The University of Texas Marine Science Institute at Port Aransas PROGRESS REPORT ' NSF-IDOE 37345 MARINE PETROLEUM POLlllTION~ BIOLOGICAL EFFECTS AND CHEMICAL CHARACTERIZATION . Section '· A The effects of a #2 fuel oil and two crude oils on the growth and photosynthesis of microalgae B Preliminary observations on the effects of crude oils on the growth of several microalgae temperature gradient plate on the light C Chemical Characterization D c;, :'·,Effects of Petroleum on marine animals and larvae •.. " .. E A study of the effect of petroleum on sand dollar eggs . THE LIBRARY OF niE UNIVERSITY OF TEXAS AT AUSTIN NSF-I DOE GB 37345 The effects of a #2 fue.l oil and two crude oils on the growth and photosynthesis of microalg·:J.e Warren 11. Pulich, Jr., Kenneth Winters, and C. Van Baalen Universit_y of Texas Marine ' Science Institute ...-/" Port J\ransas, Texas 78373 ,. :',• i.: / •. . ' ABSTRACT Sea water w11en equilibrated with a sample of #2 fuel 0:-;.1 becomes toxic in varying degrees to growth of representa_tive types of microalg·ae, two blue-greens, a d:Latom, two greens, and. a Ji11oflagellate. : . For a sensi~ive orgunism sue:-. as Thc:J.assiosirc. :)se-;.1Cvna.ha, strain 3H, ' 5 ml of sea wate1" equilibrated with ·fuel oil (cor1t::.:.ning· 15 mg/l of organics). in 20 ml of growth :nedium is :ethal, Ci!.."' ~m..-.1}\Ly in the range of ·40-400 ppb if the t:oxic mc:.·:~eria2.(s) constitc.t~ 1-10%'• of the saJnplE. This fuel oil-equilibrated seo w.3.ter also immediately s-co;>s photcsyathesis in organism 3H. ·For other microalgae tested (e.9. 580 ar.C. P:<-6), simila=effects on growth and photosynthesis were found but ::-·eq.:.ir,.:-d ~ighe:· concentrations c~ the oil-equ:.librated sea Woter. ... :",• Water solubles from Kuwait or Sou·::hern Louisiana (.!ru.des (·~e:: ·.::he . ;. straight crude was equilibrated 1: 8 with sea .H20) ~'1.0t ·~~-cj hcw2ver specific fractions made by distilla-cior. did show sara2 "1~te~ s; ~.\."'-bl e. toxicity. Growth experiments in open or closed. ~ow-er. ~ fS"{t.,fi\.S ~.t,.6wed that most organisms were ir:iliibited by varying· amountE e:f d\;ov ~ -t..wo crude oils when in direct contact wi·ch therr,.. Oi:·ganism 58J ·we.ult\ n~t grow·above 5 µl of Southern Louis:lana/25 ml of foe;di·lll.n, or 10 )Al ~t \(\lwait/ 25 ml of medium (oil in direct contact wi~~ dlgae). With both the sea water_equilibrated with fuel o·il a~".d the Cf\4de oils, the toxic activity is mainly localized in meC.i't.:.m and 1' i.q·Y,~"" boiling fractions derived from distillation cuts fro:~ \:hese Ma'tev~Al' .. ) ""'·--.. ___ . _.,/ INTRODUCTION The recent report of Gordon and Prouse (1973), togethe_r with the data reco1...ded by Kauss et al. (1973), Nuzzi (1973), and Stro.nd et c:tl. ./ (1971), all serve tc point out the potential toxicity of water solubles det'ived from pet11oleums (or refined products) on the growth and · photosynthesis of representative types of microalgae. A cor..~ervative estimate ?f photosynthesis in the oceans as compared with. all land '• masses suggests that not less than 50% and probably closer to 80% is carried out by microalgae indigenous to the marine environment (Rabinowitch and Govindjee, 1969). The extent and biochemical basis of the toxicity of petroleums to diverse microalgal types is requisite to an obje~tive assessment of effects of petroleum in the sea on primary production• .. :, . We report herein on the effects of two crude oils and of water a solubles from these oils and from~#2 fuel oil on the growth and ~ .· photos~1ti~~is of pure cultu:res of microalgae. We recognize that the experimental approach used may not allow direct extrapolation to the environment; however, it does set boundary conditions for judging the degree of toxicity of petroleums: More ~importantly, informat.ion derived from ~owth rate and photosynthetic·measurements provides a basis for more sophisticated efforts in cell phys~ology and biochemistry, dealing with the nati1re of the toxicity of petroleum. METHODS AND MATERIALS Organis~s. The microalgae ~sed, thein source, growth medium, and temperatures used ·are recorded in Table 3. Medium ASP-2 (Van Baalen, ) 1962) ,is a ~edification of the original ASP-2 medium proposed by Provasoli et al. (1957). ----.. _ 2 Three· ty[>c s of growth systems were used to exu.mine the crude oils, :/t2 fuC:~ l oil and fractions dcrived f.rom them, for inhibitory effects on growth of the inicroa1gae. rrhe first system which is termed an ffopen system" is the commonly used test tube culture method patterned after the original design of Myers (1950). In this system the algae are grown in 22~5 x 17Smm ~;rex test tubes with 1% co2 in air continuously bubbled ( 5-7 cc sec-1) through the tubes. Light intensity and temperature are '·.controlled as desired. Growth was measured tu.rbidirnetrically, using a Lumetron Model 402-E. The measured optical density (OD) is proportional to cell number over the range used. A plot of ·log10 OD versus time allows evaluation of the specific growth rate constant, k, in log10 ~nits day-1 ... :",• (see Fig. 1). For simplicity the growtb rate data reported herein are in terms of doubling time. A lag in initiation of growth; so-cal~ed lag time, wa!s measured by comparing the time that growth in the tube under question reached a certain point on the growth curve with that of a control. A lag of one generation time, 3 to 8 hours depending upon · the organism, is indicative· of a se'1.ere but temporary depression in growth rate •. Since the turbidirnetric method does not distinguish between living and dead cells, _the lags may indicate a fractional kill of the popu+ation. The second type of growth system, a "closed system", consisted .of Be~lco Glass 500 ml Nephelo-cultu1"e flasks (254-80005) with 22.5 x l 75rnrn side arms and screw cap closures. In th:is system growth rate . or lag effects we.re mainly judged by visual observat~on in comparison to CQntrols. Ho~ver, in the case of borderline effects the side arm allowed turbidimetric measure of growth rate as in the open system. ' " the screw ca ps. Tl1c; f la~:;ks w2re cJ.0rr1pc:~d onto a linear rotc-;t j_1LrJ f>&r l'cs·;:8d i .. a consto.rrt temperature water bath. Illumination was ·~roviced I ·by a bank of six F48Tl2 CW/HO fluorE~scent l amps beneath the wa-cer' bo.th. Intensity t,vas controlled by the number of lamps turned on,,and by ~ch2 distc:t:ice frora the 1amps to the flasks. Ih this type of g1'm·rch cor.d:. t:ion the limited amount of co2 originally present limits algal yield; :.owever, reasonable densitie·s of 0. 2 -0. 25mg dry wt m1-l were reached by t::-1e controls and the growth rates were the same as observed in the open occasionally system. This .flask system was alsol)run as an open system with l% C02 in air continuously bubbled through the flasks while they were shaking. A third growth system, the conventional alg·al lawn technic~-:E:.. was u~ed to test pure compounds. A given concentration of alga~ cc:~s, usually ·( 5-10, 000 cells/ml) of the organism, was added to agarized ;:ledium held at 42°; 20 ml was then immediately uistributed to plas~ic pet~~ dishes~ The test materials a~e presented to the algal cells embe~ded in the agar by absorbing them on antibiotic sensitivity discs (12. 7 ;::r:.: and placing the discs di-pectly on the agar surface. Tht~~*plates we rt then sealed with Scotch tape and incubated in the light for 5-8 day~ ~ ,' The experimental endpoint is the zone size of inhibition around the pad) judged visually and microscopically. Pnotosynthesis Measurements. Photosynthesis was meas.ured as o2 output using a Gilson 1'1edical Electronics Clark-type -electrode (OX7000 and water jacketed cell OX705) {;t (Van Baalen, · 1968 ). The tcrupcro.turc wa s c ontrolled hy a· circuJ_uting water ·11ath, with illumination provided by a Stand:1r.d projector with a 500w DAY p-1~ojection bulb. IntGnsity was controlled by a Varir..tc, sere.ens, and neutI1al filters . A Baird-At omic hot mirror limited the transmission of energies in the system to 350-800nm. Intensity measurements were . made with a calibrated Kipp and Zonen CA-1 thermopile ;with readout on a Keithley lSOA Microvolt-Ammeter. Test oils. ;, ':· The American Petroleum Institute has kindly set aside four oils which may be used by the scientific comnmnity as reference cils. They are available from Dr. Jack Anderson, Texas A&M University. Of the " four oils, two crude oils (Kuwait and Southern Louisiana) and #2 fuel .... :W,• oil were used in this work. It was observed that there was a fine suspended materia1-in our s~~ples of the #2 fuel oil. Filtration of ~he fuel oil through Gelman glass fiber type A pads removed most but not all of this mat~rial. The material appeared to be inorganic and dark purple-black in color. We have not examined the n·atur.e of this material. It has also been observed that our sample of Kuwait crude contains a residue ~t ~he bottom of the container which h~s a consistency which is different fro~ the ~verlying oil. Chemical characterization of test oils. Each of the oils was fractionated.on a column of silica gel which had been activated at 175-20o0 c for at least 12 hours. A 45 cm. x 4 cm. column of silica gel (120-200 mesh) in hexane was ) prepared.. The oil s~ple ( 2-7 grams depending on the oil ') was dilu-ced \ with an equal volume of hexane and layered on the column. Column flow rate· was adjusted to 2-3 milliliters per minute. The fract~ons of oil 5 which eluted w:~th hexane, benzene, ahd .chloroform: methanol (1: 1) were clcsiqna·cc~ci the puraffinic, aromu.tic, and aspbaltic fractions respectively (Table 1) . Fractionation of test oils. Fractions of the #2 fuel oil and Kuwait crude were pre;?ared by distillation at atmospneric pressure. The oils were d:i.st;ilj_2d through a 50 cm. x 3 cm. column packed with Raschig rings. Nine distill~te fractions were obtained; boiling point ranges are indicat~d i~ ~&ble 2A. This type of fractionation was used to obtain the data in Figs. :, 2, & 3. In an effort to avoid decomposition of high boiling compone~:s and the possible formation of toxic artifacts, we have discontinued distillation at atmospheric pressures~ We have been experimer-~tir.j : with several methods of distillation urider relatively high vacuu~. We recently prepared low ·temperature vacuum distilled fractions of ~~& ! ·' !' I #2 fuel· oil, whose boiling range and representative chemical corr~porK:~:cs are indicated in Table 2B. Bioassay· of fr.actions of #2 fuel oil pre~ared by this new method reveal a similar pattern of toxicity to the nor..-vacuum fractions when compared on the b?sis of representative components. T~e ... data in Fig. 4 for Southern Louisiana crude oil were obtained with ~h~ fractions listed in Sec •. C of .Table 2. P-.cepara.t:ion _of ~ water soluble fraction from the test oils or_ #2 ft:el oil. One part _of the oil (100 ml) to be tested was layered on the surfac~ of eight parts filtered. (Gelman glass fiber Type A) sea wata~ m· (800 ml) in a bottle containing a teflon coated magnetic stir bar. J..ne bot~le was sealed and the water· stirred at room temperature for 24 ) hours at a ~ate which would avoid the formation.of an emulsion. The water was allowed to stand undisturbed for several .minutes before being / removed by means of a stopcock at the base of the bottle. Before use 6 ,. ' .,.., in the grow.t h work the sc c1 wcJ.tc·r equilibrated with o:i.l was :filtr-1r0.d through a 0. 45nrn Millipore~ :f iltcr. on ultcrnate method was used. The same rutio of oil to water was ~)laced in a smal1 Erlenmeyer flask. The flask was sealed and shaken for 24 hours on a reciprocating shaker. The oil and water were transferred to a separatory funnel and the water soluble fraction ;, removed. Water soluble fractions prepared by .the two methods appeared similar in concentration anq. toxicity.. ·Extraction of the water soluble fraction. A continuous liquid-liquid .extractor was used to extract the organic compounds contained in the water soluble fractions prepc·2ed ... from the test oils. Extraction of more... than 150 milliliters o::::1"s~a water was carried out in a separatory funnel. The water was extracted three times with 1/10 volume portions of solvent (hexane:benzene, 1:1). Gas chromatog-~aphy. · All analyses were carried out on a Perkin-Elmer 900 gas chromatograph with a flame :l..onizatiqn detector. Peak areas and retention times determined by an I~fotronics 204 integrator were printed and paper tape punched ~y a Teletype terminal. Routine analyses were usually mad~ on both a S' x 1/8" stainless steel colunm packed with 4% Apiezon L on 80/100 mesh Gas Chrom Q and a 6' x 1/8" column of .5% FFA.P on the same support. SCOT columns (150' x 0.02") of Apiezon L and FFAP were used when greater resolution was necessary. I RESULTS ) . #2 Fuel Oil. . Table 3, surrunary growth data obtained using the open test: tube gro~lch system, shows the effects of various concentrations of sea water ~/ I .__.. . .". ·c ,-:-: tC:! cqi.d.1i,.H'atcd with //'2 ·'\wl oil. on ~r.cowt:J.1, ot Vently a.wure of any chemical or ~ioche~ical bases for this difference. Uptake experiments at letnal . o:' su~lethal concentrations cor.'°ined with assays of cellul.s.r activities such as nucj_eic acid replication, oxidative phosphorylo.~fam, cell . -\..· ..J division, etc. are needed in this area. 2ffects on Photosynthesis • . Figure 5 shows the. eff~cts of adding to suspensions of 3 types of microalgae, prior to closing the electrode chamber, water so:.ubles ' from #2 fuel oil. The algae. nseetr the· water solubles for .approXL"nately 8 'minutes before the actinic beam is turned on. Clearly the effect of the water soluble mat<~rial( s) is rapid and highly .detrimental to 02 evolution. Note the correspondence in concentrations between Table 3 (growth) and the data here. The results of Gordon and Prouse (1973) on 14co2 uptake on marine phytoplankton photosynthesis reinforce the notion that photosynLhesis may be extremely sensitive to certain components of the wa·ter solubles from #2 fuel oil. By extrapolation it could be inferred that such a situatioh may hold for materials in crude oils, particularly after emulsification which may increase surface area and hence solubility of materials. Again, it will be significant to further localize this toxie; activity to photosystems I or II of the Z scheme (Rabinowitch and Govindjee, 1969) or perhaps, but less likely, on the carboijf ixation side of photosynthesis . -1 J ..•. DI::>CUSSI Otr While tiw ?.."'c::;u1ts pr~scntt:!d here a:cc~ cs:::cnt:i.alJ.y on1y a ix::crinning, -~h0y no.vc~1--thclcss support tt1c pCJ.pcrs in the literc1.tu re dee.ling ~..1ith toxicity of potrolcums (or products therefrom) on g~cowth anc! phm:osynthesis in representative types of microalgae. f1~~"12n vie\·l•3d as a: whole, they also suggest a great deal of caution if one is making sweeping generalizations on the effect or lack of effect on even a cer~ain '· gI'oup of mic~oalgae. Witness the two green algae so far.,examined, I E. ·tertiolecta showing a high deg-.cee of indifference to water· solu~les from #2 fuel oil or crude oils ~hemselves~ while ~-autot~ophica is quite sensitive. Similar great differences in response were noted for the two blue-green algae, PR-6 and Ba-1. Obviously, the long range ...effects "' :·.· of petroleum toxic.ity may lead. to enrichment situations in a giv~n environment. Wbether such an enrichment of one or several "oi1..:'hardyn forms is desirable is difficult to argue. Sensitive forms may slowly adapt and become less sensitive, ho-wever, we have little information ' on this point. The work recorded herein nas been with pure cultures • . We can only guess at the influence of a bacterial and fungal population on the che~ical composition of an oil. T~ey may alter toxic materials and thcreby--allow algal grmvth. Additionally, microalgae may prove ! able to metabolize and possibly detoxify aromatics. Aromatics are apparently only degraded slowly and with difficulty by bacteria or ... fungi (Perry and Cerniglia, 1973) and if they form the basis for most of the toxicity we have found, then they may accumulate with chronic ) spil1s in the environment. The studies of Blumer et al. on the I environmental fate of oil strongly suggest that preservation of the more toxic aromatic materials may occur during weatheri.ng• ....._ · Tr1c C·o;·1undrum of 11conccntrotion in nl.lture:n, i.e. is a tcxic compmmd pt'c~cn-t a~1cl :i.n sufficient concentration to inriibit, can only be: on...,1:;crcd by ic..ic:!ntif icett:Lon, quantif i.cation of the amount pre sent ( inp:..it i·'l.itc c..nd s·~obility), and a detailed knowledge of its biological effects on a range of microalgac . Literature Cited Blumer, M., M. Ehrha1"dt, and J. H. Jones: ~rhc environrnerttal rate of stranded crude oil. Deep-Sea Research 20, 239-259 I (1973). Gordon, D. C. and N. J. Prouse: The effects of three oils on uarine phytoplankton photosynthesis. Mar. Biol. 22, 329-333 (1973). Kauss, P., T. C. Hutchinson, C. Soto, J. Hellebust, and M. Griffiths: The toxicity of crude oil antl i~s components to freshwat~~ algae. In: Proceedings of Joint Conference on Preve~tion fnd Control of Oil Spills, Washington, D.C., pp. 703-714. Washington, D.C.: American Petroleum Institute 1973. Mye~s, J.: The culture of algae for physiological research. ~n: The Culturing of Algae, pp. 45-51 Yellow Springs, Ohio: C. F. Kettering Foundation 1950. Nuzzi, R.: Effects of water ·soluble extracts of oil on phytoplankcon. In: _Proceedings of Joint Conference on Prevention and Control _of Oil .