.6 3,3 1. 0 Aerated 29 132 -444 204 1.9 -4,3 3.4 1.9 -4,3 3,4Composite 2.0 Composite 36 -175 -1.3 2.0 61 377 0,0 2.7 1.0 -2.7 Exposure- Recovery 3.1 Upjohn 14 321 -674 461 -2,5 1.1 -2.5 2.0 o.o 1.6 3.2 Merck 25 99 -371 163 o.o -3.1 1.5 2.2 -3.1 2.8 3.3 Bristol 13 67 -152 109 o.o -3.4 1.7 2.6 -3.4 3.1 3.4 Capri 19 86 -410 174 o.o -3.1 1.5 2.0 -3.1 2.6 3.5 Squibb 15 87 -258 137 o.o -3.3 1.6 2.6 -3.3 2,9 3.6 Pfizer 23 75 .... 332 144 o.o -2.9 1.5 2.2 -2.9 2,6 ....., U1 Table 2. Regression equations for fish respiratory metabolic responses to control and short-term exposure to pharmaceutical waste samples at various active swimming (Act) and standard (Std) rates. Values rounded to three decimal places. Time variable not included except for Equation 1.0. Equation No. Description . o.o Control 1.0 Aerated (Composite) 2.0 Exposure-Recovery (Composite) 3.1 Exposure-Recovery (Upjohn) 3.2 Exposure-Recovery (Merck) 3.3 Exposure-Recovery (Bristol) 3.4 Exposure-Recovery (Capri) 3.5 Exposure-Recovery (Squibb) 3.6 Exposure-Recovery (Pfizer) Concentration, Exposure Time (hr, min) 0 0.125%(*) 0.5%(1 hr) 0.5%(2 hr) 0.5%(25 min) 0.00625%(2 hr) 0.5%(2 hr) 0.5%(110 min) 0.5%(2 hr) N 34 29 36 14 25 13 19 15 23 • Equations A Act Y = +0.070 + 0.712 Xw +0.112 Xv A Std Y = -0.037 + 0.712 Xw A 'Act Y = +0.496 + O. 756 Xw -0.028 Xv -0.002Xt** A Act Y = +0.287 + 0.679 Xw +0.083 Xv A Std Y = +0.187 + 0.679 Xw A Act X= +0.057 + 0.740 Xw +0.178 Xv Std Y = -0.044 + 0.740 Xw A Act X= +0.397 + 0.647 Xw +0.080 Xv Std Y = +0.328 + 0.647 Xw A Act X= +0.910 + 0.392 Xw +0.059 Xv Std Y = +0.799 + 0.392 Xw A Act X= +0.311 + 0.643 Xw +0.099 Xv Std Y = +0.161 + 0.643 Xw A Act X= +0.389 + 0.635 Xw +0.056 Xv Std Y = +0.292 + 0.635 Xw A ·Act X= +0.579 + 0.550 Xw +0.082 Xv ....... Std Y = +0.465 + 0.550 Xw °' *Fish continuously exposed to aerated pollutant at initial concentration of 0.125% v/v. **Regression not run without time variable, Xt, hours. Table 3. Statistics for regression equations without time included (see Table 2). Multiple Correlation Standard Error Equation Coefficient Estimate Weight Coefficient Velocity Coefficient No. N R p Sy Sbw p SbV 0.0 Control 34 0.0790 0.92 0.1242 <0.001 0.0092 <0.001 1.0 Aerated Composite 29 0.88 0.0834 0.1469 <0.001 0.0410 n.s. 2.0 Exposure Recovery 36 0.95 0.0658 0.0499 <0.001 0.0111 <0.001 3.1 Upjohn 14 0.79 0.1400 0.3457 <0.05 0.0471 <0.005 3.2 Merck 25 0.95 0.0606 0.0527 <0.001 0.0100 <0.001 3.3 Bristol 13 0.64 0.1156 0.1888 n.s. 0.0212 <0.01 3.4 Capri '· 19 0.84 0.1278 0.1156 <0.001 0.0275 <0.005 3.5 Squibb 15 0.82 0.1042 0.1556 <0.005 0.0213 <0.025 3.6 Pfizer 23 0.86 0.0911 0.0914 <0.001 0.0169 <0.001 • ....... .......] Table 4. Regression equations with time variable for exposure or exposure-recovery experiments at active swimming respiratory rates. Values except time coefficient rounded to 3 decimal places. Concentration, Total Equation Exposure Time Duration No. Description · (hr, min) N Equations (hr) A 1. 0 Aerated 0.125%(*) 29 Y = + 0.496 + 0.756Xw -0.028Xv -0.00218Xt 130 (Composite) 2.0 Exposure-Recovery A 0.5%(1 hr) 36 Y = + ~.219 + 0.705Xw + 0.083Xv + 0.00028Xt 96 (Composite) A 3.1 Exposure-Recovery 0.5%(2 hr) 14 Y = -0.577 + l.OOlXw + 0.174~ -0.00214Xt 49 (Upjohn) 3.2 Exposure-Recovery 0.5%(25 min) 25 Y= + 0.346 + 0.661~ + 0.081Xv + 0.00047Xt 86 (Merck) A 3.3 Exposure-Recovery 0.00625%(2 hr) 13 Y = + 0.746 + 0.433Xw + 0.061~ + 0.00233Xt 70 (Bristol) A 3.4 Exposure-Recovery 0.5%(2 hr) 19 Y = + 0.397 + 0.630Xw + O.lOOXv -0.00200Xt 72 (Capri) A 3.5 Exposure-Recovery 0.5%(110 min) 15 Y = + 0.356 + 0.661~ + 0.056~ -0.00071Xt 67 (Squibb) A 3.6 Exposure-Recovery 0.5%(2 hr) 23 Y = + 0.808 + 0.473Xw + 0.080Xv -0.00128Xt 96 (Pfizer) • *Fish continuously exposed to aerated pollutant at initial concentration of 0.125% v/v. I-' Table 5. Regression statistics for equations including time (see Table 4.) Multiple Standard Errors Equation Correlation Estimate Weight Coeff. Velocity No. N Coefficient p Sy Sbw Sbv 1. 0 Aerated 34 0.88 0.0834 0.1469 <0.001 0.0410 Composite 2.0 Exposure-36 0.95 0.0666 0.0838 <0.001 0.0115 Recovery 3.1 Upjohn 14 0.79 0.1451 0.6805 n.s. 0.0496 3.2 Merck 25 0.95 0.0607 0.0545 <0.001 0.0100 3.3 Bristol 13 0.75 0.1023 0.1734 n.s. 0.0188 3.4 Capri 19 0.87 0.1215 0.1102 <0.001 0.0261 3.5 Squibb 15 0.82 0.1078 0.1704 <0.005,. 0.0220 3.6 Pfizer 23 0.89 0.0863 0.0965 <0.001 0.0161 Coeff. p n.s. <0.001 <0.005 <0.001 <0.001 <0.005 <0.05 <0.001 • Time Sbt 0.0005 0.0007 0.0047 0.0005 0.0013 0.0012 0.0015 0.0007 • Coeff. p <0.001 n.s. n.s. n.s. <0.05 n. s. n.s. n.s. • ~ ~ 20 • Fig. 1 Top Pa'nel. Active metabolic rates observed for three groups of cy-no:sci·on· nebut·?sus exposed to continuously • aerated composite industrial waste at 0.125% v/v initial concentration and at 28° C and 35% salinity. Circles denote the first group, squares the second • . group, and triangles the third group of observed metabolic rates. Corresponding larger symbols with radials denote approximate daily means. Large arrows • with same symbols between panels indicate times when successive_groups of fish were added~ Dash~d line indicates arbitrarily drawn trend of active metabolic • rates. Bottom Panel.. Maximum sustained swimming rates of same Cynosci·on ·nebulosus groups as in panel above. All ---..------.. - • --~~~ symbols same as above. • • • • • • . I • 22 exhibited declining toxicity with time~ Actually the Fig. 1 • bottom panel data indicate that the maximum swimming rates tend • to increase with time, and presumably, the lessening of toxicity to successive fish groups. What is not quantitatively measurable is the_general degree • of metabolism elevation caused by spontaneous (non-locomotory) activity, which, for resting fish that experience some degree of irritation, can be 2-3 times as high as standard levels (Fry 1957, • 1971) . In the present experiment the spontaneous activity was exceptionally high for the first two days, with considerable abatement thereafter, and with a general increase in maximum • swimming rates. The "control" level of Fig, 1 was computed from active Equation 0.0 with the average weight for the last group of fish -1 (triangles) and the single greatest X = 3.9 L sec . Similarly • v the 11 calculated" level was computed with the same weight and swimming rate using Equation 8 of Wohlschlag and Wakeman (1978) • This procedure also verified the consistency of the Equation 0.0 of this study with earlier equations, • • • • • 23 Time Series--Composite Waste Exposure-Recovery • • Experiments (2.0) Narrative Account. Initially the exposure of fish to 0.5% concentration of the composite waste was attempted for 2 hr, but excessively erratic behavior and failure to swim before 2 hr elapsed indicated that a l~hr period would constitute a severe exposure from which recovery, if any, could be assessed. • For the 1-hr exposed fish, all swam at the surface and vent ilated rapidly after about 45 min, but none showed any additional acute stre~s at the end of 1 hr. After removal to clean water of the exercise tank, a 21~hr period ~lapsed before the first le fish became supine with ·1oss of balance. At 22 hrs the maximum swimming speed was only 1.4 L sec-l for one fish, while at 26 hrs another began to show evidence of tail rot and managed to swim at a maximum rate of 1,5 L sec~l. At 45 hrs one fish that failed to swim consistently also had moderate tail rot. At 68 hrs 1.1 • L sec-l was the maximum speed attained by one fish with moderate tail rot, while at 73 hrs 1.2 L sec-l was the maximum swinuning rate for another fish with both moderate fin and tail rot. The final small fish examined at 95 hrs swam at only 1.0 L sec-l and had heavy tail rot and impaired balance. Metabolic ·~~ Activity ·Levels. The metabolic and maximum sus • tained swimming rates that were observed are plotted against elapsed time in Fig. 2. In the top panel the top solid Line (1) is calculated from the control regression at an average weight of 175 g and a maximum X = 3.9 L sec-l averaged for one group v . in the previous experiment, while Line (2) is calculated from • 24 Fig. 2 T?P Panel. Observed active (open symbols) and resting (stippled symbols) metabolic rates for three groups of Cyn·o·scion ·n·ebulosus recovering after an initial expos ure of one hciur at a composite waste concentration of 0.5% v/v. Circles, squares and triangles represent first, second and third sets of observations. Upper dashed line is arbitrarily drawn trend of observed maximum active metabolic rates; lower dashed line is arbitrarily drawn trend of observed resting metabolic rates. Maximum activity lines (1) and (2) are calcu lated for ave;age weight from control regression at X = 3.9 and 3.6 L sec-l swimming rates , respectively. v Calculated standard control line is from control regression at average we~ght (Xv = 0). Calculated resting rate is from regression based on experimental data at avera. ge weight and X = 0. . v Bottom Panel. Visually drawn trend (dashed line) of observed swimming velocities compared to maximum rate observed for controls (solid line). • • • • • • • • • • • 25 • 3.0---------------------------------------------------------r---------. 