PART I

NASA CR 114578 Avai lable t o Publ ic

A STUDY OF

MARINE LUMINESCENCE SIGNATURES

By Ar thu r W. Hornig and DeLyle Eastwood

M a r c h 1973

Distr ibut ion of t h i s r epo r t i s provided in the i n t e r e s t of information exchange. Responsibi l i ty f o r the contents r e s i d e s

i n t he au tho r s o r organizat ion that p r e p a r e d it.

P r e p a r e d under Corltract No. NASZ-6408 by

Baird- Atomic, Inc.

Bedford, Mass .

f o r

AMES RESEARCH CENTER

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

https://ntrs.nasa.gov/search.jsp?R=19730016387 2020-06-02T19:25:59+00:00Z

TABLE O F CONTENTS

Section

INTRODUCTION AND SUMMARY

Introduction

S u m m a r y

ORIGINS O F MARINE LUMINESCENCE

Luminescen:e P r inc ip l e s

Luminescent Ma te r i a l s in Seawa te r

Phytoplankton

Dissolved Organic M a t e r i a l s (Gelbstoffe)

Bioluminescence

F i s h Oils

Pol lutants

S u m m a r y

EXPERIMENTAL METHOD

Ini t ia l Approach

Development of ;. Fie ld Ins t rumen t

F i e l d Tes t ing a t VIMS

F i e l d Tes t ing a t Cape Ann (Glouces te r ) , Massachuse t t s

EXPERIMENTAL RESULTS

Compendium of Spec t r a l Date

Si te Descr ip t ions

Discuss ion of Chlorophyll Data

Gelbstoff Exci tat ion and E m i s s i o n

BIBLIOGRAPHY AND RELATED WORK

Bibliography

Related Work

TABLE OF CONTENTS (Continued)

Section

6. DISCUSSION AND RECOMMENDATIONS

6.1 Interpretation of Data

6 . 2 General Conclusions

6. 3 Recommendations for Future Work

7. ACKNOWLEDGEMENTS

Appendix A - General Principles of Fluoreacence Analysis

Appendix B - Bibliography

Appendix C - Compendium of Marine Luminescence Signatures (published separately)

Page

6-1

6 - 1

6 - 2

6 - 3

iii

Figure

LIST OF ILLUSTRATIONS

Chlorophyll a: Structural Diagram - Absorption and Fluorzscence Spectra of Chlorophyll a - and Pheophytin

The Fluorescence Spectra of Leaves Containing Varying Amounts of Chlorophyll

Mesobiliviolin (Phycocyanobilin): Structural Diagram

Absorption Spectra of the Phycobilin Pigments

Absorption Spectra of Aqueous Solutions of Phycoerythrins

Absorption Spectra of Aqueous Solutions of Phycocyanixs

Fluorescence Spectra of the Phycobilins from Porphyra Naiadum

O( -Carotene: Structural Diagram and Numberiilg Sys tem

Cell Absorption and Photosynthetic Action Spectra of a Red Alga Containing C -Phycocyanin a s i ts Principal Ac .essory Pigment

Derived Curves for the Fluorescence Spectra in Phorphyridium of Phycoerythrin, Phycocyanin, and Chlorophyll - a

Bioluminescence of Several Dinoflagellates

Multicomponent Excitation Spectra for Spanish Sardine Oil

Multicomponent Emission Spectra for Spanish Sardine Oil

Multicomponent Emis sion from Fish Scales (Smelt)

Excitation Spectra of Various Grades of Petr01er:rn Oil

Emission Spectra of Various Grades of Petroleum Oil

Multicomponent Excitation/ Emis sion Spectra of Lignin Sul- fonates (Orzon A)

Absorption Spectra for Substances in Natural Waters

Excitation/Emission Spectra for Principal Sources of Lig& Emiroion in Natural Waterr

Maximum Emir sion of Chesapeake Bay Water (9 -day- old)

Multicomponent Excitation Spectra ot Chesapeake Bay Water (9-day-old)

Psge -

LIST OF ILLUSTRATIONS (Continued)

Figure

2 3

Page - Multic.bmponen. Emission Spectra of Chesapeake Bay IJ'at e r (9 -day- old)

Multicomponent Emission Spectra of Chesapeake Bay Water (10-day-old)

Comparison of Response of EM1 9558QBand RCA C31025C Detectors

Modified Fluorescence Spectrophotometer

Site B Map: York River Entrance of Chesapeake Bay

Emission Spectra of Gelbstoff in Seawater off VIMS Pier - - Various Excitations

Emission Spectra of Gelbstoff in Seawater off VIMS P ie r - - Various Excitations

Excitation Spectra of Gelbstoff in Seawater off VIMS P ie r - - Various Monitoring Wavelengths

Emission Spectrum of Chlorophyll in Seawater off VIMS P ie r

Excitation Spectrum of Chlorophyll in Seawater off VZMS P ie r

Relative Quanta per Wavelength Interval for Field Instrument

ExcitationlEmission Spectra of Chlorophyll in Seawater, VIMS Station A

Excitation/Emission Spectra of Chlorophyll in Seawater, VlMS Station R

ExcitationlEmission Spectra of Chlorophyll in Seawater, VIAAS Station C

Excitation/Emission Spectra of Chlorophyll in Seawater, VIMS Station E

Excitation/Emission Spectra of Gelbetoff in Seawater, VIMS Station A

Excitation/Emieeion Spectra of Gelbstoff in Seawater, VIMS Station B

ExcitationlEmission Spectra of Gelbstoff in Seawater, VIMS Station D

Excitation/Emirsion Spectxa of Gelbrtoff in Seawater, VIMS Station E

Figure

3 4 ~

3 4 ~

3 4 ~

35a

35b

3 6

3 7

38

38A

39

40

41

42

43

44

4 5

Map A

LIST OF ILLUSTRATIONS (Continued)

Page - Excitation1 Emission Spectra of Chlorophyll in Seawater, VIMS Station A 3-37

Excitation/Emission Spectra of Chlorophyll in Seawater, VIMS Station B 3- 38

Excitation/Emission Spectra of Chlorophyll in Seawater, VIMS Station D 3- 39

Emission Spectrum of Gloucester Seawater Exciting at 476 nm 3-42

Excitation Spectrum of Gloucester Seawater Detecting at 686 nm

Excitation/Emission Spectra oi Gelbstoff from Gloucester Seawater 3-44

Time Decay of Seawater Luminescence 3-47

Time Fluctuations of Chlorophyll Emission in Hodgkins Cove 3-50

Comparison of a "Typical" Diatom Excitation Spectrum with a Quantun~ Counter 4-11

Excitation and Fluore. cence of Seawater Contaicing T richodeemium sp. (Traganza)

Fiuorescence Spectra of Atlantic Shelf Water and Sargasso Sea Water (Traganza)

Comparison Spectra of Incubatel: Sea Suspended Matter and Skeletonema Costatum (Traganoa) 5 -4

Normalized Spectral Distribution of Dissolved Substances (Karabashev and Zangalis) 5-4

Mean Normalized Spectral Distribution of Photolumine~cence of Sea Water (K arabashev and Zangalis) 5 - 6

Results and Measurement Conditions for the S W P Spectra (Karabashev et a l ) 5 - 6 Spectral Distribution of the Fluorescence of Seawater (Ivanoff and Morel)

Survey of Sampling Sites 4-3a

1. INTRODUCTION AND SUMMARY

1.1 Introduction

T h i s i s t he F ina l Repor t under Cont rac t Number NAS2-6048, A STUDY

OF MARINE LUMINESCENCE SIGNATURES, for the National Aeronaut ics

and Space Administrat ion, A m e s R e s e a r c h C e n t e r , Moffett F ie ld , Cal i fornia .

T h e work was p e r f o r m e d by the Applied R e s e a r c h Depar tment i n the Govern-

m e n t S y s t e m s Division of Bai rd-Atomic , Inc. , under the d i r ec t ion of Dr .

Ar thu r W. Hornig, D i rec to r of Resea rch .

T h e object ive of t h i s p r o g r a m was t o develop da t a on the luminescence

of na tu ra l w a t e r s a s a p re lude t o 'he development of a i r b o r n e and sa te l l i t e

techniques f o r the m e a s u r e m e n t of ocean product ivi ty and pollution. S u c c e s s

of the technique depends on the f ac t tha t chlorophyll a n d s e v e r a l o the r a lga l

p igments belong t o the gene ra l c l a s s of a r o m a t i c o r g a n i c s which exhibit effi-

c ien t luminescence when s t imula ted in t h e i r absorp t ion bands. C e r t a i n organic

decay products (Gelbstoffe) and wa te r pol lutants such a s oil a l s o contain

m a t e r i a l s which can be s t imula ted t o luminesce .

Remote sens ing a l lows wide-area , repe t i t ive m e a s u r e m e n t s of photo-

synthesis- induced product ivi ty which will yield a da t a base f o r evaluat ing

change6 in ocean a n d e s t u a r i n e quality. To ta l b i o m a s s m a y a l s o be de te rmined .

Measu remen t of chlorophyll concent ra t ion m a y be used as a n index of

phytoplankton concent ra t ion a n d m a r i n e food production. Regions wlth high

phytoplankton abundance c a n suppor t l a r g e concent ra t ions of he rb ivo res and

r u c c e o r i v e l inks in t h e a n i m a l food cha in . Such m e a s u r e m e n t s m a y be

useful i n del ineat ing area8 of upwelling, waterbody boundar ies and c u r r e n t s .

T h i r in format ion may , i n t u rn , l ead t o m o r e effect ive global f i sh harvest ing.

Monitor ing of pol lutants ir impor t an t not only dur ing ep isoder , but t o

check w a t e r qual i ty following aba tement action.

Lumineqcence techniquer a r e valuable b e c a u r e of t h e l r high rensi t ivi ty ,

a n d b e c a u r e of the dual rpec i f ic i ty a f f o r C ~ d by the p a r a m e t e r s of exci tat ion

and cnrrirrion wavelength.

1-1

1.2 Summary

As a result of consultations with marine biologists, I ' , rature search,

and laboratory measurements on time-dependent changes in natural water

samples, it was determined that measurements on old water samples would

not be representative of natural waters. Therefore the planned program of

laboratory measurements on samples sent from remote s i tes was modified

to field measurements zit representative si:es. This required modification

of a laboratory instrument for field use.

In the work here reported, we have demonstrated that a laboratory

fluorescence spectrophotometer, modified for field use, has sufficient sensi-

tivity to monitor the lowest concentrations of chlorophyll encountered in

coastal waters ( less than 0.2mg/l). At the sarne time, there i s enough

specificity to distinguish between water samples and establish some identifica-

tion.

We have surveyed waters on-site at five locations along the Atlantic

and Gulf Coasts. Waters from four Pacific Ocean locations were examined

in our l a b . .tory because of insufficient time aild funding for field measure-

ment, The on-site examinations included chlorophyll excitation/ emission and

Gelbstoff excitation/emission. The samples .nailed to our laboratory were

monitored only for Gelbstoff.

A broad l i terature search has been conducted, directed principally

at finding recent work in on-site luminescence of natural waters. A represen-

tative Bibliography has been assemljed and appears a s Appendix B of this

report. A8 expected, there ir very little data available an measurements

in-ritu, except for data from fil ter fluorimetcrs, The recent data a r e dis-

curred separately and rpectra reproduced for comparison with the present

work.

The excitation/emisrion data from all riteo for both chlorophyH and

Gslbntoff have been compared (both inter- and in t r s - r &a). Special attention

is given to an anaiyair of chloraphyll excitation data bccaurt of porrible use

1-2

in identifying algal types. As a resul t of this analysis , we have proposed =A

model of ?art iculates excitat ion/emission which a s s u m e s total absorption of

ultraviolet and mos t visible radiation, result ing in quantum counter response .

This concept leads to a new approach t o the identification problem which i s

discussed in detail.

The data have been assembled into a Compendium of Mar ine Lumines-

cence Signatures which i s published separately. T h e Compendium f o r m s

Appendix C of the Final Report .

#; , "

I" ' . ' , I . '

2. ORIGINS OF MARINE LUMINESCENCE

2.1 Luminescence Pr inc ip les

.4 discussion of the Genera l Pr inc ip les of Fluorescence Analysis i s

found in Appendix A. In the following discussion we focus on the application

of luminescence methods to seawater .

Pr inc ipal luminescing moiet ies in seawater will be a romat ic unsatura-

ted organic compounds, ei ther dissolved in the water o r a s constituents of

phytoplankton. In general , t hese ma te r i a l s will not phosphoresce at ambient

tempera tures ; therefore the luminescence i s uually f luorescence. An excep-

tion is bioluminescence, which is a special c a s e of chemiluminescence.

Tke excitation spect rum of a given fluorescenc mate r i a l i s usually ve ry

s imi lar t o the absorption spectrum. Hence, compar ison of excitation spec t ra

with tabulated absorption spec t ra may afford useful identification. The ex-

citation spect rum of a mix tu re of non-interacting f luorescent m a t e r i a l s will

be the weighted superposition of the individual spect ra . However, for in ter -

acting species, the excitation spect rum of a single i luorescing moiety may

show excitation cha rac te r i s t i c s of other components. The interact ion we

speak of i s known a s emrgy t r ans fe r .

Energy t r ans fe r : T of par t icular importance in algae where the in te r -

acting species a r e the chlorophylls and a c c e s s o r y pigments. In th is case ,

excitation throughout the spect rum resu l t s in typical chlorophyll emission.

The excitation spect rum for chlorophyll emis eion may contain indications of

the absorbing pigmentr and hence s e r v e to identify (however roughly) the

algal epecier. The variation6 in chlorophyll excitation s p e c t r a tabnlated in

the compendium of r p e c t r a and e lsewhere i n th is r epor t a r e one of the mos t

significant aspect6 of th i r work.

2.2 Luminescent Mater ia l r in Seawater

Seawater f o r m a t h e m a t r i x of a complex ecological rys tem. The

- inorganic portion conai r t s p r imar i ly of water and eleven ~ r g a n i c ion8

(Culkin, F1965), accounting for the predominating characterietic of salinity.

In fact, t r ace amounts of al.most al l of the elements can be found, and many

a r e involved in some of i e more important inorganic biochemical reactions

in the marine environment. However, fluorescence r.' .*ad to t r ace mater ia ls

would usually be too weak to be of intereet for ren- tt. d e t i . ' s n and identifica-

tion.

The principal fluorescent inorganic mater ia ls ar:.: dissolved r a r e

earth and uranyl salts, or suspended undissolved phosphors such a s calcium

tungstate o r zinc sulfide. These mater ia ls a r e rarely found in sizeable

concentrations in marine waters. Examination of a typical ' fart if iciallf

seawater in our laboratory has shown no significant fluorescence due to the

inorganic ions, and we have never seen seawater luminescence clearly

attributable to inorganic materials.

The living organic constituents of seawater range from large fish

and plants to the r.?inute phytoplankton and zooplankton. Fluorescence of

these "particulatas" i s related to aromatic pigments present in the surface

layer of the organism. In this laboratory we have observed the fluoreecence

of freely-swimming fish, macrascopic plants, phytoplar:,ton, and dis - solved materials. The last two a r c of greatest interest in this study.

Nor -living organic mater ia ls may be dissolved o r particulate. They

may rerul t from excretion or nhedding by living organirms, o r they may be

decay products of dead organisms. Suspended mat ter of organic origin,

permanently incapable of reproduction, i s termed detritur (Strickland, E1965).

The dissolved decayed organic mater ia ls a r e often referred to a s "Gelbstoffe"

(Kalle, B1949) because of the yellow color when concentrated. These have a

very complex comporitit?n which var ier with location.

It i r convenient to clarrify fluorercenca ar that derived from particulate

organic mat ter or f rom disrolved organic matter; however, i t ir d r o intererting

to note that the emiraionr from particulate mat ter are p r e d o m d n t l y a t wave-

lengths in the red region of t ' spectrum, \* hereas emissions from dissolved

substances a r e predominantly at wavelengths in the blue-green region of the

spectrum.

In add;tion to the above Itnaturalt ' components of sea\r,ater, there a r e

now significant components introduced by man. The most common pollutants

include pe t rdeum oil, sewage, and industrial v astes such a s lignin sulfonates.

These materials may also contribute to fluorescence backgroucd, 1- hich must

be understood if it i s to be subtracted out.

In the preceding we have discussed the general sources of stimulable

fluorescence. To these may be added a special category of luminescence

which could also be detected remotely (and misinterpreted)--name-ly

bioluminescence. This is a type of auto-luminescence v hich is properly

a form of chemiluminescence, displayed by certain dinoflagellates.

In the following subsections, the specific sources of luminescence in

seawater will he discussed by categories important to this study.

2. 3 Phytoplankton - If one isolates oarticulate metier from natural u, , ry filtration, and

then examines the fluorescence from the particulate mat etained on the

filter, the most etriking feature is the strong emission bar,d occurring at wave-

l e n g t h ~ around 680 nanometers. This emisrion i r strongest when the excitation

beam i r ceutered at 400-500 nanometerr. This emission bard i s due to the

chloroplartic pigment chlorophyll, which occurs universally in the marine

phytoplankton. Ar fa r a s can be arcertained from the mearurenrents, the

emitleion charrcter i r t ics arrociated with 6hi9 pigment a r e identical for a wide

range of organirmr. There in a wide variety of rpecies comporing marine

phytoplankton; however, most of the organirmr a r e diatom8 and dinoflageilat err.

There a r e rome exception#, and there differences rhould be noted. F i r r t

of all, red and blue-green algae, which occamionally occur in ocean waters,

have a different fluorercence emir rion rpectrum, principally due to the

presence of a group of pigment6 known ar phycobilinr. Depending upon the

type of organism, and i t s pigment content, excitation a t shor t wavelengths

will produce emiss ions general ly shor te r than that fo r chlorophyll a , - Although t h e r e has been considerable in teree t in the study of flucl:-rb$..

cence emission, principally in the connection with the emiss ion f rom chloro-

phyll a, no genera l o r clarifying statement can be made a s to the f ac to r s regu- - lating the intensity of the emission. Yet i t can be sa id that the intensity of

the emiss ion i s a .udc m e a s u r e of the concentration of the phytoplankton

organisms. T h e r e a l s o exiuts the possibility of distinguishing the type of

algae by examination of tho excitation cha rac te r i s t i c s responsib le for e m i s -

sion.

In vivo f luorescence frorr phytoplankton i s d i rec t ly o r indirect ly

re la ted to the p resence of plant pigments. All pigments will absorb light

in specific spec t ra l regions. Som !uoreece direct ly. Others will t r ans fe r

energy to another emitting moiety. The main pig men:^ t o be found in m a r i n e

phytoplankton a re : chlorophylls , biliproteins , and ca ro t enoids. Xantho-

phylls, which a r e oxygenated products of carotenes , a r e somet imes consi-

de red a separa te pigment group (Strickiand, E1965). Table I, taken f rom

Bogorad ( ~ 1 9 6 2 ) ~ r hows the distribution of pigments among algae.

2.3.1 Chlorophyllr

T h e l i t e ra tu re on chlorophylls is voluminous because of the irnpor-

tance and complexity of the subject. Almost all topics a r e considered in tne

book edited by Vernon arrd Seely (A1966). The five principal chlorophyll

typer (Bogorad, A1962) a r e labeled - a through - e. The i r concentrat ions va ry

widely among algal typer ; however, all a lgae contain chlorophyll - a. F u r t h e r ,

Yentrch (G1971) h r r de termined that only chlorophyll - a ir obrerved u hen algae

are obrerved in vivo. Siuce we are in ta re r t ed in m a r i n e luminescence in P-

natural water*, o u r attention will be r e s t r i c t ed t o chlorophyll - a.

Chlorophyll r ir built from a dihydrnporphyrin r ing which contarnr

a central non-ionirrble magneeium atom. In additiou t o the four pyr ro le

Table I. Distribution of Chlorophy'.le, Biliproteins, and Carotenoids Among Algae

Chlorophylls

Biliproteins Phycocyanin Phjcoerythrin

Carotenes d -Carotene fl -Carotene Y -Carotene Lycopene 6 -Carotene Unknown

Xanthophylls

+ = Present - = Absent ?

APn A P ~ A s Dd Dt Dn Fle Flx F L Mn M1 N

Chloro- phyta

- PD P) d

(d c o n -5 - 0 s

0

t t

t t - - *

C -

f t t

TT X u -

- 2 i g ; 3 5 % M C h 5 x

W G "

t t t

t - - - ?

- - ?

- a ? - - -

t t t

t

A s L, F N I iln L

Un

- Dd Flx, F, Dd Dt L , V Dn F P

N = Neoxanthin 0 = Os~i l loxanthin

Apn, Apl F le , i 1

? Z Mn. M1,

( ~ f t e r Bogorad, 1962)

= Insufficient Information P = Peridinin (sulcatoxant hin) = Aphanicin S = Siphonein = Asphanizophyll Sx Siphonoxanthin = Artaxanthin (eugltnarhodone) T t Taraxanthin = Diadinoxanthin V s Violaxanthin = Diatoxaqthin = Dinoxanthin = Flavacin

Z * Zerxanthin Un = Unknown

= Flavoxrnthin 8 Ooly obrerved in Tribonema bombycinum - = Fucoxanthin b Reported .Leo in Cymidium caldarium - = Lutein c Reported in Prlmellococcur miniatur - = Muxoxanthin (apbanin, ec hinmone) = ~ ~ w o w r m n t h o ~ h ~ i l s Neowrmnthin

nuclei surrounding the magnesium atom, there i s a cyclopentanone ring

with a methyl es te r group attached. A propionic sc id side-chain, esterified

with phytol, i s attached to one of the pyrrole nuclein. A s t ructural diagram

i s given in Figure 1.

In an algal cell, the chlorophyll i s bonded to a protein and t~ a lipid.

The details of the bonding affect the exact position and shape of chlorophyll

emission and excitation (or absorption). Thus spectra of extracts may be

quite different from those of intact living algae. Rabinowitch (A1951) has

noted that the absorption maxima of chlorophylls in vivo were at wavelengths

aboct 10 t o 20 nm longer than those in organic solvents. Emission maxima

may be similarly shifted, accounting for some of the apparent discrepancies

in the literature.

The absorption of chlorophyll a is characterized by two principal - absorption bands, one in the blue and one in the red region of the spectrum.

The emission is peaked in the red, just beyond the r ed absorption, with a

minor peak a t longer wavelengths. The absorption and emission of chloro-

phyll a extract in ether i s given in Figure 2, taken from Goedheer (A1966). - The peak absorption i s a t 430 nm, with a second major peak at 662 nm. The

emission is characterized by a peak a t 669 nm, with a substantial shoulder

at about 370 nm. The emission of chlorophyll a in a green leaf is given in - Figure 3, taken from French and Young (A1952). The la rger peak i s for a

partially greened leaf thought tocontain little chlorophyll and hence represent-

ing a dilute sample. The peak is a t approximately 676 nm, with a minor

shoulder a t 730 nm. The major peak i s considerably shifted u hen compared

with Figure 2. The emission of the darker green leaf of Figure 3 shows the

676 nm peak shifted t o 685 nm and reduced, whereas the 730 nm shoulder

has developed into a main peak, unshifted. French and Young attribute the

difference in rhape of the two peaks of Figure 3 to reabsorption of emitted

light due t o high chlorophyll concentrationr. (We have observed s imilar

re ru l t s in other fluorercing ryr tems as concentration has increased. ) Thus

obr erved fluorercence may depend on all parameters affecting the

too-phytyl -

FIGURE 1. Chlorophyll a. -

FIGURE 2. Absorption and fluorescence rpectra of Chlorophyll a (-) and abrorption rpectrum of phGophytin a ( - - - - 0 ) dirsolved in ether (Goedheer, ~19z6) .

concentration of chlorophyll, e. gr age, temperature, light exposure, etc.

Corresponding data on the in vivo excitation spectra for chlorophyll - a do not

exist because chlorophyll is always accompanied by other accessory pigments

in living algae, and the excitation spectrum for chlorophyll emission contains

absorption bands associated with the pigments.

The previous discussion of this section has re fe r red exclusively to

live algae. In a rea l marine sample, there will a lso be dead cells and

degradation products of chlorophyll - a. There a r e three important decomposi-

tion products, chlorophyllide, pheophytiq and pheophorbide, Chlorophyllide

is produced from chlorophyll by the loss of the phy to~ group (see Figure 1).