~pills, Washington, D. c., pp. 809-814 Wasnington, D.C.: American Petroleum Institute 1973. Perry, J. J. and .c. E. Cerniglia: Studies on the degradation of petroleum by filamentous fungi. Center· for· Wetlands Resources: LSJ,SG-73-01, pp.. 89-94 ( 19 7 3) • Pr'ovasoli, D., J. J. McLaughlin and M. R. Droop: ·'l"be development of · artificial media for marine algae •. Arch. Mikrobiol. 25, 392-428 (1957). 13 I~a;-iinm·Jitch, E. <-md Covindjcc: Photosyntr1esis, 273pp New York: J . Wiley ~nd Sons, I nc. 19G7. Strano, J· . A., VJ. L. Templeton, J. A. Licnc.:.towich and c. W. T~:.Tt:-;; : Developm2nt of toxicity test procedures for marine pnytop.:Lt.nk'con. :n: ~~oceedings of Joint Confe~cnce on Prevention and Cont""~ol of Oil Spills, Washington, D.C., pp. 279-286 New York: . Arr~rica~ Petroleum Institute 1971. j Van Baalen, C.: Studies on marine blue-green algae. Botan1ca M&rina 4, 129-139 (1902). Van Baalen, C.: Effects of ultraviolet light on a coccoid blue-g-~ee~ alga: survival, photosynthesis, and photoreactivation. Plan~ ~~y~iol. 43, 1689-1695 (1968) • .,.,..; · ., ,_ . I l\CKi-lOWLEDGL:i1ENTS \'Je trunk ~v·rs. ?.ita Qt Connell and Mrs. Connie Chc:i.rlto:-i for excellent technical assistance. T:Ois v1ork was supported by Xational Science Foundation IDOE Grant GX 3734.5. ) ,.. :',• I _,., .::· i g-. l. Grm·iCh rCtt c in units of log10-(0Dx100) of di-270 235-~85 250·-300 270-315 270-330 •" :', • 285-350 300-)385 f.: < 150-250 175-270 195-285 215-300 235-315 ~ 270-350 300-)385 or greater. '.'..', _1, ·•. )-,1_1· "'. r) ( C'""l L'-) . -\ - '·' (.. . F:c~lC·ci on n-Paraff in Ranae C27 J\ pJ1~X1X imo.tc~ Poi nt J.400 i Ta.)J.e ~5. DGubl i n temp. 300, source ( B. Wilson, Texas A&M, Galveston,). 2Fo~ty ml. of #2 Fuel Oil layered on 320 ml filtered (Gelman typ2 A) off-s!:o~e sea water, stirred gently for 24 hours at room temperature, sea wa.ter layer removed and re.filtered through 0.45 nm Millipore filter. This sea water equilibrated with #2 fuel oil has 15 mg total extractables/liter. 3N.D. =Not .determined. ) ti '\ . -... _ 'r 1~>J.e lr . U.(;1v.~~-; ;·: co~·i.c.:·'.nt·e.:~~ ·: :iun r;.:.Ji"-:c-;:r·r: o:i.l :in ;~~: ri;:;_ r.·;::.c~~. t.r:.~ of Yu·;1~ ~>: : :L(t ~CiUti :1 :J:r L~)l.Li. :·_; :i .:.11: tl Cr:\ cic..: (J_i_J_ '11/h :i..ch c.A.JJ,c·:i·~ (; ~j2()'.,'/t[; ()[ V.J:..--:~c ~ :~ i :ic·cc,.~1 l~JC11.; , vfr~ _n i r: direc·t conte1c t \-Jit:h C.J :i.J.. Cl o sr~d grm·;'th ~-; y ::~ ··(~m.. '.i.'erap . 300 . --- Stc·1in Kuwait-- Southern T..1ouisi.ana P:.<-G 150 100 Ba-1 25 25 3H .10 5 ;, Dun 200 40 Ind. 580 10 5 Organism Ba-.1 =Microcoleus chthonoplastes, a filamentous blue-green;meaium ASP-2 + B12 •" :",• i: ... ·,., • _.I ..... ........... ·;~u::lbers repr(:Sent zor.e of -:=--:. :.:.~_ .) 3. :-~~:=0ct o f f·ur2 CiY~:;:c-unds on g~--m·,;th of organisms, PR-5 and 580, using algal lavm technique. c ,)mplete ki1ling results in a zone of inhibition of 36 mm on the_ plate. ~ : ·.~1Edticr'. ( m::-,) out frcm edg·2 of filt8r pad. ... ,... ·•·: ------------·--·-··-----·---·-... ___________ -31 0 ffl.01· T"' TEB1 ':"''IB2 \1N 4 Dr-IN5 NAP6 PHEN7 Durene8 Fluorene Ws~tylene9 Biph~nyl Indar.\ C°iJ :;·.2ne i.;..:..r -u~ i. .i -_--:-.__ -_ · ~· · : :·.: :~2: ;~:2te .l ~·. Cn~sol3 ·-·-' ----. ·-----------·---.. ..;.. .•. -. -•"\ ... -~ ..., 155 36 36 36 36 36 5(155) 1 0 lJ r:.-J 3 20-22 23 2 4(9S) 0 0 0 21 1 0 12 36 ~ :-'.'".~ 2 16 10 0 6-7 •. 27 0 10 l(lOS) 4 7-8 0 4 0 0 0 4 0 ' :i .....l 0 0 0 0. s ::-.g 0 1 0 ....i I .. , .-~ -r:::: .....~ ,...,,. 5 ' . . 0 ; ·:i 10(205) 4 3 28 36 2 r...; 1 25 7-8 36 1 0 . 0 ... ·5(158) 0 0 25 36 5 15(205) 0 0. l r::; 0 15 ·-=.-·::~~ ::::/1 tsnzor1e . 7. Phenanthrene --~ : 2: ..~ -~::·.fr.-~ ~:}1yl b21:.zene 8. 1,2,4,5-tetramethyl benzene : ~.-~ -~_., 2 ~-::;.: 9. l,3,5-trim2thyl benzene ~-:: -:::-.~ ; ~ :-::.;1-..t·!-1al:-:;1~e 10. Diisopropyl benzene :--_. \.. -r_. ·: -·~, J 1 ~-, _:) :hthalene 11. Dim2thyl quinoline ·--:.. · --· -·-· ·-• ·~J:"· -. 2:::~ ~:"'i ::~ ~ 3 .~. ~n'2 --i '"' f:) t' r, d; t "Ce fr m pad • s ""'..., .....,':"', : ~~ r--· -.~.., "l" ·~ ~i,,-~d~ lfl.. ~...... ,,_.,_ D..l. S _s au 0 _ _,_,__._ .:;~ ..... ~c_. ,_ ·-~C...,~..:: l_., "' 0 -;..:. .·~ -.., l~ 14 1,... 17 18 . . 19 2 ~ . . -:-·--·--~ :: ,. --:--__ .) ' P P -2 ~1 n .1..-h ::> T'1N16 1-Napht hyl 1 ..: -.·-: 1. --:::. .. ---·-: .:2.1 . ~--\ i. ·.1en . -1· ...nL • 11-MFl DB-Th. 4,4-DMBi. 4--MBi. 0 Naphthyl 2-Naphthol Resorcinol amine ,. .. _-, I I (" 5) ,._, 0 p 36 20 36 36 -\ _._, 0 0 0 0 ; rt u 0 0 0 0 0 0 36 12-13 36 36 \ ·"' ·-~----·----- , . ::,. 3 p 0 0 0 . 0 . 5(305) 23 36 36 ' - :~ ·~· '] 1-2 p 0 0 0 0 4(255 ) 16-17 36 36... -~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ -~~~~~~~~~~~ .: -~~~ :-: . = 2-s '::th/l pl12nanthrene 17. 1-MFl = 1-methyl f luorene ._ --. ----. -~:--~ . -~-;:--.(:tl-:yJ_ 1_:;h·2~a!1threne 18. DB-Th. = dibenzothiophene ::::-:·~~1 . -~ 2.-:::ethyJ. ph2nanthrene 19. 4,4-DMBi = 4,4-dimethyl biphenyl 2-:.: .:-_,_: ::. = 2-:-:-.2thy.1 -::.r"'lthr acene 20.. 4-MBi = 4-methyl biphenyl = ::~.-::..;_,~ t l-:y l r 2p:-ttLalene .~ = -~2.'"':::·~ .':::: i:-~~,:~~i~s:::l under pad • ..... _# --. .__~... 1. -•. oH < '! l_ -------~- -.o I I : "') -; ~:~ i ~ -N j !I - 1 ('..; --; i ! I II :l o ~o I I I I li I1I CJ C'O I Q 1Ia-- i • 30. 78 126 270 3!3 HOURS AFTER Oll EXTRACT " ADDED l ,--. , ' c~ .__) -I· '·~ -'. LO li I -!!~ 0 . lI L l -:_) t r LtJ . ·iM0 <( ~ . ~. -~C\1 i ~ ~+~- ? ·.:. ...I:;..._(\J -.'"" -"'·( ""' 1 .. _.. ~.. \ J 0 (\J "t LO ~ . -.1\,,, \.;. l .........; .,<' "' '-l -:-,,...- j , <( ~~~ ~-) u -~ ·~~---~~------~-~----------------~--l ------------...I --,----·---~-~ , , d I I 0 ! ./ ·1·,..-1·c -. ·. ()l ~Q 1 V ;..-/ ~-1 ) i-1 ~ I -o c.Y -0 ""CJ rJ -:....) u·15 ~ -!_) '/ /\, Q_) 0 ~ ;:::> _ · _) '-. ~ :J J~1 ,, _,...') -c \-:),_, \ \ \ \ \ ... .,,,· \ \ \ \.0 I cc 0 u _J 0 (0 c:: ' ~··----. . ,..,,.' . u.. ..::. 0 u co w l() 0 0 " ·, .. .' (' : ; l ·: ( .\ 1 0 N - 0 1."\.! ... ~ <.i.. < !..!~ (/) l __J I / .J 1 i J i ~ ' i j I i I 0 (\i l:J (/) l _J --· 0 c~ 'i .! lt ..._ ( ; 1c' 1 0 . ., ~ N'' l ;__., i C.'.J 0C' 0 . H I ~ 0 ' 11 fro ? 1 1 f (Q l:J ~ ~ i: ~ ~ I \ " j \ 'i. c :·~ - ~ \ \·I/\ ~ '· \ ~ l \ ~ ~ l ~-·~.. · ' I...... ~ I ~ 0 ·-' I ; (\.; ........... ::~ ., ......: L~J ~~ ~ i \ ~ \ 0 N \. l- t -:\;; I 0 ~ 1. I I --{ CD I I i ~ <.D i I ~ j I i I (\J I f'\") (\) • ' 0C1 0 0 0 0 0 SSCTION B NSF-JDOE GB 37345 Preliminary obser~ations on the effects of crude oils on the growth of several microalgae on the light-temperature gradiert plate. i, R. 0' Donne11, Warren M. -Pu lich, Jr. , and C. Van Baalen University of Tex.as Marine Science Institute Port Aransas, Texas 78373 •" ~. ·,., . I • i'. } The ligi1t-tcmperatu1e gradient plate apparatus (Van Baalen & EdwaJ:'ds Handbook of Phycological ME~thods, p. 26 7) was usE!d to assess the effects of crude oiJ.s on the growth of microalgae over a range of growth conditions. The growth work r eported in Section A was larg·ely limited to measurements ·at one temperature and one light intensity and f or this reason t he results may not reflect :possible variations in toxicity of a given crude oil for a microalga under different growth conditions. i, It should be borne in mind that the light-temperature gradient plate is primarily a qualitative algal .culture device. It does not yield quantitative data of the quality of the standard test tube water type of set-up. There are unavoidable shifts in temperatures on tpe plate due to ambient changes. Shading and co2 limitations occur ~s the cultures become more dense. We also suspect that there are shading effects (lower effective light intensity) due to the crude !: oil when it is in sufficient concent~ation and spreads uniformly over ... the whole surface of the petri dish. We also cannot absolutely control the evaporation which occurs in the warmer parts of the plate • . Notwithstanding these recognizable.. limitations in the use of the light-temperature gradient plate we believe that the data are useful. The gradient plate da.ta support and in some cases extend information obtained with other algal growth devices. Figures 1 to 4 present data for two blue-greens (Ba-1, PR-6), a green (DUN) and .a diatom (3H). The organisms and growth media are identified in Table 3 of Section A. In any one experiment the region of the plate (petri dish control without oil) showing ·the best growth was given a value of 100 and growth in the other regions compared to r \I '/ :Lt ei ther tuI'bidi:netrica11y or visuc:.i11y. Th~ numbers bes ide t}v:; b.::lr -rrranh s l"P,present t he aver acrP, of temr_.~r.at:i.n~es and li(rht intensit ies · :.J -':.) '.;} ( f t-c) measured at the 1Jf2gin.ning and end of a run. vJe do not at this time wish to over-intc1"pret the data, how12ver· we b2lfr~ve the following observations are warranted. J. 0 zzz _ ~~~~...__._~~~-----------"-~~ . -o~~-~----------_._~~._._._~~I r, -~Q..____________._C~Q~~~~--~ ~ 'r' ~. . ti ~ ~ 0 ~ ~ (p I Ci 'r-(;.Q ·I\ ·r;.1 :"'-,·, .... 1 ~' : ' , ··'' , .... 1 !--------------·----··.. · ·----· -----· ~--r1 ,.; II I: 11 I ' I'I · I 1--i 1 I I l I I i ! 5U-~ 1 I I I I 1 • ! ! I I I ·' j I' I:II 1 I•· II I -370 I I 1 'I l j 1 I 1 '.:? 0 1uJI ·-Cl lUJ! ! '- I I I I I I OQ TI I lj I i 1. _ 1 l 1 j 1 r I l~ nI r I ~UT I I ,lII I -370 I Ir I n 0 ~ i, o (~...............n,...., -' I z z. 1 I /{)() il . .. ! . ~ _,I ; l j l I I' I ~ 11 ~ ~ '~ 1:-..::z~ I . SECTION C NSF-IDOE GB 37345 Chemical Characterization Section Personnel: Kenneth Winters Connie Charlton Martha Roach ... :.. i: l 4 In·~1'odu.ction Crude ,:tnd fue.l ol1s are exl1.:'()ffi'-::1y complex mixtures contu.i.ning hurld. ;~ds of chonnca.l compound.~~ . 7' J.thouah most of t:he corn~ounds are prcb2b1y C\)ntmGn to 1~11 crude oil.s, each has a unique~ co;npo::;ition. 'CDcrG ar(~ mc:my compou 1ds knovln t o occur i n pet-.rolt.:1.un vlhich have bGen proven toxic at som~ ccncorrtrc. tion to plants ard/or 12 animals . ' Some compounds are much more toxic than other s . ;, VD.:r.iations in t he conccntrr.rtions of tiiese ana ot her· yet ~midentified toxic ag·ents probabl y result in tbe variations in toxicity observed among oils. Var:i.at:i.ons in oil toxicity and uncer·tainties in the determination of environmc.;ntal concentra.tions of petroleum make I it·difficult 1to evaluate environ.rn2nta1 quality. ... TI1e goal of t he chemical section of· this project is to :",• cheracterize a f e\•J of the · mor\:: toxic agents found in several i· representative tes·t oils. Our experimental design is to use several physical and chemj_cal methods to fractionate test oils while following toxicity by the bioassays described in the biological sections of this !'eport. Accomplishment of·this: goa~ may demonst-.cate th.at a few key compounds are responsible for much petroleum toxicity. The concentration (and infe'.r·i"'ed toxicity) of a few specific compou.nds could pr'obably be measured more ?Ccurately ii.1 the environmen-'c than the concentration ( c:md infe1""red toxicity) of n oil'7 • '': Test oil s \·1hic may b~ lL-;;c:d by t~12 sciQnt:Lf:" c corru u.mity us rd~erenee:: oil s. 'J.ff' tC Lou:i.siana) and tr1-;o fuel oils ( o. 1(-2 fuc1 oil and a Bunker . C). 'T'he0e oils and some associated anal?tical dat a are cu~rrentJ.y availabl e at a n-:odest cost Leon:: Dr .. Jack Anderson, Department of BiolOSJY, Tex.:is I A[.,.J.1 University. In addition ·to t1'1e A. P.. I. oils we have studied a sample of a diesel f uel and a Ve:n.ezuelan c-.rude oil. W2 have just r ece:Lved and begun work on a sa.;ri.ple of an Alaskan crude oil. Fract:Con~tion of test oils ... :-, . Each of th(~ oils was fractionated on a column of silica ge;i_ v1id.ch ·1a.d been activated at 175--200°c f or at least .12 hours.. A i· 45 cm. x 4 cm. colurnn of silica gel (120-200 mesh) in hexane was prepared. T.t1e oil sample ( 2-7 9Tams depending on the oil) was diluted with an equal volur..·2 of hexane and layered on the column. Colurru'"'l flow rate was adjusted to 2-3 milliliters: pep .minute. The frac~ions of oil which .eluted with hexane, benzene, ·and chloroform: methanol (1:1) VvBre designated the paraffinic, a1'omat~c, and asphaltic fractions respectively .. Fractions of the #2 fuel oil and Kuwait crude also prepared ·were ·:oy distillation at atmospheric pr·essure 'Y~e oils \'Y'i:?,re distilled o through a 50 cm. x 3 cm.. column packed with Raschig rings. We are · currently experimenting with several methods of vac'.lum distillation. _,I 3 _x:_uninc~d thus fen_, err:k;loyi.::;d a 70 cm. x 2 cm.. colum..'1 of Scphr:td8X LH-20 with isoDutyl alcohol a.s the eJ.utinq solvent . A fraction collector CCf...lippc d wl+··h a d:eop count(:.!I' was s1~t to collect about 3 mil l iliters of column eluate per tube.. A sim..i.J ar column. with acetone gave less resolution . ·An J\.er og-.raph Model 202 gas ch:c'oma.l:og1'aph ~.::ith th2r!nal conduct ivity detectim1 has been us ed for rela.tively smal.l scale prepa~"'ative gas chromatogr aphy. When a compound( 0) v1as t o be collected a 15 cm. x 0. 4 cm. 0 .. D. :g·l a.ss tube ·with a right angl e bend was jnserted into · the exit pox··o of t'he gas chromatograph. The carrier gas was bubbled through several ruilliliters of hexane in p. small test tube . Th~· glass tubing was removed from th<~ gas chromatograph after the c~mpouncf(s) had elut ed and the process repeated to trap the same compound(s) from I several sample injections . A few milliliters of warm hexane were used.to transf er any rr~te~ial in the glass tubing to the test tube. Preparation of a water soJ:uble rractj_on... from the test oil• .One part of the oil to be te3t ed was layered on the surface of eight parts filtered (Gelman glass fib~r Type A) sea water in a bottle containing a teflon coated magnetic stir bar. TPe bottle was·• sealed and the water stii"red at room t emperature for 24 hours at a l"ate which would avoid the f ormati on -of an emulsion. The water was allo\.'v'ed to stand undisturbed for several minui:es before being removed by means of a stopcock at t he base of the bottle. 4 to wat2r was pl~ccd The :flask was and shc:il--?n £01 24 nours on a recj_p-'ocatin~J Shuker . The oil and watc~r wo~e transfert·co to a scpc.n1 c:tm:·y tu. ne1 anri t1JC~ (.\ia.ter ·soluble fraction ::en, vecl ·Jo.tc=r· so1ub1r:: .:=ra.c.t:i.ons prer. :tred by 'Che two rc.ethod!:; appear '· .Extro.ctj_on of-the ¥Clte::i soluble h'&ct:-i.on. 1-\ cont~Lnuous liquid-liquid ext:ractor was use:d to extract the organic compounds conta.i11.ed in the wo.t2r so1uble iractions ,prepared i! from tLe test oiJ.s. 2xtro.ction of more than 150 milliliters I of sea ~·-1cJ.tei-1 v1as carried out in a separatory funnel. The water waP-ext-.cacted •" three times with 1/10 volur.G2 portions of solvent (hexane: benzene, =',• 1: 1). Gas chromatography All analyses vleJ..'e carried out on a Perkin-Elmer 900 gas chroma~ograph with a fla~2 ionization detector. Peak areas and retention times determined by .an Infotronics 204 integrator were printed . and paper tape punched. by a Teletype 1,terniinal. Routine analys·es were usually.made on both a.sr x l/~TT stainless steel colunm packed with 4% Apiezon L on 80/100 17,esh Gas Chrom Q and a 6' x l/sn colum..-r"L of 5% FFAP on the same suppo;rt. SCQT columns (150' x 0.02"Y~of Apiezon L and FFAP were used when g-reater resolution was necessary. Results and Discussion Since hopefully a rather large number of investigators will be I involvG:d in ·the use of thE: A; 1eric~1 Pet1.'ol.eum Institu.te reference oils ,~ .) v1·c should pr1oln.bJ.y mcnc;.on .:i cou1)l ~ of obser·1.Jtions Jiktde dur:i.ng cur mat ~ r:'.al in our ~cunplr~s of tlvJ :ff. ~~ fuc~l oil. Fi1tr.tJtion of the fue l oil through G.:::lman g·l .:.ss f :i.ber type l\ po.ds removed most but not ;:-:11 . ' of th:Ls mc;te:cia.1. .. The material appr~ared t o be inorqanic arid dark pt·r 1JJ::·-black in color. 1d2 have not examined the nc:'tt:ure of. this material .. It i:,;1s al~~o Leen observ2cl 'that our Stu11ple of Kuwa.it crude contains a residue at the bottom of the containet' which ha;s a consi stency ~iliich :s different from the overlying oil. The pe-c cent by ~..:eight of the paraffinic, aromatic and asphaltic componc~nts of the test oils (determined by silica gel f ractionation) are given ir Table 1.. These l"'esults demonstrate the d:Lfference in.. : composition whic! · exists between oils. 'l'hese differences in compb?ition . probably give rise to the differences in toxicity described .. in th~: biological sections of. this report. The variations'in toxicity observed between oils suggest that to· evaluate environmental quality it is as i mportant to know the type of oil as it is to know the concen~ration.. ProiJleras are apparent in the determination of either ... of these variables in the marine environment. Uncertainties such as weathering of oils, micro.Dial degredation, and input of natural biogeni~ hyd~_ocarbons make determination of 1ow level petroleum concentrations difficult .. Also, petroleum ~esidues measu~ed in the . environtnent are rare.ly derived from a single specific oil. In view of these considerations we are attempting to characterize a few of the a.gents in oils which demonstrate the greatest toxicity to test 01"'ganisf{tS. speci.fic compound·:· fr·om p2tr,0J·;um could b.:: quite usef-ul e T11is data conCDincd \·J:ith our d(tta on whole oils ~:.:hould ena.ble us t o t ctter unclers ·-and f acto:c's ·1-.ld.ch affect toxicity and · al so allow us to better re1at::-i our data to ti·1e environmental impact of pct1."o1eu.m. Measurement of \.