0 • ~----0 ~ MAX.ACTIVITY( I) -0 _______ _ -0 ...._------MAX.ACTIVITY (2)_,_ -------- 2.8 G - ~c\\'l'..!-'< ----0 I... 0 "" ~~...:..----8 • -.c I o~_§.; _.. - -(!) ,,,,. 0 ~ OBS. REST! NG 'Ct -------- 0 N 2.6 --11 .. Ct - • 0- ----- E Ct CALC. RESTING 0 • 2.4 ~ CALC. STANDARD I I I I I • 4 I I EJ 38 ...... 0 ............ 0 ~ 8 c:J ....... • - --......... I (J 2-8 0............... 8 • Q) ' ....... ----... (/) 8 00 0 .__ ~0 ..J 0 ----0-~ ,----~,------1------,------.,_.,-----1-------,------,------1--------1...... Q_.,________ • 60 80 100 0 20 40 ELAPSED TIME (hours) • • 26 -1 the same control equation, 175 g and X = 3.6 L sec which was the maximum single value observed for the small control fish. • v The "Calculated Resting'' rate line is calculated from Equation 2.0 for all the data pertinent to swimming and resting fish, for • the average weight and for X = 0, whereas the "Calculated v Standard" line is from the control data at standard conditions (Xv = O) and at ave~age weight~ In all parts of the re~overy • periods, the amount of non--::-.locomotory, spontaneous activity exhibited by the fish was much .greater than in control experiments • • • • · I • • • 27 Time Series--Upjohn Waste Exposure-Recovery • Experiments (3.1) Narrative Account. During the first 30 min of the 2-hr exposure to the 0.5% concentration all fish appeared calm and normal. After 45 min, ventilation rates increa~d; after 55 min two fish appeared to show some equilibrium loss, while larger fish began to show · increased swimming act.tvity. During the second hr rapid • ventilation rates.were always obvious, but signs of disequilibrium were increasingly evident only for the larger fish. Some signs of coughing and he~d shaking persisted during the second hr of • exposure. After transfer to the exercise tank the fish began to appear in poor condition by about 24 hrs with the prevalence of at least • some degree of fin and tail rot. Fish tended to be sluggish and did not swim well in some cases, in which attempts to induce swimming resulted supination, a prelude to death. Death occurred in • the exercise tank after 38 hrs for one specimen with heavy fin and tail rot and body lesions. Howeve~ the 4 fish that yielded maximum sustained swimming rates did swim reasonably well and • moderately fast compared to controls. Metabolic and -A·ctivity ~~· The changes in the observed metabolic levels during recovery are ·plotted for both active and rest • ing fish in Fig. 3 (top panel). The arbitrary, visually drawn plots for the scatte~ed data probably show th~t the fish would eventually reach normal active and standard levels that are indi • · cated by the control levels calculated from the active and standard control equations of Table 2 for average ~eights (note from • 28 • Fig. 3. Top Pan·e1. Observed active '(open circles) and resti!lg (stippled circles) metabolic rates for\ cyn·os·cion nebuTosus • recovering at 28° C and 35% salinity aftei an initial exposure of 2 hours to a 0.5% v/v concentration of Upjohn pharmaceritical waste. Active ~ontrol line calculated • from control equation with aveiage experimerital fish weight of 461 g and maximum control swimming speed of -1 3.6 L sec .. Standard control level calculated at same • weight and zero velocity from standard equation for control fish. Bottom Panel. yisually drawn trend line (dashed) of • observed swimming velocities compared to maximum rate observed (solid line) for control fish. • • • • • • • 29 3.0 I I I i I I I I J I • - 0 2.8 - -....._, G -... • - ...... - e ...... ACTIVE CONTROL -- ' ...... ......... ...... e' ....... 2.6 - ....... • ' " - .t::. • -·· OI ~ ....... x • I N -• ....... - 2.4 ..._ 0 -------• 0 --- OI 0 • 2.2 -STANDARD CONTROL • • I I I I I I I I I I I I I· I I I 4 • ACTIVE CONTROL 3 .,,,,,,,. '0 _.,, --- 0 0 ...... • _, © -...... ..,.... Cl) 2 -' ....... ---0 ---- ...J I • 0 ~l..__----~-----,------..,.,-------l----~------,...-----~------,-1-----...,1-----------.1 1 0 20 40 60 80 100 ELAPSED TIME (hours) • • Table 1 that these are the largest fish studied) and for 3.6 L • sec-l at the active control level and for zero velocity at the standard control level. Apparently fish that could recover from th~ fin and tail rot would perform satisfactorily. The wide • variance among metabolic measurements results in large part from weight difference~. Such diffeiences tend to become normal if the metabolic rates at act~ve and re~ti~g are recalculated for the • average weight. • • • • • • 31 Time Series--Merck Waste Exposure-::--Recovery e Experiments (3.2) Narrative Account. Exposure to 0.5% Merck waste was for only 25 min because of severe erratic swimming and highly opaque water • even at this low conceritration. After only 5 min fish were swimming at the surface with dorsal and tail fins e~posed; after 20 min one fish b~dame supine. Upon transfer to the clean waters of • the exercise chamber,, equilibrium difficulties persisted for some fish as long as 35 min. Although all fish were at least somewhat disoriented initially, none showed any particularly obvious ab• normal behavior after 35 min t The fish terided to swim quite well for about 3 days, after which some swimming weakness started to appear along with increasingly obvious tail rot and spontaneous activity especially for the last fish at 84 his. Metabolic and Activity Leve'ls . Active and restin9 metabolic levels observed are in Fig. 4 as visual plots respectively shown • by dashed lines. Larger or sm~ller than average fish are respect~ ively denoted by L or s. The control levels at maximum activity 1 (3.6 L sec-) and at standard conditions are frbm calculated • values based on the average weight of the experimental Merck fish~ The activity as shown in Fig. 4 compared to the control level most likely tends to drop with elapsed time « • • 32 • Fig. 4. Top Panel. Observed active (open circles) and resting (stippled circles) metabolic rates for Cyno·scion • nebuloaus recovering at 28° C and 35% salinity after an initial 25 min exposure to a 0.5% v/v concentration of Merck ph~rmacetiti6al waste. Active and resting rate • trends shown by visually drawn dashed lines. Active control line calculated from control equation with average experimental fish weight of 163 g and control • 1 swimming maximum rate of 3.6 L sec-. Standard control line calculated at same wei.:cJht and zero velocity from sta.ndard equation for control fish.' • Bottbm Panel. Visually drawn trend (dashed line) of swimming rates compared to maximum rate observed for control fish ·csolid line). • • • • • • • 33 3.0 I I T • 0 0 es -----0------G----0 -ACTIVE CONTROL 2.8- -----..._ ....... ...... • 0 -----.._® ._ • ----- @ I ~ 0L --I OI -----0L e I e le .:s! 2.s-___.,.,. --------------®L .,,,_,,, 0 C\l _.,,.,.,-e • __. -~ OI 0 2.4 • -0 E> L STANDARD CONTROL I I I 1 1 I l • 4 - I ! I ACTIVE CONTROL • 3 --0 ----0 0 ----e 0 ---0 - ......... ............ 