• • STUDIES OF CHEMICAL AND BIOLOGICAL PROCESS EFFECTS OF INDUSTRIAL WASTES RELEASED INTO THE GULF OF MEXICO BY OCEAN DUMPING --DRAFT FINAL REPORT • • ·. THE*LIBRARY • OF THE UNIVERSITY OF TEXAS AT AUSTIN Supported by: Initial Grant 1 May 1977 to 31 October 1977 • Continuation 1 November 1977 · to 15 May 1978 Grant No.: 04-7~158-44053 • 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 • • CONTENTS • Page CHEMICAL CHARACTERIZATION . 4 • EFFECTS OF WASTES ON GROWTH OF MICROALGAE . 60 TOXICITY OF BIOSLUDGE ON MARINE INVERTEBRATES . 69 SENSITIVITY OF OPEN GULF FISHES TO OCEAN • DUMPING WASTES • 84 APPENDIX (SENSITIVITY OF OPEN GULF FISHES TO OCEAN DUMPING WASTES) ••••••••. 110 • • • • • • • • Report for NOAA on Ocean Dumping in the Gulf of Mexico • Chemical Characterization • by Patrick L. Parker Warren Pulich Richard Scalan • Ken Winters • • • • • • 5 Introduction • The goal of this project was to identify and quantify selected chemicals, trace metals and organic molecules, in Material to be Dumped (MTBD). The MTBD was provided by NOAA • and included substances which were suspected of being deleterious to marine life. In addition studies of the 13c;l2c ratio, 513c, of the total organic matter of the samples were • o13 done in order to evaluate the potential of c as a tracer for Dumped Material as it mixes with the dissolved and particulate organic matter 0£ the ocean• • The experimental plan had been to isolate the toxic fraction of MTBD in cooperation with the biologists, finally identifying the exact molecule(s) that were toxic. This proved to be beyond • • the scope of the research project because of the complexity of the MTBD. The results obtained point to the general problem of regulating ocean dumping, that is that the chemical composition of the MTBD is highly complex and highly variable with source ­further the toxicity of MTBD for open ocean organisms is itself highly complex and variable• The chemical studies were divided into three tasks: A. 513c of particu~ate and dissolved organic matter in MTBD • • B. Isolation and identification of selected organic compounds in MTBD. This study includes GC and GC/MS studies of the lipid soluble fraction of the MTBD. • c. Concentration of selected trace metals in the MTBD • • 6 The Materials to be Dumped (MTBD) were: • Shell biosludge # 1, received February 23, 1977 ' Shell biosludge # 2, received June 21, 1977 Shell biosludge # 3, received November 9, 1977 ' • A. al3c of Material to be Dumped (MTBD) 13 • The oc value of organic material has proven to be a useful tracer in pollution studies ·{Calder and Parker, 1968), food chain studies (Parker et al., in press) and in geochemistry {Fry, et al., 1977). It seemed worthwhile to investigate the potential of using oc as a tracer for the mixing of dumped material with • 13 the dissolved (DOC) and particulate (POC) organic matter of the sea. The ol3c value of DOC and POC are close to -20 on the PDB • scale. If dumped organic matter is significantly different (i.e. several units) then mixing may be traced. The ol3c results, measured and literature, are summarized in the following data: Typical Cone. ol3c* in the Gulf • Shell sludge, particulate (SS-POC) -24.9 • * Puerto Rico effluent, dissolved (PR-DOC) -25.5 # Sediment carbon (SOC) -19. 1% • Dissolved carbon (DOC) -20.2 1 ppm Particulate carbon (POC) -20.4 0.5 ppm • * Units are per mil vs. PDB carbonate std• * EC is 16% organic carbon # Puerto Rico effluent was not a part of the project, but was run to test the concept of a tracer. The P-DOC level is 14,600 ppm • • • • The interesting point in this data is the sharp resolution of ol3c of the SS-POC and PR-DOC from the natural carbon • reservoirs of soc, DOC and POC. Since the error in these o13c values are + 0.3 the 4 to 5 per mil resolution may be used to carryout model mixin3 calculations. Calder and Parker (1968) showed that the ratio of carbon derived from pollutant to natural carbon is a function of the c13c values of the three reservoirs: • cS = n n (1) (S cs m p where: = moles carbon derived from pollutant cP c = moles natural carbon n cS = ol3c of natural carbon op = ol3c of pollutant carbon • n ol3c om = of any mixture of natural and pollutant carbon, note that the ratio CP/Cn has no units. I • In the case of mixing Shell sludge (SS-POC) with natural POC equation (1) becomes: cSn -cSm SS -POC = ( 2) • c cnat. -POC om -os where 0 = ol3c of SS-POC and the others remain as in ( 1) • It can be seen that a value of Css/Cnat. = 1 (i.e. a 50-50 mixing • s of pollutant and natural carbon) will result if only 3 mg of Shell sludge is added to a litre of sea water. The value of om would be • -22.8 which could be resolved from -20 since the error is 0.3. Equation (2) is very sensitive to_Cnat.' the concentration of natural • • POC; if it is less than 0.5 ppm as it probably would be in open • water then the tracer works even better • • The 813c value of the pollutant DOC of the Puerto Rico effluent is very favorable to a tracer study. It is estimated that the tracer method would detect the addition of 1 ml of the • effluent to 1 litre of seawater. Further the subsequent mixing and, biodegradation (if any) and bio-uptake of the effluent could be detected by the 813c method • Our conclusion is that 813G offers good prospects as a tracer for particulate and dissolved dumped organic matter in the • natural POC and DOC reservoirs. It does not appear useful for sediment because the natural sediment reservoir is too large and too distant for necessary conditions• • B. Qualitative Analysis of Organic Components of "Biosludge" Materials • Analytical procedures used in these studies are qualitative • and no attempt has been made to quantify the methods. This is primarily because of the complex nature of the materials and the variability between samples. Two samples have been processed: • 1) "Sample II"; and 2) "Sample III ·(9 Nov '77) ". The extraction and analysis procedure for both samples was identical and is represented schematically in Fig. 1. Three fractions were prepared from each sample by a three-step process of extraction, concentration and fractionation. Each fraction • was characterized through application of combined gas chromatography-mass spectrometry-computer data reduction• • • • BIOSLUOGE SAMPLE • I Extraction W/ CHC13 I 1 . Aqueous ' + Suspended orginics • matter 1 Filter discarded filtrate residue • I • ! I Evaporate to discarded • near dryness 1 Replace solvents w/ hexanes Evaporate to • ! near dryness l Silica gel Chromatography • hexanes benzene "Saturates"..._____. -----------+---------~GC-MS • "Aromatics• Methanol ----. GC-MS •polar compounds" ' I GC-MS • Figure 1. Analytical separation scheme for "Biosludge" samples • • • • 10 A sample of "biosludge", typically 20 ml, was diluted with • distilled-deionized water of equal volume to make it more manageable in a separating funnel. The diluted sample was repeatedly extracted with pure, freshly-distilled chloroform • and the aqueous layer with suspended material was discarded. • The organic layer was filtered to remove residues and the chloroform solvent was evaporated to near-dryness. The sample extract was taken up in n-heptane and the evaporation procedure repeated. In this manner, the solvent was replaced with heptane. Care was taken to retain volatile compounds by never allowing • the sample to evaporate to dryness. The final extract was diluted to 1 to 2 ml with heptane and of was added to the top a silica gel chromatographic column 20 cm • long by 1 cm diameter. Three separate fractions were eluted from the column by successive additions of hexane, benzene, and methanol. The hexane eluates are primarily weakly polar organic • compounds such as saturated and some unsaturated hydrocarbons . This fraction is commonly termed the "saturates" fraction. Components eluted with benzene are more polar in nature and • include aromatic unsaturated, and multiple ring hydrocarbons and some halogenated and heteroorganic compounds. This is the "aromatic" fraction. The methanol-eluted fraction contains highly • polar compounds such as heterocyclic organics, alcohols, some complex halogenated compounds, etc. • gas chromatograph-mass spectrometer-computer data system. The The three fractions were characterized using a combined instrument used in this study is a DuPont Model 21-491 GC-MS with • • 11 a Model 21-094B data system. The chromatographic column was 1/8 • inch diameter, 6 foot long stainless steel packed with 5% FFAP stationary phase on Gas Chrom-Q 80 -100 mesh. The column was temperature progranuned from 700C to 26ooc at 6°c per minute • using helium as a carrier gas. The mass spectrometer jet-separator and ion source were operated at 200°c, ionization potential was 70 ev, and ion acceleration potential was approximately 1300 • volts. The magnetic sector was continuously scanned at 4 "decades per second" from mass 517 down to mass 41. Mass spectral data were continuously acquired and stored on magnetic· disc for • each scan. Data reduction is accomplished through computer software furnished by DuPont as modified in our laboratory. • Sample II (June 21, 1977) • Hexane, benzene and methanol eluates from silica gel column chromatography were concentrated to equal volumes and submitted to gas chromatographic analysis. Figure 2 illustrates the results • of analyses of equal injection volumes of the three fractions. The largest portion of extractable organics was present in the methanol eluate, or polar fraction, Figure 2C . Saturate fract~on (hexane eluate) The largest component of the saturate fraction was elementary • sulfur which precipitated during concentration. No mass spectral data was obtained for this fraction. Aromatic fraction (benzene eluate) • The reconstructed gas chromatogram (total ion chromatogram) of the aromatic fraction is given in Figure 3. Mass spectra for • • 12 • • I I • • ~B • • c • j i Figure 2~ • Gas chromatograms of saturate (A) , aromatic (B)and polar compounds lC) fractions of sample II• • • A B C -,--r..r-·r· I . 'I ....-, r..,..,..., I 0 50 100 160 Figure 3. Reconstructed total ion gas chromatogram of the saturate fraction, Sample II. • ....... w • 14 peaks "A-D" of Figure 3 are given in Figures 4 and 5 respectively . • Mass spectral data suggests that the first three major components, "A-C", are chlorinated compounds which contain nitrogen. The fourth compound, "D", has been tentatively identified as a methyl­• indole probably primarily 3-methyl-indole, skatole. Skatole is a well known product of the metabolism of nitrogen compounds by bacteria. • The presence of trace amounts of aromatic hydrocarbons in this fraction were indicated by mass chromatograms. Naphthalenes, phenanthrene and/or anthracene and alkyl derivatives of these • compounds were detected by mass chromatograms such as those given in Figures 6 and 7. Polar compound fraction (methanol eluate) • The reconstructed gas chromatogram of the polar compound fraction is given in Figure 8. Mass spectra from the major components labeled "E-L" are given in Figures 9-12. The largest • component of the fraction, "E", has been tentatively identified as a cresol (methyl phenol). The gas chromatographic retention time of the cresol is identical to that, of authentic meta and • para cresol which coelute on this column. The ortho isomer was not present. Components "F" and "G" have mass spectra which suggest they • are halogenated. Component F has an apparent molecular weight of 217 which would indicate by its odd mass that the compound probably contains an odd number of nitrogen atoms • • Components "H", "I", "J" and "L" are tentatively identified as free fatty acids with molecular weights of 228, 242, and 256 • • • • $ m cu r~ • f cS) $ t:-i cu ... QJ • ~ :J 0 rooj f Q.l .Q ·~~ t~ Cl ... Q.l :> 0 .Q ltl i.. • -9-l J : -4J 1-~ r:: r ... t: ~ a> c ~ 0 ~ ~ c.. e 0 • --CJ ""' 0 e ~ ~~ ::l ~ 1. """ CJ - Q.l c.. aJ t. 112 m tU ::t & • • Q.l M ::i C"I -~ "4 • • Q)­ > 0 .Q llS L : ~---·-. • • • ·rr-··,.