Document 6RvqEwGEwkXn72XwD6aNv4Q3E

.( 7 lifTccis of Arfx 1 oi- 1?A2^ 1--^ L x r i f` (n I'oIyrlilon'iKttcfl ISljihcnyl) > ;m<l D))T on CulluroK of in Prolo/.o.-m, P.utlumf, (Jstrncod, mid Guppy0 , ' byiAhTT R. HnilCAN fJ J /_ ' mil/<* ^ 1 'V ( *<<* m f nn>rj/g^i) : s *^ 'Mi /I '.1(I i "(t >j s>/ * d, ' f; .. <1 1 t ,* - H* Hie polychlorinated biphenyls (TCn ' cr> are <i group ol chemicals having physical and chemical properties sufficiently similar to those of DDT to warrant inven- tight,on (10). The Monsanto Company, the sole producer of mi's in the United States, markets them under the trade name of Aroclor. Aroclcr 1242 is ' a mixture of PCD compounds containing approximately A2Y, chlorine by weight. Diverse in their commercial ..pplicr.Lions, but most commonly employed as plasti- ciicn, TCD's have increased in usage dramatically since the forties (6). Jensen first reported finding roll's in pike, other fish, eagle feathers and human hair from Sweden in 1900 (1). Subsequently, scion- ticts fro.i around the world began analysing wiltllifo tissues lor the presence of Pen's, Chlorinated biphenyls v/cre detected in fish, mussels, and birds from iho Rhine Pivor and the coast of the rtctharlando (V), in :.pai`rovhaw):K, kestrels and kittiwakcs from d'V-al biitain (4) and peregrine falcons from the ' United States. (9). Cultures of the marine diatom C 'ildii1 clo*.torium have been shown capable of ' absorbing Aroclor 1242 from sea water containing 0.1 ppm and concentrating it 900 to 1000 times (G) , This concentration caused, however, a marked reduction in growth, chlorophyll index, and RNA synthesis of tho marine diatom, but no noticeable e'ffect on DNA cyn- the si s, The aim of the present study -was to investigate sor.c of the effects of Aroclor 1242 and DDT on a variety of aquatic organisms. For a more detailed report, please refer to the Master's thesis (9)* It Registered Trademark, Monsanto Co., St. Louis llo. Fisheries contribution No.350 . ii9-ill * VwMxtn tl r*ii#*np ml*) C**< wi X Tet^feUff, V*i I, J, KlA. Q fcf S^i*n|4f Ycfitj ftV*<k *!' < t ir MONO 063305 N ` Mo thod: k Pivo different or9*vuim: were used in the present investigation: the *vlgn Ch 1 unydomon-is * rr i nh,-irdt i.i , the cilinto piotoroan Trt r nhytn'-n.i vorax, the c lndoccr.in D.iphn i n the or.traced cvin i dop- vadun, and the guppy Pooci 1 i a re t i cul.H n . Media (>3 (12) wa:. the experimental medium in which the 'effects of the toxicants upon algae, cladoccr.ms, and ostracodc were studied, Pioassay media, modified i from rerncll and Por.cn (3) to contain 1/100 of tho . i . . oigame components and 1/10 of the organic r-alta, 1 w.n used for the Tetrahynena studies. Coarr.cly- fill e-rod pond viator vws used in the guppy toxicity tests. Since both DDT and PCD have a tendency to * adhere to surfaces (?, 14), disposable glassware was vised to prevent residues of the chlorinated hydrocar bons from affecting the results of subsequent experi-" mer.ts. The culture vessels were either quart or , half-gallon tr.nson jars with half of a disposable _ , plastic Petri plate for a lid or disposnble#glasa r Lost tubes for the protozoan studies. At the start of an experiment gll culture vessels and media wero autoclaved at 250w C. for 25 minutes to insure , sterility (except the guppy experiments in v/hi ch . pond water v.e.r. used), One hundred percent p.p'-DPT, designated as the "ESA pesticide reference standard" wan u-.cd in all DDT experiments and the Aroclcr 1242 used was obtained directly fro.n Monsanto in St. Louis, , Hi sscniri. Ihc test chemical was generally added to - , the growth media prior to the introduction of the , 't organisms. In order to distribute the tost chemical