- Email: [email protected]
The relation between fluorescence and dissolved organic carbon in the Ems-Dollart estuary and the Western Wadden Sea
Netherlands Journal of Sea Research G(2) : 217-227 (1982)
THE
RELATION BETWEEN FLUORESCENCE DISSOLVED ORGANIC CARBON IN THE EMS-DOLLART ESTUARY AND THE WESTERN WADDEN SEA*
R. Biological
W.
P. M.
LAANE
AND
and L. KOOLE
Research Ems-Dollart Estuary, Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, ‘Texel, The Netherlands
CONTENTS I. II. III.
IV. V.
Introduction ...................... Materials and Methods .................. Results and Discussion ........... I ...... A. Excitation and emission spectra ............. B. Relation between fluorescence and dissolved organic carbon Summary ....................... References .......................
217 218 220 220 222 226 226
I. INTRODUCTION
The dissolved organic matter in natural waters consists of a large number oforganic compounds (DUURSMA, 1965; WILLIAMS, 1975). This pool of organic compounds can be classified into 4 groups: carbohydrates, proteins, lipids and polyphenolic compounds. The concentrations of the first 3 groups of organic compounds are fairly easy to measure as described in the literature. Little is known about the last group. DAUMAS ( 1978) calculated that approximately 70% of the dissolved organic matter can be classified as polyphenolic compounds. In the Ems-Dollart estuary one can see thesegubstances by eye; particularly in the low salinity range the filtered water has a yellow-brown colour. Their maximum proportion can be estimated at 750/, of the dissolved organic carbon because approximately 18% carbohydrates, 5% amino acids (after hydrolysis) and 2 o/0lipids were found ( LAANE, 1980a, 198 1 and unpublished results). Physically, the polyphenolic compounds are distinguishable from most of the carbohydrates, proteins and lipids by their fluorescent characteristics and, using Lambert-Beer’s Law, fluorescence can be converted into concentrations. The fluorescent dissolved organic carbon (DOC,) can be calculated from fluorescence (FI) ex* Contribution
no. 42 from the Biological
Research
Ems-Dollart
Estuary
(BOEDE).
218
R. W. P. M. LAANE& L. KOOLE
pressed as units of millifluorescence ROMMETS 1961) by:
(mF1) (KALLE, 1949;
FL = A x DOC,
DUURSMA & (1)
in which A is a conversion factor (dimension mF1.1. mg C -‘). This equation is valid if certain requirements are met. Firstly, when fluorescence is measured at a fixed wave length, the use of equation (I) is only permitted if the fluorescence spectra of all samples have the same shape and maximum. Secondly, the percentage of carbon in the fluorescent matter has to be consistent over the whole estuary. In earlier work (LAANE, 1981) it was shown that the fluorescence behaves conservatively in the Ems-Dollart estuary and therefore it can be concluded that no major fluctuations in the organic carbon content of the fluorescent matter occur during transport in the estuary. Dissolved organic carbon (DOC) consists of fluorescent (DOC,) and non-fluorescent (DO&) carbon: DOC
:= DOC,
+ DOC,,
(2)
When the fluorescent spectra of all samples in the estuary have the same shape and maximum, the equations (1) and (2) can be combined, to the ratio:
DOG,, -= DOC,
(TX
A)-
1
In the first part of this paper the fluorescence spectra organic matter will be described. In the second part tween fluorescence and dissolved organic carbon from centration of the fluorescent matter in the Ems-Dollart be discussed.
of the dissolved the relation bewhich the conis derived, will
Acknowledgements.-Thanks are due to our colleagues H. Postma, P. de Wolf, W. Helder, E. Duursma, L. van Geldermalsen and J. Burrough for their suggestions and critical reading of the manuscript. II. MATERIALS
AND METHODS
The methods of investigating fluorescence and dissolved organic carbon have been described (LAANE, 1980b, 1981). Fluorescence spectra of water samples were recorded with an Aminco-Bowman SPF-500 fluorometer (excitation and emission band path 8 nm). All emission spectra were corrected for Raman scattering. The maximum excitation
DISSOLVED
ORGANIC
CARBON
219
IN ESTUARY
30’
20’
53°1d
IJSSELMEER
Fig.
1. Map of the Ems-Dollart
estuary sampling
(a) and the Wierbalg-Malzwin stations (0).
(b) showing
R. w. P. M. LAANE& I.. KooLE
220
and maximum emission wave length were determined by combining the excitation and emission spectra into a single graph (JOHNSSON, CALLIS & CHRISTIAN, 1977; WEINER, 1978; NYQUIST, 1979), and published elsewhere (KOOLE, 1980). Fluorescent matter from peat was isolated by 2 methods: In the first method 150 g dried (80” C) peat was suspended in 800 ml 0.1 N NaOH and stirred for 12 hours. After filtration, the filtrate was brought to pH 2 with 6 N HCl and the humic-like substances precipitated. Humic-like and fulvic-like substances were separated by centrifugation (20 min, 3000 g) . The second method was performed with 50 g dried (80” C) peat and 250 ml aqua-bidest. After a working-up as described above, the pH was adjusted to 7.4 with 6 N NaOH and HCl. No separation was made between humic-like and fulvic-like substances. Fluorescent matter from water was isolated by filtering a 25 litres sample (station 2, Fig. l), adjusting to pH 2 with 6 N HCl and extracting with chloroform to remove the lipid fraction. The water was passed over Amberlite XAD-8 resin (1 ml. min -l), then the column was eluted with 250 ml 0.1 N NaOH. Absorption and emission spectra of the isolated material appeared to be identical to the spectra of the starting material. Humic-like and fulvic-like substances were separated by acidification to pH 2 (6 N HCl) and centrifugation (20 min, 3000 g) . Emission spectra of the matter isolated from the peat and estuarine water were recorded on a Zeiss fluorometer (excitation 365 nm, Hg lamp). The sampling stations in the Ems-Dollart estuary and the WierbalgMalzwin are shown in Fig. 1. III.