·-·-··r-,-·1-.1--r-·rr-r--r--1 0 50 100 150 Figure 6. Reconstructed mass chromatogram of benzene eluate at mass 178, phenanthrene. • ~ -.....) 0 50 100 160 Figure 7. Reconstructed mass chromatogram of benzene eluate at mass 192, methylphenanthrene. I-' E L M J K F , ....,-·,-,-.-·1-.-r,.·..r·r..·r-r··1·..,..··rr·-r-·•""·r··--..1-·r-·1··...-·rrr-·r-T-· 1,. 1·····r......r..,....I_ a se 100 1s0 c~00 Figure 8. Reconstructed total ion gas chromatogram of the polar compound fraction, Sample II. • I-' \0 • • • & & \ll ~ CJ lU • • .... QJ :> 0 .c ia ~ 0 CD • QJ lo.I CJ • • _______c_ $ • • p·-·-·.-.-.rtt l""'~~.l~.. ~a. r••, , • .....,..l.l I I I I I~ I I I I I e 60 tea tsa eee e50 i..... H .. 1..\IJ.fl~~... r··· ·· 1··, ~•.1.,,, ~ a 69 100 tSB 200 BU0 Figure 10. Mass spectrum of component "II", a C14 fatty acid, abovei ~I" also a C14 fatty acid, below. l\J ...... ~"~~~frr~'... ' • ~ 60 tee 160 808 es0 o""•••""I..,... ......,.,._._~l'""P•t•t ,.,,. ""' •hh '" • o o\ o lo" o o,.., o o o "• • • • • .. 69 100 168 eoo e50 3ee Figure 11. Mass spectrum 0£ component "J", a C15 fatty acid, above; ~L" a C16 fatty acid below. "-> "-> • • • • • • • • • • 3 0 ..... Q) .c .. Q) > 0 .c ~ $'° : a ~ " .u c: aJ c: 0 c. e 0 tJ ..... 0 e ~ 1-4 .u CJ ~ ~ tQ tQ en ~ ~ .,. -~ which correspond to c14 , C15 and C16 acids. Component "M" has an apparent molecular weight of 254, contains • no halogens, and probably is an alcohol (strong M+-1a peak at 236). Component "K" with an apparent molecular weight of 147 probably contains one nitrogen atom and a phenolic hydroxy group • • Sample III (November 9, 1977) Saturate fraction The total ion chromatogram for the saturate fraction is given • • in Figure 13. The chromatogram is characterized by an unresolved mixture of compounds in the range of scans 150 to about 300. A few resolved compounds are observed, the most prominent of which is labeled "A" in Figure 13. The mass spectrum of this component, given in Figure 14, is typical of long chain n-alkanes and in this case the molecular ion peak at M/e = 240 indicates the component • • is n-heptadecane (n -C17H36). A whole series of n-alkanes becomes evident in the mass chromatogram reconstructed from M/e = 85 ions as shown in Figure 15. Most of the peaks in this figure are apparent • but less evident in the total ion chromatogram of Figure 13. Peak "A" of Figure 13 corresponds to Peak "A" of Figure 15. The saturated, branched, isoprenoid alkane, phytane, is • identified as component "B" in Figure 15 by the mass spectrum of Figure 16 and its elution position with respect to the normal alkanes. Other isoprenoids are probably present (pristane) • • Other homologous series of compounds are also present. An unidentified series having a prominent fragment ion at M/e = 82 is evident in the mass chromatogram shown in Figure 17. The mass spectrum of component "C" is given in Figure 18. The fragmentation pattern resembles compounds containing cyclohexyl groups but the • • • • § ~ "" ~ • I • I ~ • ~ c e .... G I ~ 6) • .... ~ .-4 Q U'l • ~ .... ~ • • • • • • • • ,.., .... s e ~ I ~ • • e i • ~ • .... ~ H ~ • • $ • 27 • • • & ui tO Cl:) (U • ~ i !a z • ! Ql • ~ 0 ~ II ...s ~~ • ~ & • I"' ID ~ ~ ri.. • • • • • • • • M ! ..-1 ~ !,\( • ~ • a::i :II. ~ & ~ .. s • ~ ~ B! & LO • ~ \D ..-1 ~ a "-< 0 • • • 29 • • • N cg • ~ i5~ 1-4 • ~I~ • ! ~ • ~ & • I ID !"­ ! ...+ • ~ • • • • • • ~ & QJ • ~ . I"') ~ 1-4 Cl. 6' U> ~ .... • I : : CJ & • ~ & ..... i ~ an s • a:a ~ ~ Cl. • • • 31 mass spectra of these relatively minor components are not • sufficiently adequate to permit specific identification. Aromatic fraction The reconstructed total ion chromatogram for this sample is • • given in Figure 19 for the "aromatic" fraction. There are three major peaks in this fraction, an incompletely resolved pair at scan 175 and a peak at scan 248. The mass spectra of peaks "D", • "E", and "F" of Figure 19 are given in Figures 20, 21 and 22 respectively. These mass spectra are difficult to interpret but all three have characteristics of polyhalogenated compounds . • Structure determination on the basis of mass spectrometry alone is probably not possible. Similar mass spectra were not found in a digitized library of 7054 compounds or in a large compendium (Stenhagen et al., 1974). Component "E" appears to be present as a major peak in both Sample II and Sample III. • The "aromatic" fraction also is characterized by a hump of unresolved components. Reconstructed specific ion mass chromatograms are able to resolve some minor components from this hump. Using this technique a component corresponding to naphthalene • is resolved in Figure 23. Peak "H" of Figure 23 has a mass· spectrum corresponding to naphthalene and on coinjection into the chromatograph coelutes • with ·naphthalene. Peak "G" also has an identical mass spectrum to that of naphthalene but is not azulene which elutes following naphthalene rather than preceding it. Component "G", which remains unidentified, is also observed in Sample II. Also present in • • s 6) (') I• I ~ • a i ~ ,. ~ I ~ ~ 0 1• ~ s ~ • I • ~ ~ ~ ra.. • • • i v • ~ • ~ cs. {') a'I ..... s Ml ~ ...... • "" ~ ~ : 6> p cu Q U'l • i ... ~ ~ & & ~ .. CJ) 1. ~ . m 0 N ~ ..... • "" • • 34 • • • "'.... ! ea. ~ • C;J • Ic e I • ~ N""" ! ea. ~ • • • • • • • • • . Cl\ ..... ~ C&o ~ .. z • I C&o ~ & & & ..... • ~ ~ tll (S) ~ ID • N N ~ .... C&o • • • 36 • • • & Ncc. lJ) ru P-4 • ~ !( i~ 1-4 Q ~ QJ • I ~ 6l e-;l LO ~ .... • ~ ::c c ~ t::i 6') • ICll 'Pt ~ G) • I LI> ,.; N ~ 1-4 • ~ • • 37 Sample III are the parent and methylated homologs of polynuclear • aromatic compounds such as naphthalenes, fluorenes, phenanthrenes (anthracenes), etc. All of these components are present in relatively minor concentrations. • Polar compound fraction The reconstructed total ion chromatogram for the methanol eluate fraction is given in Figure 24. The major component, peak • "J", is apparently an aromatic acid of molecular weight 136. A methyl substituted benzoic acid is likely. The mass spectrum of this peak is given in Figure 25 . • Peak "I" has been identified as triethyl phosphate. The mass spectrum is given in Figure 26. Alkyl phosphates are conunon alkylating agents and fuels and lubricants additives. Peak "K" • is unidentified. Some other minor components can be identified in this fraction. Phenol is readily observed at scan 145 (Figure 27) and a cresol at • scan 156 (Figure 28). Neither component is present in sufficient quantity that it is observed in the total ion chromatogram (Figure 24) as a discrete peak . • • • • • • • • • • • • • • • tn cu I & ~ ~ ~ I ~ ~ ~ s ..... I ~­ ~ N ~ ..... tr.. . --:. -= -----------::.:===~["' ' ! :.... :•. :­ - . . in N :.. • : . . ~ . . • :­ . FIGURE 26. MASS SPECTRUM OF COMPONENT "I" (ETHYL PHOSPllA'l'E) OF FIG. 24. -r-1-i1r-10-1~1.TtL~...1..,...1"'.I Ul~~I~' .~J'i'T'T 0 60 100 150 a00 ~ • 41 • • I. 1 +­ •. N. c::i 1-4 t la.. z • 1-4 • &n .-4 CJ r, ~ Ul ~ • II( ~ -3 Q z ::> 0 la.. M I 0z • ~ 1-r-• ii: f&. • 0 L 2! ' ::> I g: ~ CJrzJi::i.. • Cl] .­ Cl] en .-• ~ r-. .• N ~ . ­ ::> c::i • 1-4 .• ~ • $ • • • • • • • • • • • • •. N c. Concentration of Selected Trace Metals in the MTBD Introduction • • This report summarizes the results of chemical studies on the heavy metals component of several batches of waste sludge from Shell Chemical Co., Deer Park, Texas. The metals chosen • for analysis were Mn, Cu, Cr, Ni, Cd, Zn and Fe, based on preliminary reports which indicated that levels of some of these metals were much higher than in ordinary sediments or seawater • The project work was directed towards two main goals: • (1) Accurately determine the concentrations of the metals in various batches of sludge; (2) Characterize the chemical form • and behavior of the sludge heavy metals in seawater. This information is pertinent to understanding and predicting the transport and accumulation of heavy metals from such waste materials dumped in open ocean waters. • Methods and Materials • Samples of sludge material were obtained in either clean glass bottles or acid-washed Nalgene plastic bottles. The samples were subsequently kept in a refrigerator at 2 -4 c for the duration of the study period. All glassware for trace metals work was washed in 50% HCl overnight• • Trace Metals Analysis An appropriate volume of sludge (usually 10 ml) was measured out by graduated cylinder and placed in a porcelain Coors • crucible. The sample was then combusted at 500 c in a muffle furnace overnight (ca 16 hr). The oven was at room temperature • when the sample was first placed in it; thus heating to 500 C • was gradual. Similarly, combusted samples were allowed to cool slowly to room temperature before removing from the oven. An initial experiment was performed to evaluate methods of • oxidation of the organic material in the sludge. Dry ashing in the above manner was compared to wet digestion of sludge in a 3:1 mixture of nitric and perchloric acids at ca 100 C. Because • little difference in the methods was observed at the levels of metals which were present in the sludge, the dry ashing technique was subsequently utilized• • The combusted material was dissolved in 3N HCl and warmed on a hot plate for a few minutes. The solution was next filtered through Whatman # 42 paper and brought to volume in a volumetric • flask. This solution was then analyzed for Fe, Mn, Cu, Cr, Ni, Zn and Cd by flame atomic absorption spectrophotometry. The instrument used was a Perkin-Elmer 303 equipped with a • Recorder Readout and scale expansion and a 10 mv recorder. Matrix interference was evaluated for several elements (Cd, Ni and Zn) by using the standard additions technique and found not • to present a problem. Background corrections for non-specific absorption were regularly obtained by using non-absorbing lines for each metal. The non-absorbing lines used were: Fe, 247~3 run;• Cu, 322.9 run; Cr, 352.0 run; Ni, 231.6 run; Cd, 226.5 nm; and Zn, 220.2 run. The precision of the analytical technique appears quite• good from the variation in replicate analyses. The percent deviation for each metal was Fe, 4%; Mn, 2%; Cu, 3%; Cr, 5%; • • 45 Ni, 8%; Cd, 8%; and Zn, 7%. The accuracy of the technique was • verified by several analyses of a National Bureau of Standards sample, orchard leaves reference material # 1571. For a sample size of N.B.S. material corresponding to 10 ml of sludge (the • regular amount used), the results for Fe, Mn, Cu, Cr, and Ni were all within 10% of the N.B.S. certified values. Only for Cd and Zn were greater variations found, and these ranged from • 10 -50% lower than expected values. I. Isolation of Sludge Particulate Material I For separation of sludge into soluble and particulate phases, • Whatman # 42 filter paper was used. This paper will retain very fine crystalline precipitates. After filtration of seawater suspensions of the sludge, filters with particulate material were combusted and the residue analyzed as described above. Filter blanks were run for all determinations • • • Results Sludge # ·1 Heavy metals were measured on the settled solids fraction of sludge # 1, i.