Effects of Pollution on the Free Amino Acid Contentof Two Marine Invertebrates

RITA D. SCHAFERl

RECENT REPORTS have been received from someinhabitants of the southern California coastalarea of a change in the texture and taste of cer­tain edible marine invertebrates. Investigationshowed that the specimens so designated hadbeen taken from areas known to be polluted .The term "polluted" as used here has been giventhe same meaning as that used in the ecologicalstudy by Reish (1956) on the San Gabriel Riverarea : this meaning is the dictionary definition,"the act of making or rendering unclean."

It has been shown for some invertebratesthat a change in environmental factors willproduce a change in eith er histological or mor­phological composition. Kinne ( 1958) showedthat a change in ectodermal cell shap e can beinduced in Cordylopho ra craspia by varying theenvironment from marine to brackish. Wilsonand Armstrong (1958), after experimentation,concluded that Echinus eggs and-larvae are af­fected structurally by the properties of sea water.It has also been demonstrated (Lane and Scha­fer, in pr ogress) that a difference in diet maychange the amino acid composition of muscletissue in some invertebrates. Since th is is knownto be true under controlled conditions, the pos­sibility exists that a variation in tissue com­position might occur as a result of a pollutedand consequently altered environment.

This study was thus undertaken to determine(1) if the change in app earance and taste wasaccompanied by a change in amino acid com­position, and ( 2) the nature of the change, ifone had occurred.

A cknowledgm ents. The author is indebted toDr. Norman Mattox of the Department of Bi­ology, University of Southern California, for hisdirection and valuable suggestions throughoutthe course of this investigation, and to the

1 Allan Hancock Foundation, University of South ­ern California, Los Angeles. Manuscript received July8, 1959.

administration of the Allan Hancock Founda­tion .for the use of laboratory facilities. She alsowishes to express her thanks to Mr . Fred Zie­senhenne of the Allan Hancock Foundationand to Mr . John Fitch of the California Depart ­ment of Fish and Game, for the collections ofspecimens from the islands; and to Dr. DonaldReish for the use of his data of measured oxygenfrom the Los Angeles Harbor area. This studywas supported by a research grane-from theUnited States Public Health Service of the Na­tional Institute of Health, no. RG- 4911.

MATERIALS AND METHODS

The animals used for investigation were theabalone, Hali otis cracherodii Leach, and the crab,Pachygrapsus crassipes Randall. The abalone wasselected for study, as it is the form in whichthe greatest difference in tissue texture andtaste has been observed. P. crassipes was selectedbecause of its wide distribution and markedtolerance of polluted conditions. These twoforms also offer two extremes as to length oftime in which the animals are directly subj ectedto the polluted water. Haliotis is exposed to theair only during periods of lower low tides andtherefore is almost continuously within the in­fluence of the conditions prevailing in the pol­luted water . Pachygrapsus, on the contrary, livesin the high tide zone along a rocky shore or onfloats rather than in the water in a wharf regionand consequently is submerged only during pe­riods of high tide, and is directly subjected tothe pollutants for comparatively less time thanis the abalone. Collections were made from pol­luted waters and, for a basis of comparison, fromwaters known to be free of pollution.

Specimens of Haliotis were collected alongthe shore at W hite's Point in the Palos Verderegion of the southern California coast, fromSan Clemente Island , Anacapa Island, and Santa

49

50 PACIFIC SCIENCE, Vol. XV, January 1961

Catalina Island. Pachygrapsus crassipes werecollected from a point in the west channel ofthe Los Angeles Harbor area designated as L.A.7, the mouth of the San Gabriel River at Ala­mitos Bay, White's Point, Santa Catalina Is­land, Anacapa Island, and Morro Bay (Fig. 1 ) .White's Point, L.A. 7, and the mouth of theSan Gabriel River at Alamitos Bay can be con­sidered polluted areas; while Morro Bay, SantaCatalina Island, San Clemente Island, and Ana­capa Island are surrounded by nonpolluted wa­ter. In the polluted areas both the nature andthe degree of pollution vary.