-he co:'lcentr'a:d.ons of a fc-;w nonb::Log2nic compounds in a .·sample c.onta5nin~J an unknown lJc.ickg··round uc biogcn:Lc nyd:cocarbons should be more accurate 'them t:he nqucsstimat~dn co:i:'lcentration of t oJ6c-,,l petroleum residw;;. The total petroleum concentrc.r'cion may also:> as mentioned previously:t be a poor indicc:-ttor of toxicity (see biological sections). A numbe1"' of physical and cheinical ~ractionation procedures have 1x-en consider ed :for· the task of characterizing and hopefully isolat;i.ng ·cox:Lc materials from petroleum. In addition to its usefulness a~" a tool to compare the compositions or test oils, silica gel colur.m ;. chromatography provide s a quick and sj_mple method for the· removal of paraffins i~om a complex mixturee Paraffins are often present in large ,quantities in oils and are the least suspect class of compounds. Their removal can therefore result in a substantial concentration of toxicity. The approximate ho}ling point ranges of the distillate fractions prepared from the #2 fuel oil are given in Table 2. Gas chromatog-rams of these fro.ctions and the whole fuel oil are shown in Figures 1-4. In an ~£fort to avoi~ decomposition of high boiling compo~ents and the possible formation of toxic artifacts VJe have discontinued distillation at atmospheric pressures. We have been experimenting with several methods· of qistilla:t:ion under relatively high vacuum. We recently prepared low cemperature vacuum distilled fractions· of the 1F2 fuel oil. 7 . 2; 4 previously rcpor t:(:;.d by se vera~l. v101' i<:.e:r.s • ' S2par~tion of aromatic compound...... on Se:ph~ cir; x Li~-· 20 i s pri marily du8 to adsorption of the , coma.tic I"'infr to th~ co.turnn m,:1.terial rather t 11an gel perm~ation normally •ssoci(....ted with the use o:f Sepho.dc;x in aqueous solutions. Therefore one 1"'ing aromc:~ tic compounds g(~ne:cally eJ.ut:e prior to naphthaJ_e:nes and naphtha~.Lenes pr(::ceed thrN.! rirlg compound~.:. Alkyl subs'titution r'educes the absm:ptj_on of the aromatic ~:iny systera, hence methyl naphthalern.~ begins to elute prioI' to naphtha.1ene. C'igu:c1es 5-7 illustrate tl1e separation of com)onents from a distillate of the #2. fuel oil which ·was rich in naphthalenes. The first compounds to ~lute Figure 5). Alkyl benzr~nes (tube 76) elut~ p;."'ior to naphthalenes o Dimethyl naphthalenes (..tube 86) elute before methyl naphthalenes (tube 91).. Naphthalene b2~rins to elute in tube 91 and is the majo~ component in tube 96.. 'lT'ne three ring-cornpotinci phenanth1"'ene was iounci in tubes near 120. Difficulties we1"e encountered when several crude oils were tes·ced on. Lf-I-20 columns. A significant asnount of many crude oil9 was insoluble in isobutyl alcohol and an additional amount was bound essentially ·irreversibly to the column material. The #2 fuel oil was howeve:c soluble in and comple'teJ.y remove9 by isobutyl alcohol. Thus far our work ·with prepara·tive gas chrornat:ography has been limited to the examination of several methods of collection. This teclmique u~ed in conju~ction with one or more of the fractionation rr.ethods. p1"0viously ciesc1"ibeci allows ·che isolation oi relatively pu-re 8 Fi~JLU.'C 8 :i.J.l us t~:'ct'ce s a cl"n 'c1ma togrom 1Jbto.i ned 11y analysis 0£ ths water soluble fr.,.:ct i on prepe:U'l'.~d :h:om t f.e #2 fuel oil. Cc.lr d.::rta indicate tho.t about 15 rr.g of org·.:;m:..c rrntE~I'ia.1 p0r lit<~r of sea water remain aft er ext:."action of -he Wt":>. tel' wi t h hexarv~: benzene (1: 1) and the. solvent is allo·7ed t o evapora··::e at I'OOJ:t temperatu;::r~ . The dr2Jnati c difference in pe:e cent compo~~i·c:i.oL of cornpouncL in the water solub1e ;,fr action I ( Fig., 8) vc:.:~rsus t h2 parent oil (Fig .. 1) can be taken as evidence t :.at t he wate1"1 solubles are not simply a dispersion of the fuel oil. Li t tle work ha.s been done on t he wate1., soluble fractions of Kuwai t 0 1., Southern Lou:Lsiana crudes at this time. ... :", · Summary •· Several :techni ques have been usc.d to fractionat e test· oils 'into . a series of samples which are more manageable analytically and more informative as to the nature of pet roleum toxicity. Sepa:cations b&sed on sil:i.ca gel column. chromatography have been used to characterize gross ·.differences b~tween t est oils.. T.ne technique has also be used to remove paraffins from samples. Paraffins are often present in large quantities i!1-oils but are the least suspect with regard to toxicity. . ·~ ~ Fractionation of test oils by distillation has proved useful a'nd informative.. Analysis of lo\'P-r or higher boiling components of te st oils requires special attention and unnecessarily complicates analyses of mid-boili ng range component s . The biologico.l data indicate This fractionation t echnique may thcrefore be a useful starting point f or th~ i solatj_on of the more~ toxi c components r rom oils. i 9 :in --, few ir ejor cci: pcu11ds Q t~c·l.,1iqcw~ promises to yi eld t.illi~jram qu.am:it i es o.f pure; con-pounds mixtu:r2s ~:rcr i test oils .. -.. .. -;, . qua..L'Ca~ive a.net quant·itative ax1al_1sis o:f components of nwate-r' soluble fracti.onsa prepar2d from test oils G The \·-·:i.h::r solublE.~ COii'tponern:s of #2 fuel o:i.l prepared by our mGtnod ar'e pr2sent at a coA1centrcrt:ion of about J.5 m~J per' liti:..r of sea water. •" :", · References 1.. Boylan, D.. B. and B. W. Tripp, Determinatic:rn of hydroca1"'bons in sea ·water extracts of crude oil and crude oil fractions. i~ature, 230: 44 ( 1971) .. 2. Kauss, P. ,; et al. The To;~icit:y of Crude Oil and Its Components to . I Fresh Water" Alga-2. Proceedings of .Joint Conference on Prevention and Control of Oil Spills, 703 (1973). 3. Mair, B. J., P. T. R. Hwang, and R.. ·G. Ruberto, Separation of petroleum hydrocarbons by selective adsorption with Sephadex LH-20. Anal. ·Chem. 39~838 (1967). 4. t ,1 ..... ~ M .,. R~ . i~t~ ~ d u ~ d- v.1.L.t<., r • , u • .. vCn ..1.. ·J~ un !10 ...en e, Colum.ri. chromatography oi polycyclic aromatic nydr'o~arbons on lipophilic Sephadex LH-20. J. Chromatog. 24:414 (1966),, ·' ~ , .., .J.. -· / ,.. 1 .) !., -,r ~ ,---.., 1~· .. ~,.. l l.1. • U:"·L ;..·.i'.:.: ; : . ~ ... ·r ... . , I..L • •·1.):l ,_,,•'.·1)··· 2 ..-.J '.·;·•. °j( ~_ /t·r.•-~ c 0 I,··.;.,,, ·. .:-··.u l · ' --" · · '-C • lil 1 :;_:':i~jlll'' C!S Pec.k ideN.:ificu-ti.on '· mm = Dirr;ethyl i1aphtha J.ern:::s I'•Jl{ = Methy1 napht h.alerh.:; S N No.phthalene P = Pristo.ne TI-ill =: T1"j.methyl behzene PEHH = Phenanthrene TMJ{ = Trimethyl nap~-1thalene 'f), t ,.. .. 'J c; I r./ ':.'est oil So .rcce /o Para: fi.nic / 0 r· ,...., {1-',,_ fuci' o::i..1 "f\.• P.L 57 Bunk.~Y.' C h iel i\.~.I. 24 J\ . F\ I. 37 So . lDuisiana Cl"'llue A.P .,. SG • ..1... V-~nezuc l::-u1 crude He-:;s 38 Alaskan crude Che.;ron 42 Diet~el fuel u. .. of T. G4 .. o/ AroP:.Jtic % Ac:phaJ.tic /I) RecovP-ry 35 .5 92.5 GO 14 98 40 7 24 3.5 03.5 37 7 82 84 '· 39 5 BG 25 o5 89.5 ... =-. · ,, r' 11 11 ;· 11 I l"'-. · ..-1 -·· t 'l able. 2. · Boj .Ling PoJ.nt Hanf!es of Fractions Dist.i 11ed from k2 Fraction n-Pn.raffin Range B C' 0 ..111-v11+ c c12... c15 D C13-C1 6 /'"I C1L-v1ri ' ~ ( C15-C1s G C15-C19 H :, Boiling Point Ronge 0 c <150-1 95 215-270 250-300 270-315 270-3.3 5 285-350 •·. 300-375 :, . \ l I II I I I : --..:;,,:--) I _r. "' =---~-+--+--l-l--+-l-+-+--l--l-l--+--1-4--< t '-\ ·• I. -·-.. I I.. -,__,__.__>--+--I --._ Figure 1. Whole #2 fuel 'bil -t--t-+-l-+--1-1--f--f--+- +---+--+---+,_-!__ ---===~-=-==~-1~J="T=~=1=~==-+-t--+-l-+-1-1--1 -- _____--·- i-- ----_--------t--F------~- ' --~F i ·--+=!~ -i --+-t--t--11--!-+-l------~==-----~- -· ·---- 1-r--=t=t=--rr----_ --------1--==r~----------=i_-==--= ===-=1=-=-==~-----=!----- ---.L _..,__.._...,_ ...__,_, __, _ ------1-L---. ---- ·----- --_ffij__ 1------ ------------+,------1---{--!!--+--+-·1-----·-----------!-··-1 +--·-·---- _ ,__.__+--+--+-->-1-I ' 1 ·~+--i·--+-1-+-+-+,·-+- .---,-· - ----... ---- -----:- --- --~ -.--·--- -. --~_,___ -·---i-t--+--1-1-+---+---i---+-+--+-I-._ -· ,__,__,__ -'---· ,_,_ --'-_ ,__.__.__ --r -->-- ----;.-.-------\ - ---I-->--1-1--_ ,__,__ ----,_ _ ,__..._.._.__,___,_.. --·-·--1 _ ___ _c--------~=~~~-__,_,_ =-;===-~.=: ===::t·-.__ ---->-,__ · ,.. -= ===»~=F .,_,__ ------·-_,__,__, .... _._[_ ~ , L -1-+-1;·--t--<-I --·---- ·---r1-~·-+--!--~-+-+-+--! -l-+-l'-+--1--+-t-------·------l--l-4__._-1-1-4-1-+-+-+-~--+--+--i---f--1-__.__ --:1-+-1-+-1--1--1--1-~-+--+-J I_.__._._.__._.._....,_._..._........_....._._._._~~_._..._.............._.1.-&. 1_,_....._.._.__._"'-'__._-'-~t~..L-J~~~~-.L......L-L-L_L.. t-t--t--t-tr-t--t-+-+,-, 1--1--f-~-f ~~~~-,~~~ul~1T-1+ l , --=~gi·titLl~fE~L-ITf~!-i_~ _ ._~·-~ ,_ --+m;··_~tt~~~~~w=r~~ ~••~1~H-+ ·T-r,_ ·-11-~!-H--!-i-· -]±t~H-1-1-·-i-·-r-~ L ==-=~t-1---_._ -L-L-.=:~<--~-::. ~ =-~=-~F-= -~ ~~~ --. -1 ---t.-= ·tt'===--= j LLL -t _J±-::~~:= 13 1/ MN ·-~ -~L-~1-Jtiil~c ~ t-l-·->-1----·- --·-. ---I -+= ·---'+1 I .L _._ .__LL-. 1--I--!-' . I . ---·-• · 1-:-+-j-.-, [ ~ -__ ,_ --. 1--tt _ • 11 L 1 =-- -::.::- ---":_"_-,~:= Figure 2. Fractions pre9ued by distillation of #2 fuel oil -----::::::-__ __ ir:tE~~t . -~:... ,_,_ ---f--f-• I I I 1 ~--1----1 1 L I H , --,_.... ··-·--.. -~-~ ~..__ 1---~--~---i---1--1-->---1 -·-H--1t I I ___L__LJ_j_ ~:::___ --->------1--=~::__ __ -e->--__ := ~,_ · c__ _,_._ ' -----L+1--I l--+--l_J±t_L_L_ _,_ ->------'--·-----_,__ ____ ,__,_ __ . t f-~r---i-11·+l ·-1-~...j ,_,_.__ ='--_,_ . .__.__._ _+-~r= _:_:_ er --r-=-r i -L -:= _,__._,_L----· ------8 0 I l I I • ._ ____.____ .... ~~·-··--------~-._-_,__..._: __ -t-, _ . l [ 1.2:_ ~ ~o 1 _..... ___,__ _ __ , 1 _1 -tj·t_ :_ • • __· __:_~ ·--·--·---------· ,__ ,_·-·---~._._ ·-t-~"-~ . -. µ_,___,_,__ II T~. . L I -I-'. ---..... ~ 12 · -· --· --,_._._'-I -r · --f. t ; 14 i :::: 10 -~---: Fraction A --11 -, Fraction B r -"--H--Fraction C i :-H-rt-IT, . 1 -_Jfh-r --~ . l ... ......__ -'--1-1--H--.--1--t---1- -L-L--L-·- ----±-!---~•->-t->---~1-·~·-l-'-I-11 -~---~ ---1-'-'-'--. ·-· w . -. ----·--~ ---. -·rr-!--i-1---~ LJ-~----- r-t-t--~-1--'--~..I ' 1 I l --~--~-~ -=t --. F• f ;i' -t-1'' ->-'t-:-1-1-i-;'-'-· '---• -- --------· I - 70 r--r-r ii--j . - 1--:1--1-• . _ _ I ,_-:.-j !.. -----_,__,t__ -I I I l ,_ -1----1- 70 + .I I I I· I .·jl.· I I -...__ ___ ,_ ----~·-4-.. !....I-"-- ~ ... . 13 ------_,__ -1 i I '~--~; >--· . T. I_, __ 1--1--!l ". • l'l t j ---J:-L L JL _ *'___ . • I _ L--,__ -!L~~L _ti L.--~ ;1, 1· ... .. . .• I ; I 11I I l:ttt=H-+-+-H-t I :• i" i :i CLTlllllj-1 I I LLwn 1 i--+-H --. L __._ ._ __tt 1L.=1~.:: !l----t ., I I l I I H-H-f I I I I I I I I l-+++W-i-i-i-i-1-+-.(...+-+--l-l-1L~ ' ---1 _, w--1-·-·-----r!·-·-·-'-t· . I I , ~1 ' I _.__H_r1.' -1--HL ~"Li.!-,J ' --: _1-,._ ,._._,_.__ 1-1__ ._ .....j_ ' , , ·"" ~ ,_._ _ __,_,_,__ _ , 1 I _ I I I • i i--t=:~t-;:__~-:!I ____ -i_ i_ f ,__,_f-_,__ _._ ,_ _,_ _,__ ,_,_ .. ,,_~--60 ,1:· • ·rr-t-i='--l__ L --! • I r ",J • ,1 I ~~ ' . ..,__~ ' 1--1--L----L---~--~----·--. ·-. ~ -·1 ----l fit 1 ----~ ---1---~--'-L-~--_____.__ --i-;_ I rL . .6q -L I I .l I\ ""~ 11li I I I I -. "--!. t i----r-t-----~-~I-,~ ·,~r-t1n -r-----t·~ --r-~--~---'-~---i ' ._,__ _, ij--fL-~.L-1--i-~~, 1-l-H1· L L--t -_f ..... _: ._,:~-'"'}-'-__ ,_~T , . ~cl : , .--!-,-~ 111 1 I , -- ---·--::r-{4-----1±+:: , ·-'' · ·--1-tr 1'~!f-J;:1-_LLL-'+t--J-1++f--· - : t·--11 ~ -·1-.i-+~--~L-'---r-f--,__,__,__.__'l=Ef= --H--r-1---+1-H-ir1tid1 "·' r -·-H--1-,--l -j -1-i-r-·--1-•-l---l--1-1---1--->---"-:-:-..-11' .__I_ c+ -..., I . r__ >-1--~l-1.J_,_ -----I l~~-JC'Ui-; DI·lN _o_~_Lu ~I __ c= ---, 1-..._ f ____ -----1-->---->->-1--,__,_ __ -· ] tl '~·'' ! : 1 _LJ _j_ I! 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"---1-_, _ -.--=::-__=·=--=~~==_ __L-~ =-=~~~-L__ ··-=-~. .-1-"tt __ IT :: J.I -~..-D' -·-'-----·------1-_-,-··--·-• ------------i- 1-- -:1·· _J. -_ --,__ ----. rt I r ' I l 1 n-r[ I LL . I ' -=-= ==~==-=· Tube 120 =-=--~+ t -lf1::r-1l1 p::ll-_i=::.,r~µ--l ,1 -=~=--==: Tube 96 50 -~.=·-..:~......., lo-l -----=-=-=-= --==-~ -~=~--=1---I ~Lt r ± , ..J..... C°: - ---· ------. -""TT . ._ __._ '-----'-t . -.__._ __ ,__ -·---,_ -·-_,____,__ --_,__,__ . ---_j__I_ I -I _I --l -1 O!~J -~ -----------·-··--__,_ -----------· --·---1--,___ ~~--------'----I -I-r j 1 I [_f• .11 1 L -~ Tfl -rr1::::+ -l-L·_= ::::.:::.:::::=:-.::::.::: =.I-:::.::: --m::l=1t.l=:F:: --,"----=:::I -----+ +----r: -,--. '--, 1t~lfFfltWB=h~ r _111-;T :=== -·='=~.::: ~= i --t~===:t±f.::Jt ::==1=.:_ :==1=.::====~=~~==·::: -=::~-:= -:1 -= =-i-r T -1j1hfrt=f 1=F1~mc1 ·-.-L' ~E·-_ ~ ,---l. _-_=-=:1=~====-===·===::1_ =±==.:::-= ~ t=::~ ::=~====·= =: =~=== === ~==-: ~ _-==u~1-·--1=t-r:t+.j-~---11-I! ITl~-1=1-ni1-r-1 -_ ,______ --->-±11'" r ,-1-=-t-rt ·r I LJ I lt l ' r-t -~-1-1j..:. -r1Jm~~-I \ -m-i ~l~ ----•------.fff=1 ! h mrTIf n-rr: -::: I1-: :: -=:: \-1~-==i-,-----.-.:.:: -==1-~::1-11· F : --::::: .: -_-j:-:: .:.: ::: .::::=:: : :::_: =-: == ~=[-::·11 r--.::: 1 l . . =::l-~ -1~t~ .· :==.: lb_::::. ::::':::::: ==~=-~6-=±:frt ~r;1.~.::::::: == ~.::: ::t=~:_::::1:: _: =:_-:::::=::[@'---~~::f XIi l1rTr-t1li iTiff l~i i ----------------------r -- -- --------- . ,}-~-,_ _c ---.I .· __ __ i-~-____,__ --1-. __ 1_ _--f+ __ l 1--i I l , I . l. l , 1 I I I • I ---ir 1· -----------1-·-i-· ------+---1--,+-~-. -. --:-. ----.--[f-_]_ -~-L _ l---_J I f _FlLL I [_ i--cL.u.L(1 ·r ~ ~. iT ----· 1 --: -==: .: _-:.:: -=1::-'= · --:-=r:: -i ==--~ : [::-:::::J:=:-:=-=: -l{ -:1=-: -1 +i 1-· -=J --+1-fHFl·q'-1 rT1 1-HT1 ~~1 -_IT ·1Jt1~ 1-; ,1=:t1---1;;m,1=;,=~ff:=·~=lT1~ _l :.~k ~11=-:=~:= _-j ~l-=Rt11--~~~r-1. : ,J1 ·:~:-:--~-.2 : ! iT:T 1 ti 1:111-nn~lft1tl1UlillHt~Fitfttn~i1f1*1!1f1t1~fl_J=jJI[f_~ 1JD·ttrtri~: 1,9~F,,~ .~~.~~,,~1 J~--I ~c· · -~·~~~1~· -~·~~ l~3~~~-~~ -~ ~~~1~F-~_~,~~=,~,~~9-=,p~~,~,~1~,~, ~~~. . . ~r~ ----: J~~~l~-~f,~~ ~ -~l-~-.~1 ~~~-~h1 +-·~ -1"== ~~-___J~~ Figure 8. The wate r soluble fraction prepared fro m //2 fuel oi1 ,~~1.~: J t · ,_,.. 90 -'---j '-'--'--_J__,_~ iii 90-$~ I --I 4~ ! -~-.------,-----1-~ ---1--t->-~+------I --!+-~-ii-~ · ! t--------fl ----1--l ·--·-'----·'-f jo t ·-'-f---~---~-----·--f---t--1 + --1-H ., -----1---___,_,__ -·---__ ,___,_.---·-'-1 >--------_.__ --j-,_,_ ----L ,__-->---11 -. --->--I-,~ ,_.__,__ --'--'-'--1P. ,__ t-_._ ,__--,-r -1-___ _j__ -,_.__ -1-------r1 -i--~ --t·--t 1---·---Jt . -----1---t-~--1-l-..~ --~L--H --·------z:..I_,__ ---~.__ -· --___ ---,__ H", ~---·=--, l ---------f-·-rL i-1--_,__ -if -----------.__. _._._ ------·--,_ ----_.__ ---- -----1------~-__ ,__--r---i ----L 1---1->-_t--·-·-----t i ---. +1±-·--·->--1--1------------>-----~-'--~=-_1_ ___ ,_ ___ 1 ._ n+1--==---t-r --·-·--~t----~-:~t -1----'------q+ -=~-c=='-'------. -_ ._ ___ .______ ____ i. ,_._--rJ=-', _L_ 1 +-·--..:. --1-t ----1--1 -----------1-~~-1---,, ____,____ .:i=.---'-1J -----11----~----------,-----. ,_,_,___ -i----E.__.. ____,__ -----1----------111__ ,_._~+-1m ->------u----'--}" -----"-~1:-·------r ------1--+----~~+---'"" !-'-. -r-f-L +-->---'i·-1-----Lt Tfl.m ------'--w N r--------I l-L--->-------so ---~-u -;-,___ _ .__ .L --~so ---1 . 'JJ-1-;1---1--1-j~--'---·--. ~J ±1·---+i-1------~----'-LL_-----·----------'---~-_,__ -__u_ -----~1-'-1---U->--Ul-lifJI -+--ft'_= ;-=~j l --~-==---!L__ ,_~----;Ff::=MN ~=-=-t==-~-==~~-----~--·:=_:=::_:=::_------_,_ =J= -=: =:= =-==-= J_L =-== _J=F~=:i:=_j:_-=l±t:r-1-F1_ nl___T --·---4-~ ------------------1----4----1-----------~ -------------'--~----,__1-__ _ --!----· _1-r j _ -'-i--1-- -} -,-1----ri-,____ -+-+-t--:'--l·--H~---f-l _ --r-I ! 1-----!'_;-+ -1--- _____,__ --fI~ ---H-"·---+---~ -i1----------------·----->----------------±H-_,__ -I-' ~ j ___ .__=: -_-'-"-_._... 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' 1 :---5 0 '. ; ,' i __.......___ -·---------m -i--t=E ---- --~ 1. H J ~r·-->--;--~---;-f--1-L---·-------------------- -------------------'---i _ ,_,_ - ,_.,J,:.-~: -~ -4---1----~----___ ,_ --r---'-'-__ ,__ ---1-+-1- -_,_ _,__._._ -,___ l[ -· ~-I-,__.__ l" Ii : f" l I -~ _,_ --~ --•--_,_ ------_,,_,._.__L_ ,_,___ ± HI_ -----t-t ..t~=--=·=qil1~~t-t~ ~~:-->---- 1-1--____ -Lt ---t--;L j, -~ l1-~-~'--'-·1-------·--->---·-'----,__,__. I --->--->-->--->--->--·--- - _ _·-·-_,__ ,__ ---··1-~L--~.--., i,_,__--t:·1~ ~ ~ ~ --·-t-. ~ i 1 _ ,_ ____,_ _ ~-----L-----~-----'---'--•-_ ,__ ,_ ->->--' L I --1--1--1-l-,__,_ -- --1----:~r L 1-· :-· -'-'-r ;: !-ti ; '-'-'-~~-r(----1-I----j ----r---·----1----'--'-'-,_. --i->--!-~-,.._-_ ~-~~tt"T=--~J,±~-{f r-fit~t1Jrt::,4 <3----~I:------ll,: ===:=-~====------·~ -=~~-·: ~.:=--:---===-£-== ~"!===~~:=:i~--~-I--fl=hf. __ J ,__ --~,__ --_,____,_,_ -·----~·>-_,_ --1----: (. ---~------L-r-~-· --'-1~--'----j __' --I_,_ --•-H 1,1 ... i ". '-·t -.t,-i. ' ' ->-->--->---rj I ~ :ttt1-1-r1-===~l _L ~ ~1; -Jl~:: ,,=:;=:=:.·::_ ,-,--f:=: . :._: =-.r ---:.:-: -_:: ::::.: ==--------f=~ =~~--=-=:: -_: =-:.: ::1=r::~: l _::: ::::: = ,j; 1 ~Etl1t-=F=·~":_-1rl:--_,::::-1v1~===~J[l =~1-P-"1~11--1-=1==1 -:1::: ~=-=~=i=~= ---::-::-:.::::-.::=::::.=: ±=i~~i===*·==f~=~i~::f-:~1:::::::~ =trH~: rrrrrn:: --;-e ----!------cthi-c-§St .rr------l-~----, !-t --·---=i=::::..::11=::::· • +--·r+r==---1 pt§.Jof=j-1.. ___ --1=r~i-EE .::: t --~ ----r-+ \"Y :':-·--;-l1.l,=::r!V ~:: :: :=lf::::'::~r=r~!l::=::~::~=ti1=r==~trw~Jffw=f±Ft=~L,. -~ --.