~~' the denser material which settled out of the sample upon standing for several days. The concentrations • are listed in Table 1 on the basis of ΅g per ml of wet solids• These data, therefore, represent a severalfold concentration of the metals over the initial sludge sample • • Sludge # 2 Con.centrations of heavy metals in sludge # 2 are compared in • • 46 • • • Table 1. Concentrations of heavy metals in Shell waste # l.Values in ~g/ml of liquid sludge + S. o. for 4determinations. Samples were digested in nitric +perchloric acid• • Metal Batch i 1 Fe 99.4 -+ 9.6 Mn 1.5 -+ 0.2 cu 2.4 + 0.1 • Cr 91.4 -+ 5.4 Ni l.6 + 0.4 Cd < 0.2• Zn 8.4 + 1.0 • • • • • Table 2 on the basis of ΅g per ml liquid sludge and ΅g per g dry wt suspended particulate material ih the sludge• • Heavy metal concentrations in the particulate fraction of sludge # 2 are also presented in Table 2. Cadmium showed the most percentage in the soluble form (ca 15%), while essentially all of • the other metals were retained in the particulate fraction. Effects of washing the particulate material with various solvents can be interpreted from the data in Table 3. Seawater appeared to remove very little Cr, Zn, or Cd; at most, some 20% of the particulate-bound Ni was solubilized. Deionized water • (comparable to fresh water) did solubilize about 50% of the Cd, in addition to 20% of the Ni. Treatment with acetic acid-hydroxylamine reagent removed • varying amounts of all metals from the particulate material (same table). ~ Two groups of metals could be discerned: Cu, Cr, and Zn which were reduced 15 -20% by this treatment; and Mn, Fe, Ni, • and Cd which were desorbed some 50 -60%. • Sludge # 3 The concentrations of heavy metals in sludge # 3 are • expressed in Table 4 on the basis of ΅g metal per ml of liquid sludge and ΅g metal per g dry wt of suspended particulate material in the sludge. When the sludge was fractionated into • soluble and particulate phases, the heavy metals showed the distribution indicated in the same table. Since all metals except Cd and Mn .were predominantly (> 90%) in the particulate phase, the remainder of the studies were directed towards characterizing the nature of the association between • • • • • Table 2. Heavy metal concentrations in Shell waste # 2. Values in ΅g/ml of liquid sludge compared to ug/g dry wt particulate material in sludge. Data for particulate fractions given on ug/g dry wt basis. Values are + standard deviation for 3 determinations. • Whole Sludge Whole Sludge Particulate Metal (u~/ml) (1,Jg/2) Fraction (ug/g) • - Fe 132. 0 + 9.0 7432 7432 Mn 2.0 + 0.2 113 113 Cu 1.9 + 0.2 107 101 Cr 20.0 + s.s 1126 1126 • Ni 2.5 + 0.6 141 135 Cd 0.065 + O.Ol 3.66 2.82 Zn 9.0 + 0.7 507 507 • • I· • • • • • • Table 3. Heavy metal concentrations (ug/ml sludge) in particulate fraction of Shell waste # 2, before and after various extraction procedures. • Metal Ontreated • Fe 132.0 Mn 2.0 Cu 1.8 Cr 20.0 • Ni 2.4 Cd o.os Zn 9.0 • • • • Particulate Fraction De.ionized Water-Rinsed 18.0 2.0 0.02 8.5 Seawater­ Hyaroxyiam.ine- Rinsed Treated 56.0 0.6 l.5 18.0 16.0 2.0 l.2 a.as 0.02 9.0 a.a • • • • Table 4. Total concentration of heavy metals in sludge t 3 and in particulate and soluble fractions. Values in ΅g/g dry wt or ΅g/ml liquid sludge + standard deviation for at least 3 determinations. • Whole Sludge Metal {΅g/ml) • Fe 46.54 Mn 0.61 Cu 1.58 Cr 22.27 ' • Ni l.41 Cd 0.026 Zn 4.40 aLimit of detection was • • • • Soluble Whole Sludge Particulate Fraction (J,.Lg/2> ·Fraction (΅g/g) (1Jg/g) 1820 + 57 1540 + so 350 + 8 - 24 + 0.96 18 + a.a 6 + 0.3 - 62 + 8 58 + 2.0 2 -+ 0.2 808 + 45 796 + 27 < 10.0 56 + 6 51 + 6 6 + 2.8 - l~O + 0.2 < o.aa < o.aa 172 + ll 163 + s 10 + 3.5 - 0.8 ΅g Cd per g dry wt suspended material• the particulate material and the metals • • The effect of diluting the sludge into various amounts of seawater and deionized water is apparent from examination of Table 5. Seawater solubilized a small portion of the Mn and Zn, • while other metals were unaffected at their limits of detection. Deionized water, however, solubilized about 20% of the Ni in addition to Mn and Zn. These results hold over the seawater pH • range from ca 6 to 8.0. Two commonly-used chelating agents, ethylene dinitrilotetra­acetic acid (EDTA) and citric acid, were tested for their ability • to remove metals at pH 7.8 from the sludge particulate material. Table 6 swnmarizes these data. Only EDTA at the highest concentration showed evidence of significant activity, chelating • about 50% of the Fe and a slight amount of Ni and Mn. Cr, Cu, and Zn appeared totally unaffected. The particulate fraction was found capable of adsorbing • additional metals out of seawater. This scavenging ability was demonstrated by spiking seawater-sludge mixtures with known amounts of pure metal compounds. The data in Table 7 show that • extra Cu and Cr were readily and completely adsorbed to the particulate fraction. Manganese at the concentration occurring naturally in Gulf of Mexico bottom water (in this sample 53 ΅g/l) • was only partially adsorbed. The results with Ni are particularly interesting; Ni was poorly adsorbed when added by itself and even this small amount of adsorption was negated when extra Cu and Cr • were also present. Hence, the particulate material in sludge # 3 appeared to complex or adsorb metals selectively• • Table 5. Effect of diluting sludge I J, with sea water and deionized water. Suspensions were made by mixing 10 ml sludge with an appropriate amount of seawater or deionized water for 18 - 24 hrs. Particulate fraction was collected by filtering through Whatman I 42 paper and trace metals measured on the suspended material. Values are ~g per g dry wt + standard deviation for at least 3 replicates. - Sludge Particulate 10 ml Sludge + 10 ml Sludge + 10 ml Sludge + 10 ml Sludge + 90Metal Fraction Untreated 40 ml Seawater 90 ml Seawater 190 ml Seawater ml Deionized Water Fe 1540 1580 + 83 1560 + 60 1530 + 42 1560 + 73 Mn 18 11 + 1.9 13 + 1.4 10 + 1. 5 11 + 1.4 Cu 58 60 + J 56 + 1.0 61 + 5 58 + 4 Cr 796 770 + 42 785 + 12 793 + 50 773 + 28 Ni 51 50 + e.o 47· + 1.4 50 + 2.5 40 + 3 Cd < o.o < o.e < o.e < o.8 < o.8 Zn 163 140 + 21 142 + 32 137' + 28 140 + 35 Ul N • • • I I. Table 6. Effect of treating sludge # 3 with organic chelating agents in seawater. Values are ~g/g dry wt ! standard deviation for 3 determinations. • Particulate Fraction Treate~ with Treated with Treated with After Exposure la-M ia-5 M lo-3 M Metal to 4a ml Seawater Citric Acid EDTA EDTA Fe 1600 + 25 1600 + 22 1590 + -10 1010 -+ 51 Mn 15 + o.7' 16 + l.4 14 + 0.7 11 + l. 4 Cu 63 + 3 66 + 0.7 65 + 4.8 67 + 10 • Cr 780 + 55 738 + 31 805 + 48 790 + 42 Ni 54 + 2.8 56 + l.4 48 + 2.8 45 + l. 4 cc < 0.8 < a.a -< a.a < a.a Zn 153 + 2.8 147' + 21 158 + 12 146 + 18 • ---­ - • • • • ~ Table 7. Adsorption of extra metals added in seawater solution to sludge I 3 suspended matter.Single metal means an extra amount of only that metal was added. Combined metals means the following mixture was added to 10 ml sludge: 10 ΅g Cu, 10 ug Ni, 200 ug Cr,and 0.1 ΅g Cd. Values are ΅g/g dry wt + standard deviation for 3 determinations. Sludge Particulate Fraction After Exposure \ Adsorption of Sludge Particulate to Extra Metals Add~ MetalFraction After Exposure srngle . Combined Single Combined· Metal to 40 ml Seawater Metal Metals Metal Metals - Fe 1580 Mn 11 32 + 1.4 57 · Cu 60 100 + l.5 82 + 5 100 82 - Cr 770 1470 + 10 1440 + 10 100 97· Ni 50 57· 2 48 4 + + 15 0 Cd < o.e 1.2 + 0.2 0.9 + 0.1 100 50 Zn 140 Ul ~ • 55 Discussion • • Adsorption of heavy metals to suspended particulate material in seawater is not unexpected (Helz et al., 1975; Windom, 1976; Trefrey and Presley, 1977). The interesting observation is that the metals are so tightly bound to the particulate fraction of these sludge samples. The acid-hydroxylamine reagent was found • by Chester and Hughes (1967) to leach from clay minerals trace metals which are not part of the lattice structure of the mineral. Ferro-manganese nodules were almost completely dissolved by this • reagent. Hence, the results from the hydroxylamine leach of sludge # 2 suggest that either a considerable portion of the Cu, Cr, Zn, and Ni are in lattice positions of a mineral or that • these metals are part of a particulate organo-metallic complex . • Since the particulate material was readily dissolved by hot nitric acid (wet digestion) and almost completely combusted by dry ashing at 550 C, very little sand or clay mineral sediment • particles appear to be present. The fairly high organic carbon content (around 16% for sludge # 2 and # 3) makes it more likely that the particulate material is precipitated organic material, • possibly polymeric in nature, with a high metal-complexing capacity. This would also correlate with the apparent selectivity of the material for adsorbing metals • • Heavy metal concentrations in sludge # 3 suspended material were compared to those for suspended particulate material in natural seawater samples (Table 8). By comparing Fe and Mn values, it is apparent thatAthe sludge particulate material is highly enriched in Cu, Cr, Zn, Ni, and probably Cd. Although Fe • • • • Table 8. Comparison of heavy metalsa in naturally-occurring suspended material with concentrations measured in particulate fraction (s susp. matter) from sludge # 3 • • Miss. River STOCS Sludge t 3 Suspend!!d Nearshore Gulf Gulf of Mexico Particulate Metal Matter0 Susp. Matterb Susp. ParticulatesC Fraction Fe 47,400 50,200 15,000 l,540 Mn l,307. 1,191 567 18 • Cu 42.3 55.6 79 59 Cr 72.S 72.8 48 796 Ni 55.6 sa.s 58 51 Cd 1.4 2.0 5.4 < 0.8• Zn 184 226 300 163 a All values are ug/g dry wt. b From Trefrey and Presley (1977) •• c Average values, from Barnes (1976) • • • I • • and Mn in the sludge are only 5 -10% of the levels found in natural suspended matter, the other metals are at the same level • or, in the case of Cr, ten times higher. The impact of adding this metal-laden suspended material to the open ocean can be illustrated in the following manner • Normal concentrations of suspended particulate material fall in the range of 5 -10 mg/l for offshore waters (Spencer and Sachs, • 1970; Barnes, 1976). If the sludge were diluted immediately upon dumping to 0.1% of its full strength, the concentration of suspended particulate material would be 0.001 x 25,000 = 25 mg/l. • This means that seawater in the immediate area of dumping would then have an increased load (by a factor of 3) of suspended material which is itself enriched in Cr, Cu, and Zn. Continued • dumping of the sludge would offset the detoxifying effect of dilution by seawater, since the particulate material would gradually build up around the dumpsite. The results of mixing sludge with seawater can be extrapolated to the natural ocean situation with a high degree of relevance. Many of the laboratory tolerance studies where biological effects of the sludge are determined have utilized seawater dilutions of the sludge comparable to those listed in Table 5 (5 -20%). Since seawater desorbed very little of the potentially toxic metals • present at high levels (viz. Cu, Cr, Zn, and Ni), heavy metal effects on organisms of sludge mixed in seawater could result from exposure to or ingestion of particulate material • • • Literature Cited • Barnes, s. s. 1976. Suspended sediments: trace . metal content In Environmental Studies, South Texas Outer Continental Shelf. Henry L. Berryhill, Jr. (ed.). A report to the • Bureau of Land Management, in fulfillment of Contract AA 550-MU6-24 to U.S. Geological Survey. Calder, J. A. and P. L. Parker. 1968. Stable carbon isotope ratios as indices of petrochemical pollution of aquatic systems. Environ. Sci. Tech., July, 1968. • Chester, R. and M. J. Hughes. 1967. A chemical technique for the separation of ferromanganese minerals, carbonate minerals, and adsorbed trace elements from pelagic sediments. • Chern. Geol. 2:249-262 . Fry, B., R. S. Scalan and P. L. Parker. 1977. Stable carbon isotope evidence for two sources of organic matter in • coastal sediments: Seagrasses and plankton. Geochim. Cosmochim. Acta. 41. Helz, G. R., R. J. Huggett and J. M. Hill. 1975. Behavior of Mn, Fe, Cu, Zn, Cd and Pb discharged from a waste-water treatment plant into an estuarine environment. Water Res. 9:631-636. • Parker, P. L., B. Fry, W.-L. Jeng, R. S. Scalan. 1978. al3c food web analysis of a Texas sand dune community. Geochim. Cosmochim. Acta (in press) • • Spencer, D. W. and P. L. Sachs. 