0 0 0 ' • u C> 2 en _J I 0-4-,-------------,-------------,------1.-------,------1-------i-------------,-- • o 20 4 0 60 80 I 00 ELAPSED TIME (hours) • • • 34 Time Series--Bristol Waste Exposure-Recovery • e Experiments (3.3) Narrative Account. At preliminary levels of pollutant as high as 0.0625% v/v, the fish reacted almost immediately with complete cessation of respiratory movement and supination. At 0.00625%, however, fish appeared to be fairly normal for the 2 hr exposure used in the remainder of the studyt with little more obvious • reaction than a slight ventilation rate increase. For the duration of the recovery period in clean water, the fish showed little sign of tail or fin rot~· There was some visual evidence of a "delayed" • reaction after about 2 d~ys,, when swimming seemed to be subtly slower and less well coordinated, although the numbers of fish ;in the experiment were too ~mall and the recovery time period too short for obvious stress symptoms to be detected other than the persistence of spontaneous activity among resting fish. ~etabol·ic a~~-Activity Levels. In the top panel of Fig. 5 are I· plots of the observed active and resting metabolic rates with visually drawn dashed lines to indicate general trends. It is interesting to note that the active level stays consistently high, while the resting level is much higher than the standard control · level, calculated on the basis of the average weight. The active level for control was calculated at the same average weight and at 3.6 L sec-, the maximum observed for the control fish. In the • 1 bottom panel are plotted the observed maximum swimming rates with a visually drawn trend (dashed) line that indicates a decline with • time . • • 35 Fig. 5. Top Panel. Observed active (open circles) and resting • (stippled circles) metabolic rate~ _for Cynos~ion nebuTo·sus recovering at 28 ° C and 35% salinity after an initial 2 hr exposure to a 0~00625% v/v concentration • of Bristol pharmaceutical waste. Active and resting metabolic rate trends shown by arbitrarily drawn upper and lower dash~d lines, respedtively. Active control • line calculated from control data at avetage expeiimerital weight of 109 g and at maximum control swimming rate of 1 3.6 L sec-. Standard control rate calculated at same • weight and zero velocity. Bottom Pan'el. Visually drawn · trend among obperved sw;i,:rnming rates ·(dashed line) compared to maximum rate (solid • line) observed for control fish. · • • • • • 36 3.0 I I I I I I I I I ~ ~ 0 • ------0' ACTIVE CONTROL <:> -G - 2.8 ® L <:>-~-- • -I ----• ---... ...__ -............ ' .s= ............ - I • Cl 2.6 .:it! N 0 0 - Cl 0 el • 2.4 - STANDARD CONTROL -- I I I I I I I I I I I I I l I I I I 4 '------------------AC Tl VE C 0 NT R 0 L 0-----<=>---- C!>---e ........ 3 - • ' 0 Q) 2 "' --' • I - 0 _....~~-...-,~~-l~~---.~~~....-~~--1~~~,~~--..~~---.,~~~--,----~,---J• 0 20 40 60 80 100 ELAPSED TIME (hours) • • • 37 Time Series--CAPRI Waste Exposure,Recovery e Experiment (3.4) Narr·ative· Ac·count. A 2...;hr exposure to a 0. 5% v/v concentration of this waste produced no obvious visible signs of stress, but • within the first se~eral recovery hours in clean water of the exercise tank, the fish swam near the surface. Normal fish would swim near the bottom. Within about 12 hrs these fish all swan • normally near the bottom with little unusual evidence of excessive spontaneous activity. By 24 hrs one fish that refused to swim was discarded, and another, which had equilibrium deficiencies, • but nevertheless managed to complete a swimming run, finally performed well enough to provide suitable data. After about a 50-hr recovery period one fish tended to swim sporadically. Another at • about the 60-hr period swam.with difficulty after exhibiting definite stress, brief initial supination, and consistently slug. g i sh swimming. • Metabolic· a.nd Activity L·evels. Fig. 6, top panel, contains plots of the observed active and resting metabolic rates with arbirarily drawn dashed lines to indicate trends. Between 2 and 3 days, • it appears that the upward resting and downward active metabolic rate trends are respectively reversed and that recovery might be underway after about 3 days. The active control level for com• parison is calculated at the average experimental weight of 174 g and the control maximum swimming speed of 3.6 L sec-1 ; the standard control is calculated at the same weight and zero velocity • from the standard equation. The fish tend to be somewhat more sluggish than controls, especially after about 2 days of recovery, • 38 Fig. 6. Top Panel. Observed active (open circles) and resting (stippled circles) metabolic rates for Cynosc~on · ·nebulosus recovering at 28 ° C and 35% salinity from an initial 2-hr exposure to CAPRI industrial waste. Active and resting trends shown by repective upper and lower dashed line~. Active ~ontrol level calculated from control data ab average experimental fish weight of 174. g and control maximum activity of 3.6 L sec -1 . Standard control level calculated from standard control equation at same weight and zero velocity. Bottom Panel. ,Visually drawn trend line among observed swimming rates :(dashed line) compared to maximum observed r~te (solid line) for control fish. • • • • • • • I I • • [. 39 I I I I I I I I I I GS • 3.0 - 0 ------... ........ ....... ......_ ACTIVE CONTROL G' • 2.8 -' ,. -0 L ' • -~ ' 0 " 0 .J::. I O' ' & 2.6 ...:.: -• 0' ' ....... ' N ........ 0 - ........_ ,,,,,,.,,,. ---------#1-:· --- O' / e .:' • 0 0 / / e """' ' 2.4 - • STANDARD CONTROL tJ! L 2.2- I I I I I I I I J I • I I I I 4 -I - ACTIVE CONTROL -(!> 3 • -3...-0- ~........ ........__ ........ .._ ' 0 0 ~ Q) (,) 2 -G> ~.- en --- • ..J I O--t--------~-----------~------~------------- 1 ------_,..,--------.,------------------.---' 0 20 40 60 80 100 ELAPSED TIME (hours) • • • There is no page 40! • • ,' • • • • 41 but apparently there is a good trend toward normal swimn\ing • performance after about 3 days . • • • • • • • • 42 • Time Series--Squibb Waste Exposure-Recovery • Experiment (3.5) Narrative Account. The exposure time was only 110 min instead of 2 hr at the 0. 5% v/v level. Fish seem_ed normal just before • 110 min , after which rapid swimming at the surface and consid erable jumping began. The fish were immediately transferred to the exercise tank and within 1 to 2 hr were apparently normal • for a.bout 1 day. After about 40 hr all fish had developed at least som.e fi.n and tail rot~ At 46 hrs • 3 fish became disoriented a.nd became supine while in the Blazka chamber; all 3 had fairly severe fi.n and tail rot.· By about 40 hr and following, spontan eous a.cti.vity seemed to increase < while swimming performance • decreased.. Metabolic ~("-r ·metabolic a.rid·· Activity' Levels.. Both the active and resting ~~.-....... , . rate observations are plotted in Fig.. 7 (top panel) , with visually drawn dashed trend lines that indicate that the I 1. time trend is away from control levels shown in the solid lines. The top line is calculated from the control equation with the experimental average ~eight of 137. g and the maximum control • swimming speed of 3 .. 6 L sec"."'1 The standard line is calculated from the standard control equation for zero velocity and for the same average weight \ The bottom panel of Fig. 7 is a plot of the observed maximum swimming rates with a visually drawn dashed trend 1 line and with a comparison control also at 3.6 L sec-. The maxi.m.ufC\ swimmi.ng rate apparently tends to decline with time and • the development of tail and fin rot . • 43 • Fig. 7. Top Panel. Observed active (open circles) and resting (stippled circles) metabolic rates for Cynoscion • nebulosus redoveiing at 28° C and 35% salinity from an initial 110-min exposure to a 0.5% v/v concentration of Squibb pharmaceutical waste. Respective visually drawn • upper and lower dashed trend lines indicate active and re~ting levels. Active control level indicated by upper solid line as calculated for average experimental • fish weight of 137 g and 3.6 L sec-l maximum control fish swirnro.ing rate. Standard control level calculated .. ..: from standard control equation at zero velocity and at • same weight ... ~:o~ P~nel. Trend line (dashed)visually drawn through observed maximum swimming rates compared to control line • (solid) at maximum rate of 3.6 L sec-1 . • • • • • 44 • 3.0 I I I l I I I I I I -©S -G - - 0 ACTIVE CONTROL - -----...... • 2.8 -0 ........ .......... 0 ' --- ' - e--- --9 --® _., I _,,,,,.,, ~ .,,,,,,,. ' ..c . ' ./ / • --/ " 0 ' I / OI 2.6 .:.: e / N / - OI - 0 • 0 0 -/ ~ 2.4 - STANDARD CONTROL • - I I I I I I I I I I ! le • 45 • Time Series-:-.-Pfizer Waste Exposure-Recovery Experiment (3.6) • Narrat~ve Account. The 2~hr exposure to 0.5% v/v concentration of the Pfizer waste had no obseivable effect on the fish. However, 20 hrs after recovery in clean water had been underway, • one fish with some fin and tail rot had become a sporadic swim mer.. 1\t 22 ·~ 5 hrs another fish was observed to have some tail and fin r·otf. but r ·emained at a high ·revel of swimming ·performance . • By 42 hrs another fish with severe tail and fin rot swam well. By the 66-hr period two fish that had bad cases of tail and fin rot would weave ·from side to side in the chamber. At 70 hrs another ~-mall fish, heavily infested, was found dead in the exerct&e tank. During the entire period the level of spontane • ous activity tended to be fairly high . !!eta~o,li~, an 0 • II> 2 _J • I 0 I I f 60 100 0 20 40 80 ELAPSED TIME {hours) • • • 48 • weight and zero activity. The ~isually drawn trend line of the bottom panel is through the obse·rved swimming rates for the surviving fish and the adequate perfdrmers in reference to the 1 control rate of 3.6 L sec-. • • • .' • • • • • • • 49 DISCUSSION • General Comments The general evaluation of techniques used in this study has been given in Wohlschlag and Wakeman (1978) as well as • in reports to NOS Ocean Dumping Program from earlier studies on Gulf and Puerto Rican wastes. Inasmuch as no new physiological principles have been involved, the discussion • to follow will consist of discussions of individual portions of the study, of pertinent application possibilities in bio-monitoring, and of some badly needed future ecological • considerations for transient fish populations (or portions ~ of them) that may have been exposed to subacute toxicities for which deleterious but cumulative population effects may be • delayed. • Toxicity Attenuation Because the initial experiments with sublethal 0.0625% v/v concentrations of the composite pharmaceutical industrial wastes indicated that the metabolic scope and swimming performance of • the red snapper (Lutjanus campechanus) are reduced after a two day exposure_ (Wohlschlag and Parker, In Press) , the first preliminary experiment was to detect the degree of recovery • that a spotted seatrout might experience as the pollutant was • continuously aerated and, presumably, degraded. The choice of the initial concentration of 0.125% of the composite waste seemed reasonable on the basis that the initial red snapper experiments at this concentration indicated no mortality within 48 hours and that the metabolic scope and swimming performance were • • rather severely depressed. • groups of spotted seatrout The first group of fish active metabolic rates with • that spontaneous rates were However (Fig. 1) (Fig. 1) 50 the results of the three were unexpected• had very high initial low swimming rates, which indicated excep~ionally high. Fry (1957, • 1971) reviews the problem of spontaneous activity that can elevate metabolic rates several times above the standard rates with no swimming activity whatsoever. While swimming at • maximum sustained rates usually eliminates obvious nonlocomotory extraneous activity1· the examples with the pharmaceutical wastes are striking e~~eptions. In most ail • cases, the sublethal .concentrations o·f the composite, as well as the individual, wastes elicited some degree of non-locomotory movement and general irritability (stimulation?) at some time • interval after exposure; however, the apparent initial response often could be variously classified as "n~gative" or "anesthetized" or "stunned" at which time the metabolic rates were .also elevated• Quite possibly any initial effect of raising the metabolic rate could eventually be explained by physiological and • biochemical study of the various systems -that would be insulted or stimulated by even the lowest levels of toxic materials, but presently there is no easy observational or experimental • method of evaluating cause-and-effect relationships of spontaneous activity and elevated metabolic rates. In Fig. 1, it is rather obvious that the second and third • groups of fish functioned at a much lower active metabolic rate, while their swirruning performances increased with time • • • 51 • By the time the second group of fish had been added, there was also a much more highly variable metabolic response among the individuals, which is a common occurrence in stress experiments at the time the full impact of the stresses is • reached (Dodson and Mayfield, 1979) • In many kinds of • physiological manipulations that involve organismic stress, the variability tends to be reduced with survival time; such is the case for the third group of fish in Fig. 1 • • By the time of the third group in this experiment, 4-5 days, the fish were at a level of stress that seemed to be approximately comparable to the 0.0625% level investigated • by Wohlschlag and Parker (In Press) , whereas the maximum stress was developed after about 2~3 days. Because there could be ascertained no initial differences among the three groups of • fish in this experiment, the expected time-response pattern of starting with near normal metabolic and activity ·levels and then dropping off at progressively slower rates with each • successive group of fish was not realized. Because the Fig.I pattern prevailed and because there was no obvious initial difference among the three groups of fish, it must be concluded tentatively ~t least that (1) more than one day was required for the composite waste at 0.125% to reach maximum toxicity and • (2) there is not a clear indication whether or not the • individual fish would have suffered delayed effects had the experiments been conducted over longer periods and lower concentrations • • • 52 Because each succeeding group of fish did not show progressively less and less response in maximum sustained • metabolic rates, but did show in general a progressively increasing maximum sustained swimming rate, it might be supposed either that the quality of the fish from one group • to the next changed or that there were deiays in the efficacy · of one or more of the toxic components. Several examples of such delays and possible increases in the development of • toxicity are given by Ludke, et al. {1971), Lowe, et al. {1971), and Alley {1973). Aside from purely chemical changes • in the composition of .toxicants with time in aerated seawater . . systems other important changes may be in: {a) solubility, {b) precipitation, (c) degree of adsorption and leaching of • toxic materials to or from particulate matter. All these abiotic considerations should affect interpretations of toxicity with time. To interpret biotic effects of very low • toxic levels in terms of biomagnification through food chains or bioaccumulation in the fish themselves, however, would require types of experiments different from those of this study. • In general terms of swimming activity, maximum sustained metabolic rates, and operational and standard metabolic rates, further experimentation would be required at both higher and lower toxicities and especially at longer time intervals for a more complete evaluation of the degree of toxicity attenuation. For the present, it should suffice to note that the minimum metabolic depression at an initial concentration of 0.125% • • 53 • and 28°C occurs after about 4 days, from