throughout the media, and to ease in making dilutions', acetone was used as a yohicle for DDT, and CSji ethyl . ' alcohol was used for the PCD, A concentration of 0.2 ml solvcnt/litcr media was used, which did not appear Lo adversely affect the tested organisms as shown by . preliminary experiments and usually demonstrated by the- solvent controls. Once algal density was determined, a known volume of inoculum was added to each culture so that the initial concentration in the culture vessel would be 0.1 >; lo cells/ml. Adult Daphnia. ostracode, and fish were removed from their stock cultures, ran domized, and counted before placing them in the test cultures, Experimental cultures containing algae and/ or Paphnia were grown at 20.S C. t lc. under a bank of fluoroocont lighto which delivered 900 foot-candles .` . 130 (ac measured by a GC model 213 lightmetor with d blue filter) . tvt.r tlbyriri: I vnrax culturna, grown in tent tubes, wcie placed on a roller tuoc apparatus which rotated the tubes at 1/5 rpm, pfotozoan, ostracod, and guppy bioassayr. were conducted under the con ditions of 23.5 CilC. and 75 footcandlca of light. During tho course of an experiment, population counts of the organisms present in each culture vessel J *7 <7 i: *AV *' -J r. PL. 1. Effect of the addition of 0.2, 2, and 20 ppm Aroclor 1242 on tho Growth of Chlanydoionas roinherdtii. Each point represents tho oean of two oboervationo whoso individual values aro indicated as the rango , i Ml MOWS 06336 s/ st.iincd by adding a drop of l,-KI solution, then coun ted using a haemneytometor at'iOOX. The Tetrahymenn were stained in the snino rnannci, then ten 0,01 nil diops of tho culture were distributed on a falcon counting plate and the colls in each counted at SOX. Cnrbon-14 uptohe by nlg.il cells subjected to various concen trations of both DDT and rCD^yas determined by incu bating 5 ml samples wit'll Mali 'tO^ and using a standard filtration-planchct method. Gas chromatography anulyaifl was performed by Dr, Gamer. Saddler o the rirhericr. , Hcocarch Institute on five algal culturcs--a control, a solvent control, and three cultures to which 20, 2, . and 0,2 pp.a Aroclor had been added. Tho algal pol-- - lets and supernatant samples were extracted with occtonitrilc-pctrolcum ether; purified by olution on a Florisil column; and injected into a gac chromoto- . graph equipped with an electron capture detector, ' Observations--Discussion' ,, " Figure 1 illustrates the effect of throp dif- * `;v ferent doses (0.2, 2, and 20 ppm) of Aroclor 1242- - - ,/ alcohol on the density of Chlamydomonos over a period .*. of 22 days. Doth the control and the solvent control / , followed tho typical parabolic growth curve with no initial lag period. There was a period of rapid giowth (the log phase) followed by a leveling off of the population size as the culture approached the station ary phase. Aroclor-eontaining cultures did not follow this pattern, however, but took a much longer time to approach the cell density of the controls. In the CM ni.ydo~ona'`i cultures to which the two highest dosages/ of Aroclor were added there appeared to be not only ,,, a delay" before exponential growth began, but an initial, drop in tho algal density below the original inoculum sire. The severity of the response was dose related, After day 5, the growth rate of these inhibited cul-'j . * turcs increased so that by day 22 they had cell den- p allien similar to the controls, Dy day 22, tho standing crop of Chlamydomonas in all of the cultures* appeared to be about the same. This experiment was 1 repeated several times with similar results, although the degree of inhibition from equal additions of PCD ' varied somewhat. This variation rnay bo duo to heterogeneity of the PCD globular suspensions (14), ' or to differences in other physical factors from experiment to experiment. 