influence of ph on the fluorescence of dissolved organic matter

Marine Chemistry, 11 (1982) 395--401 395 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

I N F L U E N C E OF p H ON T H E F L U O R E S C E N C E OF D I S S O L V E D O R G A N I C M A T T E R *

R.W.P.M. LAANE

Biological Research Ems-Dollart Estuary, Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, Texel (The Netherlands)

(Received October 2, 1981; revision accepted February 18, 1982)

ABSTRACT

Laane, R.W.P.M., 1982. Influence of pH on the fluorescence of dissolved organic matter. Mar. Chem., 11:395--401

During two surveys (June, 1980 and March, 1981) the influence of the pH on the fluorescence (mF1) was studied in the Ems-Dollart estuary. In the natural pH range (7--9) the fluorescence (mF1) increased by 16.4%. To express the fluorescence (mFl) at a fixed pH, a formula was derived (see eq. 1). From the results it is concluded that an increase in fluorescence (mF1) does not necessarily include an increase in the concentration of fluor- escent matter. For areas where pH changes are possible, it is premature to infer from the increase in fluorescence that the fluorescent matter is produced in situ.

INTRODUCTION

As a resul t o f r iver discharges of organic ma t t e r , high c o n c e n t r a t i o n s o f dissolved organic m a t t e r wi th f luo rescen t p rope r t i e s are f o u n d in the Ems- Dol la r t e s tua ry (Laane , 1981 ; Laane and Koole , 1981) . The in tens i ty of the f luo rescence has been f o u n d to increase a t high sal ini ty in the s u m m e r , b u t this cou ld n o t be exp la ined b y the discharges of f luo rescen t m a t t e r f r o m the rivers en te r ing the e s tua ry (Laane , 1981) . In N o r t h Sea wate r , a seasonal f l u c t u a t i o n in f luorescence wi th h ighes t values in the s u m m e r has been de- scr ibed b y P o s t m a et al. ( 1976) and D u u r s m a (1961, 1974) . D u u r s m a (1974) suggested t ha t d e g r a d a t i o n p r o d u c t s o f p h y t o p l a n k t o n could be the source o f t he increase in f lourescence . F r o m the excess o f f luorescence in the sum- m e r in the Western Wadden sea, P o s t m a et al. (1976) have conc luded t h a t f l uo re scen t m a t t e r is f o r m e d also in situ.

These conc lus ions a b o u t p r o d u c t i o n of f luo rescen t m a t t e r are s u p p o r t e d b y earl ier e x p e r i m e n t s d o n e b y Kalle (1963) , w h o showed t h a t f l uo rescen t m a t t e r was f o r m e d in a c o n d e n s a t i o n r eac t ion of c a r b o h y d r a t e s and a m i n o acids. These c o m p o u n d s have been d e t e c t e d in the Ems-Dol l a r t e s tua ry (Laane , u n p u b l i s h e d results) and thus the wherewi tha l for this c o n d e n s a t i o n r eac t ion to t ake p lace is p r e sen t in the es tuary ; however , the reac t ion i tself has never been p r o v e d to t ake place. HOjerslev (1979) c o n c l u d e d t h a t flu- o re scen t m a t t e r does n o t f o r m in si tu in the sea. This conc lus ion con t r ad i c t s

* Contribution no. 50 from the Biological Research Ems-Dollart Estuary (BOEDE).

0304-4203/0000--0000/82/$02.75 01982 Elsevier Scientific Publishing Company

396

the suppositions of Postma et al. (1976) and Duursma (1974) about the in situ production of fluorescent matter. Before the increase in fluorescence intensity during the summer can be attr ibuted to in situ formation, all the possible sources of fluorescent matter must be closely examined.

These sources were split up into: (A) Cases in which the fluorescence intensity increases without an increase

in the concentration of the fluorescent matter. (B) Sources that produce fluorescent matter and thereby increase the in-

tensity of fluorescence in the estuary. Fluctuations in fluorescence intensity without an increase in the concentra- t ion of these substances could be caused by the temperature, pH, and formation of metal ligands (Udenfriend, 1962; Black and Christman, 1963; Christman and Minear, 1967; Ghassemi and Christman, 1968; Christman and Arnquist, 1969; Schnitzer and Kahn, 1972).