1970. Some aspects of the distribution, chemistry, and mineralogy of suspended matter in the Gulf of Maine. Marine Geology 9:117-136 • • Stenhagen, E., s. Abrahamsson and F. W. McLafferty, Editors, • "Registry of Mass Spectral Data" John Wiley and Sons, N.Y., 1974. Trefrey, J. H. and B. J. Presley. 1977. Heavy metal transport • from the Mississippi River to the Gulf of Mexico. pp 39-76. In Marine Pollutant Transfer, H. L. Windom and R. A. Duce (eds.). Lexington Books, Lexington, Mass • • • • • • • • • • Repor~ for NOAA on Ocean Dumping in the Gulf of Mexico Effects of Wastes on Growth of Microalgae • by C. Van Baalen, J. Batterton • • • • • 61 Introduction The purpose of this work was to assay samples of waste material for inhibition of growth of algae. Three samples (noted as I, II, and II) supplied to us were tested for toxicity to six microalgae. The test organisms, two blue-green algae, two green algae, and two diatoms represent three major divisions of algae • • • Methods Samples of waste material were frozen upon arrival and stored at -10°c. Just before testing, frozen aliquots of each sample were thawed and autoclaved (121°c, 15 min}. The microalgae were grown in the synthetic sea water medium ASP-2 (Provasoli, McLaughlin and Droop 1957; Van Baalen 1962), using the test-tube culture technique of Myers (1950). Green algae used were Chlorella autotrophica, strain 580 and Dunaliella tertiolecta, strain DUN (both obtained from R. R. L. Guillard); the blue-green algae used were Agmenellum quadruplicatum, strain PR-6, and Coccochloris elabens, strain 17a (isolates of this lab); and the diatoms were Cylindrotheca sp., strain N-1, and Chaetocerous simplex (isolates of this lab). All cultures were pure except £· simplex. £• simplex cultures • were incubated at 27°c, all others at 3o0 c. The cultures were illuminated continuously; C. simplex with F20Tl2-D fluorescent lamps and the others with F40CWX fluorescent lamps. All , cultures were continuously aerated with a 1.0 ~ 0.1% co2-in-air mixture. Growth was estimated turbidimetrically using a Lumetron colorimeter Model 402-E with a red glass filter (660 mm). For , simplicity the data is reported in generations per day. Autoclaved waste material was added directly to sterile growth medium. No additions were made to control cultures. Duplicate cultures were used in all assays. The culture tubes were inoculated with ~105 cells/ml and incubated immediately. Occasionally sample turbidity made optical growth measurements difficult, thus chlorophyll ~ analyses (90% redistilled acetone extracts) were done to aid in interpretation of growth data. An outline of the liquid culture method is given in Fig. 1. Algal lawn assays were done according to the method described .. previously (Pulich et al. 1974). Results • Sample I was tested in two ways, as a whole sample and as its liquid phase and solid residue obtained by settling (18 hrs). The whole sample was checked by placing material absorbed .. on a washed filter paper disc (S & S No. 740E, 12.7 ' mm) onto an algal lawn, ~105 cells/ml, in agarized medium. The petri dishes were incubated under growth conditions for 4 -6 days. There .. was no inhibition of growth (clear zones surrounding the disc) with organisms PR-6, 580, or N-1 (data not shown). However, in liquid culture assays whole Sample I somewhat .. inhibited growth of£· simplex (Table 1). The toxicity of Sample I was associated with the residue (material obtained after settling) but the amount required was rather high. The liquid phase was not toxic even at 50% (v/v) (Table 1). Table 2 summarizes the growth rate data in liquid culture • Figure l. Protocol of testing waste material samples MAIN rLE SO ML SAMPLES STORED AT -10°c i THAWED AND AOTOCLAVED (l2l0c, 15 MIN) i VORTEX MIXED i STERILE SAMPLE AOOED TO STERILE GaOWTB MEDIUM; e.g., 10, v/v MEANS 2 ML SAMPLE + l8 ML MEDIUM i .. INOCULATE, APPROX. 105 CELLS/ML i .. INCUBATE AT 30:_0.l0 c (ALL EXC~T ~·SIMPLEX, 21°c), CONTINOOOS ILLUMINATION, AND BUBBLING WITH u co2-IN-AIR i MEASURE GROWTH ~RBIDIMETRICALLY ( 66 0 ~) , AND IN SOME CASES COMPARATIVE FINAL YIELDS OETEBMINED AS CHLOROPHYLL A ---~ ~ I I ~ ~ I I W I Table 1. Effect of whole Sample I and residue on growth of Chaetocerous simplex. Lag Time in ~ype of Addition to Medium (20 ml) Generations/Day Initiation of Growth (h) Whole sample control (diec only) 5.8+0.2 0 disc +absorbed 1 sample 4.4 17' Residue, mq wet wt/disc 0 6.0+0.2 0 1.5 5.6 0 3.0 5.1 2 7:.0 4.4 16 ~20 NG-7 2 Liquid phase, \ v/v 25 5.9 0 50 5.9 0 !Filter paper disc dipped into Sample I, then added to test culture2NG-7·means no growth after 7 days incubation O'\ ~ ~ ~ I I s -e • • -------­ Table 2. Effect of autoclaved Samples II and III added to the medium (v/v) on growthrates of algae. SAMPLE II SAMPLE III 1\LGAE 0 5% 10% 0 S\ 10% Blue-greens PR6 5.0+0.4 3.0 2.0 5.1+0.4 3.6 1.6 17a 3.6+0.4 2.7 1.9 3.9+0.4 2.6 2.2 Greens 580 2.6+0.3 1.9 1.2 2.2+0.l 2.9 2.5 Oun 2.6+0.3 1.9 1.6 2.4+0.J 2.4 2.8 Diatoms N-1 4.1+0.J NG-2 2 NG-2 4.2+0.3 3.3 2.9 c. simplex 5.1+0.5 NG-2 NG-2 5.1+0.5 NG-7' NG-7 ' 1Growth rates expressed as generations/day, at 30° (C. simplex, 27°) under continuousillumination and aeration with lt co2-in-air -2NG-2 or -?· means no growth in 2 or 7 days after inoculation O'\ U1 66 of the six test algae with Samples II and III. Samples II and III were toxic to the blue-green algae, growth rate decreased about one-half at the highest concentration tested (10% v/v). This pattern of response was also seen with the two green algae and Sampl~ II. Sample III showed little toxicity to the green algae, indeed for reasons unknown, some stimulation of growth was seen. Of the algae tested, the diatoms were the most inhibited by both samples. Sample II completely suppressed growth of both species. Sample III caused only a partial reduction in growth rate of N-1 at 10% (v/v), but no .growth was obtained with C. simplex even after ? · days incubation. Further experiments with c. simplex and Sample III showed growth (4.2 generations/day) would occur at a concentration of 0.5% (v/v) but not at 2.5% (v/v). Conclusions The results of this study have shown that each of the samples were toxic to at least some of the algae tested. Samples I, II and III were especially toxic to the diatoms. It is of particular interest that Chaetocerous simplex emerged as the most sensitive test alga. Possibly this reflects a greater sensitivity (less metabolic versatility?) bf offshore diatoms to pollution. In the case of Sample I the toxicity was associated with the solid phase. Taken as a whole, the data on the microalgae suggest that caution is advisable if promiscuous dumping of samples like those assayed herein is allowed. Microscopically, the samples appeared to have high bacterial counts, their original organics were thus probably well worked over, yet the samples still evidenced toxicity. In this connection it is of interest that in Sample I the toxicity was associated with the sediment fraction, which could have a long environmental half-life. Short-term toxicity effects, such as selection or enrichment of certain species in the phytoplankton are also predictable (Table 2) with perhaps deleterious effects through the food chain. LITERATURE CITED Myers, J. 1950. The culture of algae for physiological research. In: The culturing of algae, pp 45-51. Yellow Springs, Ohio: c. F. Kettering Foundation. Provasoli, L., J. J. A. McLaughlin and M. R. Droop. 1957~ The development of artificial media for marine algae. Arch. Mikrobiol. 25, 392-428. Pulich, w. M., Jr., K. Winters and c. Van Baalen. 1974. The effects of a No. 2 fuel oil and two crude oils on the growth and photosynthesis of microalgae. Mar. Biol. ~, 87-94. Van Baalen, c. 1962. Studies on marine blue-green algae. Botan. mar. !i 129-139. Report for NOAA on Ocean Dumping in the Gulf of Mexico Toxicity of Biosludge on Marine Invertebrates by J. A. c. Nicol .. .. .. 70 Introduction .. Our previous reserach on the toxicity of industrial products to marine invertebrates dealt with petroleum oils (Lee and Nicol, 1977; Lee et al., 1977). We found deleterious action in survival, growth rate, feeding, respiration, behavior and fecundity. From our experiments with these techniques, we began a study of the effects of biosludge, destined for ocean dumping, on two marine invertebrates. For the first year we concentrated on survival, growth and reproduction of a benthic animal (an amphipod) and a pelagic one (a sergestid shrimp). Materials and Methods Four lots of biosludge were supplied in barrels by the Shell. They are designated Sample I, II, III and IV. Sample I contained much solid material and was filtered by .. the Chemistry Department of the Port Aransas Laboratory; the solid material recovered by filtration was tested. It was centrifuged, and the material in the pellet was suspended in sea .. water, 35g in 965 ml of sea water. The suspension was homogenized in a Waring blender, and subsequently stirred to maintain the suspension. From this suspension, as stock • solution, dilutions were made (see Figure 1, flow sheet) • Sample II contained much finer solid material. It remained fairly homogeneous. It was shaken vigorously before use, and aliquots were taken as required for testing. Samples III and IV were fairly homogeneous, resembling • 71 Figure l. Flow chart for the preparation of the test solution. PRE?ARATlON OF TEST SOLUTlONS SAMPLE I RAW SAMPLE CENTRIFUGED AT 3000 r?m FOR 10 min. PARTICULATES(35 g) .. ~ .. + 96~ ml GLASS -FlLTc:RED SEAWATER .. STOCKSOLUTION (3.5 °lo) HOMOGENIZED I I I .. I IS71 ml STO.CK 286,fnt sT.OcK 28.S ml STOCK 2.86ml STOCK+ + + +429ml SEAWATER 714 ml SEAWATER 971.4 mt SEAWATER 997.14 ml SEAWATER l l l ! . 20 g/1 or 20 11/o IOg/I or I 0/o I Oil or 0.1 °lo O.J g/I or O.Ot % TEST SOLUTlON TEST SOLUTl ON TEST SOLUTlON TEST SOLUTION .• 72 Sample II. They were left undisturbed for 2 weeks, whereupon solid material settled, and the clear supernatant was siphoned off for use. Offshore sea water was used as medium for the animals. It was passed through glass fiber filters, antibiotics were added, namely, penicillin G and streptomycin sulphate at 50 mg and 25 mg 1-1• The test animals were Amphithoe valida and Lucifer faxoni. They were kept in culture bowls, 19 cm diameter, containing 1.5 1 of sea water 30 o/oo. Bowls were covered with PVC film (Saran wrap), and were gently aerated. Amphipods were fed tropical fish food flakes and dried Ulva; shrimp were fed a mixture of rotifers and newly hatched Artemia. Temperatures were 24 + 2° c. Both Lc50 and TL50 values were calculated by the method of Litchfield and Wilcoxon (1946) • • Results Sample I Several concentrations (0.01 to 3%) were tested the on amphipod Amphithoe valida. Time to kill 50% of the test population for a 2.0% (20 1-l) and 1.0% (10 1-1 ) mixtures were 24 hand 33 h, respectively (Fig. 2). A later test showed that at the concentration of 2.0% the T50 was delayed to h 48 and there was no mortality at all for the population tested at 1.0% during 4 days' exposure (Fig. 3). The difference in results was probably caused by the different age groups used 73 .. SURVIVAL vrs. CONCENTRATION SAMPLE I EXPERIMENT I CONTRO~~-----------..... 20 CONTROL 16 ~ G .Q 14 e ::s c - 0.01 °lo -l 12 § O.J °lo > a: :;) 10 .Q E :s -c 12. -l ~ > -10 > a: ::> rn 6 4 2 2.0% 3.0 °lo o.._------------......------------l------------..,..----------~ 0 24 48 72 96 TIME (hrs.) !'igure 3. The survival of Amphithoe valida (6 weeks~ld) at various concentrations of bioslud.qe. Mixtures were made from the solid part of biosludqe. 