1 -.4* '' m A : \. ir ;, 'i-------- i phi1 i '* k ' - , > \ i " ' :\ -V *'V' 'v.V> HONS 0833dd I /* k i I' , I ' > ' ) ) , ' 11 .- > 1 t J \ Jn order to determine wlirUtft C uptokr by rh colls was .11 o affected by the addition oWiooioV 12 12, the experiment was repented u:.ing .five different doses of.PCU (0.002, 0.02, 0,2,2, .inti 20 ppn), incubating Nall CO^ with samples taken from the cultures at three different points in the grj^th period. On day 4, the control had the greatest C uptake and the PCB containing cultures had upLnkeo per coll and per ml in inverse relationship^}; their FCn concentration. By days 11 and 15, the C uptake ;pcr cell were all markedly reduced and showed no docoj related response. These results were undoubtedly influenced by tljjjj varying amounts of nutrients still available. The C uptake per ml tended to increase during the durotion of the experiment, the values (jenerally in inverse relation to the dose. . 1 A possible'reason for the apparent recovery of Chi at.wdomonns from the initial effects of PCB- containing media is that the PCB was degraded or otherwise made less effective during the course of the experiment. Since an experiment conducted by Hioebrough (11) indicated that PCB compounds could be broken down by ultraviolet light, the possibility that the fluorescent Lights used in growing the algae cultures were causing degradation of the added PCD was investigated. Mason jars of Media 63 containing 2 ppm Aroclor l?h2 were placed under the bank of fluorescent 1ignis for 7, 5, 2 or 0 days prior to their inoculation with Chlamydomonas Ml of the PCB-containing cultures exhibited delayed algal growth and there did not appear to be any correlation between degree of inhibition and the length of time the media was under the liqhts after - the PCB was added. This seemed to indicate that neither degradation by light nor adsorption of PCB on , the glass were significant factors in the delayed growth and subsequent recovery of the Chi nrnydoronas cul- - turos. Another possibility is that the PCB may have " been metabolized cither by the algae or by contaminating bacteria. No difference, however, was noted in the chromatograms between contaminated and axenic cultures, nor in degree of algal growth inhibition, hence bac terial degradation of Aroclor 1242 was probably not a significant factor. . Data from the gas chromatography analysis seem to indicate that^ the Chlamydomonas was concentrating the -PCB, for a much higher concentration was found in the 1J1 ' ' -'V `` f /. MOMS 083369 I I '.tl ] J oly.il pellet (which too!* up about 0.0^ ml in volume) th.in in the one liter of cupein.it.int. For example, in , cultures to which 20 ppm (my/1) PCD hod been added, 0.52 my were detected in the aly.ic, and onlyO.04 mg in- the supernatant. A large percentage of PCD that was added war. not recovered froyn either the media or tho algae, This moy have been lone because of adherence to * the glare rides of the mason jars, sinking of small PCD globule.", to the bottom, vaporisation, codir.tillation * with uthir, metabolism by the algae, or loss in tho . extraction, Florisil clean-up, or injection processes. . .. The effect of DDT or, tho growth and uptakes of ! Chi anyth ./inns cultures was studied. In the. tested con centration range of 0.2 ppm to 20 ppm DDT, the algal growth curves did not differ appreciably from those of the controls. Whi 1c both Wurr.Lcr (13) and Monjjjl ot_ al. (0) were able to demonstrate that DDT reduced c uptako in various species of marine phytoplankton, in^Jpic ' present study no significant effect of DOT on ,C u^ituko . by C.H 'rry'pmqn.is w,,<, noted. Hansel -ct al. (0) did observe that Dun.ilicl)a tc-rtiolccta, a green flagellate, did not appear to bo affected by concentrations of DDT as high as 1 ppm. It is thus possible that Chlnmyjomonaq. like ' Dunelir:) la, is relatively DDV resistant, am) yet may bo effected by polychlorinated biphenyls which may restrict 14 C, uptake by a different mechanism. , '` - -1t* \h 7- V '. . > V'- , ~'C ( J I' F ' *' * ! .T Tho toxicity of PCD and DDT to organisms other than algae was then investigated. 'The growth curves of tho proto soon Teti nhymena vornx grown in tho presence of _ ' Aroclor 1242 (0.02 to 20 ppm) and in the presence of ' DDf (0.01 to 100 ppm) seem to indicate that neither j . chronical lias any effect upon the growth rate or , population size. Aroclor 1242 appeared to be fairly to <ic to Daphnln pulr-x, however. Young Daphn ia died at levels as low as 0.02 ppm. Mortality may bo linkod with other stresses as well, however, for v.hcn the , n.inhnia were supplied with algal culture, the death ' nte was cut to one-third. A comparison of tho relative toxicity of TCB and DDT to ostracods revealed' a greater mortality of Cypridoasis vidua at 20 ppm PCD than at the same concentration of DDT. At 20 ppm there were no survivors out of 20 ostracods in the ' '' Arsilor-containing cultures while there were 13 sur- . .* vivors in tho DDT cultures. At lower concentrations, however, DDV appocred to have more of an effect than ' ) ) \ 'f or - 1M tr T* ' 1 HONS 083390 the rcfl. The guppy Pore i 1 \ a rr t- i r > 11 n ( n >1 fid ) 0071 mor tality :>t 20 and 2 ppm. but. only 2j'/, mortality ot 0.2 Hie behavior o the fish jvist prior to death in _ PCiJ-containing cultures was quite different from tha normal symptoms of hydrocarbon pesticide poisoning. 3n ;omc cniefl behavior included rapid pivoting of the body with the nose proved close to the bottom of tho jar; funguB-lii.e growLhs were also often associated with dying or rcccntly-dcad fish. ,, To explore the passage of pen through tho food chain, paphnia in rCB-frcc medio were supplied with algal cells that had been grown in Aroclor-containing . podia, centrifuged, and resuspended in PCB-free media, All of the three-day old Paphnia fed algae cells grown in media containing 20 ppm were dead by the fourth day, and there wore only three survivora from among the thirty fed algae.from the culture containing 2 ppm. The mortality rate was higher among control Daphnia that were starved than among thore that were given PCB-free algae. . t&L Suni-iarv Aroclor 1242 appeared to temporarily inhibit tho growlh rate of fh1nmydononat as evidencod by cell counts nnd C uptnho, while DDT did not appear to .have.any effect. A greater concentration of TCB waa found in the algal cells than in Lhe media, intli- , eating that concentration and passage of tho biphenyl compounds through the food chain may take* place. Neither Aroclor 1242 nor DDT in the amounts tested appeared to have any effect on the growth or cell density of Tetrahymenn vornx cultures, A summary of the results of the toxicity tents on Daphnia, or.lracodc, and guppies is presented in Table 1. According to the table, Donhnia pulex were quite sensitive to additions of Aroclor 1242 as low as 0.02 ppm. Tne toxicity of PCB and DDT to tho ` ostracod Cypndonsir. vidua appeared to be approx- ' imately the same, founy guppies died in caneen-' trations of 2 ppm Aroclor 1242. Aroclor 1242 appears to have a much lower toxicity to clado- cerans and guppies than p,p`-DDT. Although tho effect of PCB on algal growth appeared to bo tem porary, in view of itc evident capability to be passed through tho food chain and its selective ' 1U { r, t: r. ' IS*' ' * , r C. *a. *i f * ' ' I,, '> i- u, MQNS 0S3391 V;' -'s.V ,h , ` . -V - 1 I ,' '1 - ' ** * , i` i *" t ' *' ,. ' * * 1 . I C! l! HV `EHJ >,. o tV 44 > .fl *0 M 0> > VI *J 4) ffc) V V. n m oo ooo 'N >' oo o> po *1 , ." M *+f h </> W V* |4 O 0 f> > o o o o o' *, o o .! t > ;OO I t+ ' t i. H H o u, Vr< : & zoi M " * *m* +.