-1j=f-t2f.~~~L1~nr----1=1=1=-H 1-~r---_tl ~-~-==------0 -=::·=~: ===~ -~,~ ~ 1~I ---L--i_~ --~~--1~=-~-::~=~1 -:~;:)Kili' 1:~. -~:~I-~ -~ ~t _~--·-----'--'-'---~--·-----f--I_ ,__,_ ______,__ ____ --------------____ ,_,___'-_( --t -t=-t ·j -!~ ----ti-1-1----.-r I !_I ' I 'I ____-~~ ~ _:-=·=__t..._~__ j---,---'-'-:= ='""=:==.-="=-=--~ ~ --------~=-~---1-=-~ ---=-=-=~-=·-=,__:,~--= ---"t·L=--~J-1, -=-ii rlJh-,_, I-~r -_I . "l h H --•-'--'-'-'-'---'-'---~·---~~ ·'--, , --~, -_L,-.--~d-+r--,-. -~l l_~-1---t----!--.·~~ SECTION D NSF-IDOE GB 37345 Effects of petroleum on marine animals and larvae J. A. C. Nicol rv-• '· .. :, . i: "°"· '· Fe.su1ts 2.. Fer·~il:i.za.t:i.on, cJ_,:;avag·e and .le:.rval deve1opmen't •" 7, • a. Fertiliz&tion capacity b. Sperm motj_lity c. Respiration B_.. Barnacles · 1. Develop1:1en't of eggs and.. larvae ChthamaJ.us fraqi .l:U:t -----------=-- 2. Beha.vior of larv.:ie in acute expe1,irnents ·c. Cra.b ~rvae l~ Striped hermit crab Clibanarius vittab1s 2. Spider crab Libinia dubia 3., Edible stone crab :Men:i:ppe mercenaria , ... l •• _ • .,. • \,.,, ..~ \ :.... I ~\.. ...... L 1.;. (lo '· \ '! 1' .. VL .. -:', · ', ,.--, .t. ' .. 'J,'~·-l(_-, l~;;:... c]~~~.. ::. r~:L-:~~;:1 ;,:~~·S.,_\~,;:.j. t;:;(~ 'f~J.1:,J:, i, :cesea:cch q invest:i~;a.·ced., a sc-=cor..d~, a fourth, ·vith :feeding t.ehav:~or of .fish; !# cind a fifth, \d-1:n c2.:rdj.2.c X'i~Sponses under oil stress .. crabs -----~- '1 ,. ,.~ ·-i:ra.:.)2 .:i ~. . .... '_;;~.:.. .. ·_ 1:-e:i_}-")2:1 ~~ :~ ~,: ·.ls ~ ,, __.. .... ____ .__ -----"' and sand exr:~rim:~nts or ... -, \.' ~ J • : • . "".:, ": ~ .·-. \ ' ' .L .... ·.,. .. ·.i \ t I i ;_,,'' ·.. J' . t \ '.-.. •"'l 0 ." "\ r .. i•,, f l ..•. •. ~ · ' -. I ' ~ (.-! - ,,. -· .. ~ . ,' ,) .... _,_ ..... ····:::.··: ..,, . ·1 • ""'°" ... ") ' ... . • "' .• \.._.., . ~ •·, '-"' .-\••I . .. --l ,._....... ..J .. ~.. -· ~. cca.stti.l s·~:a~}2S h&.ve and Ext,~nsive studie::~ the I_:-2r1r:.22,bility o·f animal ce1ls have . ,/ ('?.>Jcl\.e and i1c;Cutcheon, 1932) .. / / p1;;.lcc:_lpods Osty(~~:: <:~1--.d ~2:.~;·<~~ITL~ ( :!:"'~e;;_((~ , t ut!l'e and -i_cc.:::., 1941) .. .. _,,... I • i ' \ ;_ , ' :\~~ : ;• ~ ' I [ ::~ \ I : l I l :( : i : j ., ·, I :I \; : I . - ,. '. ,.. ~ ," ~. I ; ) ·' I ., \, < J w ~ ~ •• .! . "I • .i i l'-\ ~. ' -. ..\ . 1 ": , . " J / I 1,. t (.. ',/ l i•...~)<'' 1 S, .i. p 6t 1, and the / ( .li1.1C l<.e 3-nd existing be·':\.·1e2:a ' the c.~:. l 1 :I..n-~e:rior :-, . '°' ·1 l ·~ 'r" • .;...y.::>c;..J.....t..i.l_._. , , icnic co;npositi.on, etc.., of the raedia; 1917) .. Eg~p and larvae oi tn"Chins are very sensitive t o slight differences b-2twe2n Allen ( 1971) has rn.s.dc cJ. s t"udy 0:2" ti.0 effects o~c p2tro.l.:~um" L a.ctions on early develor)r,1t~X'.. t of ,..-'"' / I.___i.p2 c::-gs or ::;2nd dolle:u~s are ee,sy to secm:-e. o.nd f ortilize, they develop.·:.:o the echinop1utcus stag·e quickly, anci deviat:.:.ons from normal cleavag·e and morphog·er.2sis are detected readily.. B.. Barnacle eggs 2nd l.srvae Develop:-nent ac:~.;r'egated :1.nto t v;o eJ..ongatr.:: eg·g ::'.asses or lar;:ellc..E-~ , cont:>..:i.ning· a .J .. . . . .. ; l .. .., ; ~ _, ~. ' ·' ~ ... ... L ~ .. : .} .. ( ·' .. .. : ~ ·. •' : ~ 1. ..'j ·~ ~ ...~! I/ ; :~ c ; ,r :: I 1 ! '-.. · -· ... I,, • •• '-·. L". : - I •. ~·: ,. . J _' ( ..... \.-• ~c;:·~ .. tC rt.' ~-~ ~~,J ~ !.. I ._ -, --............ ' l.l. ; I ~.c·(:'..:~1_: :. i lt~.. ~--.. · ·· ;·l~c.r~:t~1.l:L·t~t c'J7 -J.::1.. :~-''la ~ : is }--::·L0j1, c-.; s }:'~~--~ ·_~,:~ ]_J_y.. :) ,.~~/) ~~~~,-!~j t:1-ic. t:1~1:~_rlrl l, r:.~,_~:.,!~)J.:L~·;..~ st:a.;(~ 1968; 1970 )" As ,. ~.. exp21"1:i '::E":nts Larval survival a.nd behav"i.or' in a.cut<:;; e xpe·.c]Jl\er.ts Nauplius 1a::c-\1ae of ba:enacles a:r.'e r03J.e&sed in eno:r.·rr·ons nLJ.rrtbers. They pass throue;·h six ne.L~pliar sh:i.ge: s and one cypris stage before Durir:g ·the no.uplius f·2:ciod. .t-"'1.ey b2come part of the tempor&ry plankton and depend upon micro0r·ganisms for foodo This pel'iod Lists .IC\vo to t hree ~·iecks (Bassinclale, 1935; The~ larvae on hatching are·: ~strong·ly posi·tively photot:ro3_)ic, 2:i.1--.d tJ.1e cypr:J.ds become negative1y phototropic l or ., '\ Pa.Di') .. .... ..1 0..L ) i. ; ·1'..'.:I c ~ r.n rl """o ·::est l .t:. ...... -~ ::) . . ··'-"-• . _, numbers of <~a:rl/ a.nd such as muL:iJi.ty ~l ... . .... . . .. -. . ·1 • '· .. ';, ., ......... I•.... • . I \ .• ·· '•-0\_ 0 >I ., ; '· 0 ;,, , l , ·; ... ::•. '~ ~ . • •*. ", '",: t ,·I 1~:~ }j~ ::. j·~ I:, : ,, • ':/-_. ~··.... r-,·.-,r :' ,_ ! !..' ..... · ·1 ·;n) ..L.:1 /. .. l, j/j~'(2·~;2?·i ·~: s·~-;·. 1\.1~/ -r:~-~ ~J~-:,:..)~.. '\: s~.. c~c:i~~ :J c:c c·.i--:-a.t,s, ·t:·:,'t::, ~).r~2..'°"l ·;·~/u.~~lar:s G~-c~:~l c;~-1 :~ ; ;J~.. H)~-l·::i-:..··;=: ·__1, ··-,~ , .... ,...; selscting· J. l.)'..J'--'· () :food ny s02rching ,_..., ..c v.L act:~v:i:cy 2nd i•eccgLize ba.r·bels 1958; Hara., 197l) .. catfi:;h eXpOS•3d to f2eding behavior and 1:_-:xternal tis.--.;:..H~s ·v~··e:r:'e pre serveq ·fo:c· histo1ogic study., Ext2rnal E::Vc:nts aff2ct t:h~ activ'j_ty of t:h2. fish he21't tk".'ough e:a:ediac rofl.c·.)'.cs m(:cl:i.n.t.:=::cl by tl10 v::10us ncr.v~, a1x1 b:r:• :-:.dyc ·-n:dia ( s.lowin~J •4 . ·1···t·) _ ... . J a wide: Vcll':i.oty of ext~:cr~rJ.1 such as sali~ity and. ar.oxiu. 1958~ 1970; Har·vin .I Burton; 1373) I ~ To sen ·i·tivity :ceCOl"'-C~Gd . c&1'cl:!.2:c acti :ity of s2a cat:-i[;}.1 /.:.:c·ius fc:~l:i.~ t~ ds1'1 nor;::a conditions ...,..: "l ··:o ,_,.J,.. . o~:· c·:; to Cl4 C11 t:o Cl.5 r: 1 '"() ·:• f) I CJ II:C ,,_ -~..:. ... .. )•..; \) C12 to Cl7 IV (•l..._,_, _1.'ir to C19 ...... v ClG ~-0 C2/~ .--.., .., rt.,,. p~:t 11,:J. :Le r; (; ..:-J~:enanth:c·c-::n-3 ana Se:lu.r·a·:.:,__d o.Lls, and putati~ie ~ 1 0 .J . .;_ 1 Oil \'las e::ddcd to sea wa.ter :i.:n the J:'c tio 1 : 8 : ""-0 mixt.11re \·1as c cnt:i..lTl.1.ously agitrted for 24h by a magnetic stirrer, and 'fills aq~wous mi~ture, term:!d o~;.1 and o:-.1 frJ.ctions were adci.ed to s2a \·1ai:2J: ( 30 °/oo) in t:he :c'c:-.:tio .L: 8 (by vo.1.urne) a.nc"' the rn:ixt·Ln·0 1.·12s ~c it-J:t 0d on a mscht.L1:LCa1 ] ,. . .:-, ),·,·,c,.1,· ',", 0 ( \1,~(.· ,I ..,,·.1. '·')· I.·,, \ 1\, 1• .J •y . I'vi,t ') .l L', l~' "' ,, . ) ;i ~ • I • • .: .:.•: . < .': ; ;. '· :: ........, •,. .. _,..,., ' ..... ... .. .t .'.. '..... • t . : ') ~ ..' .! ".J , : : I I ~ \ l ! ~:~ I •. .i.:; ... ...' 1~ .• ( ... .h' : ·~ {' • ' ' 1. I • \ I ~ .' :..~· -. ... ) .) ~ .. .. /' -..:.(:·. .......... ' . ; .·, ·--: \ . ; , . ........ ...... .-· . .. , ... ), l ,•.i.." cc l J:..>:~·;.~ :;,:1 ·; .-~77 -~-.; I . .., ... .. ,.. .... C..-...\...~ 1. ... ( -,·~ -01 ·~ '·.. I \ \ •-'.:_) <' ··-00 .I ~" --~ ~ to r, ,~ bct::n J.. c..t ..:...":.1 ...:;·_ 35 .. S by t he of e:>:.plore .,c"he poss-ible P~yt'i'' "J.. (-'.'11, 'JT' ' OI '; ·; v --\...{.. ... :: (J .. S~d.£t: v-Son) e:yep::;..ec2 (V~'..c:~(f;j.:'S ) .. -~· 1 · I • ,; ·-., -.. ..,.... _ . . .. :r,. ...... .. .. \ .. ._, .i'. ·... '·, .. .. ...... ·' ' ...· .i \ • .. ·1 • -~ r . .... , ·, • 1 -· ·~ · ••••• . . ,.. .. • ' .....1 • • '-· ...i ···-~ (. ,.._ ... _ _. .. , .. I '\~, ,.._ ! ., \.. ;... ._ 1\jd ..L .... '... JJ! i . ~·~-:·;-l: _,, r~·· ·-~,'~~(i. .J.-_. ... ...... .. ,,..... t.. -:-... •-. . ._ ~ a_ cover :rJ..a.ss '* pip2ttc.. s .-:i ·"'r'1 microscope 0l:Ldes V . .!. l'...A. use for so.lutio~-: .. eaa-s _, _, d2v2lo1-S..tent sta,ge attai-r:.8d on :-slide ':7as compc.:ced with a s necirnen freshly dr ·a\·.ll1 r:com the cuJ.t:.~r~· bm·-.71: there 1;10.s no obs2rva0le dif:feror:ce co Ac of ':.ntil l .1.., /")~of 100 ecrcs ./ -> '"•,. \.' . .. .. .. . ... : . .. ' 1 ·, • • • • ! ... i -.' . 'J .:. ; ' . .... ~ ,.-,_ '•. '· ·'. ··' _, ' .... ~ -. ~ -~ • I • ,'_·"' •. : '·, l ,"' ' '• ... ; ~ ; .. -I (). c '\ ·'\.. '· -cid2d -Cc 20 ml of -'c11c· va.~c:10'-.1s s~c.ck · dj_lutions ( c:x1x~r:i.msn-tals ) and to 20 ~l of sea water (the 9o~trol); dilutions of oil listsd in Table I I 'l'"r. ~ --J. J."' sper·m l'smain<:;d in each solut:_o.1~. e xp·2 r:i.IT:::~ntals .s.nd co:-i trol, of eggs in se2 \·iater· (30 O/ oo) .. Obs _rv ·:'tions wer12 rr:acle or1. £0rtj_liza.tion and ( '·../ SC "' 'i·.7;::.·cc~.' . of f :I:v:_ o:E oi l ,···;:..'· ,, , l · - . -:· ... '·' .' i ·.' ..' I'.,, ... .~: r. ' 1,·· \'' -'---· ..., .. :i ''; '+ l •.. J '..: ...· ~,) :• t I '' ; ') { • l , • ( r ·. •. / . ,. ,., \ . '' .. 1_ ,: . ) " • .J : ~. ~ • ..... ~~ ~~; ·' C; o ,., ·:.... J. ,.... ····· -: 1-.~· -1 ·-· ..t ~ ......,,,.. ,,.. *' . .., ,..., .. t. 0 be no sy~chrony of in tb2 popu1.( . _•• _ ._ . .. -.-... l . ' ,I J • '-.:. _,__,, • L '~ ) .... . .. . i :\....... ~~(~ :-2·:~ u. ' -, • ..;. • .1o!. .... .. .~ . .l.Tl ~:; ·:c)C:i<. C :L .: .~\..l ·~: ~1.0:··~ I I'·~ 1/JD> .,>., ,' -J ") ·" ,")~'"'..?. l-:un<.l:t:'c::d l.c:lr."J .:,:,~:: . \.) .i.·,._-:; "'L1y o.ld:., \\\:::~ce :Js r:: ·-1. in cc..:c.n '.~;":t.' {~/t..;p, D.r:d. ~-:2--:::.. of l~(~d. i urn . !; ··..__.r" • I ·~ •• .. , . ,.., \. ~'. • • •, I~ !. j 1 • :---·~: '_' .' ! ;:·.( ·1 .. ·-,I_·.:. ~ • ·-t. 1:....... , .. • ~ ·1 • .. -..·~. , ., .... ' ··;c ... ·, :L.·'(.. ·'.. t~;":_; :.: pip:~ttcd :i.n:co le.~-='~?=~ '.Fou.1~· 2.ar·vae they vF~rc Each ~-:rr·oup was kept in m.J. of solut'ior~ .. ·~1r·oups ma i:ntainev. t he f o lJ..mdng (l) s C' .:.1 1·:.'o.·ter 30 O/oo (control); (2) oil stock-seawater 1/50; ! -( 3) 1/25; a:1d (4) Ls.I'\10.e \·12 re counted '°~:-ld tr2·~Lsi\::rred to fresh solutions; three~ drcp.s '.1: ') .. ,1... '··· l \ .)· ... \ '\ "'i ~ •••.,' • .... ... ,.,.I -·• ·' J, I •' : . • <' ....:....: .... -·· ...!.. ·.\ .. :.;: l'.·f ~ \ ; . ! -~ ~ . . . . ~'.., l.,J.. .._ IJ '<.-1...;1 "'\ .. ..... ., ;) ~3.:, f -... :)·---~:..~ . _j ,' r:..:. ., -; .' r;· ... ., . ·--1 _._; ! -;l ~:r;\:1.~::·,·:;C~ ,. c/~· f()1.~l1 d. E:.~:73 .., to cc~taining oil ~ix~1res I r.c .. ~ N.., Y ); 0~1ly f~ea .;.\. .'.t: ~ ,, '. . : 1; \ .,.... , ; .;.,, .. . ,, ' ..) ,... _ ., ........ .-.. -: '····'· ,J -··, I: : ""; \ . ~ ,_J ...... .. -·-. . . ., -.~·.. ..... -. :;:· i : ,. .. ,."j -' ~ ' _.. ~. • '• -: • I ' . -\ ... \ i_ : ·.·._,.·_ /) ·. c·_! .(; .: ·._:~ .-:_'<.: .-· • • .-, .) J... ... -. .. ' -~ .:.....:..!. .. L :_." ... r .... _.,_ A .'i ""' :..· .i. .· . _ ·:~ ·-"' · • ._ ,~t;C. :-;: ., '"/"'' ···, -· ··--/ _:_,_. (.: i "' ")-. '/ 1 Ir --1 :,) ' o.nd l /25. h .. ,-- •• -', j •1_, . ·, ·. :J.' -_ ., ' . \.},, ... '-··· · .~ \, '· :··... ,. -•-r , I t ,. -I • l ~ I ... '• ,, ;.,/. i..,) ·-··· .. \· ·.:·: ........ ~·· l. .• ',._~-l . .. ·-· '1, j ~-_., J. ~ 7 ·-..r ... C:.. .-,, ;; ·•• J.1. .!.. -.....• , .. (~ '-(-: · _:; ,,,' ~ ·,,: • : ' : 7"' .~Fl ·~---J '• :-,t./ .. ......-, .·: _.. .... ""; " l.: ...1..~ ~-_ _ ..:·:.! t V.l . -~+(20 cc:::·~~eas /:: tiss~G ·was rinsed ~nd two small pieces were reLloved, one control, ~he .., ...., "';--, ...L.. i : .... v the ~eai~Q be~~~en ~e~dings; total duraiion ot wu.s 2 ,_ on<.: ~ ·:·. , . ... '. ._'··. '· .' ..i . . ·'I ·'· i I ~. • I" • '·,I ,'I ·:.' I' .!.\ •• I • ,"j. ,.·,, !.' · ·' ,. 4 .. , . ... ; ~} . I.) -' ·'-· ~ '.. ' ·. .... -. 1,. t .......... -'..): ·.'. \. . :_~, _l... ;~ .i :.... ~.) . i. ';·~." ,. ·:~· J.t" t' :... ..:·/ l, , . ... ··! \, .. J. ~ \. ... .; ..1 . ... t . ... .i.. ,",.; S~]\·~:C i rn: .;r1f; ~v·,::~ ~c~.; c~ (\J.. ..12 ·~ t·e2autif-u.11.y :Eorrred pluteus larvae; there were ver·y few undeveloped eggs on the bottom~ ':'he results of exp.2rin~ents with petroleum oils are sum..1krized .... ·· 1 _I . .. •} ., ~I .' ... ; ~ .L ' ·'·. . . ' r "' ·' ~· -, -. ,)·," ,.., r'1-r -.. ,, ..... 1---1 ,,.... rt u. --i ' 'J :...) ~.11 ( ·--· · _,:.__ .i._..._ •.,. ... _ . -'-t,..i 1r'b (__~ .1-~ ·-;,_,.;~.: · ·'""'-1-~ .. .. .. a..~ ' '~ _,_,.. ··--' ...L--~·_ ...... . --,1 •.~,: '",·,.~ ._, .. _ rr:. :• ·.~-·_,.'-..~_,_.(J:~ /-·,\,·,··••~(! -i -·-----·'-: -~ .-.~ '·':. ·~ .· r:: ·1•• -·~ -~.,i~ :-.. -· -----~ -· · '· t o detect s rr..ilJ. ... ·r ;') +--re'.\ ·1 I c ri .~ 11 , t ~ o--1 r: r) ::· J.J. • I,__. L -:_) '-A->--.A .L. 1 ~ ,.; .._ (~ J , ..; .,.., :-i •' \ ...._ \.. ._ ~-'~-1..• .. ._ c1early ciistinr;'.._1i.sna.bls :i except for a sug~·estion of one .~:eound 01:;.1e egg 1/10.. 1/5 anci 1/2 (Table I) showed scme inte?.resting and sigTlificant l''l.~sults a:t f:i.rst cleavag·e .. In 1/25 a. renuced number of cells en"'c0red firs·: cleavage, all o:E them were norr.~l in appearancee In l/10 not only.. w~s there a r'educed number c:r eel.ls going :into fi1.,st cleavage, bu.t many o:f them doing so clove in arL abnormal fashion. In tfte J./5 dilution _on1y an insignificant of c.leo.vag·es a-pp.2ared normal but a lar~Je number shmv2d abnormalities" :. P.s rr;ight h2ve been exp2cted from 't.4te absence of fc:ctilization merabr1anes ·_i DL -'-l·v;;_ 1 • I d·i .., n "'-i'on I... .c .... . .J-..... .J....l..-l,. .. ) no cleavages occurred. in ·that mixtrJ.re. J.l.t second cleavage some effects of the oil 2.-t 1/25 dilut:i on were seen, tn2 propcrtion cleaving was slig-htly r educed and a few were . Ve--;.~y 5ti.~o:;.g e .£..~:ec·cs o.f tl12 oil ~-.'eJ.e f~scn :1n concen-U\:."ti.:_:1s I ! I . ,.... . ):: • I / ..' I ' . /.l.l) ·J··1 .. r . : i... , ... JI '../ ~-: r ,. ... ( • •1 I l .1. '···-.1 ..·..'~ .' of f .:Jtt: · cc] :.L s r:o. J..~ i"Jnci .1. )::°~ ·".' ·n.. ·~-_ c·l ..-~ ~1 ;·r~r-. ·1·.~ . ·r...c~ ._, _ l. . ' ··'"...\ ·1,-) ·1·i;·1 ·~,: it · . • --• . -I \.~ •:'· :· ,.,,•• • .• I 1~c1:i.~c;d, :Lr great n:... moers, an.:-·u~cr'e wc:r-e very fe..7 nnd.2veJ..opt~d c~f'JS cult.1u:'2s .. I~ :2ue1 o:Ll l / ·') r: _.._ £..J j~.lJ.y ca.psules .. In fue1 oil 1 /10 thr::,rc was about aJ1 equu.l divis~on a~1d :u1ck of deve.lop:nent, and 1iviEg larvae., These la:cvae we :re ccnsid.2rably retarded in developrm~nt· compared to tr.e control larvae or even ~o those in fue~ oil l/25a In fuel o:Ll 1 5 the bottom of the bowl was covered with deud e.ggs, . mos·tly undeveloped, but many stilJ. shovling various stages of deformed, aborted c.J.Bavages. At first no living la:rva.e ~vere visib).e; however, a very small number of globular, cili.atPd 1-y·v_,e ~ °'·:.. q located near the surface of the water at the edges of the culture bmvl. There were no l.arvae in the !i/2 dilution. Fractipns of No. 2 fuel oi1. Controls in clean sea water developed no1...m.ally and well formed pluteus larvae. formed in abundance., Re.sults fOJ.'." the exp~ri1:-1ental an1.maJ.s are PI'·esented j_n, TabJ_es II to VI. ~\11 fractions int0 rfered with development, some more than oth~rs, ,· 1.rn0qual.. rr,ost d:~lut::~ to l a.1vo.l c!.eve:Lcpi•.cnt; -.., -t r..;l1. ii..... to larval c3.eve1oprr.ent of all f r actions test0d" ... :-, . Fe1"tiliza.tion c apo..bLl.ity . T11e results of "!:h~ second f>G1"i·2.s• of exp2rim~nts in which the sperm were placed it1 oil-sec:;. water rnixt&.res b2fo1"e be:ing used to fer··tilize eggs in normal sea water are presented :n 'Table VII. Sperm maintained 1/2 h in sea water and in all the oil dilu-Cior..s except No. 2 fuel oil l/~ resulted in excellcrrt fertilization~ as shown by the presence of vite1.linEl membranes 1/2 h after t he s perm was added to t h e eggs. Fertilization in t he various exper::i.~n2n-to.l cultures ranged fror.L 95 •to 99~6, except for No. 2 fue1 o:i.l 1/2 where :fG'!:til·ization was z2ro. After f ertilizatio:1 the eggs p~oceeded into ! • first and second cleavages iYl a p2rfectly normal fashion. 'Obsert;ations of all cul..cures at app.L~oximately 24 h after fertilization r evealed perfectly developed pluteus larvae in the control and experimental media, and very few undeveloped eggs on the . I _ _ , ..... ~ l·7~.t::: ·f-\.1P l OJ.. _,_1 l / • .._'J Ci~J..--·!-;••1 ·~~Vi':"''<,~~ ... , of cr-.uu_·-,·.~-., -., again an exception, there we1"e no larva.e in this culture. O..it of a ::' '-i o: . ) r ). \ J,'j i t 1;··,. -r:o . : .J.. <.~nd \Jo" . 2 :·:10·l . J./') '"(': i"'-' ..; ~-·; J,. ..1 •• • L. ··'-·.1. · . r.~o·cionless. :i~.esDir'e.l:icn ., Oxy:,Jen upt:a1<.e of sperm (corrtr·..... l) c:1t th8 star t: o..· ·co /1 '"' OJ.: -r: ~ i t -o....L.i 00 '-~ u;,~ o..: ·~l1& vaj_ui~ ~...L.-. ( ,,., 1 1 IY_~ ·~ . ., ·; • mJ..n .LC:.d . .c ,.. , i· ~l~fuTe .) • Re spirato:r'y : ra.te v1as ze:oo in fuel oil 1/1 and 1/5, and one-tc.;nth of t he con~:5o.l .; ,~ ~L ;0r: ' .J. l l .. .... :) :falJ.i.ng to Z E, ro :m 40 min ('.rable IX) .. Da·ta in Neecihc.m (l.931) ;. derived f:o:: om Wa·,.,bi.ll~5"J and Sc he.a r e.r, gi~lc a ra.te of 0.18 x io-6 pl . -' oxyg~m. per sper:n h for sea. urchins (temp. ca 23°C). G:r~ay (in Needham, 1931) found highe1"' and va1"iable rates. Barnacles. Development of Eg·gs and l.ar'-'ae Cht:ho.ma.lus fra.gili[: Six pairs of lamcllae (controls anc;l experimentals) Wei.."le unsucces~ful, ChYJ.d were not--i~·1c luded in the data. 'l'hey were at starting stages 5, 6, ? cf 7, 7, 9 and i2 •· Controls and experimentals failed to develop b2.yond the starting stages a:Eter 8 days. P.11 other controls, rang·ing from starting stages 5 to 13, proceeded to hatching and yielded larvae. Not all embryos in one 1amel1a we1"'e at ) of de.vc~lopment and consf:..quently they 1Ft\..''ched over seve1a1 wJ-i.ch cl.~r:-:d witrdn orn~ day . E~;··g·s at ste..cre 12, from : la.m~::lla~~, d0ve1oped ani 1c.tch::'I( ove·"' se\leral days; the la:r.1va :, d:i.·:!d ~;Jon aft·~r; h ·1tch·· n~j., I.1 7 j_c;:.m...J.lc..i.:~ at _sta~-re ·11, sorrl~ egqs dcvc~loped and hatchei:i and t:hc lar vae soon died; other eggs were a~rested and died~ Eggs•• at -all earli~-r' r;tages, in 14· lamellae, were arrested in d.cvelorment and d:i..