Ghlorophyllide - a has an absorption spectrum very s imilar t o chlorophyll - a:

no data a r e available on i ts fluorescence. On general principles, the distant

phytol group should have little effect on the fluorescence which i s related to

the aromatic ring structure. Pheophytin a i s formed when chlorophyll - becomes acidic and the magnesium i s replaced by hydrogen. The absorption

spectrum differs markedly from chlorophyll in that the intensity of the red

peak decreases and the blue peak shifts to slightly longer wavelengths. The

fluorescence of ~ h e o ~ h ~ t i n . a is a lso nhifted to longer wavelengths. - Pheophorbide is formed by removal of the phytol group from phe0phjt.n.

The corresponding absorption is s imilar to pheophytin and presumably the

fluorescence too.

2. 3.2 Biliproteins

4 The biliproteins a r e composed of phycobilins and a protein fragment. $ ',

The phycobilins a r e tetrapyrroles, the principal clae s e s being phycoerythrins 4 4 , and phycocyanine. The phycobilins a r e not readily released from their b

associated proteins (OhEocha, A1962), unlike chlorophylls, and hence most 1

measurements a r e made on the biliproteine, ra ther than on the phycobilins.

The complexity of the protein bonding, etc., ha# made i t impossible to

precirely define the r t ructure of the phycobilins. Figure 4 depicts the

r t ructure of merobiliviolin which Lemberg and Badet (A1933) felt was the

chromophore of C-.phycocyanin. While it i s now felt that this is probably an

artifact formed from phycocyanobilin in concentrated hydrochloric acid

(OhEocha, ~ 1 9 6 0 ) , the diagram serves to indicate the general s t ructure of

phycobilins.

The absorption spectra of biliproteins in aqueous solution correspond

very closely to spectra of intact a l g ~ e . The absorption spectra of three

phycobilins f rom Prophyra naiadurn a r e given in Figure 5, taken from French

et al. (~1956) . The peaks at 545 nm (phycoerythrin) 616 nm (phycocyanin)

and 654 nm (allophycocyanin) al l l i e below the longwave chlorophyll absorption,

but well above the blue chlorophyll absorption. Figures 6 and 7, taken from

OhEocha (A1962), show more detailed zbsorption spectra of variet ies of

phycoerythrins and phycocyanins.

In aqueous solution, the phycobilins a r e strongly fluorescent.

Phycoerythrins emit in the orange (578 nm), and allophycocyanin deep red

(663 nm). Phycocanin has been reported to emit from 637 nm to 655 nm

(French and Young, A1952: Duysens, A1952). The fluorescence of intact

algae is much weaker, probably because of energy t ransfer to chlorophyll.

The solution fluorescence of three phycobilins ie given in Figure 8, taken

from French et al. (~1956) .

Biliproteins a r e generally found in Rkodophyta, Gyanophyta, and

Cryptophyta.

2.3.3 Carotenoids

Carotenoids a r e yellow, orange, o r red pigments of aliphatic o r

alicyclic s t ructure composed of isoprene units, usually eight, linked s o

that the methyl groups neares t the center of the molecule a r e in the

1,6=position, while a l l other l a te ra l methyl groups a r e in the 1,5-position.

The merier of conjugated double bonds constitutes the chromophoric system

of the carotenoid (Kar re r and Jucker, A1950). The ~ t r u c t u r e of a[-carotene is

given in Figure 9. "

FIGURE 3. The fluorescence spectrum of a leaf containing very little chlorophyll compared with that of a leaf containing a large amount of chlorophyll. Excitation, 436 nm. (French and Young, A19 52. )

FIGURE 4. Meeobiliviolin (phycocyanobilin)

FIGURE 5. Absorption spectra of the phycobilin pigments (French et a l . , 1956).

FIGURE 6.

300 400 500 600 , A b d

Absorption spectra of aqueous solutions of phycoerythrim

R-phycoer ythrin B-phycoerythrin C -phycoer ythrin cryptomonad phycoerythrin (OhEocha, A1962)

FIGURE 7, Abrorption epectra of aqueour solutions of phycocyaninr (pH 6-7):

-------- R-phycocyanin ---- C -phycoc yanin 4+

-*- -*-- Aflophycoc yanin ------ Cryptornonrd phycocymin, Plymouth #train No. 157)

i." r ,:' re . wai*.. .

600 650 700 7 50

WAVE LENGTH

FIGURE 8. The fluorescence apectra of the phycobilins from Porphyra naiadum (French, et al. , 1956).

FIGURE 9. The numbering ayrtem of carotenoida illurtrated for q-carotene according to the American Chemical Society Committee on Nomenclatura.

The carotenes do not contain oxygen, while the xanthophylls a r e oxy-

genated. In vivo ca-otenes a r e associated with proteins and lipids, which

give8 r i r e to a r tred-shiftr t of 1-20 nrn in absorption. p -carotene predominates

in all phytoplankton except Cryptophyta ( S t rickland, E1965). The abssrption

spectrum of p-carotene in acetone peaks a t about 455 nm.

The xanthophylls have the most complex distribution in algae and

hence a r e of most use for characterization of c lasses . Carotenoids do not

usually rluorcece, but a r e known to protect chlorophylls from damaging radia-

tion (Clayton, A19 66). Fucoxanthin, an important xant hophyll found in brown

algae, has an abeorption band at 530 nm (Yentsch, D1971).

2.3.4 Energy Transfer

Intermolecular energy t ransfer i s of great importance because of i t s

involvement in photosynthesis. It has now been well established (Brody,

A1962) that the role of accessory pigments in photosynthesis i s to absorb

energy and transfer i t to chlorophyll a, whose role remains that ~f the only - photocatalyst. Energy t ransfer has k e n established indirectly by oxygen

evolution and directly by fluorescence experiments. Dutton et al. (A1943)

demonstrated that some of the energy absorbed by the carotenoid fucoxanthin

in "Nitzechia closteriumtt was emitted as fluorescence of chlorophyll to

about the s ame extent a s energy directly absorbed by the chlorophyll. Brody

and Rabinowitch (A1957) measured the t ime for energy to be t ransferred

f rom phycoerythrin to chlorophyll - a (probably via phycocyanin) a s 0 . 5 ~ 1 0 ' ~ asc.

Haxe (A1960) her measured the action and absorption spectra of the red alga

Porphyridium aerugineum and compared there with the absorption spectrum

of an aqueour extract which contained principally C-phycocyanin. Figure 10

demonatrater that the action apoctrum ir very s imilar t o t he abrorption by

phycocyanin, while chlororhyllr and carotenoidr a r e relatively inactive.

Thur, the photoryntherir occurring for excitation between 420 and 445 nm

and rt 680 nm, where chlorophyll haa it# maxima, ia extremely low. F r o m

thia it would aeem that the energy ia abrorbed by phycocyanin and par red on A

to chlorophyll.

Because of the energy t r ans fe r from the accessory pigments t o

chlorophyll, the excitation spectrum for chlorophyll emission may be the

most useful single measurement which will yield information about the

type of algae present in a given seawater specimen. Since energy t ransfer

is not total, some excitation remains in the accesvory pigments which may

appear a s characterist ic fluorescence. It i s known that this fluorescence

efficiency i s not very high. Nevertheless, it may also be useful t o monitor

emission at wavelengths corresponding to phycobilins, for example ( s ee

Figure 8), when exciting in phycobilin o: possibly carotenoia absorption

bands. This last i s illustrated in Figure 11, taken from French and Young

(A1952). Here the observed fluorescence from Porphyridium excited a t

530 nm is decomposed into the sum of emissions assigned to phycoerythrin,

phycocyanin, and clilorophyll.

2.4 Dissolved Organic Materials (Gelbstoife)

Blue-green fluorescing mater ia l i s introduced into ocean waters

when organic mat ter i s decomposed, and a l so by the production of brown

tlexudatestt by aquatic plants and marine seaweeds. Spectral examination

of those yellow-brown substances, which a r e almost identical spectrally,

rhows that their color is due to a high U.V. absorption, which tails over

into the visible. Studies of C. Yentech and collaborators (D1971) have shown

that the blue-green fluorescence i s mostly associated with these yellow

compounds; that is , when the yellow compounds a r e removed from the

seawater (by extraction), there i r no fluorescence. It should be noted

that there is , apparently, no band in the abrorption spectrum correrponding

t o the excitation rpectrum.

The identity and chemical comporition of there rubrtancer have been

of great in te re r t to ocemographerr. In 1963, the German chemirt Kalle

(EU963) rhowed that fluorercent rubrtancer r imilar t o that in seawater

could be formed from carbohydrater and amino acida. It ir not c lear ,

however, how there milterialr are introduced, o r whether o r not they a r e

differantidly a l tered by Bacteria in water. Yentrch h r r obrerved that the

FIGURE 10. Cell absorption and photosynthetic action apectra of the red alga Porphyridium aerugineurn, which contains C-phycocyanin a s its principal accereory pigment (Haxo, A1960).

FIGURE ll. The derived curves for the fluorercence rpectra in Porphyridium of phycoerytkrin, phycocyanin, and chlorophyll a. Their

I rummation ir compared with the fluorercenct rGctrum of Porphyridium illuminated by a wave length of 530 nm* The rum of the tndividurl curver ir indicated by dotted liner

8 (French and Young, A1952).

amino acid tryptophane photo-oxidizes rapidly, resulting in a yellow,

highly fluurescent photoproduct. T h e rtability o r chemical nature of this

photoproduct i s not known. There a r e a number of other compounds that

can be extracted from marine organirms that a r e highly fluor~?r~cent. Most

of rhea e a r e aromatic, polycyclic hydrocarbons such a s anthracene,

phenanthrene, chryaene and fluoranthene. It should a lso be noted that some

of these a r e components of crude oils. There a r e also many intermediates

of the metabolic mechanisms in the organisms that a r e highly fluorescent.

Pigments such as pterins, flavones, and coumarins a r e highly fluorescent.

It i s not c lear that these extracts or pigments a r e observed directly in situ.

In the remainder of this report , we shall often use Kalle's t e rm

llGelbstoff" to signify the entire complex mixture of blue-fluorescing natural

materials in seawater.

2.5 Bioluminercence

Bioluminescence i s not fluorescence hut a type of chemiluminescence,

Mort of the bioluminescence reen in the oceans i s termed " rtimulablew

bioluminescence--meaning light emission after mechanical agitation of the

organirm. The most common rourcs of bioluminescence is the dino-

flagellater. Typical emirrion rpectra a r e shown in Figure 12, taken from

the ther is of M. Kelly (C1968). In general, peak emission i s in the 470-480 nm

region, rising sharply from between 435 and 450 nm and dropping off by

550 nm. While typical chlorophyll luminescence can be light-rtimulated

from bioluminercent dinoflag ellates, i t i r unclear whether a t rue fluorercencq

identical t o the bioluminorcence, can be light-rtimulated.

2.6 F i r h Oils

It i r known that rchooling firh emit characterir t ic oilr which rome-

t imer form rlicke. In a study performed in thir laboratory (Hornig, G1970),

the excitation/fluorercance rignrturer of acetone ax t r rc t r of a number of f irh

oil. were examined. Typical multicomponent rpec t r r are given in Figurer

13 and 14. It brr d r o been obrerved (Hornig, D1971) that the rkiru of live

WAVELENGTH, My

FIGURE 12. Bioltxninercence of reveral dinoflrgellatem (Kelly, C1968).

2-17

Sp

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h S

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fish fluoresce. In Figure 15, th ree different emission spectra a r e presented,

corresponding to three excitation wavelengths, for f r e sh sinelt.

2.7 Pollutants

Marine waters have become increasingly polluted by petroleum oil

spills, industrial wastes, and sewage, t o name a few common substances.

Since these all may contain aromatic mater ia ls , fluorescence may be the

rule. Typical excitation/emission spectra for thin films of a variety of

petroleum oils (Hornig, M971) a r e given in Figures 16 and 17. Sil ~ i l a r spec-

t r a for a lignin sulfonate, a waste product f rom the paper industry, a r e given

in Figure 18.

2.8 Summary

The principal natural fluorescence to be ancountered in marine water

ie f rom algae and Gelbstoff. The absorption, excitation, and fluorescence

of principal components a r e nicely summarized in Figures 19 and 20, taken

from Yentsch (D1971).

- .i' :::

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FIGURE 19 : Abwrptia Spectra for Suhtmcr in Notuml Hotus

WAVEL ENGTH (nm)

FIGURE 20 . Excitation/Emission Spectra for Principal Sourccr of Light Emission in Natural Waters

3. EXPERIMENTAL METHOD

At the inception ~f this project i t was planned to pe r fo rm a l l spec t ra l

measurements a t our labora tor ies in Bedford, Massachuset ts . Samples

from coastal a r e a s of the U. S. were t o be mailed t o Bedford by cooperating

institutions. These plans were predicated on the existence of a s imple,

rel iable sample t rca tment and packaging scheme which would p r e s e r v e sample

integrity. Consultation with m a r i n e biologists, l i t e ra tu re s e a r c h , and

measurements on r e a l samples demonst ra ted that m a r i n e samp1,es a r e too

f ragi le t o permi t shipment without d ras t i c changes in luminescing components--

especial ly those involving chlorophyll. There fo re it was decided that the

ent i re modus operandi would have t o be changed in o r d e r to ensure valid data.

Specifically, it would be necessa ry to r evamp labora tory ins t rum entation for

measurement on f r e s h samples a t field s i t e s .

Most rn easurements documented in the Compendium w e r e taken a t

field s i t e s on f r e s h samples. Since the necessa ry instrume nt improvement

was evolutionary, performance var ied a t different s i t e s depending on the

local c i rcumstances . Often the conditions w e r e l e s s than ideal, ins t rument

operation was not always up t o normal , and p r e s s of t i m e somet imes preven-

ted u s e of f i l te rs o r taking of a s much multi-component da ta a s we would

have wished, Thus t h e r e is a ce r t a in unevenness in the resul t s .

It was a l so expected t!rat a computer -correctio-, s cheme would be

available fo r presentat ion of co r rec ted c u r v e s in the Compendium. Difficul-

t i e s with the computer p rogram and dependence on borrowed instrumentat iun

delayed the availability of this facility, As a r e su l t the da ta of the Campendium

a r e not correc ted .

The inr t rumenta l evolution and typica! r e su l t s will be discc*aed in

the r emainder of th is section.

3.1 Initial Approach - Mearurementr were to b e pe r fo rmed on a n extended range ve r s ion of

a Baird- Atomic Model SF-100 F luore rcence Spectrophotometer. This

instrument employs double monochroma!ors for both excitation and emiss ion,

uses a 150-watt xenon l amp fo r excitation, and i s cal ibrated f rom 220-1400 nm

on both excitation and emission. An RCA 1P28 i s normally used in the visible,

while a cooled RCA 7102 i s used for s p e c t r a in the r ed and near in i rared .

3.1.1 Sample Degradation

The question of how to collect, p r e s e r v e , and ship a water sample

f r o m a distant source to our Bedford laboratory--and p r e s e r v e sample

integri ty insofar a s luminescence s ignatures a r e concerned- -is ve ry diffi-

cult. Fur the r , such a method mus t be s imple and inexpensive enough t o

pe rmi t volunteer personnel t o provide such samples .

As soon a s a sample has been collected, i t begins t o change due t o

s e v e r a l fac tors , including:

na ture and intensity of illumination during shipment

dissolved oxyge:, content

p r e s s u r e and t empera tu re

lack of equilibrium with a m a r i n e environment

interact ion with the sample container

If living ce l l s a r e p resen t ( a s i s the c a s e fo r samples of in teres t t o

this project), they may contiaue t o grow, and the composition the sample

m a y change i f oxygen concentration and lighting conditions a r e iavor tb le . If

cel la containing chlorophyll a r e killed, o r t h e photosynthetic p r o c e s s i s in-

hibited by lack of light o r for other reasons , the chlorophyll f luorescence m a y

i n c r e a s e by a fac tor of 2000 (Oppenheimer, E1966). If t h e eample i s kept in

darkness , the organism m?.y bleach. On the other hand, if the sample

i r exposed t o light of wavelength s h o r t e r than 400 nm, it m a y degrade in a

m a t t e r of minutes, depending on the organism.

Sampler may be stabil ized fo r a few hourr by cooling t o nea r O'C,

but ce r t a in algae m a y grow at o r just below O'C, F u r t h e r cooling may rup-

ture ce l l walls, allowing roluble pigment6 t o dissolve in the water , leaving

an a l t e red *#tern.

Chlorophylls a r e exceptionally labile and readily form green and

gray-brown alteration products when cells a r e injured or killed, subjected

to various reagents, or exposed to various unfavorable conditions (Strain and

Svec, in Vernon and Seely, A1966). Chlorophylls often isomerixe spontane-

OUR!.^ in solution, especially at elevated temperatures. They a r e oxidized

(allomerixed) rapidly when dissolved in relatively inert solvents such a s

alcohols in the presence of a i r .

For these reasons we conclude that it may be practically impossible

to preserve sample integrity tor plankton measurements o r b r any living

organisms, unless measurements a r e made immediately after sample collec - tion. This is particularly true where the measurements must duplicate those

on raw water. Shipping and handling a r e expected to have l e s s effect on Gelb-

stoff and certain pollutants such as petroleum or lignins.

3.1.2 ShiDDinfZ Methods and E x ~ e r i m e n t s

A useful shipping method must not only preserve sample integrity,

but be simple and inexpensive enough to permit practical shipment from many

remote sources. We f i r s t investigated a commercial biomailer coneisting

of a cubical styrofoam box fitted into a cardboard mailing carton. Such a

box, filled with C 0 2 powder and tightly sealed, lasted less than one dav,

Increased wall thickneslr and use of solid carbon dioxide would have increased

cold time, but hardly more than a day o r so.

Mr. J. E. Warinner of the Virginia Institute of Marine Science at

Gloucester Point, Virginia, reported that he had had rome success in mailing

eamples cooled in dry ice, with the ramples frozen after several days. He

kindly agreed to provide ur with a variety of sampler of waters from the

Cherapeake Bay, all nhipped in different container r.

Frozen ramples were rent in polypropylene bags and polystyrene

(Nalgene) pet@ dirher. Uncooled ramples were rent in the above plug a

glarr vial. Sampler were all gathered from the r ame place and a t the rame

time; bowever, three different sampler were rent in each type container to

check reproducibility. Distilled water samples were a lso sent in each type of

container (frozen and unfrozen) a s a control. The sample containers were

packed in a styrofoam box with four-inch walls top and bottom and two-inch

walls on the side. The sample/dry ice space was 4" x 6-1/214 x 4", allowing

ample space for a good load of dry ice. The uncooled samples were a lso

imbedded in thick styrofoam to maintain ambient temperature during s hip-

m ent . Despite the c a r e in setting up this experiment, it was largely disap-

pointing due to the extraordinarily slow a i r parcel post service. The cooled

samples arr ived five days after they were shipped: the uncooled eight!

Needless to say, thz dry ice was gone, and all samples were warm upon

arrival , thus vitiating the pr imary purpose of the experiment. It i s c lear

that shipping t imes of up to a week must be expected, casting further doubt

on the expected sample validity.

3.1. 3 Initial Luminescence Measurements

In order to check changes in sample luminescense,with t ime it was

decided to work in cooperation with Dr. Charles Yentsch of the Marine

Institute of the iJnivers ity of Marsachusetts at Gloucester, Mas sac hus etts.

In an effort to use a eingle photomultiplier for all measurements, and so

simplify experimental procedures when using the instrument at Y entsc h 's

laboratory, an EIAI 95580 tube with an S-20 rerponse was erripioyed. Unfor-

tunately this broke during a cooling experiment. We next returned to an S-1

tube, but ured an experimental tube, an RCA C70007A. ra ther than our 7102,

Thir tube requirer cooling and hence could not eari ly be ured in measure-

ments at Y entrch'r laboratory.

F i r s t mearurementr were on the rampler from VIMS. Examining

rompler nine o r ten dayr old we could not detect emirr ion in the 680 nm

(chlorophyll) region uring the cooled C70007A and a r tandard cuvette. Using

the 1P28 we did detect emiradon correrponding to the Gelbrtoff of Figure 20,

As expected, we found several variant emir rionr , depending on the excitation

chosen. Correspondingly, the excitation spect rum varied, depending on the

monitoring wavelength. Our initial r e su l t s a r e given in F i g u r e 21. Multicom-

pone-rt excitation afid emiss ion spec t ra fo r the s a m e sample a r e given in

F igures 22 and 23. (Raman peaks a r e identified with t h e symbol "R" in the

f igures. )

Considering the ernission spec t ra of r i g u r e 23, it is apparent the

chief emiss ion is r a t h e r s t r u c t u r e l e s s ( ins t rumenta l bandwidth, 8 nm)

peaked nea r 450 nm. The ra the r s h a r p peak at 340 nm, corresponding to

excitation at 295 nm, i s very possibly due to a light oil pollutant (cf. t r a c e s

A and B in F igure 17).

The excitation s p e c t r a of F i g u r e 22 a r e l ikewise s t ruc tu re less , a l -

though the excitation peak moves t o s h x t e r wavelengths a s the monitoring

wavelength decreases . Again, the excitatio? t r a c e when monitoring a t 340 nm

has a p e t k resembling that of the lightest oil of F i g u r e 16, suggesting ve ry

strongly that the Chesapeake Bay sample was polluted. This is not unlikely

s ince Gloucester Point is located c lose to busy docking facilit ies.

The emiss ion data of F i g u r e 24 were taken on the s a m e sample a s

F igure 23 but on the tenth day--one day la ter . Comparing re la t ive intensities

and rhapes of curves , i t would appear that the light oil has dec reased in

intensity, but the genera l shape of the remaining curves i s s imi la r . The oil

peak may have dec reased f rom evaporation, but m o r e probably it was present

a r a fine emulsion when we f i r s t examined it , but had coalesced on standing

t o l a r g e r drople ts which do not give a s in tenre a s p e c t r u m . We conclude

that the blue emi r r ion (which we a t t r ibute t o tfGelbrtofftl for convenience! is

r a t h e r r t ab le in t ime, ar opposed t o t h e r e d !tchlorophyllu emi r s ion which

we did not detect.

A fur ther conclurion of th i r experiment concerned rhipping containers.

Glar r vials , polypropylene bagr and polyrtyrene p e t r i die he# w e r e ur ed.

Examination of t h e ten-day-old dintilled water r ample r revealed that the

water r to red in polypropylene bagr had a much l a r g e r background f luorercence

than water s tored in ei ther g lass o r polystyrene. The peak background

f luorescence occur red a t approximately 415 nm for an excitation of 310 nm.

The polystyrene dish seemed equally a s good a s glass.

3, 2 Development of a Fie ld Instrument - The s tandard labora tory ins t rument descr ibed in the previous sect ion

cannot be user) for field measurements of chlorophyll. The 1P28 detector ,

which does not have to be cooled, is too insensi t ive in the 680 nm region.

The C70007A ( o r 7102) needs cooling, which is a t te r ly impract ica l in field

situations. Our solution t o the problem involves use of a new photomultiplier

with a GaAs photocathode. This new tube, an RZA C31025C, does not need

cooiing and has a f la t response in the chlorophyll region. F i g u r e 25 contains

comparat ive data on the quantum efficiencies of the RCA C31025C a ~ d the

EM1 95580. I t will be noted that the f o r m e r has a higher quantum efficiency

at 685 nm (5.970) than the la t te r (3. 370), and t h e wavelength dependence i s

l e s s severe . Thus, the C31025C should display bet ter s ignal lnoise and l e s s

wavelength distortion.

The C31025C has the s a m e geometry a s the 1P28 and hence can be

substituted in the instrument directly. Hence, it was f i r s t fel t that th is

substitution would resu l t in good performance f rom below 300 nm t o beyond

800 nm. Unfortunately, the gain of the C31025C is l e s s than that of the 1P28

by a fac tor of about 100, which puts a s t r a i n on the f i r s t s tage of amplification.

Despite u s e of specia l ampl i f ie rs , it was found by much test ing that the p e r -

formance of the C31025C in the visible was always much infer ior t o the 1P28.

Field t e s t s on wa te r s off Cape Ann, Massachuset t s (University of Massachu-

se t t s Mar ine Station) showed the sensi t ivi ty in the visible t o be unacceptably

low.