which time there should be expected a good recovery for fish newly exposed unless delayed effects (e.g. fin rot) of earlier exposures would be manifested. • The fact that the maximum active metabolic level declines • with time while the maximum swimming rate increases can be explained partially by the initially high spontaneous nonlocomotory activity-that the composite waste incites for about 2 days. A measure of the energy level of spontaneous activity could be calculated from appropriate equation (Table 2) relating oxygen consumption to fish s-ize and activity, by • calculating o2 consumption rate at the maximum control swimming rate at a given weight and by comparing the _observed o2 consumption rate at the observed slower swimming rate and at • • the same given weight. · The difference in o2 consumption rates could then reasonably be explained by excessive spontaneous activity. However, the nature of the data for the toxicity attenuation experiments does not permit a straightforward statistical regression analysis. · Also, to be kept in mind is • the possibility that the increasingly high swimming rates • may be a result of stimulatory buildup that would eventually result in physiological collapse. With the high swimming rates and the somewhat depressed active metabolic rates (Fig. 1), • it is likely that fish like the spotted seatrout could have avoided a "metabolic burnout" had they been left at these toxic levels for another day or two . • • 54 Exposure-Recovery Experiments • The metabolic and activity data for these experiments with the composite or individual industrial wastes are directly comparable to control data or to the data of Wohlschlag and • Wakeman (1978). However, throughout the following discussion, it should be recalled that the regression information for the experiments· summarized in Equations 2. 0 and 3 .1-3. 6 (Tables • 1-5) and Figs. 2-8 indicated trends. The equations simply are "averages" of the performances, while the figures illustrate arbitrarily drawn trend lines through original data uncorrected • for weight and any othe-r uncontrolled variables·. However, there are two other methods of making comparisons where the data for the exposure recovery experiments may be • compared on the basis of given body weights on the basis of standard or active .rate equations for either the overall average weight of 172.04 g for all the data (including control • data) or the average weight of fish used for each of the composite or individual industrial waste experiments, the . . data may be reduced for comparative scope comparisons. From • Tables 1, 2, and 4 appropriate data are available for calculation of both standard and active rates on a unit weight (kg) basis. The active and standard rates are calculated (per kg) directly • at an overall average weight of 172.04 g for both control and experimental groups and plotted as horizontal solid lines on the histograms of Fig. 9. The arrow between these lines indicates the magnitude of scope for activity. The lower • • 55 horizontal dashed line is the resting metabolic rate calculated • from appropriate equations using zero swimming velocity; the • upper horizontal dashed line is an estimate of the highest level calculated by using the highest observed swimming velocity from each pertinent data set. The data are replotted • in the same way (per kg) in Fig. 10, but by using separate calculations for the control rates at each average weight for the experimental groups. In Fig. 10, the upper high estimate is omitted. • For the second exposure-recovery series of experiments with Cynoscion nebulosus the control regressions of Tables 2 or 4 agree remarkably well with previous work with this species at 35 o/oo salinity and 28°C. The regressions for • the composite (2.0) and the individual industry (3.1-3.6) waste experiments, however, must be considered as arbitrary "averages" of conditions over the 2-4 days of recovery. Although • there were trends indicated in separate metabolism-time plots, the numbers of fish were quite small for good regression calculations. Even so, the lowest R value was 0.64 (P ~ 0.05) • for 10 degrees of freedom in the highly diluted Bristol (3.3) sample, in which the small b = 0.392 was not statistically w significant; the other R values were much higher, and all • the remaining b w and b v values were statistically significant, usually at P << 0.01. The relative erratic weight-metabolic response tended to be associated with the fact that the larger fish tended to be more depressed under stress than the smaller, which might be expected (Wohlschlag and Cameron,. 1967; Kloth • 56 • Fig. 9. Plots of calculated metabolic rates for Cynoscion nebulosus at 28°C and 35 o/oo salinity and at average • weight of 172.04 g for all experimental and control data. Upper dashed horizontal line is highest individual sustained active rate. Upper solid line • is standard rate; arrow represents scope .for activity. The dashed line at the intermediate level represents a resting rate (swinuning rate = 0) calculated from • active fish data. Identity designations are: Control = 0.0, Composite = 2.0, Upjohn = 3.1, Merck = 3.2, Bristol ~ 3.3, CAPRI = 3.4, Squibb = 3.5 and • Pfizer = 3.6. • • • • • • • r- I I I I -. . I I I I L • r- I i I I -lO - I f() II i L • r-- I ' I -I I I i L--I ' • ,. I I I I -I I I I I L I .~ t--I I I • ,I I - I I L-I • r--------I I I ~ ~ I I I l.------ • r------- I 1 I I ~ 0 i I C\J I L------- • r----0 ' I I 0 I I ' -I .... I I c: I 0 I 0 L - u - 0 0 0 0 0 0 0 CX) w ~ (\J >-tt,_J4 ,_f>)\ zo f> w • 0 0 w 0 •• z • • Fig. 10. Plots of Cynoscion nebulosus metabolic rates calculated at 28°C and 35 o/oo salinity and at • the average weight of fish in each experiment and compared to calculated rates at that weight for control fish. Weight averages from Table 1. • Histogram components and identity designations same as in Fig. 9. • • • • • • • • • 59 I 0 ,...... 0 I I -- t- z w Cl • I· 60 I and Wohlsch1ag, .1972) • . Accordingly, the b in Tables 2 and • . w 4 tended to be generally low. The b values lower than about v 0.1 result from some fish swimming very slowly compared to their potential in clean water; high b for the Upjohn (3.1) v • experiment indicate that the fish were swimming in a • recognizably "labored" manner with high· energy requirement for slow velocities. The Cynoscion nebulosus experiments were begun with preliminary testing of concentrations that would produce a visible effect on fish in about 2 hr. Previous experience • with the composite waste that was pa'rtially volatilized, and perhaps slightly decomposed and detoxified, provided the estimate of 0.5% for the composite, but only 1 hr was required • for the new waste to produce an effect on all subject fish . Unfortunately not all the fish in .a single trial would behave alike. There would be inexplicably different reactions to the • different individual diluted wastes as well. The reactions ranged initially from nearly no observableeffects to slightly obvious erratic swimming, minor ventilatory rate increases, • or appearance of partial anaesthesia. By the end of 2 hrs • (or 1 hr for the composite, 25 min for Merck, and 110 min for Squibb), the various reactions were quite pronounced supination, erratic swimming, and other manifestations of disequilibrium; also increasing with exposure time were pronounced coughing, head shaking, and increased ventilatory rate. In no cases • were any fish allowed to die before transfer to clean waters • • • 61 • The initial metabolic rates tended to be high and then decrease, while the swimming rates tended to be depressed • initially and then over 2-4 days of experiments sometimes improve, although generalities from one toxic waste to the next were not possible from time plots of the metabolic and • swimming activity levels against elapsed time in clean water. The averages of the Cynoscion nebulosus results are about the worst possible situation consistent with recovery, • inasmuch as the fish at the end of the