\:.=d .. s·toc"\ -s~a water 1/2.. Fifteen larncJ.J.rle WC1"8 :i.nformcrtiV'2!. Emhryo~lic d<;;velopmf~l 't in five larrK~lJ.ae or'iqino.l.y a·t stages 5 to 7 was gcne1"'c:.lly ret:arded 'in early stages of 91.'owth, with consequent: : death of the embryos. The effects, however, ·varied from embryo t'? . embryo: lamellae g(~nE-~rally showed embryos in many stages ~~ dev~J,._opment:, and t wo lamellae even hatched a few nauplii that quic}dy died. In two of .three lamellae, at intermediate stages 8 to 10, embryonic · development ceased at va1"ious stages short of hatching. In the third lamella hatching occu~red in som~ abundance, ·the .larvae were active only a short .time before dying.· Embryos"' in later stages of development, ll to 13, were s·tudied ip seven lamellae. In all of. them hatching . proceeded n~_rmally and, in two, more rapidly than in the controls. Larvae all died within one day of hatching. · ?tock -sea water .1/5. All fifteen .Pairs of lamellae provided us~ful information. In five larnellae, originally at stages 5 to 7, · development appeared to proceed· normally, and .survival was good ofter hatching. tn three of .the five groups, nauplii of both experirnentals ' ' and controls t'Jere alive four clays afte1" batching arid eight days after ,, the cuJ:ture was begun. Two groups hatched. rather small numbers of I ' "i .. ..., -. 'l -') \, ·-: ,.~ " l\. ~"\ .. ... ull. ;.l..Lch...V...... \..fl . · ., _,·1-·-· --· ~·· f:.H ..1.•.1. yc·...:. ll• .. ') .. 0.ljl:. ..; a.Ed, .:...n one g-r oup, hu.t:ch:i.ng occurrc:d two days bt.;fo:r'(:; thG con.tl"01. Lc-.. rval survival w.:1s c1~ratic: t, :i.n each cu ltur0: , several days aftsJ.:' hatching began, there were approx:i.ma'l:oly equa.1 nur.u)ers of 1iv~i.ng a1 .d dead l.arvae. All experimental larvae were dead five days after ha"tchir1.g whil e t he control s w12re st ill alive! . Oil stock -sea water 1/10. SixteE~n of nineb -Jen pairs of l anlella.e yielded infm:'mation; in th ree) both the e.x.perimentals and contro•·ls failed to develop.. In two of four lameJ.lae in ear.ly stage_s 5 t9 ~:7, development, hatching· and larval survival were all parallel to the controls. In one 9Toup, larval production and survival were less tha.n in ·th€ cmrtrol; in another no hatching took place. In two of four lamellae at starting stages 8 to.10, :there \'~as equal embryonic development: and hatching· in experim(;ntals and controls. Larval survival was as good I as that of the controls for five days after hatching. In l\vO of the four g~oups . there was no hatching, but in both cases the controls had ' ~ few larvae. E~ght lamellae starting at stages 11 to 13 were studied: ·in all. of them embryos proceeded to hatch~ng, and larval survival was about the same as in the controls. C; ·~ In a typical instance, experimental and control larvae were surviving six days after hatching had commenced. The ~sults, in sUJTu~arized form, are presented in Table X. ) / ... __ '"--.. '/ . i t... , r:~ ~~ l)r) ., ·1·· ;i{-l, r., ( l...' ·i rn t;_ l .....~ t cl ""'" \... ·'..... . .::,.. ,..' r·n Iit" ·) fm' J_S ;11.in the .l-\...--; i:on 1.."'0.L 1 · ·:. vac rncstly ~JcJ. t'ncr~< 1.n t llc i lJ.u minoted r·:i. ~:;ht c h a.mbc ... ~ Thr:: · e· plore i effective concentrations, • t, and test ed behavioral r esponses. Th::. :i.ndicat i qn of delet erious action of th~ "'--::-. ........ L.. .:, .., L.. I agent chosen wa3 l arval numbe1~s in the 1eft: chamh2r; 'anQ. the criterion) 50% of larvae remaining in the left chamber·. All tne subst<.mces tested (No. 2 fuel oil, biphonyl, naphthaJ_ene, ... · rr.ethyl naphthalene, dimethyl naphthalene=> phenanthrene, ant11raceh~, . fluo:re11e, durene), affected the la1"'vae adve-rseJ.y. Using ~!:i.e 50%;.criterion, No. 2., fuel oil was harmful at a concentration of about 15%, dimethyl naphthalene.... a~ about 11% of stock solution (Figures 6 and 7). \·le noticed in many experiments that although the larvae did not gather in the right (illuminated) chamb~r, they still were swinuning actively. . ~ The results sho\ available to t, Libl r ia dubi~ has t·1\1 · zcea.l sto.~1·es and a rnega1ops s_tagl~; in c onformit y with the developrnent a.l pat:-tern found j11 oth~r I spider crabs ( ~11aij.dat.~) (Gur'ncy, 1942). In i:he second day after hatching, t he appear -1.nce of exuv:i.ae in all the culture dishes revealed that the se·cond ,zo~al stage had begun. •._,,,.1 . \ , Between the fifth and seventh days ;. ~. moJ_·ting fr·om the second zoeal to the megalopa · stage occurred; young . crabs appeared on the tenth day. FiguI'e 10 presen-ts the survival data and indicates the progress of larval· deve.loprnent in the several media. Survival was excellent for the first fou-r dO:ys, there was a modest drop on the fifth day, and a-d1-..ima~tic drop between the fifth. and sixth days. Disregarding the 1/10 group, ~t can be seen that.the sharp drop between the fifth I • and sixth days affected .the cdntrols as well as the experimentals. At this time. the second zoeae were molting to megalops, which . cannib9.'lized many of the· zoeae. The same predatory phenomenon occurred to a less noticeable degree between the ninth and tenth days in the controls and in· the 1/50 dilution when the megalops were { / d molting to the crab stage. Tabie XIII presents a x2 analysis of the su~vival data. ) ' ,., ..; .... 0; . '.. ~-;(.' l . ::i: td . Z0(~1.. 1 (~ \'/·:~r."~ ~:() , 11:i n :·:'. '• (1I' ;1 (V~ ,..: n tl· ··; ~i ·.(~Ord d~1y dl:'l:::-~:c· n;.:)1 ·-ing w,::s corI :1pJ.ct,.!d frorn the ('.;c~c:ond ,~.oc;;r;.1 to t:l ~-~ r1l€~~.;·alor: a ~~t:aqc (),,-1 4·}1r· se"'0 'lt11 L·~·-y tl'Ul: -n~1"'1y ·}.71r\1;1 ..... .. ~ ' ,1,(.1, . ... ~_ -f-,;.i'-~,a~ ·:-.....·c""'J t}1;...; , '-"' .... Lc.J I u.~. ··· -'-1.:. p··., ..'..c-·1"'P'~S o· {-~VCJ'1c=-1 . J-\.,•.:J--'-'"-·-_'I l . ..~ .second zo;:;ae even at "'·:he i.:errnina:t ion of the~ e>cperirr..ent. ~,~1r.·vaJ. in ·l-"r•..o.. 1/~)_S. +-',n, ::in .;n -• ~ ........ -~·hr'.:)\;:. -.. -..L 1..-• --L/ 1 n n: ..;"'1ut~;on -'· ~ u...;,.. -•'-• . ' but there was good evj.dencc that it existed. Fo:r' in.:;t:a nce , on t he fifth day, the io.rvae ·i.n t he 1/25 dilution did not show qui·te as g-.ceat a decline in survivors as d.;i.d those in the J_/50 d:i.lution i.tnd t he controls. The reason f or this would seem t o be that molting to .,i.;fie rnf.:galopa stage was sJ.ightly reta.1xled i n the..~ l /25 dilution, with the result ·that the dl:°!Structivc effect of cannibalism by the megalopa ,,. u pon the zoeae l aqged slightly in this g1"oup. Delayed larval development was even more clearly demonst1"ated in the 1/25 dilution as evidenced 1 by the fact that early crab stages were abundant in the controls and the 1/50 mixture on the tenth day, but had· just begun on the eleventh day in -the 1/25 mixture. Development in the 1/50 mixtu:c:'e followed · that of the controls almost exactly. The survival rates in the controls, the 1/50 and 1/25 mixtures were very simila1"' throughout the experiment. T1he cannibalism operating . to reduce the number of survivors in the controls apparently produ~ed the same redu¢tion in the 1/50 mixture. At the termination of the • experiment there were 25 survivors in the control, ·26 in the 1/so I ''· ) mixtUr·e, 29 in the ·1/25 and 13 in the 1/10. The survivors; however, / ..... ___~- ·:rk: .../ .. ' rJ1 b •.'I '[I' ) • .l / '1 ) \. .<~ .••C A... .. cons:·d··~ t\1 i in .. on~junction with ·· ·hr.:~ obG1 days, th?.! :f=-=i:cst zoeal st·aqe. has a mea.n dtn"\ution .of 3.58 days (Pm:'b3r , 1~)60; On.q and Coct low, 1970; lkc./khout ..... ·"· :'l .L-io·71) 'J ~_' "-Om:' eX?(~rimr.mts l ast•:?d four days ' r~_• .:.; "' and sparme.d t he first Survival of l e; :eva(~ in cont::r."\ol and experimental g-..cou ps :i.s p1...esent2cl graphical ly in Figure 11. St s'tistically, the daily survival : ' ~ ~. O.iffer'er~ccs betw1:;en t he conteo1s and eYperiment:als we1..,e highl y si~ifi<"'ant at the 0.001 level except for the· difference between the cont:colsi:and larvae in the 1/10 mixture on the first day. At the end of-the third day all larvae were dead in the 1/5 and l:l dilutions •. D. Catfish 1. Behavior and survival in acute"experiments Series 1 \ (20, lO, ~· and ·f ml of No. 2 fuel oil). Thirty minutes after a~ding_.tipe oil· all the fish made an effort to stay near the ·surface, and some attemp~ed to get out of the aquaria. After 20 hours some fish died, most _of the fish exposed .to 20, 10 and 5 ml·of oil stayed at the surface, while most of the fish in 2 ml of oil stayed at the bottom. Dead fish were removed and counted. After 48 hours most fish in 20 and 10 ml of oil and about half J those in 5 ml of oil were dead, while most fish in 2 ml of oil survived . ;I ·1, ·' ·:() ' ··~ .-:-'t ·.-~ .·'l H(:!l ·ty•.· "\ L. ,_ ,..1 __ ('\· .· -,..L. -.i 1· .J:J• ·1c v... • .. l. -.. v' o~: ..:::i~n 1-'..'.11...~d \;\:~r~.us oil conc0ntrat:ion (in ppra) ); t he L.:1. ~..:f lethal .dosaqe w<.;s 140 ppn. t, Se1"ies 2 ( 2, 1 and 0. 5 :nl of l ro ,., 2 fuel oil)... Feec~:Lng V..7as observed; res•)onse.;) were c.lass.:i.fied :int o five c o:teqories .' · .Exc2.llent,. All fish actively so1J.9'11t and ingested food v Good~ All fish sought and ate f ood e Fed:·.:' .. "·lost fish sought and ate food. ·Cr--~· • ~.'• Poor. Host f ish did not seek food, _or failed to locate it,. or did not eat it. Vcrv Poor. The1"1e was no response to fcod. The l"esul·ts of the expe1"im~mts a.re. presented tn Table XIV. ·The fish survived in sea water containing l ml (38 ppm) and 2 ml ( 77 ppm) oil for 4 days, but the.ir feeding ieesponses deteriorated. Recovery of feeding responses after.exposure· to 2 ml of oil was protracted. Moreover, many p:i:eces of shrimp vv·ere ·found in the aquaria loaded. with_~ ml of fue1 oil, and likewise after. the aquaria had been I. cleaned and rGfilled. These chrimp w2re regurgitated by the fish. Histological observations of the gills and barbels of catfish treated with 2 ml of oil did not reveal any noticeable damage. On • the other hand, in the stomach of a catfish treated with 2 .ml .. of oil, the p2ripheFal JTillCUS layel"' o:f. the . Cells l:L--dng the lumen was \ defici en'i.: •.. ) ' These are preliminary observations. "·· I . •):: O(' '\ _, , .J .. records ar~ still bajng analysed. ./ E. Oxygen Consumption 1. Porcel ain crabs Oxygen consumption wa.;, no·t reduced in No. 2 fuel oil 1: 1 t'• ~ .. ( 62, 000 ppm). The data a\·1aj.t an~l.ysis. i: 2. Spiral valves of stingarec Nine Gets of experiments were ca1"'r:l.ed out involving No. 2 fuel oil s'tiock. Oxygen consumption of the controls was highest initially, t he mean being 1.69 ,ul 02 mg-1 ·11-.1, ·and_decreased to about 93% of t he initial l"ate after 120 min. The nine sets of data wc~re analysed statistically. Rates of ,. consumption between controls and expe1"imentals wene not significantly· different. 7, .,,. -· .Cornea of stingarees ~nitially the rate of oxygen consumption of· controls was o. 7 plmg-1 h-1. A.fter 3 h it fell to about 80% of the initial level. The rate in No. 2 f-uel oil 1: 1 ( 50 %) fell.i:o somewhat less than 50% in the same tim.3. (Figure 13). The r esults have not yet been an 'alysed ) \ statistically. ! .) _; ' 1• .~ l ·:~ ·' -.; l r.,,..'. · ·t-l ·»··-. j'(''l · ,...; , .. ~,-., 1, ~.":·',-; <' 0 .l.• ~ \ ·.,l.t . L J n .I J l..!. J...> .t.. 1J.nu l: .l. ., 1( 1 ~' 1.>•.l.~ '"• J _ ':he corr ' -:c .... .1 was rd.ql1 ini tid.11 Dhowed no obvious and pe:esi.stent dif fr0rercE.'~!3 f rom tCiCiSC 01~1 th(:! ccntroJ.q. r:-n.. • f ~ .l... (T , J..i 1.2 seven se·cs o c:.a.La &D...Le s XV and X.VI) were subjec;tr~d 'to stati0tical a.no.l ysis by ::reating all sc-!ven sets equally and concentrating on the initial rat«2s and final rates (aft(~l' 120 min)·. The I'c~ults of the analy:}es are shown in Tabtes XVII and XVIII. It is concluded from them that the differences between tht~ experimenta1s and -controls are nt,t significant. IV. Discussion and Conclusions Experimental solutions were prepa1"ed as saturated solutions in aqueou.s media. Oils and oil fractions were made up as oil: water 1: 8; pure :"?Ub;,:;tances, 15 mg %. These saturated solutions were designated stocl< solutions (unity' or 100%). · Dilutions are expressed as ratios, I fractions or percentages of stqck solutions, or in ppm of the original ~ amounts of material used. .., .I r•• J I ' ' ~, .. ,:·a n i·.•:)c:·; :·i::.n.;1 ·1 .1,, J. t:p r'·L. l}. l; O l DO cj,J. 1SG j :LL · tim1s o:I. stock .. r ' . ,,. ') Oil J.•9 •.,.L /1.•. 0 ..LU 1.11 x .L.:J• • 1:99 1/ 100 1 ll:l. x 10 1: 939 1/1000 0.1 111 I Pure substances 1:9 1/10 10 15 1: 99 1/100 l. 1..5 1:999 1/1000 0.1 0.15 Sand do.lla.r egsrs \ .-:.::..~~-· ,_.. , ) " c~)'ce:pt :i.n thL-~ ca.!.>c o:f .i. ·o.. 2 fu ·~J. oi.1. at: a concentr.:.iti.on of l: 1 (SW' ) .. I n the la:tt(~r mixtm_~.~ t i1e abi lity o'f it:he ~>pcrm to f e.r"l:i1iz8 the e c1gs .C " , i l•' .... ct L .....on, as was found wher1 the eg·gs were subj<~cted t:o . ..l.. eJ. -c_ i r7 -· .._ •j .. 1ncr'-··~i.s3,;1g concl~nt-i."a~c:i..ons of oil, bu·t rather a : m.dd2n r ed ict:ion to zero £r2rt:ilization in fuel oil 1: J. ( 50%). •·. Tl1e motility of sperm in Ku·wait oil 1: 1 was not !"educed afte':r; 60 min, whereas in fuel oil the proportion of sperm actively, swirrunin9' declined in the higher concentra·tions, beginning with 1/25 (4%) and after longer ....exposures ('rable VIII). About half the sperm we re still motile.and swimming in fuel oil 1/5 (20%) at 30 min, none in fuel oil l:1 ( 50%). At the £01"'mer concenb:!ation ·there was still an adequate preponderance of sperm I sufficient to fertilize the eggs. It is reasonable to assume that~the loss of motility of sperm ip fuel oil 1:1 (50%) was~a determining factor in the absence of fertilization found in that mixture. T11e parallel effects of fuel oil on sperm motility and respiration are brought out in ·Tables VIII and IX. Movement and respiration of sperm are linked, ~nd factors which enhance movement I increase the fespiratory rate (Rothschild, 1956). I \ ) I f .., ... 1•" ) c -,. •·· •.• •1-.. +·· , ,.:~ · ·.:/.~.~ ~·: ~ oLi. (' \.. L .. . ... ) ·. !l 'L·1 .,, .. :-··1· ')'~ 1\ .. ~.. J. \. ~ 1,.. • •. ~ I c:rp..o/.-.. d O ·i.·". ..L a·'-'--• l : . 111"cr"1ac ·t · \.. .1..l~p C"'n'-,_,... nt'Y''l.!..,...;On e 1nf lO .. • ~l ·-(,, ._.,~ ... c. LJ. , , .! '/AC1. ·in tr1~ e"''i')(.)rlJ ,, ..:~ ... • '-.:.. ·-•;l,rti...:. ~,-..1~L.0-,, n c, ...... n·t,ytely ~ f'· l: .::.. ~* •.111 '1. _ _;:,.. · destroyed the ability of tne c~;gs to be fm.:t:i.1ized. and ·to con-tinue dev lopmcm't •. The th1·\~e concentrei.tio:ns betwe en thc:se ext:r.:·-emc~s f;hov1ed deJ.ete1"ious effects in proportion to the amounts of o:i.1 present. Tnese effects became (~vident first of all in the reduced p~I'centages of I ~ fertiliz::l.tion, starting with the 1 25 dilution, viz. 7'C~o, I and go:rng .dmm to 0% in· the 1: 1 di1ution. The drastic drop in the last mixture f.~ may well have been caused by injur'Y to sperm (vi~~ ~~.~·J)r'J.) •. {. •"'\ E~en ·when fertilization ~vas achiev13d development wo.s not always normal. Irregular cleavages first appBa.red in the eggs in the 1/10 and 1/5 dilutions at the Itime of first cleavage. It might be assumed that those cleaving abnormally a-t early stage~s ·would not give rise to well I formed · pluteus larv~e. Abnorl'I!al cleavages we!'a evident in later stages . " in the 1/5, . l/lq and:i to a lesser extent, :Ln the 1/25 dilution. Of the five fractions of No. 2 fuel oil, Nos I to ITI were most harraful to larval developmen-t; fractions III and V produced more abnormal cell divisions. The Kuwait crude oil at the 1:1 concentration used in this experiment had almost no discernible effect on motility of sperm ), ' and fertilization; ther"e appeared to be retardation· of first cleavage / but_ce~l division' thereafter proceeded nor~ally. Consequently, ·there -· ~--------- I t. e follmv:mr. :~"'csu.l...: ::.•.. 37% t; arornatic.s i~O ·aspholtics and hGterocyclics 7 amount off coJ.umn 84 No. 2 fuel oil: saturated paraffin ... hydrocarbons ~. 57 aromatics 35 aspha1tics and hetc~rocyclics • 5 arr.cunt off column 92.5 'i"'he d:lfferent effects of l<:tJ.wa.it and No. 2 fuel oil revealed in our experiments on sand d.ollar egg·s are not to be explained by these genera1 analyses, and thc:?y are more probably referable to the presence or absence of I specific ~ater-~olubl.e substances. 1 It may be noted that thel"e ~~ \evidence in other experiments that dispersions of oils ·have a greater toxici·ty (by factors of 10 to lOO) tha.n aqueous extracts formed by allowing oil layers to rest on the water (Kuhnhold, 1970). Toxic effects of methyl phenanthrene derivatives on sea urchin / eggs were notGd by Lucke, ParpoFt and Ricca (1941), but they were using saturated aqueous solutions. . Kuwait does contain polycyclic ) aromatic hydrocarbonG (Carruthers and Douglas, 196i); any water soluble toxic components nmst be at low concentrations. ~ --.:... __ 11!,1 / 1'0 TL) I ~:,;~-; ~-:t:;d d ~~(~:-' :~_(; ~.) :)f .,,..!( .,..,,.,: J·~·,r"j c -:·1-.. '~ i • ...,, '· . .;. •••• • L i.I.:... ' .! . . ,::.