The decision was then made to mount both t h e C31025C and the 1P28 - on the ins t rument with dual electronic controls for photomultiplier high vol-

t age and d a r k cur ren t adjuet. The two side-viewing tubes w e r e mounted end

t o end in a ver t ica l tube at tached t o the s ide of the SF-100. A quar tz l ens

COMPARISON OF RESPONSE OF EM1 9S58Q B and RCA C31025C

400 500 600 WAVELENGTH ( nanometers )

. . . . ' . .._I .. . . ,., . . . I . . I., _. .. .. .4 . . . -. , . .

C

focuses the light f rom the exit s l i t onto the act ive photocathode a r e a of

whichever tube is positioned for use. The tubes a r e se lec ted by manually

sliding the des i red detector into place. The housing i s light-tight, allowing

change of de tec tors in ambient light, A switch allows u s e of normal panel

c o d ro l s for the 1P28 and auxil iary controls fo r the C31025C. Once voltages

a r e adjusted, it is possible t o switch back and forth between tubes without

fur ther reset t ing of da rk c u r r e n t and high voltage.

F igure 26 is a photograph of the modified instrument showing the ex-

t e rna l mounting of the two detec tors together with electronic controls .

The basic instrument available for field u s e was an SF-100, usable

only to 700 nm. While the chlorophyll a peak i s nea r 685 nm, the long wave- - length t a i l extends well beyond 700 nm. There fo re a camspacer was provided

to change the s tandard range to 420-900 nm when desired. This space r i s

moved into position by means of a s m a l l l eve r located within the sample

compartment. This wavelength shift i s applied only to the emiss ion mono-

chromator , although it could be applied t o the excitation s ide a s well.

The result ing modified ins t rument thus allows the use of the 1P28 fo r

Gelbstoff and other uv-visible emiss ion measurements in the 220-700 nm

range, and use of the C31025C f o r r e d and nea r - in f ra red measurements in

the range of 420-900 nm.

A eingle difficulty with the C31025C is the t i m e requi red t o achieve

low dark cur ren t a f ter the tube has been exposed t o light, o r s to red without

high-voltage applied.

While the revamped ins t rument had sensi t ivi ty enough t o monitor l e s s

than one g r a m of chlorophyll p e r l i t e r , t h e r e was s o m e indication during

measurements a t Gloucsster that s o m e amplif ier instability was evident a t

high gain. This became m o r e evident a t Nova University and finally became

a r e a l problem a t Carrabel le . As a resul t , the feedback on the high-voltage

supply f o r the photomultip?ier was revised, result ing in good stabil i ty fo r all

remaining meaeuremente.

3. 3 F ie ld Tes t ing at VIMS

T h e completed in s t rumen t was t e s t ed a t two f ie ld s i t e s , the Virg in ia

Inst i tute of Mar ine Sc ience l abo ra to ry a t Glouces te r Poin t , Virginia , and

the Universi ty of Massachuse t t s Mar ine Station a t Glouces te r , Massachuse t t s .

T h e VlMS exper iment was a r r a n g e d through Dr . P a u l L. Zubkoff, C h a i r m a n

of the Depar tment of Physiology, and M r . J. E r n e s t Warinner 111, Ass i s t an t

Mar ine Scient is t . VIMS provided l abo ra to ry fac i l i t i es f o r l uminescence

s tudies and provided s p a c e on t h e i r 47-foot boat f o r a day-long s tudy a t four

s ta t ions in Chesapeake Bay.

While t he r e s u l t s of t h e s e r r -easurements a r e included in the Cornpen-

dium, we sha l l d i scuss t hem in s o m e de ta i l h e r e as a n example of the type

of m e a s u r e m e n t s c a r r i e d out e l sewhere .

The l abo ra to ry is loca ted f ive naut ical m i l e s f r o m the mouth of t he

York R ive r and th i r ty m i l e s f r o m the mouth of the Chesapeake Bzy, a s indi-

ca t ed in F i g u r e 27. Also indicated a r e four s ta t ions , l abe led A, B, D, E,

at which samples w e r e taken.

3.3.1 Labora to rv Seawater Studies

Labora to ry s tudies w e r e c a r r i e d out on s e a w a t e r s a m p l e s taken f r o m

off t he VIMS p ie r . T h e s e s tudies w e r e m a d e t o check the in s t rumen t and

develop diagnostic exc i ta t ion /emiss ion wavelength se t t ings .

J n F i g u r e s 28a and 28b, we document t h e Gelbstoff emis s ion fo r va r ious

exci tat ion wavelengths. Both exci tat ion and emis s ion bandwidths w e r e s e t a t

approximately 16 nm. Water s a m p l e s w e r e examined in a s t anda rd 10 x 10 m m 2

cuvet te immedia te ly a f t e r collection. T h e b road emis s ion i s peaked in t h e

450 n m region, showing s o m e var ia t ion with excitation. At lowest exci ta t ion

(280 nm), t h e peak is loca ted a t about 445 nm; at a n exci tat ion of 320 nm,

t h e peak has shif ted t o 438 nm; s t higher wavelengths t h e peak sh i f t s s l ight ly

t o longer wavelengths aga in with suggest ions of a new b road peak a t 470 nm.

A s exci tat ion wavelength is inc reased , t h e wa te r R a m a n emis s ion is s e e n

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creeping up the f luorescence t r aces . At an excitation of 300 nm, i t i s bare ly

visible a t 345 nm. At an excitation of 360 nm (the highest t r a c e in the s e r i e s ) ,

the Raman line is at about 420 nm, approaching the f luorescence peak. At

g r e a t e r excitation wavelengths, the Ramail in t e r fe res with the determination

of the peak position. Thus, fo r an excitation of 400 nrrr, the Raman at 468 nm

dominates the f luorescence emiss ion which is s t i l l d iscernib le by the inflection

of 480 nm. At an excitation of 420 nm, the emiss ion i s principally Raman.

Note that t h e r e i s no indication of light oil emiss ion a s s e e n in F i g u r e 23.

F igure 29 contains severa l corresponding excitation s p e c t r a for dif-

f e ren t monitoring wavelengths, The Raman again c l imbs the excitation

spec t ra a s the monitored wavelength becomes shor te r , The excitation peak

appears to shift from 375 nm (when monitored at 510 nm) t o 355 nm (monitored

at 450 nm).

We conclude that th is Gelfstoff emission, which i s probably fa i r ly

typical, i s centered in the 440-450 nm region wit? a bandwidth of approximately

150 nm. The slight shift in emiss ion peak with excitation suggests i t i s com-

posed of many emitting moie t ies which probably exchange energy to some

degree, presenting the s t ruc tu re less spect rum. The excitation maximum

(for our uncorrected ins t rument) is in the 345-355 nm region, with an apparent t

bandwidth of about 110 nm. The m o r e s e v e r e ins t rumenta l correc t ions appli-

cable in th is region dras t ica l ly reduce excitation s p e c t r a a t shor t wavelengths,

shifting b raks to longer wavelengths and p r o d ~ c i n g n a r r o w e r bandwidths.

In F igures 30a and 30b, we show excitation and emiss ion s p e c t r a of

the uchlorophyll" emiss ion in the s a m e sample. In th is c a s e the C31025C i r I

the detector and bandwidths on both emiss ion and excitation a r e approximately

+Here and throughout th i r r epor t we r e f e r t o I1Gelbstoff emission" a s a convenient designation of t he broad emiss ion peaking in the 440-450 nm region which o c c u r r in mos t na tura l waters . WT a l s o use the t e r m "chlorophyll emi r r ion t t t o conveniently designate emiss ion occurr ing in the 685 nm region. The emi r r ion may not be due t a chlorophyll, but t o pheophytin, etc. 1

[

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24 nm. (These t r a c e s were made using a i500-watt S e a r s motor-genera tor

t o power the SF-100, p repara to ry t o experimentation aboard ship. T h e noise

is acceptable, though u s e of a one-second t ime constant could have reduced

it further . Frequency and voltage variat ions were found t o affect the r e c o r -

d e r span, result ing in some uncertainty in wavelength. )

The prominent emission in F igure 30a, with peak a t 685 nm, i s

a s sumed t o be due to chlorophjrll a. The peak a t 550 nm is the Raman assoc ia - - ted with excitation a t 470 nm. The Raman is located on a r i s ing background

which i s the t a i l of the RayleighITyndall sca t ter ing peak. Since we a r e look-

ing a t part iculates suspended in the water , we expect t o s e e scat ter ing. The

long wavelength shoulder in the 730 nm region i s r e a l and i s observed in other

samples to varying degrees.

The corresponding excitation spec t rum in F igure 30b monitoring the

chlorophyll emiss ion peak, is peaked a t 460 nm with shoulders at 400 and 430

nm. T h e r e is a l so a long wavelength shoulder a t 540 nm. The wide s l i t s

preclude examining the excitation c lose to the emission, thus miss ing the

chlorophyll absorption band a t 670 nm. The shor t wavelength termination of

the excitation curve a t 365 nm is due to the p resence of second o r d e r sca t ter .

(The emiss ion monochromator s e t t o detect 680 nm in first o r d e r will detect

exciting light at 340 nm in second o rde r . ) This s c a t t e r peak can be removed

with a f i l te r ; however, through cn oversight, we did not have f i l t e r s with us.

Unccrrected excitation s p e c t r a mus t be viewed ve ry cri t ical ly,

especially if a xenon s o u r c e i s used, because of the fine s t r u c t u r e in the

xenon spect rum between 400 nm and 500 nm. The detai ls of the correc t ion

necessa ry depend strongly on the s l i t s used. In F i g u r e 31 we show the r e l a -

t ive q a n t a pe r unit wavelength bandwidth a r r iv ing at the sample with the

s l i t3 used :or F i g u r e 30. This was obtained by inser t ing a solution of 5 g/ l

of Rhodamine R in ethylene glycol into the sample chamber in the t r ansmiss ion

mode (using a front su r face m i r r o r ) , Note that all excitation s p e c t r a will r e -

flect the s t r u c t u r e at 400 nm, 540 nrn, and par t icular ly at 470 nm.

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With the xenon source peaks in mind, it i s eas i ly deduced that the

apparent peak a t 460 nm in F i g u r e 30b will be removed, leaving the genera l

peak in the 440 nm region. This is in agreement with the action (excitation)

spec t ra for chlorophyll a f luorescence of F r e n c h and Young (A1953.) and -- Yentsch. The shoulders at 400 nm and 540 nm a r e possibly due to the

unequal distribution of source light. Despite the d is tor t ian of uncorrec ted

excitation spect ra , they can be valuable fo r the difference between them, a s

will become apparent.

3. 3.2 Chesapeake Bav Studies

A p r i m a r y purpose of the work a t VIMS was t o t ake the SF-100 (with

x-y r e c o r d e r and l amp power supply) t o s e a t o m easure f reshly collected

su r face water samples. The VIMS boat was equipped with s e v e r a l gasoline

genera tors t o provide 110 v, 60 cycle power fo r instrumentation. This oat

is taken out a t l eas t once a month in winter and weekly in s u m m e r to make

measurements in Chesapeake Bay. These measurements include t e r m p e r a t u r e

salinity, dissolved oxygen, chlorophyll (a , b, c, d, and carotenoids) , ortho- - - - - phosphate, ni trate , ni tr i te , plankton (dinoflagellates and diatoms) and light

penetration.

Water samples a r e f i l te red on the boat, and both f i l t ra te and res idue

a r e immediately s to red on d r y i c e f o r l a t e r analysis a t the laboratory. Some

chemical manipulations a r e pe r fo rmed on the boat before f reez ing samples.

The boat normally leaves S a r a h Creek, stopping success ively a t

s tat ions E, D, B, and A a s indicated in F i g u r e 27. At each station, water

samples a r e collected at the surface , 112 m , 1 m, and l a r g e r inc rements

t o the bottom. The su r face s a m p l e r cons is ts of a f r a m e with a s t a in less

s t ee l screen. The s c r e e n is placed flat on the water su r face t o pick up the

top half-millimeter of water. It is then ra i sed and dra ined into s to rage

containers . Because of lack of t i m e and experience, i t was imposs ib le t o

plot exci tat ion/emission s p e c t r a of all water samples . Instead, the su r face

samples w e r e all monitored as being of g rea tes t in t e r - . t o th is program.

F r o m laboratory work, i t was decided to use ce r t a in diagnostic se t -

t ings t o contrast water luminescence. F o r the Gelbstoff measurements , ex-

citation was s e t at 350 nm (16 nm bandwidth) and emiss ion scanned with a

7 nm bandwidth. F o r excitation scans ( 7 nm bandwidth), t h e emiss ion

monoc :romator was se t a t 440 nm (16 nm bandwidth). These m e a s u r e a nts

employed a 1P28 detector . The other s e t of measurements made with the

C31025C employed a 24 nm bandwidth f o r both excitation and emiss ion mono-

chromators . Excitation was at 458 nm and emiss ion a t 682 nm. These s e t -

t ings w e r e fel t t o cover the principal emiss ion and excitation regions (except

f o r the long wavelength excitation of chlorophyll, ctc.). The data were t o be

in terpre ted in t e r m s of relat ive s t rength of emission and spec t ra l distribution.

F igures 32*, 32B, 32D, and 3 2 ~ r e c o r d the excitat ion/emission

s p e c t r a fo r su r face waters a t the corresponding stat ions in t e r m s of chloro-

phyll type emission. F i g u r e s 3 3 ~ . . . 33~5 r e c o r d s p e c t r a f o r GelbsLoff

type emission. Before considering specific resul t s , we note that al l s p e c t r a

were taken with an instrumental t i m e constant of 0.3 seconds. Fur the r ,

ins t rumenta l gain was only medium in the wors t cases . Signal-to-noise

r a t io was, in general , ve ry good. In many c a s e s the par t icular type of noise

could be re la ted t o t k s tar t ing o r stopping of the ship 's engines, o r wave

motion. The only difficulty involved wavelength i n a c c u r a c i e ~ of the x-y

r e c o r d e r due to slow changes in the output voltage and drequency of the

gasoline generator . Low voltages a l s o caused change in s ignal gain and

sluggish response in the r ecorde r . We now consider F i g u r e s 32 and 33 in

s o m e detail .

F igures 32 have two wavelength sca les . That a t the bottom of the

page r e f e r s t o excitation, while t h e s c a l e a t the top r e f e r s t o emission. The

emiss ion t r a c e i~ always cha rac te r i zed by a signal in the 685 nm region. The

peak a t 340 n m on excitation is second o r d e r excitation and can be used as

an in ternal gain s tandard t o s o m e extent. (It will be affected if eca t t e r is

caueed p r imar i ly by turbidity r a t h e r than Rayleigh ecat ter . )

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motor. F igure 32D i s nottworthy for the high qua1:ty of the t r a c e taken at

sea.

All t r a c e s show a p r imary e m i s s ~ o n at 685 nm with a secondary peak

in the 730 nm rcgion. The lat te? has been a t t r ibu ted t o a chlorophyll aggrega-

tion effect, a different form of chlorophyll o r vibrational bar.d enhanced b y

reabsorption. The b r o ~ d emission a t 545 nm in al l t r a c e s i s Raman due to

excitation a t 458 nm. This Raman can be used a s a monitor of instruinent

gain, fair ly independent ;.f sma l l changes in turbidity. Thus, the re is nr,

other identifiable emission in the range 500-900 nm besides the chlorophyll

e n ~ i s s i o n a t 655 a m with i t s shoulder a t 730 nm.

The corresponding excitatior r aces of F igures 32 a r r s i m i l a r t o

F igure 30b, exhibiting the ( u ~ c o r r e c t t d ) pezk a t 460 nm and the (uncorrec ted)

shoulder a t 540 nm. However, no anomal- appears at 400 :1m ( a s in F igure

30b) which means the t r a c e s a r e different in that respect . We conclude that

the excitation t r a c e s a r e al l s in l i la r and show ilo indication of different ac -

c e s s o r y pigments, etc, This m.3y be expected s ince a i l samples c a m e from

the s a m e geographical a rea .

The s p e c t r a of F igures 33 were taken with the excitation and emiss ion

se t at 356 nm and 440 nm respectively. Theee diagnostic set t ings were

chosen f rom the data of F igures 28 and 29. The t r a c e s appear to be s imi la r ,

all being broad with no evident s t ruc ture . The excitation peak a t 3 R C nm and

the emiss ion peak a t 4C2 nm a r e Raman peaks. It was learned af ter the fact

that t h e r e i s a paper pulp mil l 25 mi lee up the York River. Had w e monitored

with a n exc i t a t im of 300 nm, we might have seen an emiss ion in the 400 nm

region which could be a r roc ia ted with lignin sulfonate. Such emiss ion would

have been detected a t Station 5, hut not a t Station A.

Thur, the J i scernib le differences in excitat ionieminsion spec t ra a t

all fous r tat ionr involve in tenr i t ie r r a t h e r than spect ra l flistribution. The in-

tenr i ty effects will h: dircumred in the next sect ion in conjunction with other

m e a r u r e m e n t r made by VIMS.

3- 34

3. 3. 3 Comparison of Fluorescence Intensity Data with Related \?MS Data - Before evaluating data, a few descr ip t ive comments a r t in o r d e r con-

cerning stat ions A-E. Referr ing t o Figu-e 27, s tat ion A i s located at the

head of Mobjack Bay, the confluence of s e v e r a l s m a l l r ive r s . T h e r e is no

known source of pollution; the bay is shallow (6m). Station B i s a t the mouth

of Mobjack Bay cn the north s ide of an underwater ba r , York Spit. It i s located

in a fairway channel and has a depih of 8 m. Station D i s located in the York

River channel with a depth of 11 m. Station E is located in the channel fur ther

u p the York River and has a depth of 20 rn. The York River has a paper +lp

mi l l twenty five mi les up a t i t s head, two naval installations, a power plant

and an oil refinery. Thus, s tat ions E and perhaps D a r e expected to have

the g rea tes t environmental s train.

In Table 2, we l i s t the relat ive peak heights of the 460 nm and 685 nm

emission taken f rom the data of F igures 32 and 33, together with data

measured by VIMS for each of the su r face and near su r face samples . The

luminescence data a r e normalized for the instrument gain. Some VIMS data

a r e miss ing for station D because of l o s s of cer tz in samples .

The 460 nm emission(ge1bstoff) is essential ly constant a t a l l s tat ions

except E, where it is somewhat higher. This may be expected s ince E has

the grea tes t pollutant load. The chlorophyll emiss ion a t 685 nm var i e s

among the stations in the s a m e way a s chloro$iyl l a levels , though the - relationship is not linear. Since, according t o Yentsch (D1971), the 685 nm

emission is due t o part iculates, the f luorescence will b e dependent on the

s i z e of the par t ic les , t!le distribution of chlorophyll a i n the par t ic les , - and the concentration of part icles . F o r the s a m e total chlorophyll content,

sma l l e r par t ic les a r e expected t o yield higher f luorescence readings in vivo

because of higher s u r f a c e to volume ratio.

TABLE 2. SUMMARY OF BAIRDIVIMS DATA

FOR CHESAPEAKE BAY SURFACE WATERS

Measurement Sta. A Sta. B Sta. D Sta. E

Luminescence a t 685 nm (re la t ive)

Luminescence a t 460 nm (re la t ive)

Chlorophyll - a (pg/l)(Surface)

Chlorophyll - a (pg/1)(1/2 m e t e r )

Chlorophyll - b ( ~ g l l )

Chlorophyll - c ( r g l l )

Salinity (ppth)(at 1/ 2 m e t e r )

Tempera tu re (OG)(at 112 m e t e r )

Light penetrat ion (O /o at 112 m e t e r )

Plankton

Weather (wind speed in knots)

13

8 5

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3. 3.4 Sample Decay Measurements

While the p r i m a r y purpose of the on-si te measurements was to o b t a h

data on f r e s h samples we were a l s o in teres ted in the change of spec t ra l s ig-

na ture with t ime. In F igures 34A,D# we show chlorophyll exci tat ion/emission

t r a c e s fo r one-day-old samples , s to red a t labora tory tempera ture . These

w e r e the s a m e su r face samples displayed f r e s h in F i g u r e s 32 A - 3 ' In

eve ry case , t h e r e is a distinct d rop in s ignal intensity. In F i g u r e 34A, the

chlorophyll s ignal has dec reased by a fac tor of 0.3, compared to 3ZA. The

t r a c e s a r e s i m i l a r except for the appearance of a rise in the excitation

spec t rum a1 about 560 nm. In F i g u r e 34B, the signal has dec reased by a

fac tor of 0.074 with no apparent change in s ignatures. In F igure 34D, the

d e c r e a s e is about 0.1 with no obvious change in signature. Unfortunately,

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the sample from station E was lost before the delayed measurement was

made.

All the above measurements were made without attempting to s t i r

the sample, reflecting the settling that had occurred. Other measurements

were made to determine the effect of shaking the sample just before measure-

ment. These measurements ranged from several hours to fifteen hours. In

each case, there was a measurable decrease in signal which was restored to

i ts former strength (approximately) by shaking. The broad sl i ts used for

these measurements preclude determination of ratio of pheophytin to

chlorophyll. We drew the following conclusions:

1. As the sample ages, the scattering decreases, corresponding to

settling of particulates.

2. As the sample ages, the chlorophyll signal decreases ( s s nple not

shaken).

3. If a sample i s agitated just before measurement, the scatter and

chlorophyll signal increase again.

4. The chlorophyll signal reaches the same approximate peak height as

in the f resh sample: the scattering peak i s somewhat smal ler (sugges-

ting agglomeration? ).

5. An older sample sett les out mcire quickly than a f resh sample.

6 . For the low resolution used (24 nm), the chlorophyll excitation spec-

t rum is - ery similar for a one-day-old agitated sample and a f resh

sample.

3. 3, 5 Conclusions

The principal results of the on-site measurements a t VIMS were the

more than adequate performance of the instrument on the boat with natural

samples, and the collection of typical data from an important site. The de-

tection limit for chlorophyll should be below one microgram/l i ter , which

will be uretul for most ramplee of interest.

The da t a on s a m p l e decay sugges t tha t old s a m p l e s m a y provide s o m e

useful information if shaken vigorously before m e a s u r e m e n t (i. e , , ini t ia l

ch lmophyl l concentrat ion i s suggested) . However, it is probably t r u e that

t he 24 n m bandwidth used does not allow d iscr imina t ion bet wee:^ chlorophyll

and pheophytin. T h e decay of a pa r t i cu l a r s a m p l e m u s t depend s t rongly on

t h e length of s to rage , light conditions, t e m p e r a t u r e , avai labi l i ty of oxygen,

etc. On-s i te m e a s u r e m e n t s of f r e s h s a m p l e s would s e e m t o b e the only r e l i -

ab le r e c o u r s e t o obtain r e l i ab l e da t a on identity and concent ra t ion of plankton

spec i e s . On the o the r hand, Gelbstoff s igna tu re s changed l i t t l e during s t o r -

age, indicating that s a m p l e s ma i l ed a t a d i s t ance would be useful f o r d e t e r -

mining gene ra l levels .

3.4 F ie ld Tes t ing a t Cape Ann (Glouces t e r ) , Massachuse t t s

T h e proximi ty of t he Univers i ty of Massachuse t t s M a r i n e Stat ion a t

Hodgkins Cove r in Glouces te r , combined with the excel lent working re la t ions

we have with i t s d i r e c t o r , Dr. C h a r l e s Y en tsch , have l e d us t o m a k e f requent

u s e of thz t faci l i ty . We have used that l abo ra to ry a s a t e s t -bed f o r a l l v e r -

s ions of o u r i n s t rumen t , f r o m t h e unmodified SF-100 t o o u r f inal two-detector

ve r s ion with r a n g e extended. Dr. Clarice Yentsch was a l s o v e r y helpful i n

providing a var ie ty of a lga l cu l tu re s f o r r e f e r e n c e data ,

r e s 3.4.1 Labora to ry Seawater Stud'

T h e Mar ine Station is provided with a n intake f r o m Hodgkins Cove

which provides f r e s h r ep re sen ta t ive wa te r to the labora tory . Most work was

done: on t h e s e s a m p l e s which a r e not ac tua l s u r f a c e samples . Typica l wa te r

is lower in t e m p e r a t u r e and chlorophyll but higher i n sa l in i ty a s c o m p a r e d

t o t he Chesapeake Bay water at VIMS.