75 in the two series of toxicity test. Amphipods 10 weeks old were used in .the first experiment and 6 weeks old in the later test. Moults also were recorded. There was no difference between control and experiments in test solutions at concentrations < 0.01%. There were few moults in the 0.1% test solution (Fig. 4). Sample II • For this experiment, the relative sensitivity of different age-groups of amphipods to the whole biosludge was determined. Age-groups used in this experiment were 3, 5 and 9 weeks old. Concentrations of biosludge tested varied from 4 to 10% (v/v) and exposure lasted for 96 h. We found that amphipods in either young or old stages were more sensitive than the just matured groups (5 weeks old). For example, in a 10% mixture, more than 90% of the test animals of age 5 weeks was still alive after 4 days' exposure, while for the other two ages, • under the same conditions, the mortality was > 35% • Sample III In these experiments with amphipods the supernatant of biosludge was first autoclaved and then used in the test. Concentrations used were 10%, 20%, 30% and 40%. Exposure time was extended to 8 days. Mortality was checked every day. No • mortality was observed in the 10% group. For the remaining groups {20%, 30% and 40%) the time to kill 50% of the test population was found to decrease with the concentrations and, • in order, were 165 h, 105 h, and 81 h, respectively (Fig. 5). The planktonic shrimp, Lucifer faxoni, was tested with • I --S I I I S 9 S - EFFECT ON MOLTING SAMPLE I 12 II U) 10 · .__ _J 9 0 ~ e 0:: llJ m 1 :'1 ::> z 6 _J <( 5 ..... 0 .__ 4 3 2 Ohr•. 24 48 12 96hrt. Ohrt. 24 48 72 Khn.Ohrt. 24 48 72 96hr•.Ohrt 24 48 72 Hhrt.Ohrt. 24 48 72 96hrt. CONTROL 0.005% (a) 0.005 "· ( b) 0.01 °lo 0.1% CONCENTRATIONS Figure 4. Effect of biosludge on the molting of Amphithoe -..J Cl'\ valida (6 weeks-old). Mixtures were made from the solid part of biosludge. 77 • SURVIVAL vrs. CONCENTRATION AMPHffHOE VALIDASAMPLE m CCWTROL: 20 ' 18 -... 16 ID 14 .Q c:> 12 10 ..J -------20% > a: ::> "" 4 •.. ~ E 2 300/o 0 20 40 60 80 100 120 140 160 ISO TIME (hrs) • P'iqure S. The survival of Al!lphithoe valida (3 weeks-old) in the supernatant of bioslud9e. Supernatant was autoclaved before L,imals were introduced• • •­ 78 autoclaved and non-autoclaved supernatant. The non-autoclaved solution was toxic at 10%, and all animals were dead in the 15% solution at 48 h (Fig. 6). The latter result was somewhat vitiated because of heavy bacterial growth. The autoclaved solution, even at 25%, was non-toxic at 180 h. Sample IV The data (in Fig. 4) for molting indicated that adverse effects of biosludge on amphipods may be found at much lower concentrations than that of 24 h or 96 h-Lc50 • To verify this, experiments were carried out under conditions of low concentrations (< 10%) and a long period of exposure. • Four-weeks-old amphipods were introduced into the following concentrations of biosludge supernatant, 1%, 2%, 3%, 6%, 7% and 8%. A control was also provided as a check. At each concentration, 20 individuals, with a male to female ratio of about 1.0, were added. The test medium was changed once a week; at the same time, the total number of young released within that week was also determined for each concentration. The exposure lasted 2 months. Results from this part of the experiment are shown in Fig. 7~ Two significant patterns could be recognized. The reproductive .. potential for those groups exposed to concentrations > 6% was significantly depressed as compared to that of control. Yet the amphipods which were exposed to concentrations < 3%, • released more young than did the control group. The largest number (157) of young produced was recorded at the 3% • 79 F1GURE 6 SURVIVAL vrs CONCENTRATION LUCIFER FAXON/ SAMPLE m 0% fe°lo ... - D ~ • -e ~ c 14 12 > ~ _J 2~o/o > a: • ::> (I) 6 4 0 .. 2 0 20 40 60 80 100 120 140 160 180 T1ME (h f'1) Figura 6. The survival of adult Lucifer taxoni in the supernatant of biosludqe. Supernatant was autoclaved • • • • 80 160 1~0 140 (!' 110 z ~ 100 I 0> 90 !II u.0 80 a: 70 rI w m ~ 6 z~ !iO 40 • 30 20 o-'---------..------.------.------.------,...----.,-----.., CONTROL 2 3 6 7 8 C 0 NC E NT RAT I 0 N (%) .. Piqure 7. Effect of biosludqe on the fecundity of Amphithoe valida (4 weeks-old) • Biosludqe was filtered , thrcuqh qlasswcol before used• • • • concentration and the least number (23) at the 8%. The total • number of young released in the control was 99 • Two other experiments have also been carried out in conjunction with Dr. w. Pulich, who measured the heavy metal • uptake of animals from the biosludge. Two species of marine crustaceans, Amphithoe valida, and Palaemonetes pugio (grass shrimp), were exposed to whole biosludge for 16 hours, in • concentrations varying from 5 to 20%. At the eQd of the experiment, animals were rinsed with demineralized water several times and then passed to Dr. w. Pulich. Preliminary • results showed significant changes in the total copper and iron of the treated group. Details were reported and discussed by Dr. w. Pulich• • Discussion and Conclusion There was considerable difference between toxicities of • the several samples of biosludge, perhaps because of the actual difference in the composition between samples or because of differences in methods of preparation (Siegel and Rader, 1974) . • For th~ amphipod, ~· valida, the whole biosludge was acutely toxic at about 10% and the level of toxicity varied with the age. The supernatant from the suspension was also toxic, as • shown by the responses of many marine invertebrates to pollutants (Roesijadi, et al., 1976; Rossi and Anderson, 1976), at a level of about 20%. These levels are for mortalities • significantly greater than those of controls. Results for the planktonic shrimp were complicated by bacterial growth. When • autoclaved, the supernatant was much less toxic to both the • amphipods and the shrimps. This procedure controls bacterial growth, but also affects the chemical composition of biosludge. Chronic exposure of amphipods to low concentrations, < 10%, • resulted in changes in both molting and fecundity. The changes in molting suggest that growth may be interfered at a concentration as low as 0.1%. However, data on reproduction • showed that within the sublethal levels of biosludge, more young were released at lower concentrations than at higher concentrations, when compared to the control. This pattern • was not unusual since a similar pattern has been observed on the respiration of L. faxoni when exposed to the water soluble fractions (WSF) of a No. 2 fuel oil. In Lucifer, the • respiration rate rose with increasing concentration of WSF up to 30%, then fell as the concentration increased (Lee et al., 1978) • • • • • • 83 REFERENCES • Lee, W.Y. and J.A.C. Nicol. 1977. The Effects of the Water Soluble Fraction of No. 2 Fuel Oil on the Survival and Behavior of Coastal and Oceanic Zooplankton. Environ. • Pollut. 12: 279-292 • Lee, W.Y., M.F. Welch and J.A.C. Nicol. 1977. Survival of Two Species of Amphipods in Aqueous Extracts of Petroleum • Oils. Mar. Pollut. Bull. 8(4): 92-94. Lee, W.Y., K. Winters, and J.A.C. Nicol. 1978. The Biological Effects of the Water Soluble Fractions of a No. 2 Fuel Oil • on the Planktonic Shrimp, Lucifer faxoni. Environ. Pollut. 15: 167:..183. Litchfield, J.T., Jr. and F. Wilcoxon. 1949. A Simplified • Method of Evaluating Dose-Effect Experiments. J. Pharmac. exp. Ther. 96: 99-113. Roesijadi, G., S.R. Petrocelli, J.W. Anderson, C.S. Giam and • G.E. Neff. 1976. Toxicity of Polychlorinated Byphenyls (Aroclor 1254) to Adult, Juvenile and Larval Stages of the Shrimp Palaemonetes pugio. Bull. Environ. Contam. Toxicol • • 15(3): 297-304. Rossi, S.S. and J.W. Anderson. 1976. Toxicity of Water-Soluble Fractions of No. 2 Fuel Oil and South Louisiana Crude Oil • to Selected Stages in the Life History of the Polychaete, Neanthes arenaceodentata. Bull. Environ. Contarn. Toxicol. 16(1): 18-24 • • Siegel, H. and W.E. Rader. 1974. An Analysis of Biosolid Waste from the Houston Chemical Plant Biotreater: Technical Progress Report BRC-CORP 42-74-F. Project No. 4~-83333. 32 pp • • • • Sensitivity of Open Gulf Fishes to Ocean Dumping Wastes • Donald E. Wohlschlag Principal Investigator • Faust R. Parker, Ph.D., Research Associate John R. Burns, Ph.D., Research Associate! Julie A. Kinney, B.A., Research Assistant The University of Texas Marine Science Institute Port Aransas Marine Laboratory Port Aransas, Texas 78373 • • • • • 1Present address: New York Aquarium Board Walk and West Eighth Street Brooklyn, New York 11224 • • • 85 Introduction • • The purpose of this study is to determine whether respiratory metabolic responses of a marine fish are sufficiently sensitive at sublethal pollutant levels to suggest possible biological • monitoring techniques. The rationale of using respiratory scope --the difference between oxygen consumptive rates at maximum sustained aerobic • activity and at the maintenance or standard level for the assessment of environmental quality was suggested by Fry 1947 and elaborated in general physiological terms by Fry (1957, 1971) . Since theoretical and empirical studies suggested that metabolic scope tends to be reduced under stressed systems, much research • has been related to various environmental circumstances. Brett • (1958, 1964, 1965, 1971}, Brett and Glass (1973), and Brett et al. (1969) have shown that at optimal temperatures, scope and swinuning performances are also related to optima in rations, assimilation, growth and related functions. It is important to note that most fishes generally operate• at a routine rate that lies between the standard and maximum, and is ecologically minimal at around twice the standard level to account for about 1 L sec-1 swimming (foraging), specific• dynamic action (assimilation} and other functions, excluding growth, spawning, extended migrations, etc. (Fry 1971, Kerr 1971, Mann 1969, Winberg 1956, Wohlschlag and Wakeman 1978).• Stresses also can depress routine metabolic rates (Beamish 1964, Wohlschlag and Cameron 1967, Kloth and Wohlschlag 1972, Cech and • Wohlschlag 1975), although a depressed routine rate appears to • be less definitive than scope for maximum sustained activity for species that may have a maximum swimming metabolic activity level 4-8 times standard levels (Randall 1970). • For this study the red snapper (Lutjanus campechanus) was chosen as a well known commercial and recreational species from both offshore and inshore waters. It is a relatively easy species • to maintain in the laboratory. The specific aims were to use the red snapper as a test organism: • 1. To identify metabolic effects at a very low sublethal toxicant level; 2. To utilize the metabolic results at active and standard • levels for detection of scope depression even though the chemical composition of the toxicants could be considered unknown; 3. For suggesting a possible biological monitoring system • that could operate with or without a detailed chemical knowledge of a toxicant or mixed toxicants; and 4. For retention of energetics data on a given species of • general importance in fishery and ecological considerations . • • • • 87 Methods • • Throughout the red snapper, Lutjanus campechanus was the fish of choice. Hook-and-line fishing at offshore "snapper banks" in 80-90 meters of water about 60 km offshore at Port • Aransas provided specimens for 20C experiments, while fish taken from shallower waters near the local Aransas Pass jetties and nearshore artificial reefs provided specimens for the 28C • experiments. At both locations throughout the year natural salinities were very near 35 ppt. Fish were held in live boxes with flowing seawater on board research vessels and on shore in covered outdoor or indoor tanks with flowing seawater. Frequency of feeding was sufficient to • promote growth. Before experiments fish were held in temperature controlled, filtered water tanks at 35 ppt and 20C or 28C for at least 48 hrs. Fish were fasted for at least 24 hrs before • respiration measurements . The pollutant used was a biosolid from a Shell corporation