White's Point is a sewer outfall area in whichthe pipes convey the treated sewage to a site6,716 ft. from the shore line . According to

Stephenson and Grady (1956), oxygen defi­ciency at 6,000~8,000 ft. from the outfall variedfrom 10-30 per cent, at one time of measure­ment, to 0 per cent (o r normal ) at anothertime . It was reported, further, that ammoniacontent was not normal in any area within11,000 ft. of the outfall. Measurements taken at3,000 ft . from the outfall showed an increase ofsilicates and phosphates. Although dilution un­doubtedly occurs in the remaining 3,700 ft., itwould not be safe to assume that the water inthe intertidal area where the specimens weretaken is of normal composition.

The White's Point area is divided at lowtide into a northern cove and a southern cove

FIG. 1. Areas of collecri on.

_._ .__11: ' ~ _. .__ ,, ~ •. .

by a narrow expanse of rock which extends outfrom shore for approximately 200 ft . A surveyof the fauna of these two sections shows themto be qu ite different. On the southern side ofthe rocky projections were found scattered speci ­mens of Haliotis cracberodii, an occasional Pach­ygrapsus crassipes, masses of the tubed worm,Phragmatopoma cali/ornica, and specimens ofthe limpets Fissurella volcano and A cmaea lim a­tula. Empty Olivella shells were also present inrelatively large numbers. In sharp contra st tothis paucity of forms and indi viduals was thefauna of the northern section. Here were notedspecimens of Pisaster ochraceus, Strongylocen­trotus purpuratus, S. [ranciscanas, Opbiotbrixspiculata, Bulla gouldiana, Aplysia cali/ornica,Octopus bimaculatus, Conus calijornicas, Pagu­rus samuelis, in addition to those animals foundon the south ern side. This difference can be ex­plained by the fact that the sewer outfall opensoffshore in a line with the rocky projectionwhich separates the two sides. The current flowsprimarily from north to south carrying thepolluted water in the direction of the southernsection.

Th e point designated as L.A. 7 is located inthe west channel of Los Angeles Harbor. Theterm "L.A. 7" was given to this particular pointin a pollution survey conducted in 1952 by theLos Angeles Regional W ater Pollution ControlBoard, and has been retained in this study sothat this site may be recognized and related todata taken from that point. Specimens of Pachy­grapsus crassipes tested were taken from thepiling and floats at one of the small yacht har­bors in this area. The nature of the pollutionat this point has been designated as primarilyraw sewage from approximately 500 persons.Oxygen content of the water, m ea sur ed atmonthly intervals over a period of 3 years ( 1956through 1958 ) , showed fluctuations from a lowof 2.0 to a high of 8.2 parts per million. Anecological survey of the area shows the custom­ary wharf fauna of the southern California wa­ters. Anem ones, the wharf mussel ( Mytilusedulis ) , tunicates both solitary and colonial ,hydroids, barnacles, and colonial serpulid wormsare attached in great abundance to the under­side of the wooden floats, while numerous speci-

.,

"1 u·

\

. . _ :'~~~~~l "' ( N Tl

\ .-~>,' _ ' ' ._ _l.-..-.!., • .~ ., I,'

MORRO B....Y~'-" IOO M I.N

Free Amino Acid Content-SCHAFER

mens of Pachygrapsus crassipes live on the floatsand piling just above the water line. Since thearea is maintained as a small yacht harbor, thefloats are scraped occasionally and invariablyare soon resettled by young forms of the speciesmentioned.

The conditions at the mouth of the SanGabriel River at Alamitos Bay were thoroughlyinvestigated and reported by Reish in 1956.He reports the primary source of waste dis­charge into that region to be the Dow ChemicalCompany, the Santa Fe Springs Waste DisposalCompany, two domestic sewage disposal plants,and the Los Angeles Bureau of Water andPower Steam Plant.

Since specimens of the same species living inthe same conditions are known to have a con- ·sistent pattern of free amino acids in their mus-

51

cle tissues, an investigation of these amino acidsshould indicate whether or not a change froma normal to an abnormal metabolism has takenplace. An analysis of free amino acids was thusundertaken by means of two-dimensional paperchromatography.

Muscle tissue only was used for the deter­minations. All samples studied were taken fromindividual animals. Pooled samples were notused. Specimens were quick -frozen ; muscle tis­sue from the foot of the abalone and from thelegs of the crab was excised and subsequentlylyophilized without thawing. This precautionwas taken to inhibit the activity of autoenzymesand bacterial enzymes. After lyophilization thetissue was extracted with cold 70 per centethanol. Two 50 ml. aliquot portions were used,and extraction was permitted for at least 12

FIG. 2. Chromatogram of free amino acids of Haliotis cracberodii taken from Anacapa Island, a nonpollutedarea .