T h e d a t a of F i g u r e s 35 and 36 a r e f r a m a typical win ter day with

l ight snow. Water t e m p e r a t u r e was 0. ~ O C ; sa l in i ty 31.6 ppth; chlorophyll

content 0.5 ug/ l . T h e emis s ion s p e c t r u m in t h e chlorophyll region, F i g u r e

35a, shows a typica l peak at 683 nrn, but without as l a r g e a shoulder a t

740 n m as was s e e n in m o s t s a m p l e s f r o m VIMS (cf. F i g u r e 3 2 ~ . The R a m a n

sca t t e r r i s e s off-,scale on the left s ide a t this gain. The corresponding exci-

tation spect rum in F i g u r e 35b i s unlike the Chesapeake Bay samples in the

s i z e of the long wavelength shoulder a t 550 nm, and t h e appearance of an

exci-tation peak at 430 nm.

The instrument gain fo r t h e s e t r a c e s was approximately seventeen

t imes the s tandard gain used a t VIMS: the t i m e constant was increased t o

one second. Assuming basic ins t rument performance had not changed, the

re la t ive chlorophyll f luorescence for th is sample would be 6.6 for compar i -

son with Table 2. F r o m Table 2, we would expect a much lower f luorescence

reading for such a low chlorophyll content. Ei ther the VIMS chlorophyll

values a r e too high, o r Yentsch's is too low. We tend lo believe the Yentsch

valne, (Note in Table 2 that chlorophyll values at ? .' m e t e r a r e individually

l e s s than a t the surface. ) In th is case , i t i s c l e a r that the sensi t ivi ty of our

instrument i s such that we s e e 112 m i c r o g r a m of chlorophyll pe r l i t e r of

seawater with a s ignal- to-noise of approximately for ty to one.

In F i g u r e 36, we show excitat ion/emission s p e c t r a f o r Gelbstoff e m i s -

s ion f rom the s a m e sample , The Raman peaks have been labeled with an R. -. Peak excitation and emission wavelengths a r e v e r y s i m i l a r t o those for

Chesapeake Bay waters . Using the peak Gelbetoff luminescence intensity a s

a rough index of the amount p resen t and adjusting gains t o match those used

at VIMS, the relat ive value to be compared t o l ine two of Table 2 is unity.

This suggests aGelbstoff level l e s s than 1/8Qth that found in Chesapeake Bay.

3.4.2 Sample Decay Studies

The principal r eason fo r studying decay measurements was t o de te r -

mine the utility of studying samples ma i l ed f rom distant rources . F o r th is

purpose, s tudies w e r e c a r r i e d out up t o a week. Very shor t - t e rm s tud ie l

( l e s r than one hour) w e r e a l r o made t o de te rmine whether samples could be

brought t o a nearby labora tory fo r study. F o r decay s tudier , only peak

omiroion intenait ier w e r e monitored.

With the instrument se t up for peak chlorophyll emiss ion, no notice-

able decay was noticed during the f i r s t minute. The emiss ion peak decayed

to about 2 / 3 of i t s initial value in about fifteen minutes, while being i r radia ted

in the instrument. While it i s now assumed that this decay i s largely due to

settling of part iculates, it i s a l so possible that photochemical decay occurred .

F o r tke longer t e r m measurement , a f r e shwate r sample was s to red

in a c i e a r g lass bottle and kept tightly shut for the period of a week. The bottle

was s tored in the dark between sampling per iods to s imulate mailing conditions.

Both the Gr::bstoff and chlorophyll emiss ions were monitored pcriodically.

After four days, the chlorophyll emission inh.ensity drops to about one-third

of i t s initial value (with an agitated sample) , a f ter whicri i t r emains essential ly

constant for the remainder of the week. S imi la r exper iments on Gelbstoff

showed a much s m a l l e r t ime dependence, result ing in an actual inc rease of

ebout twenty percent . These data a r e plotted in F igure 37.

The apparent longevity of the chlorophyll signal i s probably misleading.

F o r example, al terat ion products of chlorophyll in dying plankton may include

pheophytins. According t o F rench et al . (A1956), the f luorescence spect rum

of pheophytin - a i s displaced about 10 nm to the r e d a s compared to chlorophyll

a when in an e the r solution. If th;s be t r u e a l s o for the pigments in vivo, a - monochromator se t t o peak the chlorophyll emiss ion would s e e the pheophytin

off i t s peak and eo read lesc . The s a m e type of argument holds for the absorp-

tion spect ra . Fur the r , Goedheer (A1968) quotes quantum yields of f luorescence

a s 0.22 for chlorophyll a and only 0.14 for pheophytin a . Hence, if e - ~ e r y - - chlorophyll molecule became a pheophytin molecule, we wuuld expect to s e e

a t ime d \ c r e a s e in the "chlorophyll emission" which leveled out at some non-

zer o level corredpanding t o detected pheophytin emission. With thie s o r t of

possible mechanism in mind, tne reduced emiss ion in the chlorophy!l region

may not be chlorophyll; hence the use of old samples to de termine chlorophyll

and pigment concentrations i s quite suspect .

3.4. 3 F i l t e r Studies

Yentsch has shown that the m a j o r e m i s s i o n in the chlorophyll region

i s due t o pa r t i cu l a t e s whereas the Gelbstoff emis s ion i s due t o so lu te in the

m a r i n e water . F i l t e r ing s a m p l e s t o s e p a r a t e t h e s e components m a y have

bzvera l advantages. F i r s t , f i l t e r ing i s a concent ra t ion s t e p which will allow

an effective i n c r e a s e in sensitivit tr . Second, ha-ling m o r e concent ra ted s a m p l e s

wi!! allow use cjf h igher s p e c t r a l resolut ion which should al low be t t e r d e t e r -

minat ion of e i m i l a r Figments. Final ly , d r y f i l t e red s a m p l e s a r e known t o

decay m o r e slowly than eolutions.

We t h e r e f o r e began cxpe r imen t s t o c o m p a r e s p e c t r a f r o m f i l te red

plankton and or ig ina l water s amples . A goal was t o develop a p r o c z d u r e

which will a ; low d i r e c t fr ' . .% s u r f a c e luminescence examinat ion of the damp

f i l te r paper yieldin;;, semiquant i ta t ive concentrat ion iniurmation. A r equ i r emen t

i s that the vc:ume of or ig ina l water and the s i z e of the f i l t e r p a p e r be s o

chose% a s t o produce a uniform pa r t i c l e distributior. which does not cover

the sur face . In t h i s c a s e , i nc reas ing or. dec reas ing pa r t i cu l a t e c o n c e n t r a t i ~ ~ i

will have a cor responding effect on the a r e a of the f i l t e r pape r cove red , and

the luminescence s igna l should be a rough m e a s u r e of or ig ina l conrsn t ra t ion .

If too g rea t a n initial water volume i s used , the f i l t e r paper will be r.:ore

than completely covered. Since only the t c p l a y e r i s viewed, concentrat ion

information i s losl . F o r too s m a l l vo lumes , - f wate r , sens i t iv i ty is lost .

U s k g Whatman G F / C g l a s s f iber f i l t e r s ( a s sugges ted by E. Weaver)

supported on a g l a s s f r i t , we have demons t r a t ed :hat it i s feas ib le t o filto:

to d r y n e s s and then examine the r e s idue f ront s u r f a c e on o u r s t anda rd i n s t r u -

mentation. T h e rericlual d3mpnesr of the pape r i s sufficient t o ma1.e it a d h e r e

t o a flat capd placed in the sample holder . A difficulty a r i s e s with uniformity

of particulrrte deporiticrn. Thur f a r , we have been unable t o a,uure uniform

deposition of m a t e r i a l which i r n e c e l s a r y f o r quant i ta t i t e work, s ioce we

only r a m p l e p a r t of t h e f i l t e r p a p r area. Quite often the depos i t would f o r m

a n annulab r ing with the c e n t e r of t he f i l t e r p a p e r qui te f r e e of deposi t . While

we have not a e i s r m i n e d t h e r e a r o n f o r t h i r , we f ee l thir problem could oe

cvercome. By carry ing out a s e r i e s of f i l t rat ions using volumes differing by

an o r d e r of magnitude, and selecting only those p a p e r s which appeared uni-

formly covered t o the eye, we found the "chlorophyllv emiss ion i s approxi-

mately l inear with concentration of depos ' t ( o r volume of sample) .

F o r the numerous samples examined a t o ther s i t e s , the p r i m a r y ap-

proach has always been t o look di rec t ly at water samples . In one case , at

the F lo r ida State University Marine Station nea r Car rabe l l e , F lor ida , the re

was a n instrumental instability problem which did not allow reproducible

measurements at high gain. In th is case , we did u s e the f i l ter ing technique

t o obtain data on the part iculate emission.

3.4.4 T i m e Variation in Chlurophyll F luorescence - In addition t o the measurements descr ibed above, another interest ing

experiment was performed which indicates that a lga l concentrations fluctuate

s izeably due t o water mcverr. mt. The ins t rument was equipped with a flow

cel l which allowed coiltinuous monitoring of the water piped in f rom Hodgkins

Cove. Exciting at 470 nm, the chlc-rophyll emiss ion was monitored a t 678 nm

as the water flowed through a t 3 l i t e r s p e r minute. The resu l t s of a typical

scan a r e given in F igure 38. The t i m e b a s e (x-axis) is 25 s e c l c m . The s h a r p

negative signals a r e z e r o checks to a s s u r e the observed variat ions w e r e not

due to amplif ier drift. The maximum variat ion is about 20% of the average.

These re su l t s a r e a s sumed t o be re la ted t o wave motion in Hodgkins

Cove. They a l s o indicate that i t is not necessa ry t o take v e r y accura te data

on intensity a t a given s i t e a t a par t icular t ime , s ince significant variat ions

occur over shor t t imes .

i ! .. L . ..: -*-.. . ~ . - . . -. - -.-~-

;.T &. I.. ' '

+ - -...... ' j ' B

4. EXPERIMENTAL RESULTS

4.1 Compendium of Spectral Data

One of the principal purposes of this project was to assemble a Com-

pendium of SF x t r a l Data containing comparative t races of marine luminescence

from a large variety of s i tes and at various t imes. This objective has been

modified slightly by the change to on-site measurement, resulting in some-

what l ess data than anticipated. Nevertheless, a considerable body of data

has been assembled and published separately in the Compendium of Spectral

Data, which comprises Appendix C of this Final Report. The spectral data

in the Compendium a r e traced from the originals onto a standard grid for

easier comparison.

Principal data incl-~de excitation/emission spectra of chlorophyll and

Gelbstoff in natural waters taken on-site on fresh samples. Where on-site

measurements were impossible (e. g., West Coast si tes) only Gelbstoff

measurements were made on mailed samples. In one instance (Carrabelle,

Florida) where instrumental problems made water measurements untrust - worthy, samples were filtered, and chlorophyll measurements were made

front-surface on the particulates.

Also included in the Compendium a r e excitation/emission spectra of

a number of algal cultures (reference spectra) for comparison with natural

water spectra.

4.1.1 Instrumefit Settings

As was pointed out ear l ier , time was often at a premium durir.g on-site

measurements. Therefore, data were usually taken a t set excitation and

emission wavelengths to allow intercomparison. Multiple excitation1 emission

data were taken on samples from VIMS and Cape Ann, Mzs~achuse t t s , a s

discussed previously.

For llchlorophyllu data the bandpass of both excitation and emission

monochromatore was usually s e t wide open, o r about 24 nm. This was

necersary for sensitivity for the weakest samples. The excitation wavelength

was usually s e t about 460 nm, although th is was somet imes va r i ed if it was

f a r f rom the peak excitation. The monitoring wavelength was s e t on the

peak chlorophyll emiss ion which occurs about 630 nm.

F o r ttGelbstofr" s p e c t r a the excitation was s e t a t 280 o r 290 nm and

the monitoring wavelength a t 440 nm in m o s t cases . Bandpass on both exci-

tation and emiss ion monochromators was kept a t about 17 nm. ( T h e r e was

l e s s question of detectability with Gelbstoff. )

The r e f e r e n c ~ algae were usually monitored with a bandpass of 6 nm

s ince the re was ample signal. The s m a l l e r bandpass allows development of

g r e a t e r detail in the spect ra . Excitation was usually se t t o give the g rea tes t

chlorophyll signal. The monitoring wavelength was usvally the peak of chloro-

phyll emission. This peak often occur red a t wavelengths slightly longer than

680 nm, probably because of the effects of reabsorption of the chlorophyll

emiss ion in the s t rong samples .

4.1. 2 Relat ive Intensity

In the ea r ly s tages of the project , a n at tempt was made t o ca l ibra te

the ins t rument before each measurement using a ruby rod or water Raman

as reference. Consideration of the var ia t ion o: chlorophyll emiss ion a t a

given si te , not onl) with season, but even during days and hours , as shown

by the con t iwous monitoring exper iments at Cape Ann, make p r e c i s e

measurements meaningless. There fo re we have defined a "Relative Intensity"

fo r each chlorophyll curve which is proport ional t o the r a t io of the peak

height and instrument gain. Since ins t rument gain probably v a r i e s much

less than chlorophyll emiss ion a t a given si te , this fac tor can be used a s

a guide t o r e l a t e measurements made in different p laces a t different t imes .

The Relat ive Intensity i s given on each chlorophyll t r a c e f o r actual m a r i n e

wa te r s in the Compendium. The range is f rom 99 fo r a Chesapeake Bay

sample t o much less than 1 f o r a sample f rom Hodgkins Cove a t Cape Ann,

Massachuset t s,

4. 2 Site Descriptions

F o r typical on-si te measurements , s tandard instrumentat ion was sent

by a i r t o the vicinity of the se lec ted labora tory s i te , and t ranspor ted by

station wagon t o the laboratory. Standard equipment consisted of the

Fluorescence Spectrophotometer, l amp power supply, x-y r e c o r d e r , cuve'tes

and f i l ter ing apparatus for part iculates. Usually labora tory bench space

and iaci l i t ies had been p rea r ranged , allowing quick s t -up fo r measurement .

After instrument checkout on s tandard samples o r water Raman, local water

samples were examined. This would be followed by study of samples ob-

tained by shor t boat t r ips to se lec ted water s i tes . Often the water samples

were measured within hours of collection. Where possible, the water

samples were subjected t o s tandard labora tory analysis for chlorophyll,

salinity, e tc . , by the host laboratory. In the following sub-sect ions, we shal l

d iscuss each of the s i tes se lec ted and the method of collecting and handling

samples .

Samples f r o m nine different geographic s i t e s w e r e m e a s u r e d and in-

cluded in the Compendium of Data. The f i r s t five of these , covering the

Atlantic and Gulf coasts , were covere& on-si te . Here measurements were

made on chlorophyll and Gelbstoff, and on severa l algal cul tures supplied bj

labora tor ies . The remaining four s i t e s included t h r e e off the West Coast

and one s e v e r a l hundred m i l e s north of Hawaii. Lack of t i m e and funding

made it impossible t o monitor these on-si te; ther2fore samples were mailed

t o Bedford, and only Gelbstoff was monitored. The s i t e s will be descr ibed

in some detai l in the following subsections. They a r e indicated on Map A.

Fur the r de ta i l s will be found in sect ion 2. i of the Compendium of Spect ra l

Data.

4.2.1 Site A: Cape Ann, Massachuset t s (Univers:ty of Massachuset t s Mar ine Station

The labora tory is located on the wes te rn s ide of Cape Ann a t Hodgkins

Cove. Numerous measurements w e r e conducted a t the labora tory through-

out the pro jec t . Representa t ive data f r o m s e v e r a l da t e s a r e included in t he

r epo r t .

Typica l r e s u l t s v e r e obtained on both chlorophyll and Gelbstoff, a s

r epo r t ed in Section 3 . 4 and in the Compendium of Spec t r a l Data. Dr . C h a r l e s

Yentsch, D i r e c t o r of the Mar ine Station, provided a s s i s t a n c e in a l l phases

of t h i s project . The labora tory provided independent da ta on o ther a t e r

va r i ab l e s , including chlorophyll content, v henever des i r ed .

4. 2. 2 Site B: Glouces te r Point , Virginia ( 'Jirginia Inst i tute oi YJarine - Science)

The Virginia Inst i tute of M a r i n e Sc ience (VIMS) i s loca ted a t Glou-

c e s t e r Point on the York e s t u a r y cf Chesapeake Bay. M e a s u r e m e n t s h e r e

were conducted on If aqd 16 F e b r n a r y 1972. T h e s e m e a s u r e m e n t s included

l abo ra to ry water s a m p l e s [off the VIMS p ie r ) , on sh ip m e a s u r e m e n t s a t four

s i t e s ranging f r o m the mouth of the York R i v e r up into Mobjack Bay, and

s o m e algal cu l tu re measu remen t s . Th i s l abo ra to ry a l s o provided independent

m e a s u r e m e n t of water p a r a m e t e r s .

The m e a s u r e m e n t s aboa rd sh ip w e r e a n impor t an t s t e p in checking

ou r f ie ld ins t rumenta t ion and a r e c l e a r proof tha t t h i s type ins t rumenta t ion

can be m a d e t o f u n c t i m \\,ell aboa rd a s m a l l boat \i.ith s imp le moto r - g e n e r a -

t o r power. A m o r e detai led account is given in Section 3. 3, a s well a s i n

the Compendium.

The on - s i t e m e a s u r e m e n t s w e r e a r r a n g e d through the cooperat ion of

Dr . Pau l Zubkoff, Cha i rman of the Depar tment of Physiology. Mr . J.

E r n e s t War r ine r 111 a s s i s t e d in col lect ing s a m p l e s , piloting t h e boat, and

se t t ing up l abo ra to ry faci l i t ies . He a l s o provided s a m p l e s fo r o u r abor t ive

a t tempt a t mai l ing samples .

4.2. 3 Site C: F o r t Lauderda le , F l o r i d a (Nova Univers i ty Phys i ca l Oceanographic Labora tory)

Th i s l abo ra to ry is loca ted jus t south of F o r t Laude rda le n e a r t he

Atlantic Ocean. Samples w e r e ga thered 3 and 4 Apr i l 1972 and m e a s u r e d

at the Oceanographic Labora tory . Th i s s i t e i s of pa r t i cu l a r i n t e r e s t because

of the c lo se proximi ty of t he Gulf S t r e a m of fshore . Gulf S t r e a m wa te r has

low product ivi ty and high c l a r i t y a s con t r a s t ed with in shore w a t e r s having

high turbidi ty due t o yellow humic ac ids dra in ing out of P o r t Eve rg lades via

New Rive r . The s o u r c e of t h e s e ac ids i s the Eve rg lades , through the d r a i n -

a g e sys t em. Product ivi ty of i n shore reg ions i s high a s a r z su l t of domes t i c

pollution along t h e dra inage basin.

Ar rangemen t s f o r t hese m e a s u r e m e n t s w e r e m a d e by C. Yentsch, with

the kind a s s i s t a n c e of Dr . W. Richardson , the Labora to ry Di rec to r . Yentsch

a s s i s t e d in col lect ing s a m p l e s fo r immed ia t e m e a s u r e m e n t .

4. 2. 4 Site D: C a r r a b e l l e , F l o r i d a ( F l o r i d a S ta te Universi ty Mar ine Stat ion)

T h e m a r i n e s ta t ion i s located in t he panhandle of F lo r ida on the Gulf

of Mexico a t Turkey Poin t , n e a r Ca r rabe l l e . T h e water i s s ed imen ta ry and

shallow. measurement^ w e r e m a d e in mid -Apr i l 1972.

Th i s s i t e produced the fewest _good m e a s u r e m e n t s because of t he

onse t of i n s t rumen t instabi l i ty problems. A s a r e s u l t , i t was n e c e s s a r y t o

discontinue d i r e c t m e a s u r e m e n t of cnlorophyll in water s a m p l e s and t u r n

t o f i l t e r ing methods , using e l e m e n t u y appa ra tus we had brought w i i t 11s.

These f i l t e r e d pa r t i c l e s w e r e then s tudied by f ron t - su r f ace methods. T h e

exper ience shows that t h i s method i s a l s o viable , althougii l e s s quant i ta t ive

Following t h e s e m e a s u r e m e n t s , the f ie ld i n s t rumen t was r e tu rned t o

o u r Bedford l abo ra to ry where a n improved feedback c i r cu i t in the photo-

mul t ip l ie r high vol tage supply was added. T h i s m a d e the in s t rumen t p e r f o r m

stably in a l l fu ture m e a s u r e m e n t s .

Ar rangemen t s t o u s e the l abo ra to ry fac i l i t i es w e r e m a d e through Dr .

J ack Winchester , H P - , ~ of the Depar tment of Oceanography a t F l o r i d a S ta t e

Universi ty in Ta l l ahas see .

4. 2. 5 S i te E: Galveston Bay (National Mar ine F i s h e r i e s Labora to ry )

The l abo ra to ry i s located n e a r Galveston Bay. M e a s u r e m e n t s w e r e

made on 20 June 1972 on s a m p l e s f r o m nine bay s i t e s chosen t o r e p r e s e n t

typical a r e a s . Some a r e a s w e r e idea l n u r s e r i e s : o t h e r s w e r e possibly

polluted by nearby plants .

Ar rangemen t s fo r the u s e of t he l abo ra to ry fac i l i t i es and a boat t o

col lect s a m p l e s w e r e m a d e by Mr . Robe r t Temple , Ass i s t an t D i r e c t o r of t he

Labora tory . Mr . F r a n k Maru l lo co l lec ted s a m p l e s , and Mr . Neil Bax te r

provided the da t a on t e m p e r a t u r e and salinity.

4. 2 .6 Si te F: Pac i f ic Ocean--Southern Cal i forn ia (Univers i ty of Cal i forn ia a t Santa B a r b a r a Mar ine Sc ience Inst i tute)

F o r t h i s and the following t h r e e s i t e s the s a m p l e s w e r e ma i l ed t o

Bedford fo r delayed examination. Because we f ee l such m e a s u r e m e n t s on

chlorophyll a r e invalid, only Gelbstoff m e a s u r e m e n t s w e r e made ,

Th i s s i t e i s of pa r t i cu l a r i n t e r e s t because it has an abundance of

kelp beds and na tu ra l oi l seepage. F i v e s a m p l e s w e r e provided, co l lec ted

on 12 Sep tember 1972. T h e s e ranged f r o m t h r e e m i l e s of f - shore to d i r ec t ly

off the beach. They include wa te r f r o m ke lp beds and in a n o i l s l i ck region.

Sample col lect ion was a r r a n g e d by Dr . Rober t Holmes, D i rec to r of

the Institute.

4. 3 Discuss ion of Chlorophyll Data -- Before d i rec t ing o u r at tent ion t o t h e da t a t hemse lves , it i s worthwhile

to rev iew the l imi ta t ions of the da ta . F i r d t , t h e r e i c the quest ion of absolu te

intensity. Because of the evolut ionary na tu re of t he field i n s t rumen t , t he

sensi t ivi ty va r i ed f r o m s i t e t o s i t e . Thus, a s power supply p rob lems g rew

(cuiminat ing in unacceptable behavior a t s i t e D in C a r r a b e l l e , F l o r i d a ) , i t

was n e c e s s a r y to mod.ify the photomult ipl ier voltage. T h e non- l inear gain

co r r ec t ions f o r t h i s a r e not known and not i nco rpora t ed in o u r Rela t ive In-

tencrity f igurer . T h e power supply p r o b r n s ,3180 caused apparent d r i f t in

signal which was m o s t apparent in high gain s i tuat ions such a s was n e c e s s a r y

in G louces t e r , Massachuse t t s , whe re chlorophyll content was l e s s than one

mi l l i g ram p e r l i t e r . The resu l t ing background var ia t ion could be in t e rp re t ed

a s exci tat ion peaks and val leys. See , f c r example , F i g u r e C - 8 , where the

obse rved \ . a r i a t i om may not be r ea l . (Note that we sha l l r e f e r to f i gu res in

the Compendium with the pref ix C - . )

Another impor tan t l imitat ion i s that the da ta a r e not c o r r e c t e d for

i n s t rumen t r e sponse . The da ta of F i i u r e 31 show that the l a m p output v a r i e s

with wavelength. Exci tat ion peaks below about 380 nm will appea r too weak,

and t h e r e will a lways be a spu r ious r i s e a t 470 nm where the xenon l a m p

has i t s l a r g e s t peak. The da ta of F i g u r e 31 a r e based on Rhodamine B a s

a quantun. counter. F u r t h e r work has indicated that the output i s unde res -

t imated a t va l leys in the Rhodamine absorp t ion in the vicinity of 450 nm and

380 nm, and ove res t ima ted a t the peak abso rp t i cn a t 560 nm. It i s unfor-

tunate that o u r da ta co r r ec t ion s c h e m e was not completely opera t ive in t i m e

to c o r r e c t t r a c q s fo r the compendium.