•' acn.1t-.::•:i1.d! l~x.t:cc~c·-3 ( 1/ ~~o),, at con~crrtrations . .. . ;. regard the experi ments on sand oolJ..ar:3 ·:i.s b~.;ing o:z an exploratory nature. Eggs and s pe:(l;n a:ce not re9-ulo.r·.ly available arn.l c.._uantities are limited. vn1ether it be expc·;..'imentally a.dvarLtageous or r:.o, echirwder ms are very sensitive to pollu.tion (dor·th, 19·~· 7; Allen, ·1971). Sand dollars haw~ the advantage that t:hey at'e typical •.. ·ceprE:!s'entatives of an ubiquitous a.nd ahundant benthic ~r.coup, and'. c~1eir la1•vae equally good :cepresentatives of the temporary., plankton. Sperm are motile, egg·s are easily fertilized, segment regularly, and larvae are formed in abundance. The latter, in rate of development and structural features, are good indicators of normal or abnormal f . conditions in the m2dia (Wilson .and ~rmstrong, 1961). It is for future work to decide how the various deleterious materials in the oil affect the sperm and eggs; by absorption i.nto the external · membrane (D§nielli, 1950), by modifying the spindle apparatus, -, .., damaging intra~el1ular organelles, curbing enzyme activity, and so for.th. Eggs and sperm have the g~eat advantage, shared by erythrocytes, of providing homogeneous suspe·nsions of cells on ./ which the effects of petroleum upon cytological processes can be studied directly. ' : ---. T~ , ., J. j ~.t~ ~::Le fueJ. ,.·,;) .l'e ..rt an early st:..-~F;; of clev1~lo;n~e:at: ( ~) ·to 7) \':ere p.1-r:::.ced :in th ·~ o:il-se a ~-.'o.ter m:ixb.i.·22s, t h:::: ._ f:2ccts t, .v2:r;.~ pro9:'e;:;:-nve..J.y mor·(~ p!'onoi..:.nced in the hi.ghe1"' c.J!12..·=mtr a:tio:h. h: wc:.s notable that in the 1/10 mix, d~~;c. ·1 o-:Ynen7 ~---·~ • hatching and larval survival \·;er(~ about ecmal to those .l;•J ., , of the con-trols. In the 1/5 mix, deve1oprr1errt was norm.al :in most of the embryos, a.r:d lo.rva.l s:...:'v:i.val w;s.. s good but: not equal to the : ~. contro'ls. In the 1: l and stock solutions the e.. .i~fects were clearly \ disast rous . All embryos died in the oil stock, only a few--hc?.tched in the 1:1 mix, and rapid death followed hatching., ·when ,the embryos were exposed to the oil mixes at slightly lat~r stages of develop:nent, namely stc.ges 8 to 10, 1ethal effects again increased witn increasing oil concent:rations. In the 1/10 mixture, \ the experimental groups were somcwh~t erratic: in about haJ£ the cases, they ·were indi3ti..."1guisha.ble from the controls; in the other half, hatchi.rig did not occur in the experimentals, but it occurred '1 only poorly in .thG corresponding conl:rols. In the 1:1 mixture e.mbryos with:tn a larr.ella were affected to .different degrees. After they had bet~n several days in the solution it ~1as possible to find embryos at di~ferent stages, and some dead; yet some embryos completed . \I deve1oph.ent .anrl hatche~. ' In the undiluted oil stock death occurred before hatching. ------------------------, I • ' .. : ' I I ,'-.-.. '-',.... """(: I "' A '" ·~-· "., , .. ,· ,.... '' \ .... .., ··r'r· '" .. • .:~ J. <'. l. ~--• ·, __ 1_.1 • • ·~' ' :.), ) " _) J. \; . I t"L:i.'-i i.~1 th::.~ contrc 1 : .. lC-)tl1a1 ca.se ·.1"'e (~r.·1Lr~10 i ·:eorr• th0 ir11Ir£~diate eff~cts o~ the oil; with time the oil p2netrJtes ~1c cas~ •• a~d Ll1? embryonic nemb1'anes and arrest3 d\:~VE:lopment . An altern.=Jti•;e explariatj_..:,n is that the eg-g case change:~. and becom0~s less p2rm·2abl (! during later stage(_' , oJ.: development. Wi tnin the content eif these experiments it -=i~>pGt(i~. s t~nat the embryonic development of. C1rth2171~~i_t:?_ f r agi1:.Ls pr?ceeds .::00· 1t \ \ as well in the 1/10 mixtu.1~e as in the controls. It is obv:i_()Us .... i· t hat in concentrations as high as 1:1, death of nauplii is very rapid and crude oil have giv•2n results which pose some · interestirlg questions regarding the physiological effects of the oils and their specific toxic· components on e~;g ir~embranes, cuticle, sperm motility, fertilization, cleavage, respiration, and locomotion. ..some effects may be external, others the resul~ of interference with biochemical actions or ultrastructural organizat:ion. Acute -experiments of one hour duration with larvae.pf Balanus amphitrite are ·in progress. I Results on hand indicate that oils and some constituents are deleterious at c·oncentrations 15% ~nd 10%, or less, respectively, of the original stock solutions. A mortality . / level (50%) to 1 h exposure for larvae of Elminius has been1reported ) ......, ~---...-.. .._ ·t •.) :i.nd.: Cdt(~ th2 n(::eci for ~·1 ~).>::>tances; ~·u ( ·(·"e c · J...,·1..... :i -...J • j .J ...., -· L .u \.... ..; ~ +-:-. . V 1 "\ i.D t".:> .;: or· o" e ..., +-}1 ·'-'n ~..,.,(.. ·,...,' g·-., .. ~re l', ,::tl·ter ·.1· r.g· ..!.ll .... ·--,.::'. . tt.! ~-:-1..~ ,· .o..... , L c::. .i.. ::: .., _., • .!. ci.v .. J :~ .J.L~ the oppo:~'tunity for mn'vival under' nablr a.1 conditions . Crabs Larvae o.f the thT'ee crabs tested all proved by various crite:cia to he ahout ecJwlly sensitive to fuel oil, which w0s deletE:~ri.ous t~. 'tJ.1:-~velopment and survivu1. •'• The most strild~lg effects were found \·)~i.th stone crab zoeae, which were kilJ.ed in all mixes fr0m 1/25 ~to 1/2~"!' the mortality increasing with the concentration. Comparable effects \vere obtained with hermit crab zoeae, but they were somewhat J.ess prono~nced and manifested themselves more slowly. In addition to survival.., significant differences. were observed in the condition of the larvae and rate of development. Higher concentrations of oil retarded growth ·and iru~ibited. molting of hermit crab and spider crab larvae, ,. the effects being manifest at concentrations of 1/10 and 1/25• .., Catf:i.shes Sea catfish were killed by fuel oil at concentl"ations ) 77 ppm arid, prior to death, there was much damage to surface tissue. Lethal. limits for fishes in the literature (quoted below) lie in the range 4 to 167 ppm.. At a concentration of 35 ppm, feeding I'espons'e·s of ) catfishes deteriorated, and the ardmals could not retain their food. /A p~ra~le.1 study is, perhaps, that of Fostep ~al. (1966) on flagfish, \ that th~~ f :Lsh sensed o:i.1 ~rt levc:~ls of at J.c~\~~t 100 ppm. C<.Tl:f:i.0h sought to escape from oil and, under rwtural conditions:> it might be expected tht:rt t hey wou1d seize opportunities to remove thernselves from tho vicinity of u.n oil spill and .thereby avoid the damaging ef:fe-cts. Training and conditioning techniques have revealed a wide range of olfactory sensitivities to various aromatics in selected ~pecies, e.g. 500 ppm for phenol and p-chlorophenol in a minnow, 0.9 pp~ for phenol and 0.02 ppm for benzene in the roach, and 10-5 ppm for morpholine in salmon fry. General discussion and conclusions It is generally agreed that all crude oils and oil fraction~, . ~ .except for some highly purified materials, are poisonous to mari~e ·animals. Loss of marine life was observed following the wreck of the Torrey Canyon1 Tampico Maru and the tank barge Florida (at West ·Falmo1;lth, Mass.). Mortality from the last two wrecks was especially severe (North et al., 1965; North, 1967; J. E. Smith, 1968; Hampson I" - • __,.• . · and Sander's, 1969; Blumer, 1970). The o1i from the Torrey Canyon was Kuwait crude; from the Tampico. Maru, diesel fuel,. four-fifth heavy distillate and one-fifth' residual fuel oil; from the tank barge Florida, No. 2 fuel oil. . ,...._~_.oxic levels (or tolerance) depend not only upon the kind of il pollutant, but also upon the species and life history stage exposed to it. Surveys of the literatuFe (Chipman and Galtsoff, 1949; Kuhnhold, 1~70; Nelson-Smith, 1971; Lichatowich et al., 1973) reveal ) a wide range of tolerances among marine (and freshwater) animals tested. Some toxic levels reported for crude and fuel oils were: hydroids ·; · ··· ·oo· 1"' ···1,)·i-l(.-1"' -1) ... ·1 -·r"l' . ,.., . ('..) ) ~ ~·~. I 1 • • •• ..:_. • .-:~.~.::.'.:.'...:.. -,-~•• '.::'... '.:-.. · • ,)\I > :;c.~.,1 ux·crdns ~:; t.:1 )nqv :i c1cc~ ni~ .r; t;_~ s ----·----·-·-----r---·------ ,.1 • -:::..:_ .:...__ • ] ()"'("" r.. )' • Fi'_ --.. ~C0111 _~l •' •100 (' iOJ'' t '1""4 .... ··-Jl , ••• ()_. ,'~. ~l1 . °I":() ly..:.• m.oJ."•?._ ..:-:~.' ·n, .·_··, ].~ ·,.tv·~__ ' ~]_r-~t'n:-il _1• --~-·-._ c'..11 11rl.J __ u. levG1s in the .literatu:N:: are: 15, 4CO ppm :for· carp (en1d 0 __@.s 1.; 4 ppm for dace and ruff ( Baku crude ); young shud, 167 ppm (diesel fuel), 91 ppm (heavy residual fuel oil) (Veselov, 194-8; Chipman and Galtsoff, 1949; Ne lson-Smith, 1971). Concentrations having adverse effects on eggs, embryos and larvae were: toadfish Ops~ embryos 1: 200; herring Clupea, cod Gadu.s .and plaice PleuI'onectes eggs and larvae 1: 50 to 1: 25000, turbot Scophthalmus eggs 1:10,000 (Miro~ov, 1967; Kuhnhold, 1969, 1970); oyster Crassostrea, mussel Mytilus eggs 1:1000, 1:10,000 (Renzon, 1973); '· barnacle Elminius larvae 1:10,000 (Spooner, 1968). Toxic levels of oils, between 1.5 and 50% (of added ~oils),,. on invertebrate larvae, found in our experiments, are within the ranges discovered by other investigators. Some oils have been found to be more .toxic than others, Venezuelan and Iranian crudes more so than Libyan (Kuhnhold, 1970); U.S. navy crude more so than fuel, diesel and lubricating oils (Chipman and ~altsoff, 1949); bunker and heavy crude more so than highly refined petroleums; No. 2 fuel was intermediate in effect between the two latter (Allen, 1971). It is interesting that '4 I among t!ie many. oils tested by Allen, those that had a strong adverse effec~ on fertilization had the least toxic effect on cleavage, and vice versa; one was equally harmful to both processes. Young cod eggs ,, were more sensitive than older 'embry>os. and larvae (Kuhnhold, 1970). Our results. with sand dollar eggs show that aqueous extracts of No. 2 fuel oil are far more toxic than Kuwait crude. It should be noted ....___ 46 that Spoonel"' (1968) found that Kuwait crude was lethal to barnacle larvae at much lower concentrations than we employed. In all such experiments, however, when comparing absolute levels, difficulties of interpretation are introduced by the potently grossly different methods used to prepare the test media. Components of oils having inunediate toxicity are low boiling j aturated araffins and aromatic h drocarbons, both of which are water . soJuble in degree. High boiling aromatics are suspected'·as long term poisons (Nelson-Smith, 1967; Blumer, 1969, 1970). Waters along the Eastern American seaboard contain petroleum at levels of 0.001 to 0.012 ppm. This oil consists largely of nonvolatile or persistent hydrocarbons (Brown et al., ~73; Walker and Colwell, 1973). The results obtai.ned ·with invertebrate larvae are for chronic exposures to concentrat1ons equivalent to 0.0125 and o.oos of saturated solutions of the water ~· soluble components of 'the oil. These concentrations are far in excess (x 106) of oil levels now existing.in the sea; moreover the toxic fractions have mostly been dissipatedfrom the oceanic milieu (Pelpil, 1968). However, the safety margin for many fishes may be . . .considerably less (x 106 to 103) anq, for impairment of olfactory . cells; marginal or even adver~e (vide supra). Oil concentrations in excess of 1000 ppm can be expected to occur for short periods .near shore or in enclosed basins~after major spills while evaporation and photo-oxidation of volatile and toxic components are taking· place. They would harm a limited hatch of larvae; the effect on a year class would be dependent, however, on survival and health of the adults, and duration of the reproductive ) ~ . . ·).r .. I t ·~; '· · ) ·~ 47 season.. As we have shown in experiments on sand dollar gametes and barnacl~ eggs, fuel oil, in increasing concentrations,has deleterious effects on the growth and survival of eggs an~ embryos. Together, these lines of evidence point to harmful effects throughout embryonic' and larval life. Owing to the proliferic reproductive capacity of many marine animals, however, it can be expected that reinvasion ~f the affected area from neighboring regions will occur once the harmful agents are dissipated or destroyed. In conclusion, it would appear that we lack coherent information about long term 'effects of oil on larvae and post-larvae under the equivalence of natural conditions; namely, in some key species, information that can predicate at what level~ ~f oil pollution they can successfully complete the entire life .,, cycle and maintain themselves in steady numbers. •,'•. .. V. Summary 1. Eggs and larvae of sand dollars lo------quinquesperforata. Kuwait crude, No. 2 fuel oil, and five fractions of No. 2 fuel oil were tested on gametes, eggs and larvae. Criteria employed. were: permeability to water of eggs transferred to 60% sea water; motility and oxygen consumption of sperm; fe~tilizing capability of sperm; and · development of eggs (el.E?vation of vitelline membrane, cleavage,formation of echinoplutei). Fuel oil 50% (of oil-stock 1:8) did not alter egg ·~ .permeability. ·Kuwait SD°fo, and fuel oil 20% and higher dilutions did not decrease the fertilizing capability of sperm. However, sperm exposed to fuel oil 50% were rendered immobile, they ceased to respire and they . did not fertilize eggs. Kuwait 50% had no effect on cleavage and development: Fuel oil had a marked depressive effect on development, " .which e:)p(ared at a concentration of 4%, and increased at higher 48 concentrations; practically no fertilization and development took place ' .. .,,.1 at a concentration of 20%. Fractions I to III of fuel oil (C9 to c17) were ~~5~ deleterious to larval development. 2. Eggs, embryos and larvae of the. littoral barnacle Chthamalus fragilis. The water soluble components of No. 2 fuel oil were tested on the development of eggs in vitro. Concentrations used were saturated l, stock soJJ.ltion (1:8) (in sea water) and dilutions of sto~k of . 50%, 25% and 10% (in sea water, by volume). Development was arrested in the saturated (stock) solution; in the others, the effect depended on the concentration. The .50%-mix was lethal to early embryos (stages 5 to 7); later stages (8 to 10 showed variable development; terminal .. stages (11 to 13), normal or accelerated development; nauplii soon died. In the 20% mix most embryos hatched, but larval survival was poor.· the · Embryonic development and larval survival .in 10% mix was about equa~ to the controls. The egg case seemingly afforded the embryos some protection against the oil. ' The tolerance was high, some development / . still occurring at a concentration of 2.2%. The resistance of the Iembryos agrees with that of adult barnacles to envirorunentaJ. stress. · Balanus amphitrite niveus Tests_of the phototactic response of recently hatched nauplii were carried out in the ·presence of No. 2 fuel oil and petroleum oil constituents,. including biphenyl, naphthalene, methyl naphthalene, dimethyl naphthalene, etc. Concentrations used were saturated stock solutions (oil stock-sea water J..: 8;. pure compounds, 15 mg %), and various dilutfons thereof; tests lasted 1 h. )· The criteria used were positive or negative phototaxis. The larvae are normally positively --------------------------------------------1 49 phototactic; among the controls, 7. 3% L:dled to migrate towards the light. The oil had an effect by reducing positive phototaxis, at a level of 10%; the effective level for half the larvae was 15%; naphthalene had an effect at 20%, methyl naphthalene at 50%, dimethyl naphthalene at 10%. The .materials did not always kill the larvae at the concentra~ions used within 1 h; however, the loss of positive · phototaxis could deprive the larvae of a behavioral response essential to their dispersion, orientation, and survival. '· 3. Crab larvae were tested in No. 2 fuel oil, namely zoeae of striped hermit crab Clibanarius vittatus, stone crab Menippe mercenaria, and all stages (first, second zoeae, megalops and first crab) of spider crab Libinia dubia. Hermit crab. Zoeae survived and developed for 11 days almost as well or as well as controls in 2% oil stock. About half the larvae were killed in 8% oil stock. Survival and development were poor in 10% and 20% oil stock; no larvae survived in 100% oil stock. Spider crab. Development of all stages up to and including metamorphosis into young crabs proceeded as well or almost as well in 2% fuel oil as in controls (10 days) . . Development to the crab stage took place in 4% fuel oil, but· was somewhat retarded. In 10% fuel oil there was gross interference with development. Molting to second zoeae occurred, but most zoeae failed to become megalops, and none of the latter reached first crab. Stone crab. Experiments involved the first zoeal stage and lasted 5 days. Survival was reduced in.all concentrations (4% to 50%) after 5 days; many larvae died in 50% and 20% oil at the end of 1 day, and all 50 were de ad at the end of 3 duys. Levels of half mortality were: 1 day, 32% oil; 2 days, 13% oil; 3 days, 6% oil; 4 days, 2% oil. 4. Catfish Arius felis. When fuel oil was added catfish soughtto escape; attempts to escape continued in oil at levels of 190 ppmand grc!ater. Fish were killed by oil; at 48 h the concentration of oilcausing half mortality was 140 ppm. There was ITUlCh damage to flesh of the fins and to the gills. Feeding responses deteriorated at oillevels of 38 ppm or greater; food ingested was regurgitated; recoveryafter exposure to 76 ppm of oil was slow. Electrocardiography showedresponses (bradycardia) to oil at a concentr.ation of 100 ppm. 5. Oxygen consumption of porcelain crabs, of gills of pinfishand excised spiral valve of stingaree was not altered from that of r .