waste that consisted of both solid and liquid fractions with • generally known components (Anderson 1974, Siegel and Rader 1974) . Since the TLM96 was known for the liquid phase to be 20.4% (volume), a dilution to 1% (or a volume concentration of 0.2%), • was utilized throughout for comparison with controls. For experiments at 20 C, the sludge and liquid components were separated on a 0.2% v/v basis; at 28C only the liquid phase was • used. The whole sludge was suspended in a loosely woven gauze bag, while the liquid was uniformly dispersed. • Preliminary experiments indicated that 0.1% v/v dilutions would also • produce a measurable reduction in metabolic scope measurements . In each of the experiments in polluted waters, the fish were held 48 hrs under well oxygenated conditions before metabolic measure­• ments ensued. Oxygen consumption rates were measured by withdrawal of small samples for use in a Radiometer model E-5046 with a PHM 71 with • electrode equipped acid-base analyzer. Following completion of a set of experimental oxygen consumption measurements, the fish were removed and lengths and weights recorded . • Resting rates were determined by using 4 13.8 cm ID (15.2 cm OD) diameter by 61 cm long acrylic tube flow-through chambers immersed in a 450 1 insulated, temperature controlled aquarium• equipped with a filtration system. Opaque plastic shields between the chambers and black curtains around the entire mechanism prevented visually induced excitement. Measurements • of 02 at intakes and at outlets with flow rates were made over the course of 1 or 2 days to determine minimal resting metabolism rates in well oxygenated waters . • Active metabolism rates were made in a 207 1 Blazka chamber (Blazka, et al. 1960, Fry 1971) as utilized by Wohlschlag and Wakeman {1978). The entire chamber was immersed in a 3,678 1• temperature-salinity controlled system, which was a contiguous part of the circular holding tank, filtration and cooling systems. Fish were maintained for one day swimming at low velocities {about • 1 L sec-1) prior to active measurements. After swimming in the chamber at an intermediate speed for 1 hr, the velocity was • • 89 increased gradually until the fish "broke" pace. At this • instant the velocity was lowered (usually quite slightly) to the highest possible velocity at which normal swimming persisted without breaking. With this "training" regimen, the maximum • sustained swimming velocity could be reproducible for each fish . The Umax (/ Total Lengths sec-1) swimming velocity was determined at least twice to ascertain consistency, after which the fish • was tested for at least 1 hr for a consistent Umax· Following the 1 hr or longer runs, the fish were left in the chamber at intermediate or zero velocities with oxygen rate measurements • to detect any irregularities that could have resulted had the Umax been associated with any undesirable anaerobic metabolism. Along with lengths, weights, oxygen consumption rates, and • swimming rates in (total lengths)-~ sec-1, salinities and temperatures were recorded to 0.1 C and 0.1 ppm for a simple multiple regression at each control or experimental condition • in the form: where: • Y= expected 02 consumption rate in log mg02hr-1 , a = constant, • Xw = weight in grams, = oc, xt Xs = salinity in ppt, and Xv = rL sec-1. • The various b values are the respective partial regression coefficients. • • 90 The respective multiple regressions are used in this study • simply to indicate how salinities and temperatures can be reduced to 35 ppt and 20 C or 28 C, respectively, although the temperature and salinity controls remained near these values. The principal • use of the regressions as simplified in Tables 1 and 2 is to reduce data to given "averages" of weights and swimming velocities. Similar procedures have been used by Wohlschlag and Juliano (1959), • Wohlschlag and Cameron (1967), Wohlschlag and Cech (1970), and others. Regression calculation techniques are in most statistical manuals,~-~·, Snedecor and Cochran (1967) or in various pretested • library computer routines . • • • • • • Results • • Original data for the resting fish in the flow-through chambers and for the swimming fish in the Blazka chambers are in Appendix Tables 1 and 2. The multiple regression equations and ·• associated statistics are in Appendix Tables 3 and 4 respectively. By use of these equations, the "adjusted" data for temperature, salinity, weight at 250g and averages of maximum swimming • velocities are in Appendix Tables 5 and 6. The Table 1 equations are based on the adjusted averages for temperatures of 20°c or 2a0 c and salinity of 35 ppt for the • flow-through chamber resting metabolism and another additional "average" weight of 250 g for the swinuning fish in the Blazka chamber. The actual averages and ranges of the weights, temperatures • and salinities are in Table 2 for the flow-through chambers, and in Table 3 for these variables and swimming velocities in the Blazka chamber • • In order to ascertain a minimum resting rate that would be a realistic estimate of the standard rate by the Brett (1964) technique, the Appendix Figs. 1 -5 plots (based on Table l Equations 1 -5) were utilized. Similarly, for the maximum IL sec -l measurements that would be realistic, the Appendix Figs. • 6 -10 plots (based on Table 1 Equations 6 -10) were utilized• In Table 4 are equations la -Sa for the lowest estimates of the standard rates based on flow-through chamber data under • various conditions; the table also includes equations 6a -lOa for highest estimates of active fishes in the Blazka chamber under comparable conditions• • Table 1. Regression equations for L. campechanus respiration, derived from multiple regression equations and adjusted to appropriate temperatures and a salinity of 35 ppt. EquationFlow Through Chamber Equations Number Control Water: 20C, N = 21 y = -0.0347'+ 0.5602~ (1) 28C, N = 12 Y = -1.2381 + l.1973Xw (2) Treated Water: 20C, N = 08* Y = -0.7828 + 0.8965Xw (3) 20C, N = 08** y = 0.2398 + 0.6530Xw (4) 28C, N = 08** Y = -0.5214 + 0.94?3Xw (5) Blazka Chamber Equations at 250g Control Water: 20C, N = 37 · y = 1.2093 + 0.0455Xv (6) 28C, N = 29 y = 1.8993 + O.OlllXv (7) Treated Water: 20C, N = 10* y = 1.1708 + 0.0508Xv (8) 20C, N = 27** y = 1.5203 + 0.0285Xv (9) 28C, N = 23** y = 1.9243 + 0.0094Xv (10) * = Sludge Phase ** = Liquid "° Phase tv Table 2. Average values and ranges of variables used in regression equations for restingmetabolism in flow-through chambers. Condition Weight (grams) Temperature (0c) Salinity (o/oo) Average Range Average Range Average Range Control 21 232. 2 125.0 -676.0 20.0 19.9 -20.0 35.4 35.0 -35.9 12 208.0 -28.0 28.0 35.2 132.0 370.0 34.9-35.7 Treated (Sludge) 08 233.3 151.0 -290.0 20.0 20.0 35.4 35.1 -35.6 (Liquid) 08 211.0 uo.o -674.0 19.9 19.8 -20.0 35.1 34.7-35.4 (Liquid) 08 209.6 125.0 -349.0 28.0 28.0 -28.l 35.3 35.l -35.4 \0 w Table 3. Average values and ranges of variables used in regression equations for active metabolism in the Blazka respirometer. Condition N Weight (Grams) Temperature c0 c) Salinity (o/oo) Velocity Average Range Average Range Average Ranye Average Range Range (/'L sec-1 ) (IL sec-1) (L sec-1) Control 37 211.8 128. 0-676. 0 20.0 u.1~20.5 35.1 l4. 8-35. 5 14. 22 OJ.48-20.40 0.7...4.5 29 254.2 128.0-690.0 28.0 27.1-28.5 35.0 34.5-35.5 22.37 00.00-28.88 o.o-5.8 Treated {Sludge) 10 239.2 151.9-290.0 20.0 20.0-20.1 35.l 35.0-35.5 17~44 15.94-18.92 3.2-4.2 (Liquid) 21 · 368.3 129.0-787~0 20.0 19.8-20.2 35.0 34.4-36.0 12.50 00.00-19.33 o.o-4.l (Liquid) 23 229.6 123.0-661.0 27~8 27~0-28.4 35.1 35.0-36.0 20.69 00.00-29.44 o.o-s.9 \0 ~ • • • • Ta.ble . 4. Raqression equations for L. CaJm:)echanus respiration 11ainq onlythe l.owest meta.belie: ratei for how-throuqh chamber r·esults &nd only t!l• Qiqhast meta.belie: rates for Blazka chamber resul~ for a 250 q. fish adjusted to the appropriate temperature and a sa.lin.ity ot 35 ppt. (See Appendix Fiqs. l tc lO.) Equauon !!!Lw-'rhrouc;h Chamber Equations Number • Control Watu: 20C y. -0.095 + O.S602X._, (la) 28C y. -l.290 + l.l973X,,, (2a) • Treatm Water: 20C'* y • -0.830 + 0.896Sx._, (3a) 20C'** y • 0.200 + 0.6530Xw (4a) 2SC'*' y. -0.610 ~ 0.94SJx._, (Sa) • Slazka Chamber Results Control water: 20C y • l.340 + 0.0455Xv (Sa) 28C y • .l.990 + O.Olll~ (7a) Treated Water: 2oc• y • l.205 + o.osoa~ (8&) 20C'*'* y • l.600 + 0.028SXv (9a) 28C'--* y • l.970 + 0.0094= • E 200 • • CONTROL SLUDGE LIQUID TREATMENT • • • • • • • • • • • ' • • • • • • 1 • • • • • • • • • • Fig. 2. Metabolic rates for a 250 gram ~· campechanus tested • in control and treated water at 28C. Solid line represents the metabolic rate at Umax (an average U of 21.56) and the dashed line represents • max standard metabolic rates at each condition. The lightly stippled area indicates the "scope for maximum activity" for each treatment. • • • • • • • • 1000 RED SNAPPER -28° C • 800 • ~ 1L 600 • ~ • 200 • CONTROL L IQUI D • TREATMENT • • • transfer the cumulative toxins • • As noted in Wohlschlag and Wakeman (1978) the maximum swimming rates also parallel the metabolic rates at maximum sustained swimming rates. This observation suggests that • healthy, swimming fish, could be used to assess water quality directly without metabolic measurements. However, the swimming rates must be adjusted for the length of the fish (Webb 1975), • as for the U values in this study, to avoid high swimming max rates for smaller fish. Thus, with a few simple modifications and precautions, a • system of monitoring effluent effects on fish is suggested. All that would be required would be a Blazka system so that an mixture appropriate of effluent and dilution water could be adjusted to the lowest or highest desired swimming performance. For comparisons with clean waters, several seasonal sets of standard and active rate (scope) data would be required over • seasonally appropriate temperatures, salinities or other conditions. Considering the fact that toxicity of an effluent can be evaluated before the biochemistry of the effluent is • known, the system of using the scope as a measure of stress has considerable additional merit. The utilization of respiratory measurements can also have • great promise in fishery and general ecosystem studies based on energy measurements. Energy appears to be a common denominator for evaluating environmental optima and stresses in the sense • of Cody (1974), in determining population growth--foraging relationships in the sense of Kerr (1971), or in determining • • metabolic or respiration effects that are highly sensitive for • heterotrophs in ecosystem models in the sense of O'Neill (1976) • Energetics of systems with various diversity-stability relationships among major, highly iteroparous organisms also • need to be studied in order to explain species dominance and community stability (Simenstad, et al. 1978). The various open ocean fishes such as the red snapper, are relatively long-lived • and the status of their biomass and age structure would be changed appreciably by slight, chronic changes in mortality. Such changes are now suspected of having capabilities to induce • dramatic effects on the stability of man and other ecosystem components (Simenstad, et al. 1978) • • • • • • • • Literature Cited • • Anderson, J. w. 1974. Biological effects of spent caustic and biosolid wastes. Texas A&M University, processed. Beamish, F. w. H. 1964. Respiration of fishes with special • emphasis on standard oxygen consumption. Can. J. Zool. 42:177-188. Blazka, P., M. Volf, and M. Cepela. 