While lack of co r r ec t ion m a k e s r e f e r e n c e t o absorp t ion da t a difficult ,

re la t ive d i f fe rences between exci tat ion c u r v e s can be in t e rp re t ed meaning-

fully. Thus , when we examine the t r a c e s of the Compendium, we sha l l look

f o r d i f fe rences f i r s t - - t h e n r e l a t e t hem t o poss ib le c ~ m p o n e n t s .

Another l imi ta t ioa i s t h e neces s i ty f o r using r a t h e r l a r g e bagdpass

(approximate ly 24 nm) in o r d e r t o achieve o u r good senlsitivty fo r low leve l

chlorophyll m easurements . Th i s p rec ludes monitor ing of exc i t i - ion v e r y

c lose to emis s ion and ale;, l i m i t s a c c u r a t e del ineat ion of c u r v e prof i les .

On !he o the r hand, t h i s bandpass h a s r e l evance t o potential r e m o t e sens ing

application.

4. 3.1 Chlorophyll E m i e r i o n

If we page through the Compendium, concent ra t ing on chlorophyll

e m i r a i o n data , t h r e e fact, emerge . F i r r t , t h e r e ir a l a r g e var ia t ion in

r e l a t i ve in tenr i ty which c o r r e r p o n d r roughly with chlorophyll concent ra t ion

a s measured by standard labora tory procedures . (See Section 3. 3. 3. )

Second, the chlorophyll emiss ion occurs with slight variation a t about

680 nm. P a r t of th is variat ion may be due t o slightly inaccura te setting

up of the wavelengths on the x-y r e c o r d e r in the field. P a r t i s due to con-

centrat ion effects (self-absorption) which can shift emiss ion to slightly

longer wavelengtns. In any c a s e , the bandwidth of 24 nm does not allow any

cer ta in s ta tements to be made about significant variat ion in emiss ion peak

position. A t h i rd interest ing observation concerns a shoulder which appears

on most emiss ion t r a c e s a t about 74C nm. (See, for example, F igures 32B,

3 2 ~ 9 323 .1

There i s no agreement a s to the source of the 740 nm shoulder . Some

believe it i s p a r t of the emiss ion spect rum of chlorophyll A. Others believe - it is one of the many sub-types of chlorophyll being d iscovered regula*.

The variation in intensity of the shoulder i s a l s o well documented. (See

F igure 3. ) In the Compendium approximately one-third of the chlorophyll

emiss ion t r a c e s f rom on-s i te water samples did not show evidence of th is - shoulder. There i s certainly a grea t variat ion, a s evidenced for example

by F i g u r e s C-57 and C-63, both f rom the Galveston Bay a r e a .

F r o m a purely ins t rumenta l point of view, the data aliow explanation

of the 740 nm shoulder a s e i ther a separa te moiety which occurs In company

with chlorophyll a, o r as a band of chlorophyll a which suffers l e s s r eabsorp- - - tion than +he m a j o r peak. Because of our observations on excitation spect rb ,

we tend t o believe the l a t t e r explanation.

4. 3.2 Chlorophyll Excitation

It is well known that the auxil iary pigments (carotenoids, phycobilins,

e\ :,) t r ans fe r energy ve ry efficiently to chlorophyll. Therefore , the exci-

ta t on spec t rum of chlorophyll in a lgae may show peaks not associa ted with

chlorophyll i trelf but with auxil iary pigments. If this be so , i t should be

porr ib le t o make r o ~ g h determination8 1 the type of a lga f rom the excitation

spect rum.

Looking through the compendium a t excitation spec t ra , i t i s c l e a r

the re a r e variat icns in the spect ra . Yet t h e r e a r e a l so ve ry g rea t s i m i l a r i -

t ies . We shal l coneider the f ~ r m shown in F igure 32E a s "typical. This

form i s marked by i t s xenon-accentuated peak a t 470 nm, a shoulder a t

550 nm, and n shculder a t 440 nm. (The second o rde r s c a t t e r peak a:

340 nm i s to be *;eglected. ) In t.he Compendium, the following on-s i te water

t r a c e s exhibit th is typical form: C-6, 12, 16, 18, 20, 26, 28, 30, 34, 36,

38, 40, 42, 56, 60. One variant f rom tbie form has a doubie peak occurr ing

at 430 and 470 nm, e. g. , C-68 f rom Galvesto: Bay. Others showing this

variat ion include C-2, 4, 8, 24, 32, 58, 62, 66, 68, 70. In s o m e c a s e s ,

e. g . , C-32, 64, 66, 70, the 430 nm peak equals o r exceeds the 470 nm peak.

Anot;aer var iant concerns the s i z e of the peak at 550 nm. T r a c e s C - 2 , 4,

10, 22, 24, 26, 34, 40 have well developed peaks in th is region. This oeak

may be associa ted with the pigment fucoxanthin in diatoms, o r with phycoery-

thr in in other algae.

At this point, we note that according t o Yentsch a l l the naturs.1 wa te r s

sampled should have diatoms and dinoflage!lates a s the i r principal algal

components. Spect ra C-01, 8 3 and 85 are a l l diatom excitation s p e c t r a

showing the form of our Ittypicalft spect rum. Usually the 430 nm peak i s

slightly l e s s than the 470 nm peak. F igure C-87 of a dinoflagellate a l s o

shows the t l typicalv form, except : .: 430 nm peak is much l e s s than ?

the 470 n*-n peak. (Note thi.; the : . i . t . : r r spectra merltionud in th is pa ragraph

wae taken with c nm resolutron, $ 2 . ??r~5..,- ; ~ e q k r considerably, ) We !

conclude that i t i s rearonable to asbit : , , ' . : ' we a r e seeing the m a j o r com- i

ponent of t h e wa te r r in each c a r e , i f

Pre rumably the double peaked nvRe.;tra couid be iIscrpreted as due

t o the p r e r e n c e of another algal fo rm, Lor example NANNOCHLORIS ATOMUS

of F i g u r e C-72.

4 3 3 Quantum Counting

Examining a "1. pical" excitation t r a c e of any of the diatom cul tures ,

we a r e s t ruck by the similawity with quantum counter cu rves for determining

the intenuity of excitation light f r c m our aource a s a function of wavelength.

We have a l ready exhibited such a curve in F i g u r e 31, using Rhodamine B.

In this case , a l l of the energy i:, absorbed by the dye at every wavelength,

due t o the high concentration. The f l u o r e s c e ~ ~ e i s monitored at a much

10l*,~rr w,. length. If, a s is usually the c a s e with a romat ics , the quantum

vieid is independent of wavelength, the relat ive emiss ion i s a m e a s u r e of

t h e number of photons incident on the Rhodamine B, thus acting a s a "quantum

collnter. We have a l ready noted that Rhodamine B is not the ideal quantum

covnter sirtc? lt has very weak absorption in some regions, afid the f luores-

cence can be part ial ly self-absorbed. We have founa o ther super io r materiaL6

which do not show th is effect, though they do n ~ t cover a s mucL o! the spec -

t rum a s doee Rhodamine B.

In F i g u r e 38x, we have supr ?posed the "excitation spect rum" of a

concentrated dye which emi t s a t 57 ! nm and the excitation spect rum of the

diatom PHAEODACTYLUM TRICORNATUM of F igure C-bo. The dye spec -

t rum, in a heavy line, c e a s e s to a b s o r b sufficiently past about 480 nm to

ac t a s a quantum counter. However, a t s h o r t e r wavelengihr, the agreement

is surpr i r ingly good. Both r p e c t r a were taken at 6 nrn resolution. If we

examine the upper c u r v e of F igure C-50, taken on par t icula tes f rom r e a -

water in the Gulf of Mexicu, we: a l s o find ru rp r i s ing agreement . We fo rm

the tentat ive conrlurion that th i r diatom ir act ing a e a quantum couni =r,

although it i r perhapr imperfec t in the region from 340-430 nm.

Th i r conclusion, which rhould apply to tile water s - i-.:*l a! 5. a,

h a r far - reaching effectr on how t k r e r p e c t r a m a y be urec, 1 . . : c!:::r r algae,

and how the r p e c t r r are t o be in t r - prered. The babir of the r ; ', .puetation

dependr on the detailed ana ly r i r of f luotercence f rom prr t icula tcr . E s r e n -

tially, it a s r u m e r that the b I , face l aye r rrhich a b r o r b r the light is optically

dense, i. e., all the light ir absorbed. : , - tber , a lmos t all of t h i r eae rgy

i s t r a n s f e r r e d to the chlorophyll . Thus, t he exci tat ion s p e c t r u m , even of

a v e r y di lute suspension, i s the spec t rum of a concent ra ted sample . (We

have a l s o n x t t h i s s i tuat ion in o u r study of o i l s where v e r y di lute emul s ions

have spec t r a l p r o p e r t i e s of concent ra ted o i l s . ) We shal l r e t u r n t o th i s sub-

ject in ou r conclusions.

4. 3. 4 Algal Cul tures

Most a lga l c u l t r e s exhibited chlorophyll emis s ion a t 680 nm when

exci ted in ti.3 c h l o r o i ~ ~ ~ y l l excitatior, band n e a r 430 nm. Many a l s o exhibited

a va r i e ty of ~ h o r t e r wavelength e m i s s i c n s when exci ted a t s h o r t e r wave-

lengths. Thus, t he diatom THAL!,ASSIOSIRA FLUVIATILUS ( F i g u r e C-191)

exhibi ts emis s ion a t 510 nm in addition t o 680 nm when exci ted a t 354 nm,

and emis s ion a t 350 nm when exci ted a t 290 nm. In F i g u r e C-192, t h i s s a m e

d ia tom exhibits emis s ion a t 450 nm when exc i ted a t 350 nm. (Th i s l a s t could

contr ibute t o Gelastoff s p e c t r a of unfi l tered waters . ) SKELETONEMA

COSTATUM ( F i g u r e s C-189, 190) is another diatom with s i m i l a r secondary

emiss ions . The dinoflagellate GYMNODiUM NELSON1 of F i g u r e 196 a l s o

has a 450 n m emis s ion when exci ted a t 350 nrn. T h e b lue-green a lga ,

SCI-IIZOTHEUX ( F i g u r e C-90) exhibits a seconda ry e m i s s i o n a t 610 nm when

exci ted a t 400 nm. T h e g r e e n a lgae NANNOCHLORIS ATGMUS ( F i g u r e s

183, 185 and 186) and DUNALIELLA ( F i g u r e s 187 and 188) exhibit f l uo rescence

a t 340 nm and 450 nm when exci ted a t s h o r t wavelengths.

In genera l , t h e s e seconda ry emis s ions a r e m u c h weaker than the

chlorophyll emis s ion (as can be s e e n by the appea rance of Raman peaks on

the spec t ra ) . The i r ex is tence can p re sumab ly be t r a c e d t o t h e e m i s s i o n of

individual pigments as d i s c u s s e d e a r l i e r . Th i s would indicate that not a l l

ene rgy i s t r a n s f e r r e d t o chlorophyll . On the chlorophyll exci ta t ion s p e c t r a ,

i t would c a u s e low in tene i t ies it! s o m e absorp t ion reg ions as c o m p a r e d t o a n

ideal quantum counter curve . In F i g u r e 38A. we have noted that the exc i -

t a t ion t r a c e of t h e diatom is !ow in the region 340-430 nm. Thus, we c a n

main ta in a conr i r t en t p i c tu re allowing f o r s o m e indivir >la1 pigment e m i s s i o n

while maintaining t h e idea of quantum counting.

In most water samples , we did not detect emiss ions other than a t

680 nm and in the Gelbstoff region. It now appears that some of what we

have t e r m e d Gelbstoff emiss ion m a y in fact be due t o algae. (A s imple

experiment on f i l te red and unfiltered samples i s possible. ) Because of

diff icul t ies associated with on-s i te measurements and lack of t ime , we did

not always look in all spec t ra l regions and s o may have m i s s e d secondary

emis sions.

4. 3 . 5 Inter- and Intra-Site Variations

In the previous discussion, we have viewed the Compendiun~ a s a

whole in o rde r t o note s imi la r i t i e s and differences. W e now comment on

spec t ra by s i t e in o r d e r to de termine where differences occur.

It is hard to classify the s p e c t r a f rom Cape Ann, Massachuset t s

(Site A) because the chlorophyll level was so low and because our instrument

was not s o stable a s af ter pol- e r supply improvements.

The spec t ra f rom Site B (Chesapeake Bay) a r e all "typical", d i f f e r h g

only in intensity.

The spec t ra f rom the Atlantic Ocean off F o r t Lauderdale, F lor ida ,

showed significant variation, presumably reflect ing the g rea t change in

passing from the Gulf S t ream into coas ta l waters . Station 1 (F igure C-22)

has a ~ltypical" spect rum except that the 550 nm peak i s accentuated. Station

2 (F igure C-24) has a l e s s accentuated peak a t 550 nm, but now has a double

peak a t 430 and 470 nm. Station 3 (F igure C-26) is m o r e typical with the

double peak minimized and the 550 nm peak smal le r . Station 4 (F igure

C-28) i s ve ry typical, while Station 5 (Figure C-30) has a l a r g e r 430 nm

peak, though not split. Station 6 ( F i g u r e C-32), which i s in the harbor , is

"typicalt1, except that the 430 nm peak now is l a r g e r than the 470 nm peak.

The few unfiltered water eamples f rom Site C, n e a r Car rabe l l e ,

Florida, are all fair ly f'typicalw although the 550 nm peak is accentuated in

Figure C-34.

The data f rom Site D in Galveston Bay (F igures C-54, 56, 58, 60,

62, 64,66, 68, 70) showed the g rea tes t variat ion among themselves and r e l a -

tive to the other s i tes . The f i r s t t h r e e s ta t ions show an unusual s izeable

unstructured excitation extending f rom 520 nm t o the long wavelength chloro-

phyll absorption band. This i s not seen on any other t r a c e s . Otherwise,

these spec t ra a r e fair ly "typical", although F igure C-58 exhibits well-split

430-470 nm peaks. Station 4 ( F i g u r e C-60) i s "typical" except for s o m e r e -

maining long wavelength excitation. F igure C-64 of Station 6 shows a

s t ruc tured excitation with a third peak a t 410 nm. Stations 7 - 9 al l show

the split peak and a considerable long wavelength excitation.

Thus, the re i s considerable variat ion within s o m e s i t e s , a s well as

between s i tes . W e can only conjecture what the Pacif ic samples would have

shown.

4.4 Gelbstoff Excitation and Emiss ion

Gelbstoff spec t ra have a l ready been d iscussed in s o m e detai l in

Section 3.1. 3. They a r e charac ter ized by being a mix tu re of many mate r i a l s .

This is m o s t easi ly seen when the excitation spec t ra shift a s monitoring

wavelength is changed (F igure 23) and emiss ion spec t ra shift a s excitation

wavelength is changed (F igure 23). The data taken a t m o s t s i t e s were r e -

s t r i c t ed t o a diagnostic excitation a t 350 nm and m o n i t o r k g wavelength of

440 nm. In general , the l a rges t difference in spec t ra was in intensity.

At seve ra l s i t e s , an excitation wavelength of 280 o r 290 nm was used

in addition t o the 350 nm excitation. The s h o r t e r excitatior, proved to be

interest ing because it tended to excite a l a r g e r number of emit t ing moie t ies .

The p r e s s of t ime on-s i te unfortunately did not pe rmi t taking data a t many

excitation and monitoring wavelcngthe. A11 data were taken or! unfi l tered

samples except a t Carrabel le . Thus, the par t icula tes m a y have contributed

to s o m e of the s p e c t r a aa discussed in 4. 3.4.

T h e r e are hints of r t ruc tu re in some emiss ion t r a c e s such a s C-111

and C-115, both a t F o r t Lauderdale. The f o r m e r shows a shoulder on the

long wavelength side of the peak emission. This sample c a m e f rom P o r t

Everglades harbor. The la t te r t r a c e shows an emiss ion shoulder a t even

longer wavelengths, about 500 nm. This sample was taken c lose to the

coast in humic waters . It is quite probable that m o r e multicompoirent spec-

t r a would have revealed m o r e specif ic s ignatures related t o waste ma te r i a l s ,

etc.

The emiss ion s p e c t r a excited a t 280 nm in Car rabe l l e wa te r s were

s imi la r t o those excited a t 350 nm, except for lower intensity and descending

monotonically t o s h o r t e r wavelengths. (See, for example, F i g u r e C-127. ) In

Calveston Bay, on the other hand, many emiss ion spec t ra for excitation at

280 nm have the i r peaks shifted down t o below 400 nm, a s for example in

F igure C-140. This i s t rue only for s tat ions 1-5: the balance have spec t ra

m o r e l ike those of Carrabel le .

The m o d unusual excitation t r a c e occurs in F i g u r e C-151 off the coast

f rom Santa B a r b a r a at Station D. This sample is taken 100 m offshore f rom

the mouth of Goleta Slough, which is probably polluted. The excitation maxi-

m u m has shifted to approximately 315 nm.

Severa l samples f rom the E. B. Scrippe c r u i s e off Southern California

show s o m e enhanced emiss ion below 400 nm, e, g. , F i g u r e C-156, whereas

o thers do not, as in F igure C-160. The g rea tes t enhancement occurs in

F igure C-162 fit Station 8. This peak is augmented by Raman, but yet i s

unusually la rge , being peaked c lose t o 350 nm.

5.

5.1

biblio

BIBLIOGRAPHY AND RELATED WORK

Bibliography

A second principal objective of this pr0gra.n was t o a s s e m b l e a

graphy of re la ted work, with par t icular emphasis on recent lurninescenc

studies of water samples in s i tu . As expected, t h e r e is a l a r g e amount of

published work on samples which have been manipulated in the laboratory,

o r on cultures. Also a s expected, t h e r e i s a paucity of luminescence data

on m a r i n e samples in situ.

The Bibliography will be found a t the end of th i s r epor t a s Appendix B.

It is organized under r.he following subheadings:

A. Chlorophyll and Other Plant Pigments: Photosynthesis

B. Gelbstoff

C. Bioluminescence

D. Genera l Marine Luminescence

E. Related Marine Biology

F. Related Marine Chemis t ry

G. F i s h Pigments and Oils

H. Pollution

I. Optical P roper t i e s of Seawater

J. Miecellaneous

The Bibliography does not pretend t o be exhaustive. We have reviewed

much m o r e mate r i a l but have se lec ted i teme which w e r e useful to the project

o r which we wished t o reier t o in o u r repor ts . In the following sect ion we

rhal l d i scuss published work on m a r i n e luminescence in eitu.

5.2 Related Work - Aa we anticipated at the inception of t h i r p rogram, v e r y few lumines-

cence m e a r u r e m e n t s brve been m a d e in aitu on su r face waters . On the o ther -

hand, considerable numbers of papers have appeared on the luminescence

of ext rac ts , o r labora tory cultures. Quite commonly f luorescence m e a s u r e -

ments utilizing s imple f i l te r ins t ruments a r e made in s i tu. Most m e a s u r e -

men t s a r e made with f luor imeters which lack specificity. Duursma and

Rommets (B1961) adapted a Ze i s s spectrophotometer , giving them higher

specificity, and Traganza (B1969) used a Baird-Atomic instrument aboard

ship. According t o Duursma, Traganza used ' 'the most developed laboratory

equipment", s ince he could investigate both the excitation and emiss ion spec-

t r a . Also mentioned is the Fraunhofer Line Discr iminator developed under

the aegis of the Geological Survey by Perk in E l m e r Corp. This las t device

examines sun-stimulated iuminescence and demands detection only in

Fraunhofer lines.

Traganza ' s work s e e m s to be the only at tempt a t full excitation1

emiss ion s ignatures before the p resen t work. Traganza used a n e a r l i e r

model Bai rd instrument which cer ta in ly lacked the sensi t ivi ty of our

present model for chlorophyll, and probably a l s o f o r Gelbstoff. His r e su l t s

a r e reproduced in F i g u r e s 39-41, f o r compar ison with the r e su l t s of our

study.

Kullenberg and ~ ~ ~ % r d (B1971) desc r ibe an advanced vers ion of an

instrument designed by J e r l o v (11968); however, it i s s t i l l a f luor imeter .

Th i s par t icular instrument employs a modulated excitation source t o allow

easy automatic background subt rac t of sun sca t t e r . The authors ' r e su l t s

f rom measurements in the Baltic suggest a relat ion between pa r t i c l e content

and i luorescence, except nea r the su r face layer .

Z a r ~ b a s h e v and Zangalis (A1970) have wri t ten on the ' 'F luorometr ic

Determination of Chlorophyll In Vivow and noted that the Lorenzen method of -- exciting in the 430 nrn excitation band of chlorophyll a l s o excited other

m a t e r i a l s (including Gelbstoff) whlc h cause an e r r o r in f luor imetr ic methods.

The i r inatrumentation consis ts of a l ine source ( the Hg l ine a t 436 nm) and a

monochromator detector . F i g u r e 42, f rom th i s paper, shows the i r resul t s .

I

LkC -1011 PEAKS CLUORESCENCE PEAKS

E W I ' A ~ I O N WAVELCYG'H (me) EMISSION WAVELENGTH 1 ~ 1 ~ 1

FIGURE 39. Left, excitation spectrum of sea water collected in a suriace concentration of Trichodesmium sp. near 3s025, 8'N. 6799'W; right, fluorescence spectrum of same. (The irregular t races were caused by the roll of the ship. ) ( Yentsch, B1969.)

FLUORESCENCE

FIGURE 40. Left, moderate, broad-peaked fluorercence epectrum of Atlantic Shelf water collected a t 30 m near Nantucket Shoclr (40%5'N, 69'37' W); right, weak, broad- banded fluoxercenca rpectra of Sargarso Sea water (35P25.8'N. 6799'W) collected a t depth8 of 1, 8, 17, 32, 57, 82, 107, 132, 157, end 206 m. (Yentrch, Bl969. )

, . ' . . ' , . ,; . , , . , , . , , ,' ' ' ,

.J . . . . . ' \ ..' , . , . . . ' .. I .

: . + . . , , J: 1.k . , . , . . * , ' , ' j ; ',, . (. .

. % . - ." . .. - - A". ~ . ~. -

EMISSION WAVELENGTH ( w + I

FIGURE 41. Left, fluorescence s p e c t r ~ m of a 7-day culture of con- centrated suspended matter, incubated at sea. The sample consisted of surface water from the Continental Shelf near 40°43'N, 69011tW. Right, fluorescence spec- trum of a 6-day culture of Skeletonema costaturn at the Woods Hole Oceanographic Institution. (Yentsch, ~ 1 9 5 9 . )

300 600 b0 WAVELENGTH

FIGURE 42. Normalized spectral distribution of sea water luminescence (BH) after excitation by the 436 nm mercury line:

I) luminercence spectrum of the dirsolved sub- rtances; XI) luminercence band of chlorophyll. Fur ther explanation in the text. (Karabar hev and Zurgalis, A1970. )

They advocate getting a basel ine f rom f i l te red seawater in o r d e r t o be able

t o de termine actual chlorophyll signal.

In a m o r e recent paper, the s a m e authors (Karabashev and Zangalis,

B1971) have "discovered that the photoluminescence spect rum of seawater de-

pends on the spect ra l composition of exciting radiation. f t The i r instrument

had now been m,odified t o allow excitation by the 313, 365, 436 and 546 l ines

of mercury . The resu l t s of th is study a r e given in F igure 43, where changes

in the emiss ioc a r e noted as a ictlction of excitation wavelength,

In yet another paper, t i t led "New Data on Sea Water Photoluminescence, "

Karabashev, Zangalis, Solovtyev and Yakubovich (11971) d iscuss r e su l t s

of the measurement of what we ca l l Gelbstoff f luorescence when excited with

the 365 and 436 nm l ines of mercury . These re su l t s a r e given in F i g u r e 44.