-, control's ·in various oils ( 2-3 h). But it was depressed :in corne'-as of stingarees in 50% fuel oil. The cornea is a delicate. tissue exposeddirectly to the environment. 51 VI. References Allen, H. 1971. Effects of petroleum fractions on the early development of a sea urchin. Marine Pollution Bull. ~' 138-140. Bassindale, R. 1936. The developmental stages of three English barnacles, Balanus balanoides (Linn.), Chthamalus stellatus (Poli) and Verruca stroemia (O. F. MUller). Proc. Zool. Soc. Lond. 1936, pp. 57-74. Blumer, ·M. 1969. Oil pollution of the ocean. Oceans, 15, 2-7. _,, Blumer, M. 1970. Oil contamination and the living resources of the sea. PAO Technical Conference on Marine Pollution and its Effects on Living Resources and Fishing. Rome, Italy, 9-18 Dec. 1970. Boekhout, c. G. 1972. Larval development of the hermit crab Pagurus r .., /ala.tus Fabricius, reared in the development. Crustaceana, ·:·_22, 215-238. Boekhout, c. G., Wilson, A. J., Jr., Duke, T. W. and Lowe, J. I. 1972. • I Effects of Mirex on the larval development of two crabs. Water, Air and Soil Pollution, 1, 165-180. Carruthers, w. and Douglas, A. G. 1961. 1,2-Benzanthracene derivatives in a Kuwait mineral oil. Nature, _U:>nd. 192, 256-257. Chipman, W. A. and Galtsoff·, P. S. ·1949; Effects of oil mixed with carbonized sand on aquatic animals. Spec. Sci. Rept. Fisheries .No. l, U.S. Dept. Int., Fish and Wildlife Service~ 52pp. Costello, D. P. and Henley, C. 1971. Methods of obtaining and handling marine eggs and embryos. Marine Biological Laboratory, Woods Hole, Mass. · Costlow, J. D., Jr. and Bookho~t, c. G. 1957. Larval development · of Balanus eburneus in the laboratory. Biol. Bull. Woods Hole 112, 313-324. ----------------~-~--------- 52 Cost low, J. D., Jr. and Boekhout, ~. G. 1958. Larval development of Balanus amphitrite var. denticultil:a Broch reared in the laboratory. Biol. Bull. Woods Bole, 114, 284-295. Crisp, D. J. 1954. The breeding of Balanus porcatus (da Costa). J. mar. 0iol. Assoc. U.K. 33, 473-494. Crisp, D. J. 1959. The rate of development of Balanus balanoides (L.) embryos in vitro. J. Animal Ecol. 28, 119-132. Danielli, J. F. 1950. Cell physiology and pharmacology. 1Blsevier Publishing Co., New York, Ams·terdam, London, Brussels. Foster, N. R., Scheier, A. and Cairns, J., Jr. 1966. Effects of ABS on feeding behavior of flagfish, Jordanella floridae. Trans. Amer. Fish. Soc. 95, 109-110. ... Gurney, R. 1942. Larvae of decapod crustacea. The Roy. Society, ... London • . Hampson, G. R. and Sanders·, H. L. 1969. Local oil sp:Lll. Oceanu~, 15, 8-11. Hara, T. J. 1971. ~hemoreception. In Fish physio"logy (eds. W. s. Hoar and D. J. Randall), Vol. v, pp. 79-120. Academic Press, New York and London. Harvey, E. B•. 1956. The American Arbac.i"a arid other sea urchins. Princeton Univ. Press, ·Princeton, N.J. Herz, L. E. 1933. The morphology of the later stages of Balanus crenatus Brugniere. Biol. Bull. Woods Hole, 64, 432-442. Kleer~koper, H. 1969. Oliaction in fishe·s. Indiana Univ. Press, Bloomington, London. " Kuhnhold, W. W. 1969. The inf11lence of water soluble compounds of crud~ oils and their fractions on the ontogenetic development of herring fry (Clupea harengus"). Ber. dt Wiss. Korrunn. Meeresforsch. ·~, 165-171. 53 Kuhnhold, W. W. 1970. T11e influence"'of· crude oils on fish fry. FAO Technical Conference on Marine Pollution and its Effects on I1iving Resources and Fishing. Rome, Italy, 9-18 Dec. 1970, lOpp. Lichatowic~, J. A., O'Kecfe, P. W., Strand, J. A. and TempJ£ton, W. L. 1973. Development of methodology and apparatu.sfor the bioassay of oil. In Proceedings of Joint Conference for Prevention and Control of Oil Spills, March 13-15, 1973, Wash.'· D.C., I pp. 659-666. American Petroleum Institute, Wash. D.C. /' Iilcke, B. 1940. The living cell as an osmotic system and its permeability to water. Cold Spring Harbor Symp. Quant. Biol. ~' 123~132. I' Lucke, B. and McCutcheon, M. 1932. The living cell as an osmotic•.·.system and its permeability to water. Physiol. Rev. 12, 68-139. I:uc~. B~, Parport, A. K. and Ricca, R. A. l94l. Failure of choleic 1~ I . acids of carcinogenic hydrocarbons to alter permeability of . .marine eggs and of mammalian erythrocytes. Cancer Res. 1, 709-713. I• Lucke, B., Hartline, H. K. and Ricca, . R. A. 1939. Comparative permeability to water. and to certain solut~s of the egg cells of three marine invertebrates, Arbacia, Cumingfa and Chae'to'pteru·s. J. cell. comp. Physiol. 14, 237-252. , Lucke, B., Larrabee, M. G. and Hartline, H. K. 1935. Studies on osmotic equilibrium and on the kinetics of osmosis in living cells by a diffraction method. J~ gen. Physiol. 19, l-17. Marvin, D. E., Jr. and D. T. Burton. 1973.· Cardiac and respiratory responses of rainpow trout, blue gills and bro\'ffi bullhead catfish during rapid hypoxia and recovery under normoxic conditions. Comp. Biochem. Physiol. 46A, 755-565. S4 Medes, G. 1917. A study of the cu.uses and the extent of variations fn the larvae of Arbacia punctu1ata . J. Morph . 30, 317-432 . Mironov, 0. G. 1967. Effects of low concentrations of oil and petro.leum products on the development of eggs of the Black Sea turbot. Vop. TI:.· :: 1/25 100 0 68 28 4 96 4 0 Excellent Good . l/10 lOO o. 14 25 61 12 10 78 None tion:; 1/5 100 . 0 l 4 95 0 0 100 None I·;o:-te j\7.'""\.....,>:l 1/2 ·100 0 0 6 94 0 5 95 .None •'VJ. .,...._ .::" :•-:-. \ 'i Table V Gt'oups Vitelline membrane First cleavage Second cleavage LarvaePresent Absent Present Absent Present Absc~t Nurri.ber Develop::-.2:~.i.: Normal Abnormal Normal Abnormal Control . 100 0 95 1 4 95 1 4 Excellent Excell2:-tt No. 2 fuel oil Fraction IV 1/50 100 0 99 0 1 100 0 0 Excellent Exc2 lJ.cr, ~ 1/25 100 0 100 0 0 100 0 0 . Excellent Excell,=~.t 1/10 100 o· 91 3 6 86 1 13 Excellent 2xcell2~-~ . 1/5 100 0. 84 6 10 84 9 7 Excellent Very s-2:,:l 1/2 100 0 0 1 99 0 1 99 None No~.e ::" .,~~ ---·-------___,. -~--~-~---~~---~-~.----~------~----~--~-----= ~~-~~---~---~ -"-~~--~-------~ ( Table VI , qroups . . Control No. 2 fuel oil Fraction V 1/50 1/25 1/10· 1/5 1/2 Vitelline membrane Present Ab.sent 100 0 100 0 100 0 100 Q. 100 . 0 100 ,.Q First cleavage Present Absent Normal Abnormal 99 0 1 86 14 0 69 31 0 95 3 2 73 21 6 0 7 93 Second cleavage Present Absent Normal Abnormal 95 2 3 96 4 0 88 11 1 98 0 2 75 8 17 0 4 96 Larvae ~umber Deve lo~!:1.:: '.: t , ·) Excellent ExcelJe:-:".: Excellent Excells~.t Exc~lle11t ExcelJ_e!",~ Excellent Very g20~. Excellent p .. -j (' -0.-None r·;orle : ·.· ~.. To.blc VII Effects of Vi~troleum oils on Sund dollar sperm, u.s revealed by their I capacity to fertilize eggs. Numbers of eggs at several stages of development were determined from samples of 100 eggs in each culturebowl. Eggs were examined under a compound microscope (magnificationx80) for the presence of a vitteline membrane after insemination, forfirst cleavage a 1 h, and for 16-32 cell-stages at 2 1/21• h. Culturebowls were examined under a stereomicroscope at 24 h to determine thenumber and condition of larvae. ..,, .rI __,,I Table VII Vitclline First 16-32 Larvae membrane cleavag·e cells Numbr::r Development Control 99% 99% 99% Excel1ent Excellent Kuwait 1/2 95 96 96 Excellent Excellent No. 2 fuel oil 1/50 95 94 95 Excellent Excellent 1/25 99 94 95 Excellent Excellent '· 1/10 99 100 99 Excellent Excellent 1/5 97 97 97 Excellent Excellent 1/2 0 0 0 None None Table VIII Effects of petroleum oils (No. 2 fuel oil and Kuwait crude) on motility of sand dollar sperm. '· Tc1ble VIII Time Nedium Dilution ..,. 5 min 30 min 60 min r-i • ·" Fuel oil , 1/2 0.5 swimming 0.1 moving all immobile" moving 0.9 immobile Fuel oil 1/5 a11 swimming 0.5 swirnming 0.1 swimming 0.5 moving 0.4 moving 0.5 immobile Fuel oil 1/25 all swimming all swimming 0.2 moving 0.8 swimming '· Fuel oil 1/125 all swimming all swimming all swimming Kuwait 1/2 all swirnrning . all swimming all swinuning Control all swirruning all swimming all swimming sea water .. Table IX O~ygen uptake of spermatozoa. of sand dollars in sea water and oil s,. ·" stock-sea, water mixtures at 2s0c. '· ... Table IX Time (min) Conditi on 02 uptake (pl/sperrn-h) 4 Cont rol 0. 36 x io-6 7 fuel oil 1/2 0 10 fuel oil 1/5 0 15 fuel oil 1/25 0.38 20 Control 0.27 1, 25 fuel oil 1/2 0 30 fuel oil l/5 0 40 fuel oil 1/25 0 45 Control 0.22 65 Control 0.18 100 Control ~ ·. 0.14 Solubility of oxygen in sea water (30 °/oo) at 2s0c is 48 pl ml-1. ~ Table X Generalized summary of the effects of petroleum (No. 2 fuel oil) mixtures '...' on embryonic development and larval survival in Chthamalus fragilis \l ' ) ~~~~~~~~~~~~~~~, /r--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Original Stages Controls Oil -sea water mixtures 1/10 1/5 1/2 Stock 5-7 Embryos developed, Embryos hatched. Most.embryos Most embryos died All er. ,~:."\/~ s hatched in 4-7 Larval development hatched. Larval before hatchir:.g, died b2£0:-2 (median 6) days. about equal to survival good, a very few hatched. hatching. Larvae lived several controls not equal to Larvae died ra~idly. days •. controls. 8-10 Embryos developed, Hatching and Most embryos Variable eD.bryonic Embryoshatched in 3-7 la~val survival hatched. Larval development . S0~2 de velo;-·:?d(median 5) days. equal to controls survival poor, hatching . R.apid sli;ht:y,Larvae lived several in half la~ellae. not equal to larval death. died.days. controls. 11-13 Embryos hatched in Normal hatching. Normal or rapid Normal or rapid Rapid0-6 days (median 1). Larval survival hatching. Larval hatching. Rapid . t .. n a. c::i:--~g . Larvae survived about equal to survival erratic, larval death. Rapid larva~ several days. controls. not equal to death.controls. ::" ·~-:-::.-~ - Table XI Behavior of barnacle larvcie in a test charnber(Figure 4) ·Agent No. of larvae in chambers Percentages Observations L LC RC R L R Sea water 55 46 35 465 9.7 82.2 Swimming Controls 10 18 12 62 9.8 61.1 Swimming1 1 10 119 0.7 91.1 1, Swimming8 5 10 150 4.6 86.9 Swimming No. 2-;fu~l oil • r . .. " . First ... Se.t·ies10% 42 19 12 69 29.5 48.620 309 56 13 5 85.0 1.430 368 23 4 2· 95.0 0.5so 114 22 0 0 84.0 0 Second series .. 1% 9 3 3 176 9.9 92.0 Swimming2 0 0 1 132 0 98.2 Swimming4 1 1 2 200 0.5 98.0 Swimming10 22 4 0 119 15.2 82.0 Swirruning50 141 2 0 0 98.S 0 Mostly dead Biphenyl10% 42 32 42 246 11.6 68.230· 59 59 121 415 9·~2 61.9100 668 29 4 0 95.0 5.0 Naphthalene20% 411 47 199 79 56.1 10.8 Swimming30 413 8 0 0 98.5 0 Swimming100 537 6 . 0 1 98.S 0.2 Swimming Methylnaphthalene1% 16 17 30 326 4.1 83.9 Swimming2 13 21 23 282 3.8 83.4 Swimming 4 . 11 17 20 312 3.1 86.4 Swimming10 12 7 67 335 2.9 79.8 Swimming50 492 3· 0 0 99.4 0 Swimming Dimethyl naphthalene 2% 67 14 52 362 13.S 73 Swirruning __,,, 4· 45 7 43 295 11.6 75.8 Swimming10 130 4 6 106 52.8 43.0 Swinuning50 164 0 0 0 100 0 Mostly dead ' Table XI (cont·.) Agent No. of larvae in chambers Percentages Observations L LC RC R L R Fluorc-.me 1% 14 42 27 243 Swimming2 0 13 42 309 Swimming 4 16 11 46 276 Swimming 10 75 79 31 148 Swimming so 48 119 146 0 Swimming -'· Phenanthrene 50% 32 102 125 318 5.6 55~2 78 136 39 4 1 75.8 0.1 Anthracene 100% 92 31 180 121 21.7 28.6 l l } I Durene 30% 43 33 49 121 9 63.S I. ... 100 185 64 42 38 56.2 11.6 Table XII ·'I Re sults of two sets of experiments testjng No. 2 fuel oil on 1arvae of the striped hermit crub Clibanariiu s vitt:itus. Experiments in both ·series involved first and subsequent zoeae; they lasted 11 days. Exp. N, Obs. N., expected, observed numbers; NS, not significant. 1, .. ... ·-" Table XII ~ So.luti.on Original Si.rrvivor~;(oiJ. 0tock-N °Exp. lJ Ou!>. iT x2sea water) Series 1 Sec1 WaterControl 100 37 1/10 100 37 24 7.24 1/5 100 37 4 \ 46.71 1/2 100 37 0 '• 58 .63 Series 2 Sea WaterControl 100 90 .,:.. 1/50 100 90 .89 NS 1/25. 100 90 79 13.44 1/10 100 90 28 427.11 .. Table XIII Experiments with larvae of the spider crab Libina dubia, testing No. 2 fuel oil. The data show survival of controls and experimentals (exposed to oil dilutions 1/50, 1/25 and 1/10) for 11 days. x2 analyses. NS, not significant. '· I '\ F i l I .. 1 I I i ·~ ,._,/ . Table XIII Day Control 1/50 x2 1/25 x2 1/10 x2 100 100 NS 100 NS 100 NS 100 100 NS 100 NS 99 NS 98 100 NS 98 NS 97 NS 97 98 NS 97 NS 87 34.36 82 78 NS 91 5.48 82 . NS'· 44 44 NS 52 2.59 71 29:57 41 38 NS 32 3.34 48 2.02 39 38 NS 31 2.68 41 NS 39 37 NS 29 4.19 30 3.39 25 26 NS 29 NS 18 3.32 .. 25 26 NS 29 NS 13 8.78 'rable XIV B2havior of catfish exposed to No. 2 fuel oil. '· ... ,, Table XIV Days in oil or sea water Oil concentration Cont rol2 ml 1 ml 0.5 ml 0 Excellent Excellent Excellent Excellent 1 Excellent Excellent Excellent Excellent 2 Poor Fair Excellent Excellent 3 Poor Fair Excellent Excellent '· ... 4 Very Poor Fair Excellent Excellent I Recovn:;?y;', Days ~ 1 Poor Fair Excellent Excellent 2 Fair Good Excellent Excellent 3 Fair Good Excellent ..Excellent 4 Good Excellent Excellent ExceP.ent .5 Good Excellent Excellent 'Excel) :mt 6 Good Excellent Excellent Excellent 7 Good Excellent Excellent Excellent ·4 Tab1c XV Initial Rate of Oxygen Uptake by Pinfish Gills Cont ·:'ol Experim~ntal 'lype of oil pl/mq h-~l pl/mg h-1 1.4-6 1.53 Diesel fuel 2.34 2.06 Diesel fuel '· 2.11 1.69 Diesel fuel 2.40 2.05 Southern La. crude 1: 1 dilution 1.41 1.25 Southern La. crude 1.89 1.41 Bunker C -1:1 dil. 1.94 1.97 Bunker C 1.94 1.60 No. 2 fuel oil I 1.94 1.53 No. 2 fuel oil 1.93 2.15 No. 2 fuel oil n = 10 10 x-= 1.94 1.72 ()= 0 • 319 = 0 • 32 . 0.31 S.E. = 0.11 0.11 S.E. =~= 0.32n-1 10-1 Table XVI Final Rate of Oxygen Uptake by Pinfish Gi1ls r:::.. Control ~ Experimental Type of oil pl/mg h-1 Time pl/mg 11-l Time (minutes) (minutes) 1.09 84 1.13 92 Die·sel fuel 1.44 80 2.06 90 Diesel fuel 1.58 97 1.35 104 '•Diesel fuel 1.15 95 1.25 102 Southern La. crude 1:1 dil. 1.01 96 1.17 102 Southern La. crude 1.15 97 1.03 104 Bunker c -1:1 dil. 1.51 104 1.56 ill Bunker c .. 1. 36 129 1.28 137 .No. 2 fuel .oil 1.04 122 1.11 130 No. 2 fuel oil 1.45 127 1.35 119 No. 2 fuel oil n = 10 10 x = 1.28 1.33 &= 0.21 0.30 S.E. = 0.07 0.10 Table XVII Oxygen Uptake by Pinfish Gills Statistical analysis of initial rates Control Experimental 1.46 µl/mg-h 1. 53 pl/mg-h 2.34 2.06 2.11 1.69 '· 2.40 2.05 1.41 1.25 1.89 1.41 1.94 1.97 .. n = 7 7 - x= 1.94 pl/mg-h l.71 µl/mg-h c:= 0.39 0.33 S.E. = 0.15 0.12 -· ' Table XVIII I ~· Oxygen Uptake by Pinfish Gills )J Statistical analysis of final rates Control Experimental 1. 09 pl/mg-h 1.13 pl/mg-:-h 1.44 2.06 "' 1.58 1.35 '· 1.15 1.25 1.01 1.17 1.15 1.03 1.51 1.56 .. n= 7 7 - x= 1.28 pl/mg-h 1. 36 Jll/mg-h er= 0.23 0.35 S.E. 0.09 = 0.13 . . . '! 1l ,, l .. '1 ........... 1( _,, Ii I· ; ,1 .I' I ,1 ' !k ., Dc~scriptions of Figure s Figure 1. Result of an experime nt· showing-progre ssive swelling of sand dollar eggs when transferred from sea woter 33.3 °/oo to dilute (60%) sea water. Ten eggs were used and each point is the mean of 9 to 10 measurements. Temp. 2s0 c. Figure 2. Time course of swelling of sand dollar eggs when placed in dilute sea water. Left curve, controls, means of 10 cells. Right, experimentals, means of 12 cells'. Experimental eggs were treated w~th No. 2 fuel oil stock-sea water 1:1 for · 2 to 3 h. At the start of the experiment, control and experimental eggs were transferred from a medium 35.8 °/oo to 6 0% sea water, ·· and diameters were measured. Controls: y _= -5.442 + 0.00889 x. Experimentals: y =-6.041 + 0.00877 x. I!I I Temp. 24-2s0 c. ~ • .I t· Figure 3. Rate of oxygen consumption of sand dollar sperm (contra.ls) in sea water 30 °/oo. Temp. 25~C. '· a Figure 4. Drawing of the apparatus used ~01, testing the behavior of burn0cle larvae in acute experiments. A, side view. B, top view. Scale X2 • ...... .. • Figures S to 7. Behavior of barnctcle 1arvae in the upparutus illustrc.1tcd i n Figure 4. The apparatus is designed to test the ability of larvae to migrate towards the light (positive phototaxis). Larvae are added to the l ·eft chamber' and the right chamber is illuminated for 15 min. Figure 5. Distribution of larvae in the four chambers after 15 min illumination. . '·· Control and an experiment involving durene at the concentrations (%) shown. Figure 6. Experimental larvae, in No. 2 fuel oil for 1 h. Proportion remaining in the left chamber. Figure 7. Experimental larvae, in dimethyl naphthalene for 1 h. Proportion remaining in the left chamber. .,,. J ,, ''1 Fi~ures f3 and 9. Lcirva(~ ( zocae ) of the stri ped hermit crab Clj.b.:jnnrj_us vitt.-itus. Survival in No. 2 fuel at the concentrations shown on the curw~ s. 'Figure 8, first experimGntal series. Figure 9 , second experimental series. '· .. 7. Fig-ure 10. Ilistograrns showinq the ~;urvivcil of l arvae of the~ spid'?r crab Li~ini~ dubia in No. 2 fuel oil ~t t he concentrations shown. Duration of expcrim·2nt, 11 days. Zl, Z2, first and second zoeae; M, rnegalops; Cl, first crab. '· .. ,, Fiourc 11. Surviv'-11 of stone crab l arvoe ( :::oc~ac~ ) cxpo0cd to Ho. 2 fuel oil. Controls and f~Xpc!rirnr.~ntols ( x10-l). Concentrations of oil shown on the curves. '· Figure 12. Survival of catfish in No. 2 fuel oil. '· Fig11re 13. Rl:-:! spiration of corneus of stin~;o.rees Dasy....0_ti~ snbina (oxygen consumption in pl mg-1 (dry we ight) h-1. Controls · 0 and experim2ntals 6 , the latter in No. 2 fuel oil ( 50%). '· t ·.;) '· l II ~ .. .. . ·~ ! ~ ! 'i d l. I I [ t f ~ I I ~ : . \ : ,I :~. -: Fi ti ~ {J ·~ t ·---- I I' iC '° ·s-01 cwrf ~wn10A ~ i ,.......... ;. J. t sl • / • '· • ..,. I ,,\ \: o.1..__~_6_.o-·o--'----6s..._o_......___70-o_...__7___,as_o_ _...__a_..oc I!i/ VoIum e µ." m3 x Io-3 .~ tf ....../ :.!1I \ Figure 2 0 '· I Q':? r-• -...,} ~ .,._ 0 t - : 0.2 ~ o_ N 0 - ~ 0.1 50 100 0 150 Time min -·. -·· ------·-· ... -------· . Figure 3 ·_J\ -----··' I ~ ;:~ IIi II nI 1 l .en : ~ ! I --~ ; !I ·1 ~ --'I iI---------: ~ ~--------~ ------1 ~ ;---...------------------... , ~ J i ! .L ;. . ii! ! .I ! I~ : I1 ~ '· I -t_J •, l " l u 1::.-_-_-_-_-_---=::::.::: L "'-l ~ I lI +-> II I c ~ I i I u I I.+> i l I "+-I I ~ II I _J I J ,. 1----·-; I +-' i I I~ I I i ~ ; I '_J l I _____1, IiI f l .,.// i <( I I co f I }I ' ~~·I 41 ,,.. c::' t.,,.. __.. -) ·-G'l LL ---·--. ---·- 100-rI 50: . ...--I Ii 2 '---·-·-· _Ij - ) r====7 ·, ')-·.:. .. -J 10 I ) ~--- 1 .i 1 -'c..---.. · -I C· I. --:: ) . • I ) . I I I ) L--·--. .l.'100i );--· • I . /Control 1·L II LC ij ·RC R I L LC\ j RC .·fR --1 ---' ---·I---------1 I 1 Fioure 5 r---------------------0 ·---·--·· _J Q '· ~= 0 LO ' ' ' ' 0 ' ' ' ' ~ t 0 ' ' i 0 I i c ' l 0 ' ' I ' I ' +-' ' 1' '., \ +' rJ 'O 0 d c ' ' ~I I u "' I I () l I c ' ' ' , 0 '. lDt \·u \ I I . I I • .o "~----' ~------------------- /too,-~-------~~~---~---~-------------------------------------- 1 L: II_ :~. I _aE:l - -cru ;:oo1 , +> I ~ ~I / ·' ~: /-·! / ·-C i! / I I / I 0...' , c' '/ o; ./ ~t·Aol / / L :L.} ~ / / / . o;o..o l: ./ L/ o_ 1lI / / ___1 ·~ . \ o· //o/ . / / 1 5·-· ______~10•.• . 50 100 --· •...t.._ Concentration °/o ,------- · i Figure 7 -----, r. '; . _., (J. r, .,._' · ;- • i I ' 0 0 '~ . ,. L . ~-J; . ...__ . -r-> • ~ ~ !~,, • ---r j 1· c 'J. ( -;\ ..J 0 :1~ u • c) c . l ,{ JOJ I i i---- . . I . . • '· 0 I J _t(j) -----·- . . . . .. •• . 1 ,. ' I I 0 0 -0) .• . I . • ...• I I s> . . f'.. ~ , . ""'• ! (\1 lu .. -