The dependence of fluorescence on pH has been described in two types of curves, one peaking at pH 5--7 (Christman and Minear, 1967; Christman and Arnquist, 1969; Smart et al., 1976) and the other a sigmoidal curve (Black and Christman, 1963; Smart et al., 1976). If the intensity of fluorescence changes, wi thout a change in the concentration of dissolved fluorescent matter, two possibilities have to be distinguished if the fluorescence is meas- ured with a filter--fluorometer:

(1) An increase or decrease in fluorescence intensity without a shift in the emission maximum.

(2) A bathochromatic or hypsochromic shift of the emission maximum. In the second case it is no longer permissible to convert the fluorescence in- tensity into the unit millifluorescence (see definition in Materials and Methods).

An increase in temperature and pH can also alter the equilibrium between the particulate and dissolved state of the fluorescent matter in favour of the dissolved state. Other sources that can increase the concentration of fluores- cent matter in an estuary are: rivers, marshes, seepage from the bot tom of tidal flats, rainfall, release from living and dead organisms and in situ forma- tion. Usually, a combination of these sources is present. In this paper the influence of the pH on the fluorescence intensity and the emission spectra of dissolved matter in the Ems-Dollart estuary is discussed.

MATERIALS AND METHODS

All water samples were collected at a depth of 1.5 m with a pumping sys- tem. After filtration over a precombusted (4.5 hours at 480°C) Whatman GF/F filter, the filtrate was stored in the dark at I°C and analysed within a week. No loss or formation of fluorescent substances occurred during this storage procedure (Van 't Hof, 1972).

Fluorescence is expressed in the absolute unit, millifluorescence (mF1), as defined by Kalle (1963); a solution of I mg quinine bisulphate in a litre of

397

0 .01N H2SO4 has a fluorescence intensity of 700mF1. Fluorescence was measured at room temperature , on a T u r n e r - I l l f luorometer equipped with a primary filter 7-60 (maximum transmission 358 nm) and a secondary filter combinat ion of a Wratten 48 and a 2A filter (maximum transmission 457 rim). F luorometer readings were corrected for the absorption of the excited and emit ted light, following the method of Duursma and Rommets (1961), modif ied for use on a T u r n e r - I l l .

Samples were analysed in duplicate, with a precision of 1.8%. The varia- t ion in the intensity of f luorescence that resulted from a change in pH was studied as follows: 10 pl of a solution with a different pH value was added to 10 ml of filtered water. The resulting pH was measured with a Radiometer PHM-64, equipped with a glass electrode. Experiments were performed in duplicate with a precision of 0.7%.

The reversibility of the relation between fluorescence and pH was studied as follows: 10p l 5 N NaOH was added to the samples that were already at pH of ca. 2. Similarly, 10p l 3 N HCI was added to samples with a pH of ca. 9. The resulting pH and fluorescence were measured and compared with the f luorescence obtained in the first experiments.

Emission spectra were recorded with a Zeiss spectral f luorometer (excita- t ion 365 nm, Hg-lamp). The absorpt ion of the water samples was measured on a Hitachi 200 spec t rophotomete r at 358 nm and 457 nm.

AREA INVESTIGATED

The Ems-Dollart estuary receives its fresh water f rom the river Ems and the Westerwoldse Aa. The hydrography of the estuary has been described by Dorrestein (1960). From these studies it can be concluded that the estuary has no salinity gradient and can be considered as well-mixed.

i~-~:" ~ ~

< , . . . . . ii 9 • 8 , \ I

I \ , • - 2 ( ~ ' , ~ + .

t / ° " " ~ '< - "

Fig. 1. Map of the Ems-Dollart estuary, showing location of sampling stations.

398

The locations of the sampling stations are given in Fig. 1. Sampling sta- tions 1, 3, 4 and 5 lay within the Dollart, whereas stations 6, 7, 8 and 9, 10, 11 were in the middle and outer part of the estuary, respectively.

R E S U L T S A N D D I S C U S S I O N

The seasonal variation in the mean pH in the three parts of the Ems- Dollart estuary are plotted in Fig. 2. It is not the aim of this paper to give a full explanation of the fluctuations of the mean pH. However, Fig. 2 shows that the mean pH is lowest in the Dollart and highest in the outer part of the estuary. During the summer the pH increases in the whole estuary, as a result of primary production. The highest pH value (8.84) was measured in the outer part in June 1979 and the lowest pH (7.10) in January 1979 in the Dollart.