As if to confirm our s tatement on the paucity of in s i tu luminescence data on

Gelbstoff (o r chlorophyll), the authors s tate , ". . . neither in th is nor in any

other work published abroad have we found any information about the spec t ra l

distribution of SWP, (SWP is t h e i r t e r m fo r seawater photoluminescence. )

A very important prac t ica l r e su l t of the i r measurements i s that the lumines-

cence s p e r t r u m of SWP is independent of salinity, oxygen concentration, and

pH in the l imi ts 0-13W, 0-8 m l / l , and 7.0-8. 5 respectively.

Ivanoff and Morel (11971) have a l s o wri t ten on the "Spectral Dist r ibu - tion of the Natural F luorescence of Sea- Waters. l t Again, they a r e p r imar i ly

in teres ted in what we t e r m Gelbstoff emission. T h e spec t ra l data published

in th i s paper a r e reproduced as F igure 45. (P ro fessor Jer lov informs m e

that the labeling on curves 2 and 3 is reve r sed . ) These authors a l s o use

m e r c u r y l ines fo r excitation. The i r r e su l t s a r e cer ta in ly l e s s complete tharr

those of Karabarhev e t al. s ince they do not rhow the decline in the shc@ri

wavelength region. Jer lov has also informed m e that lvanoff and Morel have

m o r e recent and bet ter resul t6 which are not yet publirhed.

During r t r i p to Europe in t h e Fall of 1972, i t war por r ib le t o d i r c u r r

o u r work and tbat of o tke r r with P r o f s r r o r Jer lov in Copenhagen. It war

FIGURE 43: Mean normalised spectral distribution of photo- luminescence of sea water for excitation by the 313 nm (curve I), 365 nm (curve a), and 436 nm (curve IfI) lines of mercury. IV) Absorption spectrum of "yellow substancett in water sample i r o m Gulf of Riga. I i s the relative apectra! spectral intensity, k is the absorption coeffieient of substances dissolved in the sea water. (Karabashev and Zangalis, 81971. )

FIGURE 44: Rerultr and measurement conditions f o r ~ t h e S W P spectra:

1) average over all rtationr, excitation using filter with transmiiiaian F1; 2) the r ame wing filter with tronrmirrion F2; 3) SU'P rpectrum from (4) a through d are the poritionr and relative ititenritiefi of Hg liner in the rpecttum of the exciting rource, and a' through d' axe the poritionr of the Raman line8 from water for the Hs line8 a through d, rerpectively (from (19); lporm ir the normalired rpectral intenrity of S W P and T 18

the t ranrmtrr ion of the filter8 in tbe exciting beam. (K8rabarhev et 81. , 11971. )

EXCITATION : 366nrn

400 hnm

FIGURE 45: 'spectral clirtribution of the fluctescence of meawater (the fluore8cence Fe. dirt. of pure wate.- ham been rubtrrcted from that, F, of the conridered reawater. and the difference F-Fe. dirt. i r compared to the Raman affect of pure water at 418 nrn).

Curve. nO1, 2. 3 correrpond rempectively to coartal water. to rurface Msditerraaeur water, uad to deep 500 m Mediter- rra8.a wator. (Ivunoff and Morel, U971. )

Je r lov ta impress ion that the re was no work in p r o g r e s s anywhere which

had proceeded s o fa r a s ours in collectlor. of specif ic data. Jer lov a l s o

indicated that his group was about t o purchase i rs t rumenta t ion which would

allow scanning of bott? excitation and emiss ion in o r d e r t o fur ther investigate

the extraordinary blue f luorescence in the Baltic. This f luoreecence i s

noticed a s a peak in the upwelling light. It has a na r rower bandwidth than

normal Gelbstoff emission. (It i s quite possible he was seeing an oil such

a s we saw i:i F i g u r e 23. )

We a r e a l s o a w a r e of cu r ren t work bein3 performed by SPARCOM, Inc.,

on "Lase r Induced Fluorescence of Algaer' with the support of NASA/ Wallops

Island. This work has used a. tunable l a s e r t o examine algal cul tures . F o r

eventual a i rborne detection no dotlbt l a s e r s will be ext remely useful; however,

it s t tsrnr cumbersome to do point by point measurements on labora tory cul-

t ,ures which a r e very easy t o scan automatically with m o r e than adequate

sensitivity with ccntinuous instruments. This work, when available, should

be compered with the da ta of the second and focrth sect ions of our Compendium

of Spect ra l Data.

6. DISCUSSION AND RECOMMENDATIONS

6.1 Interpretation of Data

In Sectio~l 4. 3. 3, we formed the tentative corrclusion that algae par t i -

culates were totally absorbing over much of the near ultraviolet and visible

spectrum and acted approximately a s quantum counters. This conclusion

would have a far-reaching effect on how epec t r aze to be interpreted and

how algae a r e to be identified.

In most cases, plant pigments absorb energy and transfer a large

fraction to chlorophyll. Chlorophyll i s a lso excited by direct absorption.

The energy i s now used for photosynthesis, but some fraction is emitted

a s chlorophyll fluorescence. If the same fraction is emitted, regardless of

whether excitatioa was direct or came from energy t ransferred from other

then quantum counting may be observed, resulting in the "typical""

excitation spectrum of Section 4. Further, this would be a maximum spec-

trum: any deviations would be expected to fall below this curve.

If a particulate i s not optically dense in some spectral region because

of the lack of a pigment, this w ~ l l cause a relative dip i n the "typical" cpec- - trum. If the abrorption in a rpectral region i s caused by the p r e s r x e of

an absorbing pigment which does not t ransfer energy to chlorophyll or any

other pigment--an internal filter effect--then this too will cause a dip in

the lltypicall' excitation rpectrum for chlorophyll ~!uorercence.

AS a rerul t of these conclusionr, the interpretation of excitation

spectra for identification must depend on negative deviations from a "typical"

spectrum and the abrence of certain pigments, ra ther than their presence.

Also important may be the presence of pigments *-qhich cause total abrorption

in a spectral region, but which do not contributc to chlorophyll emission.

W e note that the data of Figure 38A only suggert quantum ccrunter

action of algal particulates t o about 480 nm, due to a limitation of the com-

pariron dye. The Rhodunine data of Figure 31, which extend to 580 nm,

suggest that in many c a s e s quantum counter action may extend pas t 550 nm,

where the re i s a cha rac te r i s t i c peak. It i s quite possible that in diatoms

and dinoflagellates which contain no phycobilins quantum counting may c e a s e

a t longer wavelengths. Thus, i t may be useful to examine excitation s p e c t r a

between 550 nm and the chlorophyll absorption a t 670 nm.

Gelbstoff data a r e not expected t o exhibit quantum counter action

because of the low concentration and b e c a u s e they do not a r i s e from par t i -

culates.

6.2 General Conclusions

Surveying the r e su l t s of th is project , we can make the following

general s tztem ents:

Luminescence data on natural wa te r s can be useful in roughly quan-

titating and identifying algal concentrations.

Luminescence d a ; ~ on n a t , ~ r a l wa te r s can a l s o be useful in de te r -

mining Gelbstoff concentrations and establishing the existence and

i d e ~ t i t y of pollutants such a s oil.

The sensitivity of luminescence methods sugges ts that chlorophyll can

be monitored remotely f rom an a i rc ra f t , par t icular ly a t night.

The interpretat ion of chlorophyll excitation spec t ra should be based

on a quantum counting scheme based on total absorption throughout

mos t of the ultraviolet and visible spect rum by each part iculate.

Since absorption throughout the spect rum contr ibutes t o chlorophyll

emission, maximum sensi t ivi ty f o r chlorophyll detection will be

obtained by wideband excitation up t o and including the l a s t chloro-

phyll absorption a t 670 nm. T h i s may be m o r e sens i t ive than

narrow-band l a s e r excitation.

. . . ' I . . ' . . . , .

6. 3 Rec~mmendations for Future Work

The reorientation of the approach to on-site measurements led to

certain incompleteness of data, due to shortage of t ime and funds, and field

instrument evolution. The concept of particulate quantum counting redirects

the approach to data interpretation. The following i tems a r e recommended for

further study and measurement:

A dedicated field instrrlment, essentially similar to the tlnal

instrument used in this project, but equipped with a continuous

on-stre.im monitoring system, should be built for field measure-

ment s.

Laboratory measurements on a variety of algal cultures should

be carr ied out to verify the quantum caunting concept and estab-

lish limitations of applicability.

On-site measurements should be conducted at a few carefully

selected s i tes with sufficient time to c a r r y out a considered pro-

gram of experiments, and where a ~ x i l i a r y and independent

measurements of important parameters czn be carr ied out.

More detailed multicomponent spectra should be taken at selec-

ted si tes, both to seek evidence of other non-chlorophyll algal

fluorescence, and to develop more detailed Gelbstoff spectra.

Measurements of both sor t s should be made m natural waters,

filtered p a r t i c i ~ l ~ t e s and filtrates.

Calculations should be performed comparing the utility of

bl . ad-band a r c excitation versus narrow-band laser e x i t ation

for remote sensing.

ACKNOWLEDGEMENTS

The au tho r s wish t o thank the numerous m a r i n e sc i en t i s t s who con t r i -

buted t o t h i s p r o g r a m by m e a n s of l e t t e r s and pe r sona l conversa t ions . Spec ia l

thanks go t o t hose r e spons ib l e f o r a r r ang ing on-s i te m e a s u r e m e n t s who a r e

acknowledged e l sewhere . Dr . C h a r l e s Yentsch was a consul tant t o t h i s p r o -

g r a m and provided a s s i s t a n c e both in l abo ra to ry fac i l i t i es and in in te rpre ta t ion .

Mr. Lu the r Campbel l was r e spons ib l e f o r t he succes s fu l modif iciat ion of a

l abo ra to ry in s t rumen t f o r f ie ld use. F ina l ly , we wish t o thanh M r s . Gera ld ine

Garnick f o r a i d in taking l abo ra to ry da t a and in organizing t h e l a r g e amount

of da t a accumulated.

Appendix A

General Principles of Fluorescence Analysis

In figure A-1 we relate and define absorption and fluorescence by means

of a generalized energy level diagram which would apply to a typical aromatic

organic molecule in dilute solution. Light i s absorbed by the molecule in i ts

ground state - - usually a singlet - - here designated by S . The absorption of 0

energy raises it to one of a number of higher electronic singlet levels, designated

S1, S2, etc. These levels a r e further split by small vibrational differences into

sublevels, indicated by fine lines in the diagram. Further rotational splitting

i s usually masked by the broadening caused by molecular collisions in the matrix

which may be solid o r liquid.

The population of any level depends on the Roltzman f a c t ~ r , e -E/kT

9

where E i s the energy of a particular level, k i s the Boltzman constant and T is

the absolute temperature. The vibrational spacing i n organic aromatic molecules

is typically 700 c m l . whereas the thermal energy a t room temperature (300' K) -1

i s only about 210 cm . Thus the ratio of molecules in the f i r s t vibrational state

to those in the zero vibrational state of S will be given by 0

-a EjkT = e -7OO/Z 1 0

Nol/Noo ' e = 0.04 a t room temperature,

Thus about four percent of the molecules will be in the f i r s t vibrational state and

about ninety-six percent in the ground state. (Higher s ta tes have negligible

populations. ) Absorption then occurs predominantly f rom the zero vibrational

of S to various vibrationals of the higher singlets. (Absorption from the poorly 0

populated higher ground vibrational give r i s e to "hot bands. ) This selective

absorption i s the basis of identification by absorption spectroscopy.

Once excited, a molecule may return to the ground state by radiating

light, fir by radiationless transitions (aided by. molecular collisions) which result

in dissipation of energy in the form of heat. In dilute solution (or solid) the

populations of the vibrationals

As a result the large majority

of S a r e a lso determined by a Boltzman factor. 1

of transitions f rom S to S occurs f rom the zero 1 0

A- l

Vibrotiono' levels of sec~nd excited stste

Figure A-1 .

Vibrat io~al ievels of first excited state

is,;

I Vibrotisqal levels 3f 3 r ; ~ n s

1 state

T -ansitions Giving Rise to Absorption and Fluorescence Emission Spectra

vibrational state of S to various ground vibrationals. These processes a r e 1

indicated in figure A-1 where radiationless processes a r e designated by zigzag

lines and absorption and emission processes by solid lines.

Because fluorescence, originating in the zero-vibrational of S may 1 '

terminate in any >f the ground vibrational states, the iluorescence often displays

vibrational structure which i s a m i r r o r image of absorption.

Comparison of possible absorption and fluorescence transitions reveals

that the transition between zero-vibrational levels, the so-called 0-0 transition,

i s common to absorption and fluorescence, resulting in self-absorption a t the

common wavelength. All other fluorescence transitions occur a t lower energies

(and longer wavelengths than the corresponding absorption. As a result ,

fluorescence i s found to occur a t wavelengths just greater than the longest wave-

length absorption.

A third possibility exists for a molecule in the S state - - and this i s 1

not depicted in figure A-1. It may undergo a radiationless intersystem crossing

to an excited triplet level, TI , lying below S . Such a triplet may also radiate, 1

resulting in phosphorescence a t even longer wavelengths. Because the radiative

lifetime of the triplet i s so much longer than the singlets (typically one second

compared to 1 0-' seconds), non-radiative processes usually dominate over

emission at room temperature and phosphorescence is not observed. Since we

a r e interested in emission a t ambient temperatures, we shall disregard triplets

except a s further non-radiative paths for deexcitation from S . 1

To continue our contrast of absorption and emission processes , note

that in an absorption experiment one measures an incident intensity and a t rans-

mitted intensity, both a t the same wavelength, which a r e almost equal in

magnitude. The small intensity difference i s the desired information about the

eample. In a fluorescence experiment the incident (exciting) light i s a t one

wavelength and the emitted light (fluorescence) i s at another wavelength. Since

the incident light can be made monochromatic, i t s contribution to background

at the fluorescence wavelength can be made very small. As a result, the

fluorescence, which contains the desired information, i s measured against an

almost zero background. It i s this difference which makes fluorescence methods

so much more sensitive than absorption for materials which fluoresce with

reasonable efficiency. Considering a typical organic having an extinction 4 2

coefficient of 10 cm /m-mole and a fluorescence quantum yield of 0.4, 1 -1 2 -1 3

Fbrs te r calculates a detection limit of 10 mole l i ter o r 5 x 10 g for low

spectral resolution. With the newer high intensity light sources available this

could be exceeded. The practical limit to sensitivity i s scattered light and

background emission. Standard laboratory instrumentation, such a s the Baird-

Atomic " Fluorispecu Fluorescence Spectrophotometer allows measurement of

sub-nanogram quantities of material corresponding to concentrations of l ess

than Gne part per billion.

Fluorescence spectroscopy has another advantage, in addition to great

sensitivity. This results from the double specificity of excitation and fluores-

cence wavelengths. In absorption spectroscopy a mixture will have a unique

absorption spectrum which is the simple sum of the absorbances of the components.

In fluorescence spectroscopy a single organic compound, in dilute solution in a

non-absorbing, non-interacting matrix, will have a unique fluorescence spectrum

and a unique excitation spectrum. A mixture of fluorescing compounds, possibly

concentrated, in a matr ix which may be absorbing (but not fluorescent) will no

longer have a unique fluorescence spectrum o r a unique excitation spectrum.

(Seawater is just such a mixture. ) Rather a particular fluorescence spectrum

will depend on the wavelength and spectral bandwidth of the excitstion and a

particular excitation spectrum will depend on the wavelength and bandwidth of the

observed fluorescence band. Thus it i s necessary to take ca re :r. in te rp~et ing

observed results.

This complicated interrelationship between excitation and emission i s

cac.sed f i r s t becauae of the overlapping of the simple excitation and fluorescence

spectra of the individual components. It is further complicated because of the

possibility that one type of excited molecule may transfer i ts energy to a second

type, reeultirrg in absorption by the f i r s t and fluoreeccnce by the second. The

m a t r i x (solvent) m a y itself absorb without f luorescing, thereby acting a s an

internal f i l ter to reduce excitation of f luorescent species. The mat r ix o r a

non-fluorescing component may ac t t o quench normal f luorescence of a spec ies ,

i. e. , deexcite by changing energy into heat without radiation. Finally, high

concentrations of the s a m e o r differing spec ies m a y resul t in quenching of

emission. High concentrations of one species m a y resul t in formation of d i m e r s

which m a y o r m a y not f luoresce. Dimer f luorescence usually h a s i ts f luorescence

shifted to longer wavelengths than the monomer .

In addition t o the effects of interact ions between f luorescing components

and the ma t r ix , observed excitation and f luorescence m a y be affected by the

geometry and dimensions of the sample. If the sample i s a n optically thick l aye r

which absorbs a l l of the incident radiat ion over a broad excitation region, tk-.

excitation spect rum of the f luorescence m a y mimic the photon flux v e r s u s wavd-

length distr ibution of the source and a c t a s a quantum counter. A f i lm may be

optically thick fo r a s trong absorbe r and optically thin for a weak absorbe r . In

a n emulsion a l a rge droplet m a y be optically thick f o r a l l excitation wavelengths

result ing in only surface f luorescence which does not ref lec t the t rue volume.

The g rea t possible complexity of observed excitation and f luorescence

f rom such a complex sample prevents a p r i o r i predict ion of resul t s . Never-

theless, t c e ve ry complexity of r e su l t s impl ies high information content in

the method, allowing a ski l led exper imenter not only t o detect but t o identify

components.

1. Th. F b r s t e r , Cronache Di Chimica l4, 3 (1 966).

Appendix B

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Clayton, R o K, (1966). "Physical P roce ree r Involving Chlorophyll in Vivott in The Chlorophyk (L. P. Vernon arid G. R. Seely, edr.) Academic Prerr, New York.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued) - Cole, W. J. , OhEocha, C . , Moecowitz, A. and Krueger , W. R. (1967).

"The Optical Activity of Urobiline Derived f rom Phycoerythrobilintt European J. Biochem. - 3: 202-207.

Das, Rabinowitch, M. E. and Szalay, L. (1968). "Red Drop i r r the Uuantun-4 Yield of Fluoreecence of Sonicated Alga" ~ i o ~ h ~ s i c ~ l Journal 8 (10): - 1131-1137,

Dougherty, R. C. , Strain, H. H. and Katz, 3. (1965). "Hydrogen Exchange in Chlorophyll and Related Compounds, and Coorelation with Molecular Orbital Caiculationstt "Journal of the American Chemical Societv 87: . - (1) 104-109.

D o q h e r t y , R. C. , Strain, 11. H. and Katz, J, J. (1966). "The Uee of Fully Deuterated Pigments to Studv the i r Funct ionw Biochemistrv of - C h l o r o d a e t s 2: Academic Press. New York. 427-430.

Dougherty, R. C., Strain, H. H., Svec, W. A, , Uphaus, R. A. and Katz, J. J . (1969). "The Structure, P roper t i e s , and Distribution of Chlorcphyll ct ' J o ~ r n a l o i the American Chemical Society 92 (9): - 2826-2833.

Dutton, H. J . , Manning, W. Me and Duggar, B. M. (1943). vChloropnyll F luorescence and Energy T r a n s f e r in the Diatom Nitzschia CloeteriumH J. Phys Chem 47: 308-313. -

Duysens, L. N. M, and Sweers, H. E. (1963). "Mechanism of Two Photo- chemical Reactions in Algae a s Studiesr by means of F luorescence t t in Studies cln Microalgae and Photosynthetic Bacter ia , ed. by J a p a n e r e Society of Plant Physiologists, Universi ty of Tokyo Press, T okyq Japan.

Duyrenr, L. N. M. (1952). T r a n r f e r of Excitatioq Energy in Photosynthesis, Kemink, Utrecht, Netherlands.

Ebrey, T e G. (1971). t tAnomalous Gnezgy Trans fe r Behavior of Light Abrorbed by Bacteriochlorophyli i n Severa l Photorynthetic Bacter ia t t B i ~ c h i m Biophyr. A c t s 253: - 385- 395.

A. CHLOROPHYLL AND OTHER PLANT PTC'VIENTS (Continl ~ d ) - Emerron, R. and Lewis, C. Me (1942). "The Photosynthetic Efficiency of

Phycocyanin in Chroococcue and tLe Problem of Carotenoid Participa- tion in Photosynthesis" J . Gen. Phyriol, - 25: 579-595.

Flciechman, D. E. (1971). "Luminescence in Photosynthetic Bacteria" Photochem. Photobiol 14: 277-286. -

Frackowiak, D. and Salamon, 2. (1970). "The Protective Action of Carotenoids on Fluorescence of Chlorophyll bt' Photochem Photobiol 11: 559-563. -

French, C. S., Michel-Wolwertz, M. R. , Michel, J. M., Brown, J. S. and Prager , L. K. :1968). "Naturally Occurring Chlorophyll Tv aes and Their Function in Photosynthesis" in Prophyrlns and Related - Corn- - pou..dr (T. We Goodwin, ed. ), Academic P r e r s , New York.

French, C. S., Smith, H. C., Virgin, H. I. and Airth, R. L. (1956). "Fluorercence Spectrum Curves of Chlorophylls, Pheophytins, Phycoer ythrinr , Phycocyanins and Hyper icin" Plant Fsly s i -- nl - - 31: 369 - 374.

French, C. S. and Young, V. K, (1952). "The Fluorercence Spectra of Red Algae and the Transfer of Energy from Phycoerythrin to Phycocyanin and Chlorophyll" J. Gen. Phyriol - 35: 873-890.

Fuller, R. C. (1971). !!The Arror,;.ation and Activitier of Pteridines in ~ h o t o r ~ k t e t i c Syrternr" Photochem Photobiol - 14: 359 - 371.

Geacintov, N, E., Wan Nortrand, F,, Pope, M. and Tinkel, J. B. (1971). "Magnetic Field Effect on tho ~ h l o r o ~ h ~ l l Fluorercence in Chlorella"

Gert, H,, San Pietro, A. and Vernon, L. F., edr. (1963). Bacterial Photo- ryntherir, Tim Antioch P r e s s , Yellow Springe, 9hio.

Geirrman, T. A., ed. (1962). The Chemirtry of Flavonoid Compwndr, - The Macmillan Company, New Y ork.

Giraud, George (196i). "La F l u o r e ~ c e n e e in Vivo de Quelqubr Alguer MarinerM in Seaweed Synaparium Biarr i tz -1961, D. D. Davy de Virville and 3. Feldrnan, edr , , The Macmillan Company, New York, 1964.

Goedheer, J. C. (1966). ' W r i b l e -4brorption and Fluorercence of Chloro- phyll a d ito Aggregrter in SolutionH in The Chlorophylla (L. P. Vernon and G, R, Sedy, edr. ) Academic Pr*ms, New York, 147-172.

A. CHLOROPHYLL AND CTHER PLANT PIGMENTS (Continued)

Goedheer, J. C. (1969). "Energy Transfe r f rom Carotenoids to Chlorophyll in Blue-Green, Red and Green Algae and Greening Bean Leavesu Biochim. Biophys. Acta 172: 252-265. -

Goedheer, J. C. (1968). "On the Low-Temperature Fluorescence Spectra of Blue-Green and Red Algaett Biochim Biophys Acta 153: 903-906. -

Goedheer, J. C. (1970). I fon the Pigment System of Brown Algaeft Photo- synthetisa - 4 (2): 97-106.

Goedheer, 3. C. and Gulyaev, B. A. (1971). "Fluorescence Polarisat ion of Photosynthetic Pigments" F i r s t European Biophysics Congress, 14 t o 17 September, 197 , Baden near Vienna, Austria, E. Broda, A. Locker, H. Springer-Lederer, eds. , 43-47.

Goodwin, T. W. , et; (1965). Chemistry and Biochemistry of Plant Pigments, Academic P r e s s , New York, 1965.

Goodwin, T. W. , ed. (1968). Porphyrins and Related Compounds, Academic P re s s , London, 1968.