+ '* i * + n + ... i + 0 t: CV V 0 :* - *- >* r. Cf'V; ** 5 r"' * ' _ , wu ^4 >. -**/' m u J i ':;Jt iV7..i :M1 x I + w o > t ... ,.T , .* .* in .i . . * M c* w> oi * :* oi ++ ^D U <A 41 o o #. 4I i - - +. + . - 4d> ?t o ^' *4 * 0 ** * w fc* , - 4 4 1 40=1, V (J t/A: ii i ( I*. I *; 1 ** 4 ii > i i i i -oV 1 l, 1 L4 v. O4 CJ OJ 4- ,4 * V) o 34 'i ,i . -t i ryt* ' , CP *' 6 ** ^ tr * 3 CP ev nc 3 *4 4J 4J H HH 3 3^ , ' 4J ' #-44 9 Cp t3> cr 3 Cl JJ * ................ o+ 0 00 *0 13 Ui - ;. *o 0 O *4 o 4 4oJ ra o fW , m 0 U rj 7s. ,? tt * 4-r ' >% 't f J >4 /4 6# IV * *4 4 44 * 4 4 " * . *4 41 f ft H . ' f ** ja * **4 < (H * * ", t*v c*> , w - . 4 IA IA >v fN V' 40'. 4 CD V. 'rt 4* c 0 4 * d 4. .r " . t *_ '` 4 * c CP > o c 0 -H #0 9 CA 4J 41 M fr* Ml 0 nj 4^ C r4 TS H5 0 *0 o to V 1 ro m H C o u* 4c &H 0 O Mto pC Hi H4 10 o i u 4J 1-1 u AS 41 C 0 c U Ro A U 0 At CP (A cU W r. 3 *r4 +J r4 JJ 3 r* o o u 10 CQ CP LI H p* 4( C <4 rHc O u --1 3 IV *J tn *i *- o C 3 00u uo uw 1 r-t 10 4J y tp w S rH O 19 h-4 r-l 4-' 0 'el c; o o o A9 n (A a1 o crt D Ud Oud 0H 3 *0 o0 01 li ft *T-< o to to u f 4J cq H| O0 Pk V) cd H or4 10 V> *"1 <3 c r as a* a (A rt> g aM * f " t y." * < :* ' / t' w * I pi ! * . .-44 -* ^ , 08339i HONS I> r * * . * ` * A,-. toxicity to certain xooplankton, it wauld bo unwiaa,. . to attempt to predict its effect on tno ococyotcm ^ . without further study. ' . ** - ' v 1 1 *. \ ` . * ." rft,:.- t" ..................' ` ` `v. . Acknow] cdqomcnt s ' ; ' ' ' \ ' ' 1 f/r For assistance with the research and constructive; . ' criticism of the manuscript, X wish to thunk *. Dr. Frieda Taub and the members of )ier laloxatory ; .*. staff. . This research vns supported by the Eivviron- mental'Protection Agency, Grant 16050 DXM. .. | . : , '' i \'i v . ' ....... .. , 'i 'ji. J* t * `: he fercnccs > - >- * ANONYMOUS, New sci., 32, 612 {1966). ' * -V J I* 2.' jwstmah, E." and L. ST1CKEL, Ann. N. If. ,, " '*' " * " ` Acad, set., 160. 162 (1969) ' .*. ** * It'/.*/) . J... 3.* FEkNELL, U. and G. ROSEN, Brit. J. Nuts.,' . ^ jo, 143 (1956). . "* /', " Pi. , .. ` holmes, d. . shimons,* and j. tatton, .* h'uturc, 216, 227 (1967). . *' 5. 1 JENSEN, S., A. J05INn,S, M, OI.fCON, ar.d ' * ? ' *. G. OTTERUND, Nature, 224, 247' (1969) ` - : hi'.,,. 6.' KEIL, J,, L/pRIESTCR, and S. SANDIFER, '7* ,V ?'* . \ ", , ` Dull. Environ, Contam. To6, 156 (1971), ' T ' 7. . KOilMAN, J.,M. TEN NOEVER DE BRAUW, and . R. DE^ Vp$, Nature, 221, 1126 (1969) '" < . :*> .r 7" * ' 0.- MEN/.LJJT D.. j. ANDERSON, and A. RANDTKE, * * - * '7';'., i , Science, 167, 1724 (1970). -" _ 9. J'.ORGliN, .7. Uftpub. Maatcr'u thesis. Fish- ' `J.* " v Ocean. Lib., U. of Viash,, Seattle (1971) *\ 10. REYNOLDS. L., Bull, Environ. Contam. Tox.i . 4, 120 (1969) .. ''V ` ' 11. RISEOROUv!!, R., P, REIGIE, and H. 0LC0P7, . ' `' Bull. Environ. Contam. Tox., 4, 192 (1969) - : ' 12, TAUS, P. and A. DOLLAR, Limnol. Oceanogr., . 607 (1968) a . 13. WRSTER, C., Science, 159. 1474 (1968). * 1 *\ 14. ZXTKO, V., Bull. Environ., Contain. Tox., ' */. * ... 5, 279 (1970). .. _ *.` t'f * '' * " 1 1 ' j* 1 ' * iW* * -* . - \ i ;j\v * -4 f * ,. fc* j. , J *4 * t- *. 1 *.- ; ' * ", '-.I, .11, wd v,t wr,*' i \ .. > 1 . .. .*`i ^ f <>. ,(>, * v ** I1' >* ','iT. S.'> t .'c : : * o-v r'l.1! 117 b A `t.. `3 i .. - r. v.j, 1 J r V' 11 ll> +*** .J.-*-- .> ,!7 r oa 33**'