exposure-recovery experiments were approaching morbidity with high incidences of fin and tail rot, although too few~ metabolic measurements • were possible at the end of the experiments for a clearcut assessment of the metabolic and swimming rates at that time. These averages are expressed in the histograms of Figs. 9 and • 10 as calculated from the appropriate regressions, average weights, and swimming# resting, or standard conditions. At the exposure-recovery conditions plotted in Fig. 9 • with all data adjusted to the . same weight, the composite waste recovery levels indicate a great reduction in scope both by a drop in the active level and by an increase in the standard • level. (Possibly the relatively small scope of the composite waste experiments, compared with the other single waste experiments, is due to interactions among all the different • wastes.) All other standard metabolic levels in Fig. 9 are higher than the control level. Why the active levels of the Upjohn (3.1) and Merck (3.2) are higher than the controls, • • 62 • even though the subject fish swam more slowly on the average than did the controls (Table 1) is not altogether clear, but • it is possible that the wastes did contain stimulatory substances. Such stimulation by low level toxicants have been known for some time. Webb (1978) has summarized several instances from the literature to show such stimulatory effects. However, in the C. nebulosus experiments, the concentrations of toxic components were all sufficiently high enough to make • • ultimate recovery doubtful, judging from the amount of fin rot as evidence for morbidity at the termination of the recovery periods. Thus there is reason to believe that the high metabolic scope for the Upjohn (3.1~ experiment and the high active metabolic rates (with poor swimming performance) for both Upjohn and Merck (3.2) were simply transitory manifestations • • or "burnouts" that would eventually be followed by depressed active rates and scopes. The unusually high standard rate of the Merck experiment represents a stress level that w6uld be difficult to maintain for c. nebulosus under normal 28°C conditions (Wohlschlag and Wakeman, 1978) . When the controls and the experimental exposure-recovery • • results are _compared on an equivalent weight basis as in Fig. 10, the active rates appear to be stimulated by the Upjohn wastes (as in Fig. 9) and additionally by the very low concentration of the Bristol (3.3) and the Pfizer (3.6) wastes. In both Fig. 9 and Fig. 10, however, the scopes for all but the Upjohn • (3.1) experiment are reduced . • • 63 Population Effects of Ocean Dumping • For open ocean dumping of such toxic materials as used • in these studies two kinds of population effects are important if dumping occurs at intervals of about one week at a given locality. The first is when the low level toxicities at • somewhat lower than the levels indicated in the earlier Lutjanus campechanus experiments would have a depressing effect on such functions as growth--assuming no cumulative effects leading • to direct lethality. This first effect would be persistent depending upon dispersion over the area where the sublethal dilution level persist. It should .b~ kept in mind that even small changes in instantaneous rates· of growth, metabolism, death, etc. can have great population effects with their • continuous accrual on long-lived organisms. The second · • effect is when all the fish subjected briefly to concentrations of the orders illustrated in the Cynoscion nebulosus experiments or higher shortly after the time of dumping would • experience a deleterious effect for up to at least 4 days even after there was no trace of waste. Should such dumping continue with regularity, it is conceivable that entire fish populations could eventually be decimated over wide a area. Future application of such metabolic stress data, as • exemplified in this study, is indicated for the various fish population dynamics models. Additional values of acquiting data in metabolic rate, • swimming rate, or in energy units are numerous and are related • • 64 • to evaluation of niche theory in terms of growth, assessment of growth-foraging relationships, and in clarification of various fishery models (Wohlschlag and Parker, in press). Similarly, all environmental variations, as pointed out by • Fry (1971), are ultimately expressed at the total metabolic • level. Fry (1971) relates an animal to environmental response categories~ which are the factors: lethal, controlling, limiting, masking, and directive. No doubt all of these physiological response categories were involved in the two types of studies of toxic waste effects on fishes that are • reported here, but a real and practical ecological and • physiological challenge remains to separate the effects into these categories in future studies. Because it is difficult to relate separate chemical • components of complex organic wastes to specific physiological effects in an oceanic environment, Wohlschlag and Parker (in press) suggested that a simple, modified Blazka respirometer system be utilized to study marine fish at a site where sea water and a continuous supply of test wastes could be added • or diverted from the respirometer at will. In all of the • above experiments, the swimming velocities and both the degrees of stress and the metabolic scope values tend to be closely correlated to the extent that even an open flume into which a regulated clean sea water flow rate would suffice to provide swimming rates as a measure of stress. The needs for, and the • rationale of, biological monitoring as early warning systems have long been advocated by Prof. John Cairns, Jr. and his • • 65 associates, e.g. Cairns and Gruber {1979) . • • • • • le · • • • 66 ACKNOWLEDGMENTS • We are grateful for the support from NOAA Grant No • 04-8-MOl-54 and for the ·aid of the NOAA Ocean Dumping Program personnel who shipped the Puerto Rican pharmaceutical wastes • and provided advice and encouragement • We extend special thanks to research assistants Mr. Dobbs and E. Findley, graduate student assistants M. Gunter and • R. Ilg, boat captains D. Gibson (R/V LONGHORN) and E. Wingfield (R/V LORENE), the R/V crew members and typist H. Garrett. · We also extend our gratitude to Dr. T. w. Duke of the E.P.A• • Gulf Breeze Laboratory for adding his unusually keen insight to the interpretations of our experiment • • • • • • • • 67 REFERENCES • ALLEY E.G. (1973) The use of mirex in control of the imported fire ant. Journal of Environmental Quality, 2, 52-61. BRETT J.R. (1964) The respiratory metabolism and swimming performance of young sockeye salmon. Journal of the • ------- Fisheries Research Board of Canada, 21, 1183-1226. CAIRNS J. and D. GRUBER (1980) A comparison of the methods • and instrumentation of biological early warning systems • Ameri~an Water Resources Bulletin, 16, 261-266. CAIRNS J. and D. GRUBER (1979) The coupling of mini-and • microcomputers to biological early warning systems • . · Bioscience, 29, 665-669. DODSON J.J. and C.I. MAYFIELD (1979) The dynamics and behavioral toxicology of Aqua-Kleen (2,4-D butoxyethanol ester) • R as revealed by the modification of rheotropism in rainbow trout. Transactions of the American Fisheries Society, • 108, 632-640. FRY F.E.J. (1947) Effects of the environment on animal activity. University of Toronto Studies of Biology, Publications • of the Ontario Fisheries Research Laboratory, 68, 1-62. FRY F.E.J. (1957) The aquatic respiration of fish. In: The Physiology of Fishes, M. E. Brown, editor. Academic • Press, New York, pp 1-63. FRY F.E.J. (1971) The effect of environmental factors on the physiology of fish. In: Fish Physiology, Vol. 6, w.s . • Hoar and D.J. Randall, editors, Academic Press, pp. 1-98 • • • 68 • KDOTH T.C. and D.E. WOHLSCHLAG (1972) Size-related metabolic responses of the pinfish, Lagodon rhomboides, to salinity variations and sublethal pollution. Contributions in Marine Science, 16, 125-127. LOWE J.L., P.R. PARRISH, A.J. WILSON, P.O. WILSON, and T.W• • DUKE (1971) Paper presented at 36th North American Wildlife and Natural Resources Conference, as cited by ALLEY (1973) . • • LUDKE J.L., M.T. FINELY and L.LUSK (1971) Toxicity of mirex to crayfish, Procambarus blandingi. Bulletin of Environ metal and Contamination Toxicology, 6, 89-96 . SNEDECOR G.W. and W.G. COCHRAN (1967) . Statistical Methods. 