    I I 1tc:: ~l / ..v'V Z2 I & & z .._7"'1 l"'.1 1--1 i Z2 IZ2 M M M M \....t, ' \...... . 0 100 M &ivl 1&.titJ ~ 0 Y-' t- 50-1/25 Z2 Z2 o & & a.. Z1 Z1 Z2 z2· M·· &M C1 ;0 M M M M .&M 0:: 1001 50-Z1 Z1 Z1 Zl -1/10 & & & & Z1 Z1 Z2 Z? Z2 Z2 i ~) 7 0 9 ~o i 1 1---------------· \ Fioure 10 \ ;J -----·-------------wl ·"""',:.•·., -·-· :· "-:-:---=-------I ~-" \.,._, ~ .. -~• .:.._:..-. ~-> -~ ' ·. . --....:. .._. -x . I.. ' ' • • ..., . ..... .......0 ' ' ~·,\ --..._ I -·;!; ·.·. \ . ·"'-·, .............. ~....____ 1·-,,... .~....\.~.. ii.. .~e ' ....... Cl •._,v ' ' ... ' . ..., ' t '\ i! ... \ ' c\ , . I ·. . ' \ 1(,;-1 ": \ \ \ ' \ ~ : \ \ '· •• \ ' ! ~?'9 " \ 0\1: ~::i \ \ \ . . \ \ . u \ \ . \ \ \ \ ,·. ) ' ·. \ \ \ :. : \ \ I~ . \ \ 'o :. \ I: I0 . \ ' \ . . \\. ~-\ \ \ \ '. \ .I, .• . . \ 1 ·. ·.•. \ .. \ . .. \ . x ()-L--------,,------~~~----~o~~----~4~ I· 2 3 O Days, _ ,/ ,I . Fiaure I I ;; SECTION E NSF-IDOE GB 37345 A study of the effect of petroleum on sand dollar eggs W. H. Donahue and J. A. C. Nicol 1, .. Tr trc·rl.uci~.;on tolerance. In studies of oil pol.luttur: f:lE.:: h&ve bc:cn utilizing S(JJr.e of •, the CO!i"'JLOnl:/ availab1f:! animu.ls of the ri.1cxas coastal I'egi oT,l tmd, to t est developmental st(-lges we have used, int~ _&lia, gar.;2tes of the sand dollar. Investigations · have dealt with the effects of petrolet2.m on sperm, egg permeability, f~rtilization, cleavage, and early development of embryos. I Extensive studies of the permeability of .animal cells have, / exploited marine eggs and marrrrnalian erythrocytes (Lucke and M.cCutchcon: 1932). Among marine eg·gs. used have been those of sea stars l\st crias, urchins Arbac:La, polycnaetes Chactoptc...:rus, and pelecypods Os trea and / Cumingia (Lucke, Hartline and Ricca, 1939; Luckle and Ricca, 1941 . . ) A method frequently used to measure ·permea]?ility to wate~ has been to expose the egg to a dilute medium and to measure the change of diameter which ensues with time. Eggs have been exposed to test substances and possible changes of permeability resulting therefrom ~xplored by the same method. Marine eggs are invested by semipermeable membranes and behave as good osrnometers when transferred to anisosmotic media. In hypotonic solutions water enters the cel ls owing to the driving force of osmotic pressu:-e. ' T~1e r ate of p(:rr:12.:... ~ility of tiie cell to ·.-ctter is ca.lcula'tc~ · from the equation p I #v ,,.,1 _.... \,.J ..... ,,/ \.,.. }...,) s~ ~~ L·:J .~ ·' -_,.__ ~. .._ ) _ l. c·:--,,"1;-..:. t. 0.r. '": r)_JL·; ./ • .-·-• •.1.....:>) v . u. J ·1' .c..;:.,..,(, ·--· rn (;' ,. ~ "--i.-·1'1(l, _J._'/_ J._c'tU~ :l-. 1. ('''j l c "I' ·oy .. .J..1. .. c.4_LJ., )i 1·ii~-i::. ~..-r· ( T.,.,....,,·S ·· -.,·l , ,, o,.1 __ /\.. .•. , ,_1j .:.·. ""' ~o·· ...lP lf.1,_ .._ ,::., ' ..__ ,_ a.ri.d E.:rrtlinc, l~J.55). Ripe eggs of sand dol1o.T'S u.re easy to secure ()J',d fc;r"'cilize , t'i:ey develop to the echinoplut2us stc1 Experiments with sperm In this series the -same experimental solutions and controls were used. The eggs, which had been shed into a culture bowl of sea water, were agitated and allowed to settle~ The supernatant was decanted and replacedwith clean sea w~te~this was repeated twice. Sperm was collected in a 25 rmn culture bowl· containing a little sea water. Fpur drops of this concentrate were added to 20 ml of the various oil stock dilutions (experi'raentals) and to 20 ml of sea water (the control). The sperm remaine;0. ~n each solution, experimentals and control, for 30 min. At that time · so ml of sperm solution was pipetted into 50 ml culture bowls . each con~ cec.: i :l ;:· ~--~c<;c~ f~:1-~ ":n..::> v:(~Y"2 ::o. ~ ft.~ :--:1 c.;1.:.. ~-,.. , / 'I'his mixture, :ceferred to as oil stor:k (con(' ' '-~I~\.":.':'o~~l •-LI._,,.._ u,.._ -i n~1· '~':'"\U J •• L ..: ,-I.. y·) L ••• ~ ! ' wt:~s d:U.uted with sec-. wat er as r equired.. l, Oil stocks were Tf\0d£2 up week.ly and stm.'ed in a ref::-igc~ator ( 4°C). Results Permeability to w2.-ter ... I 'E.1.e average di2_ m~ter of 40 eggs in 33.31°100 sea water was l.06 µm, "the rar.ge ·was ·94. 6 to 114.8 J.Un, the average volurne was 623763 µm3. The average diameter of 92 eggs in 35.8°/oo sea water was 106.7 µm, the range was 95. 5 to 120.1 µm) the average .volume was 636022 pm3. The osmotically inert volUJ.le of the cell was 11% and k, the permeability ! I of the cell to water (Lucke, Larra~ee and_ Hartline, 1935; Lucke, ...:,, Hartline and Ricca, 1939_), was _P. l~~ cubic µm of -water per square p.rn of cell surf&ce per min for a pressure difference of 1 atmos. Values of k to 0 .1 to 0. 4 -for stu1dry species of urchins(sand dollar.s and sea I stars have been published (Lucke, 1940). Trarisferred fro~ full strength (33.~0/oo) to dilute sea water (60%) the eggs swelled , reaching full equilibriUiu in 7 to 8 min (Fig. 1). The curve for swelling rate is muc:'l. like those presented for othe'r J etc. , Luc~e, Lcn""'ti.i::-ie ar;d :-<.icea, 1939) ," &:ad ,. ·• .,/I / I r /' t.:1\~ :··(.L hi c[<)l.°~_ ._1:1 ;,_·.· • :tr:; ,-,>~·~:_· ,~ir. .-·1 ·t:c; I~ ~~ l.1c~·j~ .· , \/~; r~~r .~1:·~ q~,~(,;~ \')t~;; ~·,·H ;··· .·r~ r·:--,c Ci ~ .L ' I~ -·. l • • . , •. '' ,-,;·l ~t '' ., . . (• .,., / ...-, ..., c/"> j ,-,r)) -'•; • I Y ...J, .~ ~ ' •-• I ¥ • • \_• sp:1erical, ari.d t:r.e surface crc:a ::>os cli~ ; c-:.qpear2d .-s thG egg-:; bPcc?;J~ .-:C .turgid.. m..1.hP u._0c~ ' ~ • .._ ~(3_\,fl·L~-_:o-~l l.£r0~JU ~p~P~~c -1 _.. l ._'} · l:~.l.•.l 0.- sl-:.ape intrc6uci:::y' som~ v.:n."' j_ J ticns /.-:J·~-rT'C...- into the data for rate o:: S\· (~lling o~c indivj_dual cells, which ~;.~~· mutually conterbaJ.:·nced in the means. An experiment involved 10 t:o 12 eggs: with these numbers, m2an ini tie:1l volumes w2re noi... i 6entical. The rate of swelling.of eggs in No. 2 fuel oil stock -60% ?G:.ter 1:1 is .4' ... r show.l1 in Fig. 2, compared with a control series;· It is apparent that the rates of swelling are the samev Fertilization, cleavage and io.rval ctevelopm_ent All the controls in untreated sea water developed normally. Approximately 1/2, 1 and l 1/2 h after insemination, samples of 100 eggs showed·100% with vite.lline membrane; first and second cleava.ges, respectively. After approximately 24 h the control bowl contained a large swarm of beautifully formed pluteus larvae; there were very fe;w undeveloped eggs on the bottom.. The results of experiments with pet~oletL-n oils are sumrna:cized in Table I .. Kuwait oil stock-sea wate~ l:~~ In general Kuwait oil, at this high conc·:·nt1;'ation, had the least ef ~ect of all rr:i xD;res tried on fertilization and develo~ment. The red·...iced ·1u:1ilier of eg~7s unrie, qoj_r,g first cle· vo.ge wa.s probciJ)l~' sisnific rrt; S(~cor:.d cleavage .Jnd lurvSl?J. .... -· e I'~1Jr-1 1"irnr~-r1tc l: _,,..., . 2 fuel .1.\-t. "-J e.ffect~; on the fertilization ,3n~ dr~volopiTle:rc of sand dol1ar egg·~:; thatr 1 &d been i:.::xposed to it for an hour :be:Zore the a.dd:ixion of c.perm, and "'che:t r cma:ineci in it througout developmsnt. On the other· hand, No . 2 :CuEd oil at the hi9l·,cst concentration employed in the experiments completely destroyed the ·~ ability of the eggs t:o ·be fertilized vnd to continue d<.:?Velopment~:·. The three concen'c-.rations bctw~en these extremes showed deleterious effecJcs in proportion to the ~nounts of oil present~ These effects becari.e evident first of all in the reduced percentages of fertilization, starting with the 1: 25 ·dilution~ viz. · 78%, o.nd going down ~o 0% in the 1:1 dilution. The drastic drop in the last mixture rnay have been .. caused by injury to sperm (vide infE.E_). .The· data are equivocal on this matter; however, the progressive decline of successful fertflizations~ as the concentrations of fuel ·oil increased, indicate. that an even greater ·eff~ct on the eggs rnight be expected at a five-fold increase of coricentration above 1:5~ :Sven when f ertiliz&tion was achieved, development was not always no:r1((aL -··· A.unormal cleavag·es firs~ appeared in tbe eggs in the 1:10 aJ1d 1.: 5 dilutions at the ti1.1e of first cleavage ii It might be assumed th t t~1ose c'leo.ving ;;.\0r:o:c·mal1y ,'' ,. , ~£~;·~·~~;_--;h~~eus larv~e.. ~bno_mal cleava1es·were evident in later .' i of sand dollar eggs. Since no effects at this concentration were dis cove;:-ed, the::-:0 wa.s no incontive to test lm;2r concc~ntrc.t:i.ons, as wa.s dvri(c: vdt:n .ho.. ·2 fuel oil.. Th~ experiments r evealed a great dif.ierencf~ ::.I i "l:he effects of these 1...wo types of oil on ·-he sand dollar eg·gs: the Kuwait, seeraingly, wa s h2~rrnl2ss, vihil e a comparable concentratiori.'· of No. 2 fuel oil was totally lethal-. Experiments with sperm r "· . \ Exposure of ·sperm cells of the sand dollar to the Xuwait ~d. oil to the various concent rations of tne No. 2 fuel oil for a half l1our'· prior to fertilization had appd.rently no e.i:fect on the sperm;;; . except for No. 2 fuel oil at a .concentration of l _: L. In the latter mixture the ability of the sperm to fertilize the eggs was completely destroyed. There was rw change of fertilization percem:age, as was found when th~ eggs ware subjected to the oil, but rather a sudden reduction to zero fertilization.· It is not clear if t:iis was caused by loss 0£ sperm h\otility~ or by·some othe~ factor.. / ; .,~~l~~\:.'~~ .. / ....... 'T ., iit.~2~0' 1). j ! l. J\. (' -· .).;.. _____..:,,. __ c~1;nj~;-~ c~ ~ a c1~l~~~;.~...~':.JtJtr.:~::'11 :=; •..\J v ------_:....._~-- P11ysioL ~LLl-, 237--252. / )), Lucke, B., H. 8. Larra.her~ c\nd H. K.. IIartlin.-2;, 19:~5. St-cdiGs on osn1ot:; .~ " equilibrium and on the k:ineti.cs of osri1osis 1:1. liv~-~1g c~lls by c: diffraction method. J. ge:.n. TY1y""'i.0J J.. .I ~ -·. 1° -i-17 _.:::.., ..l. -'-• Luck~, B. and H. Mccutcheon. 1932. The· .living cell u3 an os'11otic sys ·terr~ and its permeability to water. Physiol. Rev. 12, 68-139. ... ~ ~:. Lud'-e, B., A. X. Parpor·t and R. l\. Ricca. 1941. Failure of choleis ac~..ds of carcinogenic hydroca·rbons to alter permeability of ·marine eggs ario of mabrnalian e1"\ythrocyles. Cancer Res .. 2:, 709-713. I Lucke,. B. and. R. A• .Ricca. 1941. Os;rlotic properties of the egg cells 0£ the oyster (O.strea virginica). J. ·gen. Physiol. 25, 215-227. Mccutcheon, .M. and B. Lucke. 1926: The kinetics of osmotic swelling in living cells. J. gen. Physiol.~2' 697-707. ·, ' ·~ I. . .. ' ; t •I I • / / ,-.2,lbJ (; I ·.·:', , ·(·:--.'_~) r __ c: _~.'~·: -~ ._~ "') -~ ,-.. , -~ .... " ---:. . : .. ,) '.... J J t. _ \._ c tl ::-ures 24 h after insen'J.n2i.:ion .. ~ '. ~ .* ._.) ( ) Con·lro1 . Kuwait J :l No .. 2 Fue1 l~ 50 1: L~5 J.:10 . 1: 5 . l.: 1 ) Ta! I Vitelline membrane First cJ.eavo.ge Sec:ond cJ_t;avasrc: T ~.. .. \1' _.,, '."""'\ .....; C.... L ,., -:"· Present Absent Pre.sent l~lis ;2nt P:cc sc:n.t Ah~; e;rt: Hu: .·:,:)cr· fJ r? \: C J_ C· ~-•~ ~: ..... : · t -1 Nonnal Abnormal Horir.al l\.bno.:··~na.1 ) "" 100 .o 100 0 0 100 .' 0 0 Exc0}J_ 2:-~ c .t:xc c:·J_ J. :~ ·-_ .. 100 0 65 0 35 99. ---0 Ir ,, l 100 ' ·o 100 0 0 100 0 0 u I ~ ..; .. 78 22· 67 0 33 35 · It 5 10 \/cry g.:.: .~~ ._: s 95 :?n 23. 31 46 3 -J L 65 .Fail"' FE:i:.~ 1 99 3 23 74 2 .. 0 S·8 E>-:tI'\ 2~:·.c: 2-~/ s-: i.~ :_--:_· ~·~--.:.; -~~, 1 yco:_l. I)C.~.::-· . .. ·o .100 0 0 100 0 0 J_lJQ l-~Cn•2 ;:sr~s :'~.. '· :~ '·, ,,/ .. 2 1/2 h. exa.r.1ined '· L :;::o <:..~·~cr;~ri.,.,i12 and condition of lcTI'va_. ... ~ . .'. I 'I I, ·1 '4 _ .,. , IC·-::;;~ c c .'i.. J.s Cnnt,_ol Q (~~/ -· -,~) '.JS 9G SS 11 '· 99 94 i1 95 1:10 99 100 99 l .; 5 97 C.7 97 _, . 1 .: O· ...... 0 0 li"one ,. ,, . . ·· · .. ~. ~ •... --,-! -''-'' .......; I.>i g. -. ·. "". --J..4 ..c..,.... .,__ 2 fuel oil stoc~-s -a water I • f ..J.-I) -L... .l•...).;.. 2 OT the mental eggs were transferred from 60% Controls: y ::: -5 .. 4l~2 ,·. 0.,00889 )(~ Exper·irr.entals ~ y = -6.. 04.:. -I O,,COB77 x" • . ... .,.-./' ··"'~·-..• •f'·••--...... ~ .. .. :-~-....-.-.. •••.Pr..._·~·_ ... ..__ __...._...,,..,.,.. ••.•.., .. .,..,.....--...-...,., _..,_._,,..~ ...... -......... ~....... •••1 ••""'"••' ., ,,, ,, • ... ,,.. ·• • .. _ •., •. , ••• ; )· \ ', ! \ ··.,, \ I i, \ ,,.~~, ~. r · ,~ " •4\~· ~\ \ ..... . .. 1·1 I 'I ,.,, ~' . .-..~ ~ ,: ...,, ~ ~ .~-r.: .'.) ) ; I' ......... )' ·~.....) t ,:• • ~.,,,...... ll ·. --~ · 11 0 [1 uv .: I t .. 200 ~-·co. ooo :. c6-nC-Ci1ta(i6i1_,_ppm Fi our~ .) :J c ~ I n ...!. ' • .._ '-C0'~1...,4L-..·0t-,~_.... Q, n11m·o· eY'c;.. ~t.;-i. .,, _ __, -,1-:~-1 ·1""\rg O""" -'<....,.Jh~-L " settli~g by un au~oma~ic counter. Materials a-:1.d EeJchods Barnacles 3alan-...1s amD!-litrite niveus were collected daily from the ~.:~< ::...\:.. •.•d side of the south jetty at Port Jl.ransas. Placed in a bowl of sea water, they soon released great numbers of nauplii. The bowl .was illuminated on one side, to which the larvae swam because they were pos~t{v~;f~p~ototactic. Larvae were collected and transferred to a f:cesh bowl of sea water, unilaterally illuminated, and the procedure was :'e~eated (two transfers). _,. I This procedure provided a suspension of larv~e -:.::ree from other animals.and _extr&1eous material. Stock solutions of oil and pu~e compounds were prepared in the manner described previousiy. Stocks were rated l00% and a series of 19 concent-~ations (lOG% to 0%) were made by adding stock to sea water. Quantities of 2 ml each were added to Pasteur capillary pipettes. ':Lhe bottoJ.ls of the pipettes were covered with sleeves of tygon tubing, which \·1ere sealed at one end (see Figure 1). Pipettes were placed on . a rack, which was partially enclosed in such a way that the lm"-er h&lves of the· ~Jh~s we~e j_n da:".':c·.ess, ·whi .2.e the up:;).2-c· halves were in the :..io-l~t. 0~2 drop of sea water co:-ltaining several hundred barnacle 1;•1 .. -·, ' -4. ~\ .l .. ' .1.·1c.s collected in aEoth,:;1: tube; this was the top fractio:i .. '· W2ll over a million larvae were assayed. The larvae in each fraction 1.·1ere passed through an o.utomatic counter built by Dr. I/Jang. The larvae moved singly through an illui:i.in-::.t:;d capillary lying over a photoelec~ric cell, and interx1uption of the ste&dy signal was recorded on a digital multimeter. The percentages of 1ar·vae in the botto:n fractions w2re plotted against. the log .of percentag~ saturation of pollutant, and the percentage saturations at which 50% of the larvae were sent to the bottom were ·read off the curves .. Results The results are presented in Table I (oils) and Table II (single cora~ounds). The values for 50% of larvae in the bottom fraction (deject D~) were inversely proportional to the potency of the pollutant~ Discussion and Conclusion ':'he h';ethod allows rapid assay of a large nui:lber of oils, fractio~s and constitt.:ents, rela~ive to each other. The test animals are no~r.~l •r-r T_, ._i"f'V.~.(1 -·-......... . . l ........... ; ,_ ' Se2 previous report. '· -. .. -, .. , ') r~ _L :...: ~~ \ :• \.: : -, ~.) I_ n _, u 2'z·:, ). , . ~-, l."\ '-lC t.iotl C trom :C1..ic: l oil r : s~~ · ' _.,, ,: n l't"·I L' ·.-/o 1 I• 22% 15% 6. Fl"C.C~ion A4 16% 7. F:-'action Ji.5 '· 23% 8. Fraction AG 16% 9. Fract-ion A7 ! • 15% from #2 fuel oil ! 10. Kuwait 28% l l 11. Southern Louisiana crude )! 93% it '1 ... 12. Bunker c 33% 13. Alaskan crude 70% 14. Ve~ezuelan crude oil 56% 15. Diesel fuel oil 62% 16. Drained crank case oil (Texaco Havaline 10W40) 3% ... i ! ...;,_ . ? • .) . s. G. 7. r, 0. 9. 10. 11. 12. 13. ?r i 2t hyl benzene ;(.:iphthal~ne l-Y~eic:1yl-naphthal2:--.e Din:ethyl-naphthalene Durene Bipnenyl ?l-lenanthrene ~· 4% In prog-~ess '· 1 3% 6.4% 60/_ ,o 69% ... 44% 27% 70% -..._/ . / ___.__ ;. .:..;: . ----1: Li