Govindjee, Munday, J. C. and Papageorgiou, G. (1966). "Fluorescence Studies with Algae: Changes with Time and Preilluminationf : From

-

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Griffiths, M., Per ro t t , P. S. and Edmondson, W. T. (1969). ftOscillaxanthin in the Sediment of Lake Washington" Limn01 Oceanog 14 (3): 317. --

Halldal P. (1966). "Induction Phenomena and Action Spectra Analyses of Photosynthesis in Ultraviolet and Visible Light Studied in Green and Blue-Green Algae, and in Isolated Chloroplast Fragments t t Z. - Pflanzenphysiol. Bd. 54: S. 28-44. -

Halldal, P. (1967). "Ultraviolet Action Spectra in Algology" Photochem Photobiol 6: 445-460. -

Halldal, P. (1968). "Photoeynthetic Capacit: se and Photoeynthetic Action Qpectra of E;ndosoic Algae of the Maseiv. Coral Favia" The Biological Bulletin 134 (3): 41-424. --

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Halldel, P. (1969). "Automatic Recording of Action Spectra of Photobiological Processes , Spectrophotometric Analyses, Fluorescence Measure- ments and Recording of the F i r s t Derivative of the Absorption Curve in one Simple Unit" Photochemistry and Photobiology - 13: 23-24.

Haxo, F. T. (1960). "The Wavelength Dependence of Photosynthesis and the Role of Accessory Pigments" in Comparative Biochemistry of Photo- reactive Systems (M. B. Allen, ed.) Academic P r e s s , New York. -

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Henry, B. R. and Hunt, R. V. (1971). "Triplet-Triplet Absorption Studies on Coumarin and Related Molecules" 3. Mol Spectry 39(3): 466-470. -

Holm-Hansen, O., Lorenzen, G. J., Holmes, R. W. and S t r i c ~ l a n d , 3. D. H. (1965). l lFluorometr ic Determination of Chlorophyll" 3. Cons. perm. int. explor. mer . 30(1): 3-15. --

Houssier, C. and Sauer, K. (1969). "Optical Proper t ies of the Protochloro- phyll Pigments. I. Isolation, Characterization, and Infrared Spectrafr Biochim. Biophys. Acta 172: 476-491. -

Houssier, C. and Sauer, K. (1969). "Optical Proper t ies of the Protochloro- phyll Pigments. 11. Electronic Absorption, Fluorescence, and Dir- cular Dichroism Spectraw Riochirn. Biophys. Acta 172: 492- 502. -

Houssier, G. and Sauer, K. (1970). l lCi rcu la r Dichroism and Magnetic Circular Dichroism of the Chlo +ophyll and Protochlorophyll Pigments" Journal of the American Chemical Society 92 (4): 779-791. -

Japanese Society of Plant Physiologists (1963). S t u d i e ~ on MicroAlgae and Photosynthetic Bacteria, The University of Tckyo Press .

Jeffrey, S. W. and Allen, M. B. (1967). "A Paper Chromatographic Method for the Separation of Phytoplankton Pigments a t Sea. " Reprinted from Limnology and Oceanography - 12 (3): July 1967, 533- 537.

Jeffrey, S. W. (1968). "Photosynthetic Pigments of the Phytoplankton of Some Coral Reef Watereu Limn01 Oceanog 13: 350-355. -

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Jeffrey, S. W. (1969). "Properties of Two Spectrally Different Components in Chlorophyll c Preparations. " Reprinted from Biochim. Biophys. Acta 177(3): 456-467. --

Kahn, A, Boardman, N. K. and Thorne, S. W. (1970). "Energy Transfer between Protcrchlorophyllide Molecules: Evidence for Multiple Chromophores in the Photoactive Protochlorophyllide-Protein Compiex in Vivo and in Vitro" J. Mol. Biol. 48: 85-101. -

Kamrin, M. (1966). "Changes in the Capacity of Algae t o Fluorescence during Steady State Photosynthesis" Biochim Biophys. Acta 126: - 262-268.

Karabashev, G. S. and Zangalis, K. P. (1970). "Fluorometric Determination of Chlorophyll IN VIVO P, F. Jhirshov Institute of Oceanclogy, USSR Academy of Sciences, Kaliningrad, 619-622, November 2, 1970.

Kar re r , P. and Jucker, E. (1350). Carotenoids, Elsevier, New York, 1950.

Katz, J., Strain, H. H., Leussing, D. L. and Dougherty, R. C. (1968). "Chlorophyll-Ligand Interactions from Nuclear Magnetic Resonance Studies" Journal of the American Chemical Society 90(3): 784-791. - -

Katz, J. 3. , Norman, G. 3., Svec, W. A. and Strain, H. H. (1968). llChlorophyll Diastereoisomers. The Nature of Chlorophylls a' and b1 and Evidence for Bacteriochlorophyll Epimers from Proton Magnetic Resonance Studies11 Journal of the American Chemical Society 90 (24): - 6841-6845.

Killilea, S. D. and 0 Carra , P. (1968). I1Amino Acids Involved in Chromo- yhore-Protein Linkages in Phycoerythrins" Biochem. J. 110 (2): 14-15. -

Kochubei, S. M. (1970). "Fluorescence Spectrum of Chlorophyll a in Chloroplasts a t 4*~ ." DoMady Akademii Nauk SSR 192 (2): 38-39. -

Krey, A. and Govindjee (1964). "Fluorescence Changes in Porphyridium Exposed to Green Light of Different Intensity: A New Emission Band at 693 nrn and Its ~ ign i f icance t o ~ h o t o s ~ n t h & i s ~ ~ Proceedings of the National Academy of Sciences 52 (6): 1568-1572, -

A. CHLGROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Krey, A. and Govindjee (1966). "Fluorescence Studies on a Red Algae Porphyridium Cruentum" Biochem Biophys. Acta 120: 1-18. -

Krinsky, N. I. (1971). "Functions" f rom Carotenoids, eds. Otto I s le r BirkhYuser Verlag Basel, Switzerland, 670- 711.

Lavorel, J. (1971). "Fluorescence and Luminescence Studies of in vivo Chlorophyll with a L a s e r Phos?horoscope" Photochemistry and Photobiolonv 14: 261-27 5.

Leff, J. and Krinsky, N. I. (1967). "A Mutagenic Effect of Visible Light Mediated by Endogenous P i g m e ~ t s in Euglena gracilis" Science 158 - (3806): 1332-1335.

Lemberg, R. and Bader, G. (1933). Ann. Chem. Liebigs. 505: 151-177. - Lorenzen, G. J. (1965). "A Note on the Chlorophyll and Phaeophytin

Content of the Chlorophyll Maximum" Limnol. Oceanogr. 10 (3): - 482 - 48 3.

Lorenzen, C. J , (1966). "A Method fo r the Continuous Measurement of in vivo Chlorophyll Concentration" Deep-sea Res. 13: 223-227. -

Lucatu, E. and Vasilache, M. (1970). "Quenching and Polarization of the Fluorescence in Solutions of Chlorophyll a Containing Quenchersvl - J. of Luminescence 3: 132-136. -

Mar, T. and Govindj ee (1971). "Thermoluminescence in Spinach Chloroplasts and in Chlorella" Biochirn. Biophys. k c t a 226: 200- 203. -

Margulis, L. (1968). l*Visible Light" Mutagen o r Killer?" Science 160: - 1255-1256.

, I : , Mathewe-Roth M. M. and Krinsky, N, I. (1969). lSCarotenoid

Pigments and the Stability of the Cell Membrane of Sarcina lutea 1 8

Biocbim. Biophys. Acta 203: 357- 359. - i 1 !

Mathews-Roth, M. M. and Krinsky, N. I. (1970). Photochemietry and Photobiology - 11: 419 -428.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Mauzerall, D. snd Malley, M. (1971). "The Light-Induced Increase in Fluorescence Yield in Chlor ella is Complete in 60 Nanoseconds" Photochem Photophysiol 14: 225-227. -

Mayne, B. C. (1965). "The Formation of a Quencher of the Fluorescence of Chromatophores from Photosynthetic Bacteria" Biochim. Biophys. Acta 109: 59-66. --

Mayne, B. C. (1967). "The Effect of Inhibitors rxd Uncouplers of Photo- synthetic Phosphorylation on the Delayed Light Emission of Chloro- plaststl Photochemistry md Photobiology - 6: 189 -137.

Mayne, B. r3. (1368). "The Light Requirement of Acid-Base Transition Induced Luminescence of chloroplast^^^ Photochemistry and Photo- - biology 8: 107 -113. -

Mayne, B. C. and Clayton, R. K. (1966). "Luminescence of Chlorophyll in Spinach Chloroplasts Induced by Acid-Base Transition" National Academy of Science 55(3): 494-497. -

Millard, J. P. azd Arvesen, 3. C. (1971). I1Effects of Skylight Polarization, Cloudiness, and View Angle on the Detection of Oil on Water, " Joint Conference on Sensing of Environmental Pollutants, Palo Alto, California, ALGA Paper No. 71-1075, November 8-la, 1971.

Mohanty, P. , Papageorgiou, G. and Govindj ee (1971). l lFluorescence Induc - tion in the Red Alga Porphyridium Cruentum" Photochemistry and Photobiology - 14: 667- 682.

Mohanth, P. , Munday, J. C., Jr. , and Govindjee (1970). "Time Dependent Quenching of Chiorophyll a Fluorescence from (Pigment) System I of Photosynthesis in Chlorellafl Biochim. Biophys. Acta 223: 198-200. -

Mohanty, P., Mar, T. and Govindjee (1971). rtAction of Hydroxylamine in the Red Alga Prophyridium Cruentum" Biochim. Biophys. Acta 253: - 213-221.

Moore, T o A. , Xarter, M. L. and Pill-Soon Song (1971). " Ultraviolet Spectra of Coumarins and Psoralenev J. ~ o l Spectry - 40: 144-157.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS - (Continued)

MOSS, B. (1967). "A Note on the Estimation of Chlorophyll a in Freshwater Algal Communities1' Limn01 and Oceanog. 12: 340-342. -

Muller, A., Lumry, R. and Walker, M. S. (1969). "Light-Intensity Depen- dence of the In Vivo Fluorescence Lifetime of Chlorophyll" Photochemistry and Photobiology - 9: 113-126.

Munday, J. C., Jr. and Govindjee (1969). "Fluorescence Transients in Chlor ella: Effects of Supp!ementary Light, Anaerobiosis, and Methyl Viologen" Progress in Photosynthesis Research 11: 913-922. -

Murty, N. R., Cederst, C. N. and Rabinowtich, E. (1965). "Chlorophyll a Fluorescence Yield in Live Cells and Solutions" Photochemistry and Photobiology - 4(5): 917-921.

Murty, N. R, and Rabinowitch, E. (1965). t tFluor escence Decay Studies of Chlorophyll a In Vivo" Biophysical Journal - 5(5): 655 - 661.

Myer, F. and Cook, A. H. (1943). The Chemistry of Natural Coloring Mat- ters . The Constitutions, Proper t ies and Biological Relations of the Important Natural Pigments, Reinhold Publishing Company, New York.

Nishimura, M. and Takarniya, A. (1965). "Analyses of Light -Induced Bacteriochlorophyll Absorbance Change and Fluorescence Emission in Purple Bacteriaw Biochim Biophys Acta - 120: 34-44.

0 Carra , P. (1970). "Algal Biliproteins" Biochem. J. - 119 (3): 2-3.

0 Carra , P. , OhEocha, C. and Carrol l , D. M. (1964). "Spectral Proper t ies of the Phycoerythrobilin" Biochemistry 3: .1343. -

0 Carra , P. and OhEocha, C. (1966). "Bilins Released from Algae and Biliproteins by Methanolic Extraction" Phytochemistry - 5: 993-997.

0 Carra , P. and Killilea, S. D. (1970). "Mesobilirhodin, An Isomeride of i-Urobilin with the Spectral Characterist ics of Phycoerythrobilin" Tetrahedron Let ters 48: 4211-4214. -

OhEocha, C. (1960). %hemica1 Studies of Phycoerythrins and Phycocyaninstl in Comparative Biochemistry of Photoreactive Systems (M. B. Allen, ed.), Academic Prers, New York.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

OhEocha, C. (1962). "Phycobilins" in Physiology and Biochemistry of Algae (R. A. Lewin, ed.), Academic P r e s s , New York.

OhEocha, C. (1960). "Spectrophotometric Studies of Some Red Algan Consti- tuents" Du Centre National de l a Recherche Scientifique 15: 121-134. -

OhEocha, C. (1966). "Biliproteins" reprinted from Biochemistry of Chloro- plasts I: (T. W. Goodwin, ed. ) Academic P re s s , London, 407-421.

OhEocha, C. (1971). "Pigments of the Red Algae" Oceanogr. Mar, Biol. Ann. Rev. 9: 61-82. -

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Olson, J. M. (1966). "Chlorophyll-Protein Complexes P a r t I1 Complexes Derived from Green ~ h o t o s ~ n t h e t i c Bacteriat ' The Chlorophylls, - Academic P r e s s , Inc., New York, 413-425,

Olson, J. M. and Stanton, E. K. (1966). I1Absorption and Fluorescence Spectra of Bacterial Chlorophylls in Situ" The Chlorophylls, Academic P re s s , Inc. , New York, 381-398.

Olson, J. M., Fi lmer , D., Radloff, R., Romano, C. A. and Sybesma, C. (1963). "The Protein-Chlorophyll-700 Complex from Green Bacteriav Bacterial Photosynthesis (H. Gest, A. San Pietro, L. P. Vernon, eds. ) The Antioch P re s s , Yellow Springs, Ohio, 423-431.

Papageargiou, G. and Govindjee (1969). "The Second Wave of Fluorescence Induction in Chlorella Pyrenoidosal' P rog re s s in Photosynthesis Re- search 11: 905-912.

Papageorgiou, G. and Govindjee (19 71). PH Contr 01 of the Chlorophyll a Fluorescence in Algaew Biochim Biophys Acta - 234: 428-432.

Parsons, T. R. (1963). "A New Method for the Microdetermination of Chlorophyll c in Sea Wateru J. Marine Res. - 21: 164-171.

Patterson, J. and Parsons, T. R. (1963). "Distribution of ' ~ h l o r o ~ h ~ l l a and Degradation Products in Various Marine Materials1' Limnol. and Oceanog. 8: 355-356. -

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Pennington, F. C. , Strain, H. H., Svec, W. A., and Katz, J. J. (1967). "Preparation and Proper t ies of 10-Hydroxychlorophylls a and btl Journal of the American Chemical Society 89 (15): 3875-3880. -

Philipson, K. D., Cheng Tsai, S. and Sauer, K. (1971). t 'Circular Dichroism of Chlorophyll and Related Molecules Calculated Using a Point Mono- pole Model fo r the Electronic Transitionml' Journal of Physical Chemistry 75 (10): 1440-1445. -

Rabinowitch, E.I. (1951). Photosynthesis and Related Processes . Vol. I1 (11, Interscience, New York.

Rabinowitch, E. and Govindjee (lrL.9). Photosynthesis, John Wiley and Sons, New York .

Ruby, R. H. (1971). "Delayed Fluorescence from Chlorella Pyrenoidosa: Effect of Background I l l ~ m i n a t i o n ' ~ Photochem Photobiol 13: 97-111. -

RUdiger , W. and 0 Carra , P. (1968). "Studies on the Structures and Ak~pro t e in Linkages of the Phy*:obilinsl' European J. Biochem. - 7: 509 -516.

Sauer, K., Dratz, E. A. and Coyne, L. (1968). "Circular Dichroism Spectra and the Molecular Arrangement of Bacteriochlorophylls in the Reac - tion Centers of Photosvnthetic Bacteriav Proceedinns of the National " Academv of Sci.?nce 61 (1): 17-24.

Sauer, K., Smith, J. R. :,, and Schultz, A. J. (1966). "The Dimerization of Chlorophyll a , Ct!orophyll b, and Bacteriochlorophyll in Solution: Journal of the Americ.zn Chemical Society 88: 2681-2688. -

Scott, A. I. (1964). Interpretaticn of the Ultraviolet Spectra of Natural Products, The Macmillan Company, New York.

Seely, G. R. and Jensen, R. G. (1965). "Effect of Solvent on the Spectrum of ChlorophylltlSpectrochimica Acta 21: 1835-1845. -

Seely, G. R. (1969). "The Quenching of Pyrochlorophyll Fluoree cence by Nitro Compoundott Journal of-the ~ m e r i c a n Chemical Society: - 72 (1): 125-129.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Seely, G. R. (1970). "Chlorophyll- Poly (vinylpyridine) Complexes. 11. Depolarization of Fluorescence" Journal of the American Chemical Society 74 (2): 219-227. -

Singhal, G. S. and Hevesl, J. (1971). "The Corre la t ion between the Absorp- tion and the Fluorescence Energy Spect ra , and the Quantum Yicld of Chlorophyll a in Different Solventsu Photochem Photobiol 14: 509-514. -

Singhal, G. S., Williams, W. P. and Rabinowitch, E. (1968). "Fluorescence and Absorption Studies on Chlorophyll a in Vitro at 77OW' J. Phys. Chem. 72 1121: 3941.

Stacy,

Strain,

Strain,

Strain,

Strain,

Strain,

Strain,

W. T. , Mar, T. , Swenberg, C. E. and Govindjee (1971). "An Analysis or' a Tr ip le t Exciton Model f o r the Delayed Light in Chlorel la" Photochemistry and Photobiology - 14: 197 -219.

H. H. (1954). "Oxidation and Isometrizat ion Reactions of the Chloro- p h y l l ~ in Killed Leaves" Agr. Food Chem 2 (24): 1222-1226. - H. H. (1965). "Chloroplast P igments and the Classif icat ion of Some Siphonalean G r e e n Algae of Aust ra l ia" Biological Bulletin 129 12): 366-370.

Ha H. (1966). "Fat-Soluble Chloroplast Pigments: The i r Identifica- tion and Distribution in Various Austral ian Plants" Biochemistry of Chloroplasts (T , W. Goodwin, ed. ) Academic P r e s s , 387-406.

H. H., Cope, B. T. , Jr., McDonald, G. N., Svec, W. A. and Katz, J. J. (1971). wChlorophylls cl and c2" Phytochemis , r y 10: -- 1109-1114.

H. Ha and Katz, J. J. (1969). "The Chemical Immutability of i

Chlorophyll a in Photosynthesis1' P r o g r e s s in Photosynthesis Resea rch 11: 539-546.

H. H., Sherma, J., Benton, F. L. and Katz, J. J. (1965). "Radial i 1

f Paper Chromatography and Columnar Chromatography of the Chloro- 1 p las t P igments of Leaver t ' Biochim. Biophyr. Acta 109: 23-32.

7 i

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

Strain, H, H., Sherma, J. and Grandolfo, M. (1967). "Alteration of Chloro- plast Pigments by Chromatography with Siliceous Adsorbents" Analytical Chemistry - 39 (8): 926-932.

Strain, H. H., Thomas, M. R., Crespi, H. L. and Katz, J. J. (1961). tlDeuterio-Carotenoid Pigments from Fully Deuterated Green Algaett Biochim. Biophys. Acta 52: 517-526. -

Strain, H. H. , Thomas, M. R. and Katz, J. J. (1963). IIS?ectral Absorption Properties of Ordinary and Fully Deuteriated Chlorophylls a and b: Biochim. Biophys. Acta 75: 306- 311. -

Strain, H. H., Svec, W. A., AitzemUller, K, , Grandolfo, M. C., Katz, J, J. Kjb sen, H., Norgard, S., Liaaen-Jensen, S., Haxo, F, T. , Wegfahrt, P. , and Rapoport, H. (1971). "The Structure of Peridinin, The Char- acterist ic Dinoflagellate Carotenoid" Journal of the American Chemical Society 93: 1823-1825. --

Sun, Alexander S. K. and Sauer, K. (1971). "Pigment Systems and Electron Transport in Chloroplasts I. Quantum Requirements for the Two Light Reactions in Spinach Chloroplaststt Biochim. Biophys, Acta 234: 399-414. -

SUzet Sefik and Sauer, K. (1971). "The Site of Photoconversion of Proto- chlorophyllide to Chlorophyllide in Barley SeedlingsI1 Plant Physiol. 48: 60-63.

Thomeon, A. J. (1969). "The Polarized Fluorescence Spectra of Some Naturallv Occurring Corr inef l Journal of the AIT~erican Chemical Societv 91 (lo): 276-2785,

Thomar, Joseph, Phondke, G, P., Tatake, V. G. and Gopal-Ayengar, A. R. (1970). tlMicrospectrophotometric Studies on the Pitmente in Vivo of Single Algae Cells - -I. Pigments of C hlorella Pyr enoidosa" Photochem. Photobiol, 11: 85-92. -

Thornber, J. P. (1970). ltPhotochemical Reactions of Purple Bacteria as Revealed by Studier of Three Spectrally Different Carotenobacteri- ochlorophyll-Protein Complexes Isolated f rom Chromatiurn, Strain DM Bicchemirtry - 9 (13): 2688-2698.

A. CHLOROPHYLL AND OTHER PLANT PIGMENTS (Continued)

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Thorne, S. W. (1971). "The Greening of Etiolated Bean Leaves 111. Multiple Light / Dark Step Photoconverr ion P r o c e e s e ~ ~ ~ Biochim, Biophys. Acta - 253: 459-475. -

Thorne, S. W. and Boardman, M. K. (1971). "The Effect of Temperature on the Fluorescence Kinetics of Spinach Chloroplaststf Biochim. B i~phys . Acta 234 (1): 113-125. --

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Trosper, T., Park, R. B. and Sauer, K. (1968). tlExcitation Transfer by Chlorophyll a in Monolayers and the Interaction with Chloroplast G l y ~ o l i p i d r ~ ~ Photochemirtry and Photobiology - 7: ,451-469.

Troeper, T. and Sauer, K. (1968). l'Chlorophyll a Interactions with Chloro- plast Lipids in Vitro" Biochim. Biophys. Acta 162: 97-105. -

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Vernon, L. P. (1964). ttSpectrophotom etr ic Determination of C hlorophyllr and Pheophytinr in Plant Extracts I t Anal. Chem . - 32: 1144-1150.

Vernon, L, P. and Seely, G. R., ads. (1966). The Chlorophyllr, Academic P r e r s , New York, 1966.

Vernon, L. P., Shaw, E. R., Ogawa, T., and Raveed, D. (1971). "Struc- tu re of Photoryrtem 1 and Photoryrtem 11 of Piant Ch lo rop la r t s~ Photochem Photobiol 14: 343- 357.

Vernotte, C. (1971). "Separation and Caracterirat ion Spectrorcopique du Monomers et der Poiymerer de l a C, Phycocyminev Photochem Photobiol 14: 163-173. -

A. CHLOROPZYLL AND OTHER PLANT PIGMENTS (Continued)

Wang, R e T. and Clayton, R. K. (1971). "The Absolute Yield of Bacteri- ochlorophyll Fluorescence in Viva" Photochem Photobiol 13: 215 -224. -- -

Welch, E. B. and Isaac, G. W. (1967). t'Chlorophyll Variation with Tide and with Plankton Productivitv in an Estuarvtf J. Water Pollution Control Fed. 39: 366. --

Will iam~, W . P., Murth, N. R. and Rabinowitch, E. (1969). "The Complexi- tv of the Fluorescence of Chlorella Pvrenoidosa" Photochem Photo- biol 9: 455-469. --

Wclf, F. T. and S t e v e n ~ , M. V. (1967). "The Fluorescence of Carotenoids1! Photochem Photobiol 6: 597- 599. -

Yamaguchi, K. (1970). Spectral Data of Natural Products, - V. I. Elsevier, New York, 1970.

Yentsch, C. S. (1960). Deep-sea Res. - 7: 1-9.

Yentrrch, C. S. and Lee, R. W. (1966). "Astudy of photosynthetic light r e - actions, and a new interpretation of sun and shade p h y t o p l a n k t ~ n , ~ ~ J. Mar. Res. 24 (3): 319-37. -

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B. GELBSTOFF

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C. BIOLUMINESCENCE

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Clarke, G, L. and Backus, R. H. (1956). "Measurements of Light P e n e t r a - tion in Relation t o Ver t ica l Migration and Records ot Luminescence of Deep-sea Animals" Deep-sea R e s e a r c h - 4: 1-14.

Clarke , G. L. and Backus, R. H. (1964). "Interrelat ions between the Vert ical Migration of Deep Scatter ing L a y e r s , Bioluminescence, and Changes in Daylight in the Sea" Bulletin de LIIns t i tu t Oceanographique No. 1318: 1-36.