6th ed. Iowa State University Press, Ames, 593 pp. STEELE J. (1974) The structure of marine ecosystems. Harvard • University Press, Cambridge and London, pp 128. • WAKEMAN J.M. (1978) Environmental effects on metabolic scope and swimming performance of some estuarine fishes • Ph.D. Dissertation. University of Texas . .PP. 146. WEBB P.W. (1978) Partitioning of energy into metabolism and growth. In: Ecology of freshwater fish production, • S.D. GERKING, editor, Wiley, pp. 184-214. • WOHLSCHLAG D.E. and J.N. CAMERON (1967) Assessment of a low level stress on the respiratory metabolism of the pinfish (Lagodon rhomboides) . Contributions in Marine Science, 12, 160-171. • WOHLSCHLAG D.E. and F.R. PARKER (in press) Metabolic sensitivity of fish to ocean dumping of industrial wastes. In: • • 69 Ocean dumping of industrial wastes, B.H. KETCHUM, D.R. KESTER and P.K. PARK, editors, Plenum Press, pp .. • WOHLSCHLAG D.E. and J.M. WAKEMAN (1978) Salinity stresses, metabolic responses and distribution of the coastal spotted seatrout, Cynoscion nebulosus. Contributions • in Marine Science, 22, 171-185. WOHLSCHLAG D.E. and Associates (1978) Sensitivity of Marine Fish to Organic Pollutants. In: Biological Process • Effects of Organic Wastes at Puerto Rican Ocean Dumping Sites. Draft Report to NOS, Ocean Dumping Program. pp. 6-64 . • • • • • • • I. Appendix Table 1. Control (0.0) raw data used in the calculation le of regression equations. · Fish No. = fish identification number Wt. (g) = fish weight in grams • log. Wt. = log10 weight in grams V (L sec-1) = velocity in lengths per second mg02h-l = milligrams q,xygen consumed per hou~ • log mg02h-l = log10 milligrams oxygen consumed per hour mg02kg-lh-l = milligrams oxygen consumed per kilogram • per hour * following a velocity measurement indicates a swimming speed at Umax for that fish. • • • • • " Fish No. Wt. (g) log. Wt. V (L sec-1 ) rng02h-l -1 -1 log. rng02h-1 mg02kg h Control (O.O) PRT050 174.1 2.2408 1. 2 62.4 1. 7952 358.5 PRT050 174.1 2.2408 3. o";~ 83.2 1. 9201 477.8 PRT050 174.1 2.2408 o.o 58.2 1. 7649 334.5 PRT051 117.8 2.0712 1. 3 49.9 1. 6981 423.7 PRT051 117.8 2.0712 2.9";': 79.0. 1. 8976 670.9 PRT051 117.8 2.0712 0.0 33.3 1. 5224 282. 5 PRT052 71. 7 1. 8555 3. 2";': 54.1 1. 7332 754.1 PRT052 71. 7 1. 8555 o.o 20.8 1. 3181 290.1 PRT053 155.6 2.1920 3. 5";': 121.1 2.0831 778.3 PRT053 155.6 2.1920 o.o 62.4 1. 7952 401.0 PRT054 119.5 2.0774 3. 3-;': 79.0 1. 8976 661. 3 PRT054 119.5 2.0774 0.0 33.3 1. 5224 278.5 PRT055 97.8 1. 9903 3. 4«': 70.7 1. 9903 723.0 PRT055 97.8 1. 9903 0.0 25.o 1. 3979 255.2 PRT056 97.1 1:9872 3.P': 62.4 ·i.7952 642.5 PRT056 97.1 1. 9872 2.0 37.4 1. 5729 385.5 PRT057 90.5 1. 9567 3. 6";': 91. 5 1. 9614 1011.1 .~ .j-1 rv PRT057 90.5 1. 9567 0.0 33.3 1. 5·224 367.7 Fish No. Wt. (g) Control (0.0) -Continued PRT057 90.5 . PRT058 92.1 PRT058 92.1 PRT059 118.2 PRT059 118.2 PRT059 118.2 PRT060 105.4 PRT060 105.4 PRT060 105.4 PRT061 149.2 PRT061 149.2 PRT062 154.3 PRT062 154.3 PRT062 154.3 PRT063 83.4 PRT063. 83. 4 log. Wt. V (L sec-l) 1. 9567 2.5 1. 9643 3. 3-;': 1. 9643 0.0 2.0726 3. 0-;': 2.0726 0.0 2.0726 2~1 2.0228 3. 2-;': 2.0228 0.0 2.0228 2.3 2.1738 3. 5-;': 2.1738 0.0 2.1884 3. 2-;': 2.1884 0.0 2.1884 2.0 1. 9212 3. 5-;': 1. 9212 0.0 • mg02h-1 74.9 66.6 33.3 79.0 33.3 49.9 91.5 45.8 62.4 ·104. 0 39.6 99.8 33.3 49.9 66.6 2.5.0 -1 -1 -1 log. mg02h mg02kg h 1. 8745 827.3 1. 8235 722.6 1. 5224 361. 6 1. 8976 668. 6 1. 5224 281. 5 1. 6981 422.3 1. 9614 868.2 1.6609 434.1 1. 7952 591. 9 2.0170 696. 9 1. 5977 265.5 1. 9991 647.0 1. 5224 215.7 1.6981 323.5 1. 8 235 798.0 1.3979 299.2 . ~ ....... . w • A. 2 .1 • • Appendix Table 2. Composite a~ration (1.0) raw data used in the calculation of regression equations. · Fish No. = fish identification number • Wt. (g) = fish weight in grams log. Wt. = log10 weight in grams V (L sec-1) = velocity in lengths per second (all Umax) • Tl (hrs) = time from pollution of water • 2 T2 (hrs) = time since fish placed in polluted water mg0h-l = milligrams oxygen consumed per hour log. mg02h-l. = logmilligrams oxygen consumed per 10 hour mgokg-1h-1 =milligrams oxygen consumed per kilogram 2 .• per hour . • • • • -1 -1 Fish No. Wt. (g) log. Wt. V (L sec-1) Tl (hrs) (hrs) mg0h-l log.mg02h-1 mg02kg h -T2 2 Composite Aeration (1.0) PRT064 . 190. 5 2.2799 3.4 6.4 6.4 174.7 2.2423 917.1 PRT065 192.0 2.2833 2.4 6.5 6.5 133.1 2.1242 693.2 PRT066 358.5 2.5545 1.9 8.3 8.3 224.6 2.3514 626.5 PRT067 287.4 2.4585 2.0 8.4 8.4 183.0 2.2625 636.7 PRT068 149.2 2.1738 3.1 25.6 25.6 124.8 2.0962 836. 5 PRT069 175.8 2.2450 3.8 25. 7 . 25.7 98.6 1. 9939 560.9 PRT070 155.7 2.1923 3.1 27.6 27.6 115.3 2.0618 740.5 PRT071 222.4 2.3471 3.9 29.8 . 29.8 119.4 2.0770 536.9 PRT072 169.0 2.2279 3.3 29.6 29.6 108.2 2.0342 640.2 PRT073 267.0 2.4265 3.1 31. 9 31. 9 174.7 2.2423 654.3 PRT074 187.0 2.2718 3.3 32.2 32.2 106.9 2.0290 571. 7 PRT075 191.0 2.2810 3.2 50.0 so.a 114.0 2.0569 596.9 PRT076 444.5 2.6479 3.3 54.4 0.9 206.7 2.3153 465.0 PRT077 163. 0 2.2122 3.3 55.9 6.4 66.6 1. 8235 408.6 PRT078 206.0 2.3139 3.3 56.3 8.3 69.5 1. 8420 337.4 PRT079 250.0 2.3979 3.2 58.0 8.5 104.0 2.0170 416.0 . ~ PRT080 180.0 2.2553 4.0 73.6 25.6 61. 2 1. 7868 340.0 N . N PRT081 201.8 2.3049 3.5 75.3 25.8 116.5 2.0663 577.3 -1 -1 Fish No. Wt. (g) 1 log. Wt. V (L sec-1 ) Tl (hrs) (hrs) mg0h-l log.mgo2h-1 mg02kg h T2 2 Composite Aeration (1.0) -Continued PRT082 206.2 2.3143 3.8 75.4 27.4 69.5 1. 8420 337.1 PRT083 132.0 2.1206 3.8 77.2 27.7 58.2 1. 7649 440.9 . PRT084 169.5 2.2292 3.8 77.2 29.2 82.0 1.9138 483.8 PRT085 175.0 2.2430 4.2 79.0 31.0 82.0 1.9138 468.6 PRT086 160.3 2.2049 3.9 79.2 29.8 95.7 1.9809 597. 0 PRT087 315.7 2.4993 4.0 102.8 5.8 115.2 2.0615 364.9 PRT088 253.5 2.4040 3.3 -104. 7 7.7 111.1 2.0457 438. 3 PRT089 193. 2 2.2860 3.8 121.9 24.9 65.3 1. 8149 338.0 PRT090 206.7 2.3153 3.7 123.7 26.7 73.6 1. 8669 356.l PRT091 189.4 2.2774 4.3 126.7 29.7 82.0 1.9138 432.9 PRT092 152.5 2.1833 3.8 129.7 32.7 65.3 1.8149 428.2 .~ f\..) . w A.3.1 • • • Appendix Table 3. Composite ~ecovery (2.0) raw data used in the calculation of regression equations . • Fish No. = fish identification number Wt. (g) = fish weight in grams • log. Wt. = log10 weight in grams V (L sec-1) = velocity in lengths per second T (hrs) . = time from removal of fish from polluted • water mg02h-1 = milligrams oxygen consumed per hour log mg0h-l = logmilligrams oxygen consumed per hour 210 mgo2kg-1h-l =milligrams oxygen consumed per ~ilogram per hour. • * following a velocity measurement indicates a swimming speed of U for that fish. max • • • • Fish No. Wt. (g) log. Wt. V (L sec-1 ) T (hrs) mg02h-l log. mg02h-1 mg02kg-1h-1 Composite Recovery (2.0) PRT043 256.2 2.4086 2. 5~·--1. 3 146.0 2.1644 569.3 PRT044 200.1 2. 3013 1. 4~·--23.2 99.8 1. 9991 498.9 PRT044 200.1 2.3013 0.0 24.2 58.2 1. 7649 291.0 PRT044 200.1 2.3013 1. 2 24.5 74.9 1. 8745 374.2 PRT045 141.0 2.1492 1. 5~·, 26.3 91. 5 1. 9614 649.0 PRT045 141.0 2.1492 o.o 27. 3 58.2 1. 7649 413. 0 PRT045 141.0 2.1492 1. 3 27.6 74.9 1. 8745 531.0 PRT046 92.5 1.9661 1.0 45.9 37.4 1. 5729 404.7 PRT046 92.5 1. 9661 1. 3~·: 47.2 58.2 1. 7649 629.5 PRT046 92.5 1.9661 o.o 49.2 35.4 1. 5490 382. 2 " PRT047 124.2 2.0941 / . /~.0.,£1-70.1 108.1 2.0338 870.7 PRT047 124.2 2.0941 1.0 70.5 74.9 1. 8745 602.8 PRT048 76.3 1. 8825 1. 3~·: 74.3 54.1 1. 733·2 708.7 PRT048 76.3 1. 8825 o.o 75.3 37.4 1. 5729 490.6 PRT049 61. 3 1. 7875 0.8 94.6 25.0 1.3979 407.1 PRT049 61. 3 1. 78 75 1. o~·, 95.7 41.6 1.6191 678.5 ::i:=i PRT093 376.6 2.5759 1.1 0.2 124.8 ~.0962 331.3 . .w PRT093 376.6 3.6 187.2 2.2723 2. 