1960. A new type of • respirometer for the determination of metabolism of fish in the active state. Physiol. Bohemoslov 9:553-558. Brett, J. R. 1958. Implications and assessments of environmental • stress. p. 69-83. In: P.A. Larkin (ed.), The investigation of fish-power problems. The H.R. MacMillan lectures in fisheries. Univ. British Columbia, Vancouver . • Brett, J. R. 1964. The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Bd. Can. 21:1183-1226 • • Brett, J. R. 1965. The relation of size to rate of oxygen consumption and sustained swimming speed of sockeye salmon (Oncorhyncus nerka). J. Fish. Res. Bd. Can. 22:1491-1501 . Brett, J. R. 1971. Energetic responses of salmon to temperature. A study of some thermal relations in the physiology and • freshwater ecology of sockeye salmon (Oncorhynchus nerka) . Am. Zoologist 11:99-113. Brett, J. R. and N. R. Glass. 1973. Metabolic rates and critical • swimming speeds of sockeye salmon (Oncorhyncus nerka) in relation to size and temperature. J. Fish. Res. Bd. Can. 30:379-387 . • Brett, J. R., J. E. Shelbourn, and C. T. Shoop. 1969. Growth • rate and body composition of fingerling sockeye salmon, Oncorhynchus nerka, in relation to temperature and ration size. J. Fish. Res. Bd. Can. 26:2363-2394 . • Cech, J. J., Jr. and D. E. Wohlschlag. 1975. Summer growth depression in the striped mullet, Mugil cephalus L. Contr. mar. Sci. 19:91-100 • • Cody, M. L. 1974. Optimization in ecology. Science 183:1156-1164. Fry, F. E. J. 1947. Effects of the environment on animal activity. Univ. Toronto Studies Biol., Ontario Fish. Res. Lab., 68:1-62 . • Fry, F. E. J. 1957. The aquatic respiration of fish. pp. 1-63. In: M.E. Brown (ed.) The Physiology of Fishes. Academic Press, New York . • Fry, F. E. J. 1971. The effect of environmental factors on the physiology of fish. p. 1-98. In: W.S. Hoar and D.J. Randall (eds.) Fish Physiology, Vol. 6, Environmental Relations and• Behavior. Academic Press, New York and London. Kerr, S. R~ 1971. A simulation model of lake trout growth. J. Fish. Res. Bd. Can. 28:815-819 . • Kloth, T. C. and D. E. Wohlschlag. 1972. Size-related metabolic responses of the pinfish, Lagodon rhomboides to salinity variations and sublethal pollution. Contr. mar. Sci. 16: • 125-137. Mann, K. H. 1969. The dynamics of aquatic ecosystems. Advan. Ecol. Res. 6:1-81 • • O'Neill, R. V. 1976. Ecosystem persistence and heterotrophic regulation. Ecol. 57:1244-1253 • • Randall, D. J. 1970. Gas exchange in fish, p. 253-292. In: W.S . • Hoar and D.J. Randall (eds.). Fish Physiology, Vol. 4, Academic Press, New York. Siegel, H., and w. E. Rader. 1974. An analysis of biosolid waste • from the Houston chemical plant biotreater. Tech. Prog. Rept. BRC-CORP 42-74-F. Shell Dev. Co., Houston. Simenstad, C. A., J. A. Estes and K. w. Kenyon. 1978. Aleuts,• sea otters and alternate stable-state communities. Science 200:403-411. Snedecor, G. w. and W. G. Cochran. 1967. Statistical methods . • Iowa State Univ. Press. Ames. Webb, J. W. 1975. Hydrodynamics and energetics of fish propulsion. Bull. Fish. Res. Bd. Can. 190:X + 158pp• • Winberg, G. G. 1956. Rate of metabolism and food requirements of fishes. Fisheries Research Board of Canada Translation Series 194:1-253• • Wohlschlag, D. E. and J. J. Cech. 1970. Size of pinfish in relation to thermal stress response. Contr. mar. Sci. 15:22-31. • • Wohlschlag, D. E. and J. N. Cameron. 1967. Assessment of a low level stress on the respiratory metabolism of the pinfish (Lagodon rhornboides). Contr. mar. Sci. 12:160-171 . Wohlschlag, D. E. and R. O. Juliano. 1959. Seasonal changes in bluegill metabolism. Lirnnol. Oceanogr. 4:195-209. • Wohlschlag, D. E. and J. M. Wakeman. 1978. Salinity stresses, metabolic responses and distribution of the coastal spotted seatrout, Cynoscion nebulosus. Contr. mar. Sci. 22:In press . • • • • APPENDIX Tables and Figures • • • • • • • • • 111 • • • • Appendix Table l. Raw data used in the calculation of • regression equations for fish tested ill flow-through c:ham.bers (Appendix J) • Fish No. • fish identification number log. wt. • log10 weight in grams • Temp. (OC) • temperature in degrees centigrade Sal. (o/oo) • salinity expressed in parts per thousand log. mq02hr-l • respiration, loq10 mqo2hr-l • • • • • • • • I• I • • • • • • Fish No. log. wt. TemE· (OC) Sal. (o/oo) los:. ms:o.,hr-1 Control (20C) 001 2.40654 20.0 35. 0 l.36059 002 2.29885 20.0 35.0 l.30856 004 2.35984 20.0 35 .,o l.32449 005 2. ll727 20.0 35.0 l.10072 006 2.36922 20.0 35.0 l.28217 007 2.54654 20.0 35.0 l.37535 008 2.18752 20.0 35.0 1.17522 010 2.82995 20.0 35.S l.50974 Oll 2.28103 20.0 35.S l.19562 012 2.39445 20.0 35.5 1.31154 013 2.11727 20.0 35.9 l.14145 014 2.37291 20.0 35.9 l.28338 015 2.09691 20.0 35.9 l.08350 016 2.32428 20.0 35.9 l.30492 018 2.17898 20.0 35.l l.19535 039 2.48714 19.9 35.6 l.21748 040 2.36922 19.9 35.6 l. l 7260 041 2.48714 19.9 35.6 l.24969 042 2.57287 19.9 35.6 l. 29181 043 2.42488 20.0 35.0 l.35851 045 2.45939 20.0 35.0 l.27554 • • • • • • I • • • • • 113 !i!h No • loq wt. Temo. c0 c) Sal. (o/oo) loq. :nq02hr-l Control (28C) 046 2.13672 28.0 35.0 l.28400 047 2.36549 28.0 35.0 l.55255 048 2. 20140 28.0 35.0 l.34811 049 2.29003 28.0 35.l l.54295 050 2.46389 28.0 35.l l.68851 051 2.12057 28.0 35.l l.36568 053 2.36361 28.0 34.9 l.62603 054 2.29447 28.0 34.9 l.52943 ass 2.56820 28.0 35.5 l.81578 056 2.24055 28.0 35.S l.37199 057 2.4l500 28.0 35.5 l.60788 058 2.35603 28.0 35.7 l.53567 Treated, Sluds;e (20C) 027 2.17898 20.0 35.6 l.22453 028 2.38739 20.0 35.6 l.33985 023 2.42488 20.0 35.l l.38364 024 2.39445 20.0 35.l l.34908 025 2.25042 20.0 35.l l.23477 026 2.45332 20.0 35.l l.46494 019 2.39094 20.0 35.6 l.44716 021 2.46240 20.0 35.6 l·. 46165 Treated, Li~id (20C) 037 2.~6140 20.0 35.3 l.27623 038 2.26482 20.0 35.3 l.28533 • • • • I• • • • • • • 114 Fish No. los: wt. Temc • (OC) Sal. (o/oo) los:. mq02hr-l Treated, Li~id (20C) Cont. 033 2.26717 20.0 35.4 1.13925 034 2.11394 20.0 35.4 l.08422 035 2.39445 20.0 35.4 l.25042 036 2.14922 20.0 35.4 l.09026 030 2.82867 19.8 34.7 1.55206 031 2.38202 19.8 34.7 l.26055 Treated, Li~id (28C) 059 2.23045 28.0 35.3 l.40019 060 2.35411 28.0 35.3 . l. 46613 061 2.36549 28.0 35.3 l.61542 062 2.25042 28.0 35.l l.49248 063 2.36173 28.0 35.l l.70817 064 2.54283 28. l 35.4 l.79029 065 2.09691 28.l 35.4 l.41280 066 2.36922 28.l 35.4 l.68789 • 115 • • • • Appendix Ta.l:>le 2. Raw data used in the calculation of regression equations for fish tested in the Slazka chamber (Appendix 3) . Fish No. • fish identification number loq. wt. • loq10 weight in grams • Temp. (OC) • temperature in degress centigrade Sal. (o/oo) • salinity in parts per thousand V ({I; sec-1) • ~elocity in square root of lengths per second • log. mc;o2hr-l • respiration, log10 mg02hr-l *by a respiration measurement indicates that the reading was made at an actual Omax for that fish• • • • • • • • le • • • • • • 116 Fish No. lo9:· wt. Temc. (OC) Sal. (o/oo) V (.rt sec-l) loa. ms:o.,hr-1 Control (20C) I umax ave;. a 17.70 119 2.10121 20.0 34.9 15.20* l. 7l450 120 2.12057 19.7 35.0 16.50* l.79064 121 2.12057 19.7 35.0 17.80 l.84942 122 2.12057 19.7 35.0 14.20 l.64365 123 2. l4613 20.0 35.0 14.SO l.60767 124 2.14613 20.0 JS.a 20.40* l.83651 125 2.18752 20.0 35.0 15.60 l.72558 126 2.18752 20.0 JS.O 17.90* l.79900 127 2.18752 20.0 35.0 17.00* l.79518 128 2.19312 20.0 35.0 13.90 l.79211 129 2.19312 20.0 35.0 19.50 l.89642 lJl 2.27416 20.5 35.0 19 .10* l.97230 132 2.. 31806 19. 9 35.0 18.58* l.88835 133 2.31806 19.9 35.0 14.29 l.7SS57 134 2.32015 20.0 JS.O 14.54 l.67825 135 2. 31806 20.l 35.5 OS.24 l.56820 136 2.31806 20.l 3S.5 04.29 l.54220 137 2.32015 20.0 35.0 19.88 l.97179 138 2.35984 20.0 34.8 16.97 l.97722 139 2.8299S 20.2 3S.S 15.15 2.37157 l40 . 2.55388 20.0 35.0 lS.87 l.98981 141 2.55388 20.0 35.0 19.05* 2.14919 l42 2.42651 20.0 35.0 17.60* l.97722 • 117 • • Fish No. log:. wt. Tem~. (oC) Sal. (o/oo) v (/L sec-1) loq. ms:02hr-l Control (20C), Oma.v avg:.• 17.70 (Cont.) 143 2.40l40 19.9 35.0 14. 73 :2. 01941 • 144 2.40140 19.9 35.0 15. 71* 2.05507 146 2.38917 20.0 35.0 13.63 l.76253 147 2.38917 20.0 35.0 17.67* 2.04653 148 2.38917 20.0 35.0 15. 22 1.97987 • • 149 2.38917 20.0 35.0 18.16* 2.07780 150 2.37291 20.0 35.0 14.lS l. 87967 151 2.37291 20.0 35.0 l8. OS* 2.00359 152 2.55388 20.0 35.0 03.70 l.46687 153 2.42651 20.2 35.5 04.02 l.44420 154 2.37291 20.0 35.3 03.90 l.32675 155 2.36922 20.0 35.0 03.48 l.34064 157 2.35984 20.l 35.0 03.99 1.26387 160 2.25285 20.4 35.5 16.85* l.87927 Control ( 28C) , !!max a.:!9;• a 26.06• 059 2.83885 27.3 35.0 00.00 2.25310 • 060 2.83885 28.0 34.9 16.97 2.49799 061 2.83885 28.0 34.9 23.76* 2.47780 063 2.34635 27.8 35.0 19.03 2.18041 064 2.34635 27.8 35.0 23.42* 2.20243 065 2.34635 27.l 35.0 23.42* 2.17569 066 2.57403 27.9 • 34.S 17.37 2.23355 067 2.57403 28.0 34.5 27.37* 2.38819 068 2.57403 28.2 34.5 27.37* 2. 34143 069 2.41497 28.0 34.5 23.18 2.17863 • • • • Fish No. los;. wt. Temo. (OC) Sal. (o/oo) V (/"L sec-l) loa. mqo2hr-l Control ( 28C) I aVS[ • :a 2 7 . 0 6 (Cont.) • • Omax 070 2.35793 27.9 35.0 17.82 2.01611 07l 2.35793 28.0 35.0 27. 72 * 2.15247 072 2.35793 28.l 35.0 20.29 2.17455 073 2.35793 28.l 35.0 28.21* 2.18233 074 2.64738 28.2 35.0 00.00 2.06066 075 2.10721 28.0 35.0 25.08* 2.06288 076 2.39967 28.0 35.0 26.00 2.17534 I 077 2.39967 28.0 35.0 27.00* 2.24667 078 2.27416 27.5 35.0 18.02 2.07203 • • 079 2.27416 27.5 35.0 27.99* 2.09764 081 2.30535 28.4 35.S 25. 63 * 2.04999 082 2.15229 28.2 35.S 26.58* l.99021 • 083 2.46538 28.l 35.0 21.34 2 .13111 084 2.46538 28.l 35.0 25.Sl* 2.21519 085 2.31175 27.S 35.0 23.03 2.16699 088 2.20140 28.2 35.S 23. 72 l.87967 089 2.13672 28.4 3S.O 25.86 l.89470 090 2.13672 28.4 35.0 28.21* l.95554 091 2.35025 28.5 35.0 28.88* 2.10483 Treated Sludge ( 20C) I Omay avg. -17.28 ~ • 161 2.18169 20.0 35.5 18.92* l.8l023 162 2.26007 20.0 35.0 l6. 09* l.87558 163 2.37291 20.0 35.0 lS. 78* 2.11069 164 2.38202 20.0 35.0 16.90* 2.00557 166 2.38561 20.0 35.0 17. 04 * l.98682 • • • • Fish No. lo~:-wt. Temo. (OC) • Treated Sluds:e (20C) I 167 2. 40312 • 168 2.43775 170 2.44716 171 2.45484 172 2.46240 Treated Liguid (20C), 185 • ,. • 187 199 190 191 192 194 196 197 199 • 200 201 202 203 204 206 • 207 208 • • 2.48144 2. 51188 2.51188 2.51055 2.74974 2.74974 2.89265 2.89265 2. 57287 2.57287 2.572_87 2.89597 2.89597 2.89597 2.89597 2.76268 2.76268 2.76268 Omax av9:· 20.l 20.0 20.0 20.0 20.0 2mu avg:. 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.l 20.l 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 Sal. (o/oo) V • 17.28 (Cont.) 35.0 35.S 35.0 35.0 35.0 • 17.05 35.0 35.0 35.0 35.0 35.0 j·5. o 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 ( rL sec-1) lo5l· m5102hr-l 15.94* 1.98191 17. 50 * l.97433 18.87 2.20238 17.89* 2.09423 16.43* 2.09947 00.00 l. 50325 10.14 l.80828 14. 4l l.95525 00.00 l.63124 14.06* 2.26060 00.00 l.82556 10.63 2.22300 18.90* 2.40157 00.00 l.59627 13. 68 l.95751 17.89 2.11032 00.00 l .• 99782 10.78 2.19524 13.18 2.31481 14.98 2.34573 ll. 88 2.04739 14. 7l 2.23967 l.9.23 2.41576 • 120 • Fish No. log:. wt. Tem:;:!. (OC) Sal. (o/oo) V (/L sec-l) log:. mg:O:zhr-1 Treated Liguid ( 20C) , !!max avg:. • l7.05 • l73 2. ll059 20.l 34.9 11. 36 l.56573 • l74 2.11059 20.l 34.9 16.61* l.70200 175 2.15836 20.0 35.0 19.33* l.89120 177 2.25285 20.2 36.0 17.12'* l.95463 179 2. 27875· 20.0 35.0 17.78* l.94758 • lSO 2.31597 19.8 35.0 18.0l* l.98046 181 2.37840 20.0 35.0 12.55 l.89878 182 2.37840 20.0 35.0 18.34* 2.04052 183 2.38739 19.S 34.S 16.20'* 2.07361 Treated Liguid ( 28C) , !:!ma~ avg:. • 26.lO 092 2.37107 2.8.2 35.0 • 24.34 2.13599 093 2.36549 27.8 35.0 29.44* 2.15637 094 2.23805 27.8 • 35.0 20.16 l.96303 095 2.36736 28.0 35. 0 24.78 2.11621 096 2.24551 27.0 35.0 23.61 l.96123 097 2.24551 27.0 35.0 27.39 l.98218 098 2.24551 27.0 35.0 25.97• 2.01828 100 2.38021 28.2 35.0 27.83* 2.15978 101 2.38021 28.2 35.0 • 25.30 2.19145 102 2.82020 28.l 35.0 oo.oo 2.30638 103 2.82020 ' 27.8 35~0 lS. 67* 2.48636 104 2.82020 27.8 35.0 • 00.00 2.26867 106 2.56110 28.2 35.0 25.40'* 2.29108 107 2.08991 28.4 35.0 22.20 l.85352 108 2.08991 28.4 35.0 27.40* l.87806 • • •• 121 41 Fish No. l~. wt. Terni?. (oC) Sal. (o~oo) v Cw + l.09653Xt -O.Ol928X8 (le) 28C, Na 12 : Y = -2.48221 + l.19729Xw + 0.14117Xt -0.07739X8 (2c) Treated Waters •2oc, N ... 