52 PACIFIC SCIENCE, Vol. XV, January 1961

FIG. 3. Chromatogram of free amino acids of Haliotis cracherodii taken from White's Point , a polluteda rea.

hours for each portion. Extracts were then con­centrared and stored under refrigeration as a 4.4ml. solution of 10 per cent isopropanol.

Chromatograms were run on 18Y2- by 22}-4­inch Whatman no. 1 filter papers. A mixture ofbutanol, acetic acid, and water (4: 1:5) wasused as the first phase; water-saturated phenolwas used as the second. Development was car­ried our by dipping in a 0.2 per cent solutionof ninhydrin in acetone.

RESULTS

The abalone, Haliotis crscberodii, from bothpolluted and nonpollured areas contained theamino acids alanine, arginine, aspartic acid,cystine, glutamic acid, glycine, histidine, leucine­isoleucine, tyrosine, and valine. Specimens taken

from the nonpolluted areas (Santa CatalinaIsland, San Clemente Island, and Anacapa Is­land ) contained asparagine in addition to theabove amino acids. The specimens taken fromWhite's Point showed no asparagine, but didshow a definite spot identified as aspartic acid.This acid was either absent or only very faintlydiscernible in the specimens from the non­polluted areas. Phenylalanine and three uniden­tified spots were present in the specimens ob­tained from the White's Point area (Figs. 2,3; Table 1).

All specimens of the crab, Pachygrapsuscrassipes, contained the free amino acids alanine,arginine, aspartic acid, cystine, glutamic acid,glycine, histidine, leucine-isoleucine, lysine, me­thionine, proline, serine, threonine, tyrosine, and

Free Amino Acid Content-SCHAFER

valine. Specimens taken from Santa CatalinaIsland, Anacapa Island , and Morro Bay con­tained asparagine as well. Those from SantaCatalina and Anacapa islands contained an un­identified amino acid located to the right ofarginine on the chromatographic pattern (Figs.4,5; Table 2).

DISCUSSION

Both Haliotis cracberodii, which occurs in onemarkedly polluted area, and Pachygrapsus eras­sipes, which was collected from a variety ofpolluted areas, have a free amino acid composi­tion different from that of the same animalscollected from nonpolluted areas. Haliotis fromclean water gives a more consistent pattern thandoes Pachygrapsus. Both forms taken from pol-

53

luted waters are marked by the absence of as­paragine. This indicates that in some mannerthe metabolism dealing with this amino acidhas been altered. In the case of Raliotis fromWhite's Point, phenylalanine and three uniden­tified spots appeared, indicating a more exten­sive change in metabolic pattern. The changein the abalone is greater than that in the crab.This may be due either to a difference in re­sponse to polluted conditions because of thedifference in the animals; or it may in someway be associated with the difference in lengthof time during which the animals are submergedand are subjected to the polluted conditions.

The factors of pollution common to theareas considered were a depletion of availableoxygen and an increase in nutrients resulting

FIG. 4. Chromatogram of free amino acids of Pachygrapsus crassipes from Anacapa Island, a nonpollutedarea.

54 PACIFIC SCIENCE, Vol. XV, January 1961

TABLE 1

FREE AMINO AC IDS PR ESEN T IN SPEC IMENS OF THE A BALON E,Ha!iotis cracberodi i . FROM A P OLLUTED AND T H REE NONPOLL UT ED AR EAS

.

I I '" I I lit0 z :it tic u '" ZU -e Z -e '" Z Z Z-c z u '".... Zsa '" Z -e '" l< l< l<"' z u s "' if: '" z "' 0 .... "' Z ~ 0 0 0Z Z ;:: -c Z -c Z is z "' s: l- Z "' 0 '" Z Z ZZ '" '" ;:: .... u § ti z z ::; z "' (3 Z

<: ;:; <: < :0 l- " '".... '" 0 ;;; '" '" ::; l<i l<i l<i

.... '" e; e; ~ .... .... '" l- "' X '" ::; x ,. <: Z Z Z-c -e <: -e u o o x .... .... ;:;: '" '" .... .... > " " "- :-- - - - .- - - - 1- - - - - - f-- -

Santa Catalina 15................ ....._ IX X Ix · X :~ ·x X X X X X X X X X Xx 'l- - - - -

Anacapa Is............ ................ ... IX X X X X X X X X X X X X X

San Clemente Is.................... ...TxX 1= - - - - - - -

X ~ x ix X X X X ~.~ X X X

White's Point.........____ ...... ..__ .__ . X ><T>< X x i x X X X X X X X X X X X X X

• Very faint .