5759 0.5 497.0 N -1 -1 -1 Fish No. Wt. (g) log. Wt. V (L sec-1) T (hrs) mg0h-l log. mg02h mg02kg h 2 - Composite Recovery (2.0) -Continued PRT093 2. 7~·: 376.6 2.5759 1. 2 143.5 2.1569 381.0 PRT093 376.6 2.5759 a.a 2.2 95.7 1. 9809 254.0 PRT094 308.0 2.4886 2. 4~·: 4.3 133.1 2.1242 432.1 PRT094 308.0 2.4886 a.a 5.4 93.1 1. 9690 302.3 PRT095 169.8 2.2299 2. 6~... 20.3 120.6 2.0814 710.4 PRT095 169.8 2.2299 a.a 21. 3 70.7 1. 8494 416.4 PRT095 169.8 2.2299 2.0 21. 6 99.8 1.9991 587.9 PRT096 118.2 2.0726 2.3* 24.1 70.7 1.8494 598.2 PRT096 118.2 2.0726 1.8 25.7 68.8 1.8376 582.4 PRT097 205.0 2.3118 2.Pt-45.1 99.8 1.9991 486.9 PRT097 205.0 2.3118 0.0 46.2 80.5 1. 9058 392.7 PRT098 2. 5~._. 243.5 2. 3865 49.5 137.3 2.1377 563.7 PRT098 243.5 2. 3865 0.0 50.5 95.7 1. 9809 392.9 PRT098 243.5 2.3865 2.0 51.1 128.0 2.1072 525.6 PRT099 300.0.' 2.4771 2. 2~..-1. 5 137.3 2.1377 457.5 PRT099 300. or 2.4771 a.a 2.7 107.4 2.0310 358.2 PRTlOO 292.0 2.4654 1. 6~..-4.1 124.8 2.0962 427.3 .~ .w PRT101 180.0 2.2553 2. 5~..-24.3 133.l 2.1242 739.4 w A. 4 .1 • • • • • • • • • • • Appendix Table 4. Individual pharmaceutical company sample recovery (3.1 -3.6) raw data used in the calculation of regression equations. ' Fish No. = fish identification number Wt. (g) =fish weight in grams log. Wt. = logweight in grams 10 V (L sec-1) = velocity in lengths per second T (hrs) = time from removal of fish from polluted water mg02h-l = milligrams oxygen consumed per hour log mg02h-l = logmilligrams oxygen consumed per hour 10 mgo2kg-lh-l = milligrams oxygen consumed per kilo~air~ . per hour * following a velocity measurement indicates a swirraning speed of U for that fish. max Fish No. Wt. (g) Upjohn Recovery (3.1) PRT001 320.5 PRTOOl 320.5 PRT001 320.5 PRT002 674.0 PRT003 415.0 PRT003 415.0 PRT003 415.0 PRT004 461.7 PRT004 461. 7 PRT004 461. 7 PRT005 510. 6 PRT006 627.4 PRT006 627.4 PRT006 627.4 Merck Recovery (3.2) PRT007 122.5 PRT007 122.5 • log. Wt. 2.5058 2.5058 2.5058 2.8287 2. 6181 2.6181 2.6181 2.6644 2.6644 2.6644 2.7081 2.7975 2.7975 2.7975 2.0881 2.0881 • V (L • sec-1 ) 1. 6 2. 5.." 0.0 1. 3 1.0 1. 5-.': o.o 0.9 1. 5-.': o.o 1.1 1.1 2. 2-.': 0.0 3.P" o.o • T (hrs) 2.5 3.5 4.8 22.4 23.2 24.4 25.3 26.8 27.7 28.6 46.3 47.1 48.3 49.4 2.5 4.0 • mg0h-l 2 149.7 193. 4 79.0 274.5 149.7 216.3 54.5 224.6 318.2 144.5 199.7 129.8 262.0 144.3 99.8 49.9 • log. mgo2h-1 2.1752 2.2865 1. 8976 2. 4385 2.1752 2.3351 1. 7364 2.3514 2.5027 2.1599 2.3004 2.1133 2.4183 2.1593 1.9991 1.6981 -1 -1 mg02kg h 467.2 603. 5 246.6 407.3 360.8 521.2 131.2 486. 5 689. 2 312.9 391.0 206. 8 417.7 229.9 814.9 !l;:i . 407.4 ~ . rv Fish No. Wt. (g) - Merck Recovery (3.2) PRT007 122.5 PRT008 626.0 PRT008 626.0 PRT009 142.3 PRT009 142.3 PRT009 142.3 PRTOlO 122.~ PRTOlO 122.5 PRT010 122.5 PRTOll 226.5 PRTOll 226.5 PRTOll 226.5 PRT012 131. 5 PRT012 131. 5 PRT0l2 131. 5 PRT013 115.3 PRT013 115.3 • log. Wt. Continued 2.0881 2.7966 2.7966 2.1532 2.1532 2.1532 2.0881 2.0881 2.0881 2.3551 2.3551 2.3551 2.1189 2.1189 2.1189 2.0618 2.0618 V (L sec-1 ) 2.2 2. 2~·: 0.0 1.2 3.P'>" 0.0 2. 9~': o.o 2.0 2. 9~·: o.o 1.9 1.4 2. 9~·: o.o 3.P': o.o • T (hrs) . 4.4 6.4 6.9 20.8 22.0 23.0 : 24.7 25.7 26.0 42.5 43.6 44.0 45.5 46.7 47.6 65.3 66.3 • mg0h-l 2 87.4 266.2 149.7 87.4 104.0 62.4 116.5 41. 6 62.4 149.7 78. 4 124.8 74.9 99.8 59.9 79.0 66.6 • log. mgo2h 1.9415 2.4252 2.1752 1.9415 2.0170 1. 7952 2.0663 1.6191 1. 7952 2.1752 1. 8943 2.0962 1. 8745 1.9991 1. 7774 1. 8976 L8235 -1 -1 -1 mg02kg h -713~0 425.2 239.2 613.8 730.7 438.4 950.7 . 339. 5 509.3 661.1 346.3 550.9 570.2 760.3 456.2 685.4 577.2 . ~ .i:::.. . w Fish No. Wt. (g) Merck R~covery (3.2) PRT013 115.3 PRT014 371.2 PRT014 371.2 PRT021 98.7 PRT021 98.7 PRT021 98.7 Bristol Recovery (3.3) PRT015 67.2 PRT015 67.2 PRT016 152.1 PRT016 152.1 PRT016 152.1 PRT()l7 123.3 PRT017 123.3 PRTOl7 123.3 ··-·. PRT018 125.5 PRT018 125.5 • log. Wt. Continued 2.0618 2. 5696 l 2.5696 1.9943 1.9943 1.9943 1.8274 1.8274 2.1821 2.1821 2.1821 2.0910 2.0910 2.0910 2.0986 2.0986 • V (L • sec-1 ) 1.9 2. 3~-: o.o 1.2 2. 6~'. o.o 3.1~.. o.o 3. 3~·.f). 0 . 2.3 3.P'"' 0.0 · 2.4 · 3~4~': o.o • T (hrs) 66.7 68.4 69.4 83. 8 85.0 86. 2 3.9 4.9 19.7 20.7 21. 2 24.0 25.0 25.3 43.4 44.5 • mg0h-l 2 74.9 149.7 133.l 74.9 83.2 49.9 49.9 37.4 83. 2 41.6 62.4 83. 2 66.5 74.9 104.0 76.8 • log. mg02h-1 1.8745 2.1752 2.1242 1.8743 1.9201 1.6981 1.6981 1. 5729 1.9201 1. 6191 1. 7952 1.9201 1.8228 1. 8745 2.0170 L8·854 -1 -1 mg02kg h 649.3 403.4 358.6 758.5 842.8 505.7 742.6 556.5 547.0 273.5 410.3 674.8 539.3 607.5 828.7 612. 0 . ~ .~ ~ Fish No. Wt. (g) Bristol Recovery (3.3) PRT020 87.4 PRT020 87.4 PRT020 87.4 Capri Recovery (3.4) PRT022 410.4 PRT022 410.4 PRT022 410.4 PRT023 92.5 PRT023 92.5 PRT023 92.5 PRT024 85.7 PRT024 85.7 PRT024 85.7 PRT025 248.0 PRT025 248.0 PRT025 248.0 PRT026 163. 0 • log. Wt. -Continued 1. 9415 1. 9415 2. 6132 2. 6132 2.6132 1.9661 1.9661 1.9661 1.9330 1.9330 1.9330 2.3946 2.3946 2.3946 2.2122 1.9415 V (L sec-1) 2.0 2. 5-.\ o.o 1. 2 2. g·l: o.o 1.4 2. 9-.\ 0.6 1.8 3.P\ o.o 1.4 2. 8..... o.o 2. 4-:: • T (hrs) 67.4 68.5 69.5 90.8 1. 9 ; (3. 0 4.0 5.2 5.9 20.2 21. 5 22.5 25.1 26.3 27.1 45.9 • mg0h-l 2 74.9 83. 2 41. 5 99.8 . 208.0 70.2 74.9 99.8 55.5 49.9 74.9 24.9 124.8 174.7 99.8 74.9 • log. mg02h 1. 8745 1.9201 1.6181 1.9991 2.3181 1.8463 1.8745 1. 9991 1. 7443 1.6981 1. 8745 1.3962 2.0962 2.2423 1.9991 l~B745 -1 -1 -1 mg02kg h 857.0 952.0 474.8 . 243. 2 506.7 171.0 809.4 1079.2 599.5 582.4 873.6 291. 2 503.2 704.4 402.5 459.3 . ~ . ~ Ul -1 -1 Fish No. Wt. (g) log. Wt. V (L sec-1 ) T (hrs) mg02h-l log. mg02h-1 mg02kg h Capri Recovery (3.4) -Continued PRT026 163.0 2.2122 o.o 46.9 66.6 1.8235 408.3 PRT027 295.l 2.4700 2. o~"" 51.8 116.0 2.0645 393.2 PRT027 295.l 2.4700 o.o 52.3 89.8 1. 9533 304.4 PRT027 295.1 2.4700 1.7 52.6 112.3 2.0504 380. 5 PRT028 104.5 2.0191 2. 4~·: 71.0 49.9 1.6981 477.6 PRT028 104.5 2.0191 1.9 72.3 37.4 1. 5729 358.2 Squibb Recovery (3.5) PRT029 86. 7 1. 9380 2. 9~·: 4.4 66.6 1.8235 767.6 PRT029 86. 7 1.9380 o.o 5.4 33.3 1. 5224 383. 8 PRT029 86. 7 1.9380 2.1 5.7 ... 37.4 1. 5729 431.8 PRT030 257.8 2. 4113 2.8~'" 23.3 145.6 2.1632 564.7 PRT030 257.8 2.4113 o.o 24.3 87.4 1.9415 338.8 PRT030 257.8 2. 4113 2.0 24.6 99.8 1.9991 387.2 PRT031 115.4 2.0622 1.7 21.3 74.9 1.8745 648.8 PRT031 115.4 2.0622 3. 3~·: 22.5 83.2 1.9201 720.9 PRT031 115. 4 2.0622 o.o 23.5 58.2 1.7649 504.6 PRT032 149.l 2.1735 2. 8~'" 41. 7 95.7 1 .. 9809 641. 6 .~ .i::.. . "' Fish No. Wt. (g) Squibb Recovery (3.5) PRT032 149.l PRT033 95.l PRT033 95.1 PRT034 184.9 PRT034 184.9 Pfizer Recovery (3.6) PRT035 331.8 PRT035 331.8 PRT035 331.8 PRT036 190.9 PRT036 190.9 PRT036 190.9 PRT037 74.6 PRT037 74.6 PRT037 74.6 PRT038 167.6 PRT038 167.6 • log. Wt. -Continued 2.1735 1.9782 1.9782 2.2669 2.2669 2.5209 2.5209 2.5209 2.2808 2.2808 2.2808 1.8727 1.8727 1.8727 2.2243 2.2243 V (L sec-1) o.o 3.P-: o.o 1.3 2. 5-.-: 2. 9-.·.. 0.0 2.1 1.4 2. 2--·. o.o 2. 9-.': o.o 2.5 1.5 2. 9-.': • T (hrs) 42.7 46.9 47.9 65.3 66.6 2.2 3.2 3.6 21. 6 22.8 23.8:.' 23.7 24.7 25.0 41.8 42.9 • mg02h:..1 74.9 74.9 49.9 49.9 74.9 178.9 7-9. 0 149.7 99.8 141.4 91. 5 74.9 49.9 62.4 49.9 104.0 • log. mg02h-1 1.8745 1.8745 1.6981 1.6981 1. 8745 2.2526 1.8976 2.1752 1.9991 2.1505 1.9614 1. 8745 1.6981 1. 7952 1.6981 2.0170 -1 -1 mg02kg h 502.1 787.3 524.8 269.9 404.9 539.0 238.2 451. 3 522.9 740.8 479.3 1003.6 669.1 836.3 297.8 . ~ 620.4 ii::. . ~ Fish No. Wt. (g) Pfizer Recovery (3.6) PRTa38 167.6 PRTa39 9a.8 PRTa39 9a.8 I:>RTa39 9a.8 PRTa4a 183. 2 PRTa4a 183.2 PRTa4a 183. 2 PRTa41 165.l PRTa41 165.l PRTa42 88.2 PRTa42 88.2 PRTa42 88.2 • log. Wt. -Continued 2.2243 1. 9581 1. 9581 1. 9581 2.2629 2.2629 2.2629 2.2178 2.2178 1.9455 1.9455 1.9455 • V (L sec 2.1 i.a 2. 9~·: a.a 2. 3~': a.a 1. 7 2. 4~·: a.a 2. 9~t: a.a 2.a • 1) • T (hrs) 44.4 46.4 47.6 48.6 67.2 68.2 68.5 71.4 72.4 94.3 95.3 95.7 • mg0h-l --2 74.8 62.4 83.2 49.9 99.8 49.9 87.4 la8.l 74.9 74.9 37.4 49.9 • log. mg02h-1 1.8739 1. 7952 l.92al 1.6981 1.9991 1.6981 1.9415 2.a338 1.8745 1.8745 1. 5729 1.6981 -1 -1 mg02kg h 687.l 916.2 549.7 544.9 212.5 476.8 655.a 453.5 848.8 424.4 565.9 :x:=i . .i:::.. .