From these data it was inferred that the seasonal pH in the Ems-Dollart estuary f luctuated between 7--9, and therefore the effect of the pH on the fluorescence was closely studied in this range.

The absorptions at the two wavelengths and the fluorometer readings were both dependent on the pH of the solution. In both cases the intensity in- creased with increasing pH. The relations between pH and the absorption at 358 nm and 457 nm for all samples were very similar: two examples are given in Figs. 3A and 3B. The relations between millifluorescence (mF1) and pH for all samples were also almost identical: three examples are given in Fig. 3C. There was a mean increase of 16.4% in millifluorescence in the pH range 7--9.

By least square treatments (r 2 = 0.96; n = 29) the following equation was

pH

86-

85-

84-

83- 82-

81 ~

80 ~

79-

78- 7'7- 76-

75- 7'4-

Z3-

J ' F ' M ' A ' M ' J ' J ' A ' S ' O' N ' D IJ ' F ' M ' A ' M ' J ' J ' A ' S ' O ' N ' DIJ ' F ' M' A' MIJ ' 1978 1979 1980

Fig. 2. Sea sona l va r i a t i on o f t h e m o n t h l y m e a n p H va lues in the E m s - D o l l a r t e s t u a r y dur- ing 1978 , 1979 and 1980 . ( A n o n . , 1978 , 1 9 7 9 a n d 1980) . ( . ---- o u t e r pa r t , - - . . . . . m idd l e par t , - - - - Dol la r t . )

399

ABS

GO60

0.050

0.04,0-

O.050-

(DO 20-

QOIO-

O"

__.j.~. 9

ABS 0 0 0 9 0.008

0 0 0 7

0.006-

0005 -

0 0 0 4 -

Q005-

0.002-

(100 I-

0

B ,. 5

/

/ .-'-'9 /

mFi

140 C

L20

I 0 0 -

8 0

6 0

4O

2 0 -

0

/ /

f.~" 9 ./

.~°~' . ~ . Ii

Fig. 3(A)Relation between absorption (358 nm) and pH at stations 3 (June, 1980) and 9 (March, 1981) in the Ems-Dollart estuary. (B) Relation between absorption (457 nm) and pH at stations 3 (June, 1980) and 9 (March, 1981) in the Ems-Dollart estuary. (C) Relation between the fluorescence (mF1) and pH at station 3 (June, 1980) and stations 9 and 11 (both from March 1981).

derived from the results, in which the measured fluorescence (mFlmeas.) is expressed in fluorescence at a fixed pH (mFlpH=7).

mFl,,eas. - 0.082 (PHmeas.- 7) + 1 (i)

mFlpH=7

This equation is only applicable if the emission maxima of both fluores- cences are the same.

Emission spectra of all samples at various pH values were recorded and no shift was observed in the emssion maximum. From these results it can be concluded that the fluorescence at different pH values can be expressed in the unit mFl.

The results on the reversibility of the relation between fluorescence and pH reveal that there was no change in the fluorescence intensity and the position of the emission maximum after two changes of pH. The conclusion is that pH changes do not drastically change the structure of the dissolved fluorescent matter. Probably, ionization of the fluorescent molecules, result- ing from changes in pH, alters the molecular orbital of the excitable electrons and this could cause the intensity and/or position of the emission maximum to change.

From the relation between millifluorescence and pH (Fig. 3C) hypotheses can be put forward about the structure of fluorescent matter. In a closer investigation, the relation between pH and millifluorescence is characterized by a curve with two slopes: one in the alkaline part and the other in the acid part. The slope around pH = 8 suggests an ionization of an ammonium and/or

400

phenol group and the slope around pH = 3 could be the ionization of sul- phonyl and/or carboxylic acids.

The uniformity in the relation between millifluorescence and pH in the Ems-Dollart estuary supports Laane's (1981) conclusion that the fluorescent matter is mainly al lochthonous and behaves conservatively during transport through the estuary.

It is impossible to say anything about in situ formation of the fluorescent matter until the other sources of fluorescent matter and interactions be- tween these sources have been studied.

ACKNOWLEDGEMENT

I am indebted to my colleagues P. de Wolf, D. Spitzer, R. Mulder, P. Ruardij and H. Postma and Mrs. J. Burrough, for their valuable discussions and for critically reading an earlier draft of the manuscript.

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