FI G. 5. Chromatogram of free amino acids of Pachygrapsus crassipes from the mouth of the San Gabr ielRiver at Alamitos Bay, a polluted area.

Free Amino Acid Content-SCHAFER

TABLE 2

FRE E AMINO ACIDS PRESENT IN SPECIMENS OF THE CRAB ,Pachygrapsus crassipes, FROM FOUR NONPOLLUTED AN D T HREE P OLLUTED A REAS

55

I ! I I '"0 Z0 U '" Z *Ieu '" -c Z '" '"-c Z u "' Z .... z z

"' '" -c '" l<'" z u (3 '" i '" z "' 0 .... '" Z~ 0Z Z ;:: -e Z -e z Q z "' i >- z "' 0 ZZ '" '" S ... u ~

u z z :l z "' 0< (3 -c ..,

" >- " ~... "' 0 ;< '" '"

.:.... '" e; e; .... .... i "' "' :I: '" ::; :I: >- Z.., -c .., -c u o o .... .... ::;: '" c, ... ... "xF<TxXX- - - 1-- - - - - - xIX-Santa Catalina Is.____ _______ __ _____ ___ __ ____ __ ____ ___ X X X X X X X X X X

Anacapa Is.___ ________ __ __________ ___ ___ ___ ___ ___ ___ ____ X x~x xlx X X X X X X X X x i- X

~~=x lxt- xlSan Clemente Is.____ __ ______ ___ _______ ____ _____ ______ X X X X X X X X X--

Morro Bay__ ___ _________ ___ _____ ___ _______ ________ ______ X x l x l x ~I~ X X X X X X X X ~I- X

j- == - --

x fxi= x,-

~:?;~~:~ ~ : ~ ~ : I~xlxl xix X X X X X Xc--___ _ ._

x l x l ~x X X X X X X X X xl"Xixllx X x l-x I"- -Xi-X X X X X X

from sewage. Since the role of the free aminoacids in the animal body is as yet not completelyunderstood, it is not possible to state whatphysiological mechanisms have been altered bythese environmental changes or whether one orboth of the factors have operated to bring aboutthe altered free amino acid metabolism. Thata biochemical change as well as a morphologicalchange has occurred is evident .

SUMMARY

1. Specimens of the abalone, Haliotis era­cherodii, and the crab, Pachygrapsus crassipes,were collected from clean and polluted waters.

2. Chromatographic analysis of the free aminoacids of these forms were made by two-dimen­sional paper chromatography .

3. Specimens from polluted areas were foundto be lacking in asparagine. Th is amino acidwas present in specimens from clean waters.

4. Phenylalanine and three unidentified aminoacids, not found in Haliotis cracherodii fromclean water, were present in this species takenfrom polluted water.

5. Polluted waters differed from nonp ollutedwaters in that they showed a depletion of avail­able oxygen and an increase in nut rients .

REFERENCES

KINNE, OTTO. 1958. aber die Reaktion erbglei­chen Coelenreraten-ge webes auf verschiedeneSalzgehalts und Temperaturbedigen , II. Mit­teilung tiber den Einfluss des Salzgehaltes aufW achstum und Entwicklung mariner, brack­ischer und limnischer Organism en . ZooI.Jahrb. Abt. ZooI. PhysioI. Tiere 67 (4 ) : 407­488.

LANE, C. E., and R. D. SCHAFER. 1959. Workin progress.

REISH, D. J. 1956. An ecological study of lowerSan Gabriel River, California, with specialreference to pollut ion. Calif. Fish and Game42(1 ) : 51-61.

--- 1959. Unpublished data. Personal com­munication.

STEPHENSON, R. E., and JOHN R. GRADY. 1956.Plankton and associated nutrients in the wa­ter surrounding three sewer outfalls in south ­ern California. Report to Hyperion Engineers,Inc. Un iv. of Southern Calif. Press. 48 pp.

WILSON, D. P., and F. A. J. ARMSTRONG. 1958.Biological differences between sea waters :Experiments in 1954 and 1955. Jour. MarineBioI. Assoc. Un ited Kingdom 37 (2): 331­348.