08: Y = -J.11759 + o.e9645Xw + o.oo·oooxt + o.06671X8 (3c) **20C, N • 08 : Y c -46.86570 + 0.65339Xw + 4.77874Xt -l.38491X9 (4c) **28C, N • 08 : Y = -29.44410 + 0.94532Xw + l.76070Xt -0.58220X8 (Sc) Blazka Chamber Eguations Control Waters 20C, N = 37 : Y = -6.81392 + 0.81048>Cw -0.12605Xt + 0.24548X8 + 0.04562Xv (6c) 28C, N -29 I y = 5.43911 + 0.73691Xw -0.09023Xt -0.07301X8 + O. 01109Xv (7c) Treated Water: *20C, N • 10 : Y = 8.23674 + 0.91953Xw -0.03468Xt -0.24507Xs + 0.05080Xv (Oc) **20C, N • 27 : Y = 4.46818 + 0.76345Xw -0.30058Xt + 0.03531Xs + 0.02822Xv (9c) **28C, N = 23 a Y = -1.21438 + 0.89113Xw + 0.02223Xt + 0.0108JX9 + 0.00942Xy (lOc) * = Sludge Phase1 ** = Liquid Phase. t-' I\..) I\..) Appendix Table 4a. Regression statistics for L. campechanus respiration tests in flow-through system. Original equations (Appendix Table 3) with all variables included. Equation N Multiple Std. Error Standard Errors of Regression Number Correlation of Coefficient (sb) and Probability (P) Coefficient Estimate p p p R By BbW Bbt sbs (l~) 21 0.94 0.0373 0.0509 0.005 0.2336 0.005 0.0243 n.s. (2c) 12 0.97 0.0442 0.1098 0.005 o. 0041 n.s. 0. 0517 n.s. (Jc) 08 0.93 0.0403 0.1532 0.005 0.0000 n.s. 0.0576 n.s. (4c) 08 0.99 0.0194 0.0426 0.005 0.5657 0.005 0.1676 0.005 (5c) 08 0.94 0.0670 0.1946 0.005 0. 7367 0.025 0.3059 0.05 • 1--' tv w L. campechanus respiration tests in the Blazka Appendix Table 4b. Regression statistics for system. Original equations (Appendix Table 3) with all variables included. Equation N Multiple Std. Error Standard Errors of Regression Number Correlation of Coefficient (sb) and Probability (P) Coefficient Estimate p p p p R Sy sbw sbt sbs Bbv (6c) 31 0.96 0.0757 0.0902 0.005 0.0974 n.s. 0.0889 0.005 0.0028 0.005 (7c) 29 0.94 0.0535 0.0692 0.005 o. 0328 0.005 0. 0473 n.s. 0.0018 0.005 ' (Be) 10 0.98 0.0123 0.1293 0 • .005 0.3839 n.s. 0.0591 0.005 0.0119 0.005 (9c) 27 0.97 0.0632 0.0471 0.005 0.1903 n.s. 0.0607 n.s. 0.0020 0.005 (10c) 23 0.99 0.0287 0.0354 0.005 0. 0132 n.s. 0.0189 n.s. 0.0007 0.005 • ....., tv ~ • • • • Appendix Table 5. Fish identification number, weiqht and respiration for Lutjanus camcechanus tested in flow-through chambers. Adjusted rates. • Fish No. • fish identification number Log.·.Wt. • log10 weight in grams loq mqo2hr-l • respiration, loq10 mqo2hr-l • • • • • 126 • • Fish No. Loa. Wt • Log:. ms:02hr-l Fish No. Log: Wt. Loa. mg:o2hr-l • Control (20C) 001 2.40654 l.36059 014 2.37291 l.30065 002 2.29885 l.30856 015 2.09691 l.10085 004 2.35984 l. 32449 016 2.32428 l.32227 • 005 2. ll727 l.10072 018 2.17898 l.19723 006 2.36922 l.28217 039 2.48714 l.33870 007 2.54654 l.37585 040 2.36922 l.29382 008 2.18752 l.17522 041 2. 48714 l.37091 • 010 2.82995 l.51938 042 2.57287 l.41303 Oll 2.28103 l.20526 043 2.42488 1.35851 012 2.39445 l. 32118 0.45 2.45939 l.27554 013 2 •. ll727 1.15880 • Control ( 28C) 046 2.13672 l.28400 052 2.36361 l.61829 047 2.36549 l.55255 054 2.29447 l.52169 048 2.20140 l.34811 055 2.56820 l.85448 • 049 2.29003 1.55069 056 2.24055 1.41069 050 2.46389 l.69625 057 2.41500 1.64658 051 2.12057 l.37342 058 2.35603 1.58984 Treated, Sludg:e {20C) 027 2.17898 l.18450 025 2.25042 l. 22810 028 2.38739 l.29982 026 2.45332 l.45827 • 023 2.42488 l.37697 019 2.39094 l.40713 024 2.39445 l.34241 021 2.46240 1.42162 • • • • Fish No. LoS:. Wt • Los;. ms;02hr-l Fish No. Los;. Wt. Los;. ma02hr-l • Treated, Li~id (20C} 037 2.20140 1.69170 035 2.39445 l.80438 038 2.26482 l.70080 036 2.14922 l.64422 033 2.26717 l.69321 030 2.82867 2.09234• 034 2.11394 l.63818 031 2.38202 l.80083 • Treated, Liauid (28C) 059 2.23045 l.57485 063 2.36173 l.76639 060 2. 35411 1.64079 064 2.54283 l. 84710 061 2.36549 1.79008 065 2.09691 l.46961 062 2.25042 l.55070 066 2.369.22 l. 74470 • • • • • • • • • • Appendix Table 6. Fish identification number, weight, swimming velocity, and respiration for Lutjanus campechanus tested in the Blazka chamber. Adjusted rates• • Fish No. • fish identification number Log. Wt. =log10 weight in grams • V (/!; sec-l) • swimming velocity expres~ed as the square root of total length per second• (adj. 250 g} • respiration, loglO mq02hr-l, Log. mg02hr-l adjusted to the average weiqht (250 grams) of fish tested. (adj. tJ.max) • • respiration, log10mg02hr-l,Log. mg02hr-l adjusted to the average Omax for each experimental condition. Omax for each condition listed with heading for that group of experiments• • • • • • • (adj. 250 g) (adj. °'max) Fish No. Lo~ Wt• V Fish No. LoS:· Wt• V {l'°L sec-l) Los:. mao,hr-1 Los:. mao,hr-l • 144 2.40140 15.71 2.03966 2.13324 146 2.38917 13.63 2.02280 2.08760 l47 2.38917 17.67 2.05364 2.04790 148 2.38917 15.22 l.98698 • 149 2.38917 18.16 2.08491 2.05681 l50 2.37291 14.15 l.89996 151 2.37291 lS.05 2.02388 l.98762 152 2.55388 03.70 l.34048• 153 2.42651 04.02 l.32351 154 2.37291 03.90 l.27340 155 2.36922 03.48 l.36392 157 2.35984 03.99 l.30736 • 160 2.25285 16.85 l .·9 2454 l.84573 Control (2SC) , 26.06 2max • 059 2.83885 00.00 l.85943 060 2.83885 16.97 2.16578• 061 2.83885 23.76 2.14559 2.49601 • 063 2.34635 19.03 2.19878 064 2.34635 23. 42 2.22080 2.21206 065 2.34635 23.42 2.12530 2.11656 066 2.57403 17.37 2.05746 • 067 2.57403 27.37 2. 22192 2.33715 068 2.57403 27.37 2.19481 2.29039 069 2.41497 23.18 2.12957 2.17406 070 2.35793 17.82 2.03577 071 2.35793 27. 72 2.18195 2.13406 • • • • Fish No• • 072 073 074 075 ~ 076 077 078 • 079 081 082 083 • 084 085 088 089 • 090 091 Loa. Wt. 2.35793 2.35793 2.64738 2.10721 2.39967 2.39967 2.27416 2.27416 2.30535 2.l5229 2.46538 2.46538 2.31175 2. 20140 2.13672 2.13672 2.35025 V (IL sec-1) 20.29 28.21 00.00 25.08 26.00 27.00 18.02 27.99 25.63 26.58 21. 34 25.51 23.03 23. 72 25.86 28.21 28.98 • Treated Sludg:e ( 20C) I C'ma x • l 7 • 2 8 161 2.18169 18.92 162 2.26007 16.09 • 163 2.37291 18.78 164 2.38202 16.90 166 2.38561 17.04 167 2.40312 15.94 168 2. 4377?' 17.SO 170 2.44716 18.87 • • (adj. 250 g)Los;. mc:ro2hr-l (adj. Omax) Log. mgo.,hr-l 2.21385 2.22163 2.16831 l.89650 2.27712 2.07375 2.17407 2.24540 2.23625 2. ll412 2.13973 2.02712 2.19402 2.13056 2.22739 2.04060 2.09123 2.17531 2. 23111 2.18138 2.08066 l.96178 2.12649 2.18733 l. 97099 2.18909 2.12268 2.13162 l. 84946 2.00236 l.93603 2.13371 2.03449 2.02021 2.02487 1.99816 l.99901 1.98062 2.05345 2.06026 2.08569 2.15712 • • (adj. 250 g) (adj. Uma?C)-l • Fish No. Los:. Wt. v en sec-l) Los:. ms:O:zhr-l Loq. maO:znr 171 2.45484 17.89 2.04191 2.06324 172 2.46240 16.43 2.04020 2.14265 Treated Li~id (20C) I O;max = 17.05 185 2.48144 00.00 l.43950 • 187 2.51183 10. l4 l. 72129 189 2. 5ll88 14. 41 1.86826 190 2.51055 00.00 l.54527 191 2.74974 14 .06 l.99202 2.34498 • 194 2.89265 10.63 l.84531 196 2.89265 18.90 2.02388 2.34936 197 2.57287 00.00 l.49278 199 2.57287 13.68 1.85402 • 200 2. 57287 17.89 l.97677 201 2.89597 00.00 l.61760 202 2.89597 10.78 l.81502 203 2.89597 13.18 1.93459 • 204 2.89597 14. 98 l.96551 206 2.76268 ll.88 l.76893 207 2.76268 14. 7l l.96121 208 2.76268 19.23 2.13730• 173 2.11059 ll.36 l.81870 174 2.11059 16.61 l.95497 l.74801 175 2.15836 19.33 2.07411 l.82686 177 2.25285 17.12 2.• 09021 • l.97746 179 2.27875 17.78 2.03858 l.92698 • • • • (adj. 250 gl(adj. Umax) 1Fish No. Log. Wt. V • E ......... z 50 0 • ,,,,,. ;I' ~ / 0:: / ........ a.. ....... V> / w 25 .A+ / / ~ ....... ++ ·V.·/ ........ • c:: /';/· ~ .,,,,,,, ......... / /·/ • 10 // 1 2 3 4 5 WEIGHT ( g X 102) • Appendix Fiq. l.. Respiration and weight plot at 20C and JS ppt. for Lutjanus campechanus in control water. crosses represent observed data• • Solid l.ine drawn from equation la in Table l. Cashed line is for estimate of the standard level drawn parallel to the restinq resp~ation line through the lowest measured• values. Resting measurements made in flow­throuc;h chambers• • • • • • • I.• • • • • 500 400 300 200 ~ ~ / / 't... / .J::. ON 100 / / CJ) / E / / '-"' z / / 50 0 / ~ +/ ~ +~/ a_ +/ tJ) + w 25 + / / ~ /+// / 10 / / / / 1 2 3 4 5 WEJGHT ( g X 102) Appendix Fi9. 2. Respiration and wei9ht plot at 28C and 35 ppt. for Lutjanus campechanus in control water. crosses represent observed data• Solid line drawn from equation 2a in Table l. Ca.shed line is for estimate of the standard level drawn parallel to the restinq respiration line throuqh the lowest measured values. Restinq measurements made in tlow-t.brou9h chambers • • 136 500 400 • 300 200 ,,,,-.. • I ~ L .!: ON 100 / • ~......_,,,, / z 50 / / ~ / a::: / • 0 / / a.. + / I./) + + / w 25 +/a:: ~/ /+/ • / + / • 10 1 2 3 4 5 WEIGHT ( g X 102) • Appendix Fig. 3. Respiration and weight plot at 20C and 35 ppt. for Lutja.nus campechanus in polluted• (sludge phase) water. Crosses represent observed. data. Solid line drawn from equation Ja in Table l.. Cashed line is for estimate ot the standard level drawn parallel to the• r~st.inq respiration line through the lowest measured values. Restinq measurements made in flow-through chambers • • • 500 -400 • 300 200 ......... ~ • ./ 'L / ~ 100 / / ON / O> / • .._, E .... / / 0 50 // z /./~/ / ~ 1. a:: a.. (j) / 25 w 0:: • 10 • 1 2 3 4 5 WEIGHT ( g X 102) • Appendix Fiq. 4. Respiration and weiqht plot at 20C and 35 ppt. for Lutjanus campechanus in polluted {liquid phase) water. Crosses represent • observed data. Solid line drawn from equation. 4a in Table l. Cashed line is for estimate of the standard level drawn parallel to the restinq respiration line t.brouqh the lowest • measured values. Restinq measurements made in flow-throuqh chambers• • • 138 500 400• 300 200 / / ~ 0 • ,... / 'L. /.c /~ 100 / O> E +/ • .._., / ++ /z + / 50 Q / ~ ;'+ + 0:: / • (l. (j') w 25 0:: //+ / • / • 10 1 2 3 4 5 WEIGHT ( g X 102) • • Appendix Fiq. 5. Respiration and wei9ht plot at 28C and 35 ppt. for Lutjanus campechanus in polluted (liquid phase) water. Creases r~present observed data. Solid line drawn from • equation Sa in Table l. Dashed l.ille is for estimate of the standard level drawn parallel to the restinq respiration line t:hrouqh tlie lowest measured values. Rastinq measurements made in flow-t:hrouqh c:.hambers • • • • • • • • • • • • • 500 400 / / 300 / / // 200 / ~ ~ / 'L / J:: / ON / 0>100 + E 4 + '-' / z / / 0 / + / ~ 50 / / ~ / Cl. /+ l/) /+ w / a:: 25 / /~: 0 5 10· 15 20 25VELOCITY ({L sec-1) Appendix Fiq. 6. Respiration and swimminq velocity plot at 20C and 35 ppt. for Lutjanus campechanus in control water. crosses represent observed data. Solid line drawn from equation 6a in Ta.b1e l. Dashed line is for estimate of maximum sustained level drawn parallel to the active respiration line throuqh the n.iqhest measured values. Active swimming measurements made ill Blazka respirometer• 139 / ~ 30 • • • • • • • • • ' 140 500 400 300 200 --------.-.-.----­ ·----­ + -------- + 50 25 0 5 10 15 30VELOCITY Appendix Piq. 7~ Respiration and swimminq velocity plot at 28C and 35 ppt. for Lutjanus eampechanus in control water. Crosses ·represent observed data. Solid line drawn from equation 7a in Table l. Dashed line is for estimate of ma.~imum sustained level drawn parallel to the active respiration line throuqh the hiqhest measured values. Active swimmi.nc; measurements made in Blazka respirometer. • • • • • • • • • • • • 500 400 300 200 ..-.. ~ 'L ..c ON 0>100E .._...... / + /.. +/ + ++ + t / / / / / / / z 0 ~ 0:: CL U) wcc: 50 25 0 5 10 15 VELOCITY 20 ( {[ sec­1 ) 25 30 Appendix Fiq. 8. Respiration and swimmi.nq velocity plot at 20C and 35 ppt. for Lutjanus campechanus in polluted (liquid phase) water. Crosses represent observed data. Solid li.ne drawn fr?fJl equation Ba in Table l. Dashed line is for estimate of maximum sustained level drawn parallel to the active respiration line through the highest measured values • Active swimmill.g measurements made in the Blazka r.espirometer • • 500 400 300 • 200 ~ ~ •t­ .!::. ON • 0100 E ....... z 0 • ~ 50 a::: a..