Clarke, G. L. and Breslau, L. R. (1959). "Measurement of Bioluminescence off Monaco and Northern Corsica ' ! Bulletin de LIInst i tut Oceanographique NO. 1171, 1-32.

Clarke, G. L., Conover, R. J., David, C. N. and Nicol, J.A. C. (1962). "Comparat ive Studies of Luminescence in Copepods and Other Pelzgic Marine Animals" J. Mar. Biol. Assn. U. K. 42: -541-564. -

Clarke, G. L. and Denton, E. J. (1962). "Light and Animal Life" Ch. 10 in The Sea (M. N. Hill, ed. ) Vol. 1: 456-468, In tersc iance Publ ishers , Ltd., London.

Clarke, G. L. and Hubbard, G. J. (1959). "Quantitative Records of the Luminescent Flashing of Oceanic Animals at G r e a t Depthsw Limnology and O c e a n o e r a ~ h v 4: 163-183.

Clarke, G. L. and Kelly, M. G. (1964). "Variation in T r a s s p a r e n c y and in Bioluminescence on Longitudinal T r a n s e c t s in the Western Indian Ocean" Bulletin de LIInst i tut Oceanographique No. 1319: 1-20.

Clarke, G. L. and Kelly, M. G. (1965!. "Measurements of Diurnal Changes in Bioluminescence f r o m the Sea Sur face t o 2000 Mete r s Using a New Photometr ic Device" Lirnnology and Oceanography Vol. 10 Supplement, 54-66.

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C. BIOLUMINESCENCE (Continued)

Gormie r , M . , Eley, J. M . , Abe, S. and Nakano, Y . (1969). "On the Require- ment and Mode of Action of Long Chain Aldehydes during Bacter ia l B i o l ~ m i n e s c e n c e ' ~ Photochem. Photobiol. - 9: 351- 358.

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Hastings, J., Gibson, Q. H. and Greenwood, C. (1965). "Evidence for High Energy Storage In termedia tes in Bioluminescence" Photochemis- t r y and Photobiology 4: 1227 - 1241. -

C, BIOLUIVIINESCENCE (Cont inued)

Hastings, J. W. and Gibson, Q. H. (1967). "The Role of Oxygen in the Photoexcited Luminescence of Bacter ia l Luci ferase" The Journal of Biological Chemis t ry - 242 (4) , Februa ry 25, 1967, 720 - 726.

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Johnson, F. H. and Haneda, Y. (1966). ! 'Chemistry of the Luc i fe rases of Cypridina hilgendorii and Apogon ellioti " Bioluminescence in P r o g r e s s , Princeton University Press, Princeton, N. J.

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I C. BIOLUMINESCENCE (Continued)

Newton, H. E. (1940). Living Light, P r i r ~ ~ t o n University P r e s s , Princeton, N. J.

I Seliger , H. H. , Biggley, W. H. and Swift, E. (1969). "Absolute Values of Photon Emiss ion f rom the Marine Dinoflagellates Pyrodinium Bahamense

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I Seliger , H. H., Fas t ie , W. G., Taylor , W. R. and McElroy, W. D. (1962). "Bioluminescence of Mar ine Dinoflagellates. I. An U n d e ~ water Photometer f o r Day and Night Measurements" J. Gen. Physiol. 45: - -

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Tsuji, F. I. and Haneda, Y. (1967). "Luminescence in the Pacif ic F i shes . Apogon ellioti and Parapr iacanthue ransonetti" Science Repor t of the Yokoeuka Citv Museum No. 13: 12.

G. BIOLUMINESCENCE (Continued)

Turner, R . J . (1965). Notes on the Nature and Occurrence of Marine Bio- luminescent Phenomena. NIO Internal Report No. B -4, July, 1965, 1- 30.

Wilson, T . and Hastings, J . W. (1970). "Chemical and Biological Aspects of Singlet Excited Molecular Oxygen" reprinted from Photophysiology 5, Academic P r e s s Inc., New York, 49-95. -

D, GENERAL, MARINE LUMINESCENCE - Cata la , R. , Carn iva l Under t h e S e a ( f luo rescence of coral), (R. S i ca rd , ed. ),

30 r u e Jouber t , Paris.

Duursma, E. K. and Rommets , J. W. (1961). " In te rpre ta t ion mathamat ique d e la f luorescence d e s eaux douces , s a u m a t r e s e t m a r i n e s " Netherlands J. S e a Res . 1 (3): 391-405. -

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Mil la rd , J. P. and Arvesen , J. C. (1972). "Ai rbo rne Optical Detection of Oil and Water" -4pplied Optics 11 (1): 102-107, J a n u a r y 1972. -

Noble, V. E. and Ayer s , J. C. (1961). "A por t ab le photocell f l uo rome te r f o r dilution m e a s u r e m e n t s in na tu ra l water sM Limnol . Oceanoer . 6 (41:

Van Norman, R. W., F rench , C. S. and MacDowall, F. D. H. (1948). "The Absorpt ion and F l u o r e s c e n c e S p e c t r a of Two R e d Mar ine Algae" P lan t Physiol. 2 3 (4): 455-466. -

Yentsch, C. S. (1971). "The Absorp t ion a n d F l u o r e s c e n c e C h a r a c t e r i s t i c s of Biochemical Subs tances i n Na tu ra l Watersu f r o m Remote Sensing in Mar ine Biology and F i s h e r y R e s o u r c e s , Symposium proceedings, T e x a s A&M Universi ty , Col lege Station, Texas .

E. RELATED MARINE BIOLOGY

Berman, T. and Rodhe, W. (1971). "Distribution and Migration of Peridinium in Lake Kinneret" Mitt. Internat. Verein Limnol. 19: 266-276.

7

Boden, B. P. and Kampa, E. M. (1963). "An Inshore Sonic -Scattering Layer ' ' Proceedings of the XVI International Congress of Zoology, Washingtci~, D. C., August 20-27, 1963.

9

Boden, B. P. and Kampa, E. M. (1967). "The Influence of Natural Light on the Vert ical Migrations of an Animal Community in the Sea ' ' Symp. Zool. Soc. London, No. 19, 15-26.

Boney, A. D. (1966). A Biology of Mar ine Algae, Hutchinson Educational.

Brown, M. E. (1957). The Physiology of F i shes , Academic P r e s s , Vol. 1 and 2.

Carlucci , A. F. and Williams, P. M. (1965). l 'Concentration of bac te r i a f rom s e a water by bubble scavenging" J. Cons. P e r m . int. explor. mer . 30 (1): 28-33. --

Chapman, V. J. (1962). The Algae, Macmillan and Company Ltd. , London.

De Virville, Davy, A. D. and Feldman, J. , eds. (1964). Seaweed Symposium. Ba i r r i t z 1961, The Macmillzn Company, New York. -

Emery , A. R. (1968). "Pre l iminary Observations on Cora l Reef Plankton, Limnol Oceanog. 13: 293- 303. - -

Frolander , H. F. (1968). "Statistical Variat ions in Zooplankton, Numbers f rom Subsampling with a Stempel Pipet te" J . Water Pollution Coatrol - Fed. 40. R82.

Halldal, P. (1964). "Zur F r a g e dea Pliotoreceptors bei d e r Topophototaxis d e r Flagellaten" ~ e r i c h t e D e r Deut schen Botanischen Gessel lschaft Bd. LXXVI, Heft 8, S. 323-327.

Hart , T. J. (1966). "Some Observations on the Relative Abundance of Mar ine Phytoplankton Population in Nature" Some Contemporary Studies in Mar ine Science (H. Barnes , ed. ) George Allen and Unwin Ltd., London.

E. RELATED MARINE BIOLOGY (Continued)

Hulburt, E. M. (1962). "Phytoplankton in the Southwestern Sa rgasso Sea and North Equatorial Cur ren t , F e b r u a r y 1971" Limn01 Oceanog - 7 (1): 307- 315.

Jackson, 3. F., ed. (1964). Algae and Man, Plenum P r e s s , New York.

Johnson, R, M., Schwent, R. M. and P r e s s , W. (1968). "1 he Charac te r i s - t i c s and Distribution of Marine Bacter ia Isolated f rom the Indian Ocean" Limn01 Oceanog 13: 656-664.

Levring, T , , Hoppe, H. A. 2nd Schmid, 0. J. (1969). Mar ine Algae, C r a m , DeGruyter and Co., Hamburg.

Meadows, P. S. and Anderson, J. G. (1966). "Micro-organisms attached to mar ine and f reshwater sand grains. " Nature 212: 1059. -

Oehler , J. H. and Schopf, J. W. (1971). "Art if icial Microfossi ls : Exper i - mental Studies of Perminera l iza t ion of Blue-Green Algae in Silica" Science 174: 1229-1232, -

Oppenheimer, C. H., ed. (1966). Mar ine Biology 11, New Y o r k Academy of Sciences, New York.

Pringsheim, E. Go (1964). P u r e Cul tures of Algae, Hafner Publishing Go., New York.

Raymont, J. E. G. (1963). Plankton and Productivity of the Oceans, The Macmillan Company, New York.

Reisch, D. J. (1969). Biology of the Oceans, Dickenson Publishing Co. , Inc., Belmont, Cal ,

Round, F. E. (1964). The Biology of the Algae, Edward Arnold Publ. Ltd., London.

Strickland, J. D. H. (1965). "Production of Organic Matter in the P r i m a r y Stages of the Marine Food Chain" in Chemical Oceanography (J. P. Riley and Go Skirrow, eds. ) Academic P r e s s , New York.

Wileon, D. P. and Armetrong, F. A. J. (1952). " F u r t h e r exper iments on biclogical differences between natura l s e a w- r e" J. Mar. Biol. Ass. U. K. 31: 335-349. -

Wimpenny, R. S. (1966). The Plankton of the Sea, American Elsevier , New York.

Wood, E. J. F. (1962). "A Method for Phytoplankton Studyv Limnol. Oceanog. 7 (1): 32-35. -

Zenkevitch, L. (1963). Biology of the Seas of the -- USSR, In tersc ience , New Y ork.

F. RELATED MARINE CHEMISTRY

Blumer, M., Guillard, R. R. L. and Chase, T. (1971). "Hydrocarbons of Mar ine Phytoplankton" Marine Biology 8: - 183-189.

Blumer, M., Souza, G. and S a s s J. (1973;. "Hydrocarbon Pollution of Edible Shellf ish by an Oil Spill" - Marine Biology 5: 195-202. -

Blumer, M., Mullin, M. M. and Guillard, R. R . L. (1970). "A Polyunsatu- ra ted Hydrocarbon (3, 6, 9, 12, 15, 18-heneicosahexaene) in the Marine Food Web" Marine Bio%y - 6: 226-235.

Chave, K. E. (1965). "Carbonates: associat ion with organic m a t t e r in surface s e a water" Science Wash. D. C, 148 (3678): 1723-1724. - -

Chet, I., Fogel, S. and Mitchell, R. (1971). "Chemical Detection of Micro- bial P r e y by Bacter ia l P r e d a t o r s f f Journa l of Bacteriology 106 (3): - 863-867.

Culkin, F. (1965). "The Major Constituents of Sea Water" in Chemical Oceanography (J. P. Riley and G. Skirrow, eds. ) Academic P r e s s , New York.

Duursma, E. K. (1965). "Dissolved organic carbon, nitrogen and phosphor- us in the seav Netherlands J . Sea Res. 1 (1): 1-148. -

Duursma, E. K. (1965). l tDissolved organic constituents of s e a water" in Chemical Oceanography (J. P. Riley and C. Skirrow, e d s . ) Vol. 1, - Academic Press Inc., London, 433-475.

Duursma, E. K. (1966). "Note on chelation and solubility of ce r t a in me ta l s in s e a water a t different pH values" Netherlands J. Sea Res. 3 (1): - 95-106,

Faust , S. J. md Hunter, J. V. , eds. (1971). Organic Compounds in Aquatic Environments, Marce l Dekker, Inc. , New York.

Fraga , I?. (1966). "Distribution of part iculate and dissolved nitrogen in t h e wer tern Indian Ocean" Deep-sea Res. 13: 413-425. -

Golterman, H. L. and Clymo, R, S., eds. (1966). Chemical Environment in the Aquatic Habit, N. V. ~ o o r d - ~ o l l a n d s i h e Uitgeverlr Maatrchaapij -Amrterdarn 1971.

F. RELATED MARINE CHEMISTRY (Continued)

Gorham, E. (1957). "The chemical composition of lake wa te r s in Halifax County, Nova Scotiaw Limnol. Oceanogr. 2: 12 -21. -

Hoelzl Wallach, D. F. and Steck, T. L. (1963). "Fluorescence techniques in the ni icrodetermination of me ta l s in biological ma te r i a l s " Anal, - Chem. 35 (8): 1035-44. --

Holm-Hansen, O. , Sutcliffe, W. M., J r . , and Sharp, J. (1968). Limnol. Oceanog. 13: 656-664. -

Hood, D. W., ed. (1970). Symposium on Organic Matter in Natural Waters, Institute of Marine Science, Occasional Publication No. 1, June 1970.

Johnston, R. (1955). "Biologically act ive compounds in the sea" J. Mar ine Biol. Ass. U. K. 34: 185-195. -

Johnston, R. (1964). "Sea. water , the natural medium of phytoplankton. I T r a c e meta l s and chelation, and genera l diacussia-i" J. Mar. Biol. U. K. 44 (1): 87-109. -

Jones,

Kalle,

R. F. (1960). "Accumulation of ni trosyl ruthenium by fine par t ic les and m a r i n e organismsf ' Limnol. Oceanogr. 5: 312 -25. - K. (1953). "Der Einflusz des engliechen KUstenwassers auf den Chemismus d e r Wasserk8rper in d e stldlichen Nordseel ' Ber. Dtsch. Wise. Komm. Meereeforsch. 13: 130-!35. -

Kent, F. and Hooper, F. I?. (1966). "Synt hctic detergents: the i r i n f l ~ e n c e Lpon iron-binding complexes of na tura l warerr" Science 153: 526-527. -

Krey, J. (1959). t 'Chemical determinations of net plankton, with special reference to equivalent albumin content1' J. Mar. Res. 17: 312-314. -

! Laevartu, T. and Thompson, T. G. (1958). "Soluble i ron in coas ta l waters" i ,

J. Mar. Res. 16: 192-198. .- - Martin, D. F. (1968). Marine Chemis t ry , Vol. 1, Analytical Method*;

Vol. 2, Theory and Applications, Marce l Dekket , Inc., New York.

Mitchell, R. (1971). "Role of P r e d a t o r s in the R e v e r s a l of Imbalance in Microbial Ecosys temaH Nature 230: 257-258. --

F. RELATED MARINE CHEMISTRY (Continued)

Riley, J. P. and Skirrow, G. (1965). Chemical Oceanography, Academic P r e s s , New York.

Sackett, W. M. and Arrhenius, G. 0. S. (1959). "Aluminum content of ocean an6 other na turs l waters" Int. Oceanogr. Congr. l s t , New Yotk, prepr in ts .

Schcenes, ,r. and Rowe, G. T. (1970). "Pelagic Sargaesum and i t s P r e s e n c e among the Deep-sea Benthos" Deep-sea R e s e a r c h -- 17: 923-925.

Skopintsev, B. A. (1958). "Study of the content of suspended par t ic le3 and coloured organic compounds in the Azov and Black Seas" Akademiia Akad. Nauk. SSSR. Morekoi Gidrofizicheskii Inatitut, Trudy. 13: -- 113- 129.

Trueedale, V. W. (1971). "A Modified Spectrophotometric Method for t h e De- termination of Ammonia (and Amino-acids) in Natural Waters, with Pa r t i cu la r R eference to Sea Water" Analyst 96: 584-593. August,

w-

1971.

Vollenwc bder, R. A. , ed. (1969). A Manual f o r Methods fo r Measur ing P r i m a r y Production ia Aquatic Environments, - Blackwell-scientific Publ., Oxford.

Wattenberg, H. (1937). "Die chemischen Arbeiten auf d e r 'Meteor1-Fahr t" F e b r u a r bis Mai 1937, Ann. d. Hydrogr. b5. -

Youngblood, W. W,, Blumer, M., Guillard, R, L. aad F io re , F. (1971;. "Saturated and Unsaturated Hydrocarbone in Mar ine Benthic Algae" Marine Biology 8: - 190-201.

G. FIEH PIGMENTS AND OILS

Cockerel:, T . D. A. (1914). l lObservations of F i s h Scales" Bull. U. S. Bureau of F i she r i e s . 117-174.

Fontaine, M. et Busnel, R. G. (1939). "Nouvelles r echerches s u r l a repart i t ion des flavines e t de quelques a u t r e s pigments f luorescents dans l a peau et les yeux des Tcleosteens" Bulletin de L'Institut -, No. 782, 30 Decembre 1939.

Fox, D. L. (1953). Animal Bichromes and St ructura l Colours, Cambridge University F r e s s .

Fox, H. M. and Vevere, C. (1960). T h e ?Tature of Animal Colours, The - Macmillan Company, New York.

Goodrick, E. S. and Tower, R. W. (1902). "The Organic Constituents of t h e s c a l e s of Fish1' Bull. of U. S. Bureau of Comm. F i s h e r i e s Vol. 21, 97-102.

Hornig, A. W. (1970). "Fish Oil F i l m s on the S e a Surfacet ' F inal Report , Contract N62306-69-C-0354 f o r U. S. Naval Oceanographic Office, October, 1970.

Kampa, %. M. (1955). "Euphausiopsin, a New Photosensitive Pigment f rom the Eyes of Euphausiid Crus taceans" Nature 175: 996. -

Kushibiki, K. , Hama, T., e t Gotto, T. (1954). llQuelques p ter ines de l a peau ou les Czailles de l a C a r p e et l eu r t ransformat ion photochimiqueI1 Societe de Bi ologie Comptes Rendus 148: 759 - 762. -

Polonovski, M, , Busnel, R. G., et Pesson (1946). " ~ r o ~ r i e t 6 ; biochimques des pterinest ' Helvetica Chimie ACT A 29 (171). -

Summer, L'. B. (1940). "Quantitative Changes in the Pigmentation Result - ing f rom Visual St imuli in F i s h e s and Anphibia" Bio. Rev. C a m - bridco Phil. Soc. 15: 351.

Von Hiittelund, S. C. (1943). ItUber Ichthyopterin einen blaufluorescierenden Stdf aus FishautIt Annalen d e r Chemie 554: 69-83. -

Ziegler-GUnder, 1. (1956). "Pigmente und Wirkstoffe Im Tierre ichl l Bio. - Rev. 31: 313-348. --

H. POLLUTION

Boucart, J. and Maliet, L. (i965). "Marine pollution of the s h o r e s in the cent ra l region of the Tyrrhenian Sea (Naples Bight)" Academie des Sciences, P a r i s , Comtes Rendus - 260 (13): 3729- 3734.

Greve, P.A. (1971). "Chemical Wastes in the Sea: New F o r m s of Marine Pollution" Science 173: 1021-1022. -

Hood, D. W. (1971). Impingement of Man or. the Oceans, Wiley In tersc ience , New York.

Hoult, D. P. (1969). Oil on the Sea, Plenum P r e s s , New York.

Hornig, A. W. (1971). "An Analytical Method for the Detection, Identification and Tracing of Lignin Sulfonates" A proposal p repared by Baird- Atomic f o r the Environmental Protect ion Agency, January 1971.

Hornig, A. W. (1971). "Model Oil Study ' Pre l iminary Report , Contract No. 68-01-0146 fo r the Environmental Protect ion Agency.

Masuda, Y. and Kuratsune, M. (1966). "Photochemical oxidation of benzo- a-pyrene" Air & Wat. Pollut. Int. J. - 10: 805-811.

Mosser , J. L. , F i she r , N. S. and Wurster , C. F. (1972). "Polychlorinated Biphenyls and DDT Al ter Species Composition in Mixed Cul tures of Algae" Science 176 (4034): 533- 536. -

I. OPTICAL PROPERTIES OF SEA WATER

Badgley, P. C. , Miloy, L. and Childs, L. , eds. (1969). Oceans f rom Space (Symposium), Gulf Publishing Company, H o u s t o ~ , Texas. -

Clarke, C. L. and James , H. R. (1939). "Laboratory Analysis of the Selective Absorption of Light by Sea Water" J. 0. S. A. 29: 43-55. -

Clarke, G. L. (1969). "The Significance of Spectral Changes in Light Scattered by the SeaH Remote Sensing in Ecology (Phil ip L. J ohnson, ed.) University of Georgia P r e s s , Athens, Georgia, 164-172.

Ivanoff, A. and Morel, A. (1971). "Spectral Distribution of the Natural Fluorescence of Sea- Waters, " Proc. Joint Oceanogr. Assembly (Tokyo, 1.970), 1971.

Jerlov, N. G. (1951). "Optical Studies of Sea Water" Rep. Swedish Deep-sea Exped. - 3 (1).

Jerlov, N. G. (1955). "Factors Influencing the Transparency of the Baltic WatersH K. Vet. 0. Vitterh. Samh. Handl. F. 6 Ser. B. Bd. 6 No. 14.

Jerlov, N. G. (1957). "A t ransparency-meter f o r ocean water. " Tellus 9 (2): 229-233. -

Jerlov, N. G. (1963). "Optical Oceanography" Oceanogr. Mar. Biol. Ann. Rev. 1: 89-114. --

Jerlov, N. G. (1968). Optical Oceanography, Elsevier Publishing Co., New York, 140-151.

Karabashev, G. S., Zangalis, K. P., Solovtyev, A. N. and Yakubovich, V. V. (1971). "New Data on Sea Water Photoluminescence~ Izv., Atmospheric and Oceanic Physics 7 (1): 60-68. -

Malmberg, S. A. (1964). "Transparency measurements in the SkagerackH K. Vet. 0. Vitterh. Samh. Handl. Shatte Foljden. Ser. B. Band

(1).

National Academy of Sciences - -National Research Council (1965). "Report of the in eitu light measurements Working Group of the Committee on Oceanography" Limnol. Oceanogr. - 10 (1): 161,

Ogura, N. (1965). t'Ultraviolet Absorbance of Sea Waters of Tokyo Bay, Sagami Bay and Off Shore Waters ir, the Western North Pacific" J. Oceanogr. Soc. Japan - 21 (5): 1-244.

I. OPTICAL PROPERTIES O F SEA WATER (Continued)

Ogura, N. and Hanya, T. (1966). ! 'Nature of Ultraviolet Absorpt ion of S e a Water" Na tu re 212: 758. -

Ramsey , R. C. et al. (1968). "Study of t he Remote Measu remen t of Ocean Co lo r t t Cont rac t No. NAS W-1658. NASA. F ina l Repor t , J a n u a r y 1968.

R e m o t e Sensing in Mar ine Biology and F i s h e r y R e s o u r c e s (1971). Symposium proceedings, Texas A&M Universi ty , Remote Sens ing Cen te r , College Station, Texas.

Skopintsev, B. A. (1952). "Optical c h a r a c t e r i s t i c s of o rgan ic m a t e r i a l i n s e a waters" Akad. Nauk. SSSP. Invest i ia . Se r . Geofiz. 1: 57-60. - -

Sournia , A. (1965). "Mesure d e l ' absorp t ion d e ; 'u l t raviolet dans l e s eaux o b s i e r e s de Noss i -Be (Madagasca r ) " Bull. Inst. Oceanogr. Monaco 65 (1348).

Yentsch, C. S. (1962). "Measurement of Visible Light Absorpt ion by P a r t i - cu la te Ma t t e r in the Ocean" Limnol. Oceanogr. 7 - (2): 207-217.

J. MISCELLANEOUS

F r e y , D. G., ed. (1963). Limnology in Nor th A m e r i c a , U. Of Wisconsin Press, Madison, Wisconsin.

G a r r e t t , W. D. (1967). "The Organic C-mposi t ion of t h e Ocean Suriacetl Deep-sea Res . 14: 221-227. -

Hood, D. W. (1971). Impingement of Man on t h e Oceans , Wiley In t e r sc i ence , New York.

Kinne, O., ed. (1958). Mar ine Ecology, Wiley, New York.

Koe, B. K., Fox, D. L. and Zechmei s t e r , L. (1950). "The Na tu re of Some F luo resc ing Subs t ances Contained in a Deep S e a Mud" Archs . Bio- chem. 27: 449-452. --

Moore, H. B. M a r i n e Ecology, Wiley, New York.