RESULTS
A. EXCITATION
AND DISCUSSION AND EMISSION SPECTRA
Four surveys were carried out in the Ems-Dollart estuary between February and July 1980, and one survey in the Wierbalg-Malzwin in July 1980 (Fig. 1). E mission spectra are plotted for different sampling stations in the Ems-Dollart (Fig. 2a) and in the Wierbalg-Malzwin (Fig. 2b), both during one survey. The variation in the emission spectra at sampling station 3, as recorded during 4 surveys are also plotted (Fig. 2~). It can be concluded that for all samples taken the emission maximum (excitation 350 nm) of the organic matter in the Ems-Dollart and the Wierbalg-Malzwin does not deviate much outside 420 to 440 nm in time and space. It is difficult to compare the maximum excitation and emission wave
DISSOLVEDORGANICCARBONIN ESTUARI
221
lengths with those previously published (BROWN, 1974; HALL & LEE, 1974; WILANDER, KVARN~ & LINDELL, 1974; SMART et al. 1976; NYQUIST, 1979) because all these spectra were not corrected for the spectral sensivity factor of the equipment used. GIENAPP (1979) described “true” emission spectra of which the maximum wave lengths were around 450 to 460 nm (excitation 367 nm). CHRISTMAN(1970) and GIENAPP (1979) observed that the emission spectra of different natural waters are indeed very similar. The results of the emission spectra in the Ems-Dollart and Wierbalg-Malzwin agree with these
Fig. 2. Emission spectra (relative intensity) of fluorescent material in the Ems-Dollart estuary and the Wierbalg-Malzwin (excitation 350 nm). a. Stations 1 to 6, 21-04-1980. b. Station 3, 4 surveys. c. Stations 7 to 12, 09-07-1980.
222
R. W. P. M. LAANE & L. KOOLE
observations. When the wave length of the maximum emission does not vary, it is permissible to use the unit millifluorescence as a measure of the concentration of fluorescent substances. But it is not true that the spectra in a series of investigations will not vary in wave length; every spectrum has to be checked individually. This is illustrated by the following example in which emission spectra of the estuarine water of the Ems-Dollart and of the organic matter obtained from peat from Wadden Sea sediment are compared (Fig. 3). Different spectra were obtained showing that in this case it is not permissible to express fluorescence in the unit millifluorescence. B. RELATION
BETWEEN FLUORESCENCE ORGANIC CARBON
AND
DISSOLVED
An asymmetrical relationship between fluorescence and dissolved organic carbon in leachates from leaves and in lakes is described by STEWART & WETZEL (1981). SMART et al. (1976) described a linear relation for various surface waters, all showing a small (positive) intercept on the dissolved organic carbon axis. They concluded that some non-fluorescent carbon was present in these waters. Because the fluorescence and the dissolved organic carbon in the Ems-Dollart estuary and the Wierbalg-Malzwin behave conservatively, they also must be linearly related (LAANE, 1980b, 198 1). The regressions for all 14 sur-
Fig. 3. Emission spectra (excitation 350 nm) of aqua-bidest peat extract (Aj, fluorescent substances in the Ems-Dollart estuary, pH 7.0 (B), in the humic fraction, pH 7.3 (C), and in the fulvic fraction, pH 7.3 (D).
DISSOLVED
ORGANIC
CARBON
TABLE
223
IN ESTUARY
I
Linear relation between fluorescence Cy) and dissolved organic carbon (x) in the EmsDollart estuary and the Wierbalg-Malzwin, with number of samples taken (n) and correlation coefficient (r’), The Wierblag-Malzwin relation of 1957 calculated from results published by DUURSMA (196 1). Date
y=a-bx
of suruty
Ems-Dollart 22-10-1978 7-12-1978 26- 3-1979 14- 5-1979 9- 7-1979 17-10-1979 29-l 1-1979 27- 2-1980 21- 4-1980 3- 6-1980 7- 7-1980 14- 8-1980 Wierbalg-Malzwin 27- 8-1957 27-10-1978 9- 7-1980
Y Y Y Y Y Y Y Y Y y y Y
= = = = = = = = = = = =
+ + + + + + + $ $$~ $$-
- 30.08 - 20.08 5.68 - 20.00 - 22.83 5.32 17.74 - 26.13 - 50.50 -156.61 11.25 18.00
Y = Y =
n
r2
19.96x 20.13x 12.96x 13.75x 12.83.x 9.29x 12.93x 23.18x 21.63.x 38.46x 8.68x 15.89x
13 11 7 4 6 8 5 11 11 6 9 8
0.75 0.98 0.98 0.89 0.96 0.97 0.98 0.91 0.98 0.99 0.98 0.99
8.75x 10.10x 6.07x
12 8 14 __~_____
0.89 0.85 0.94 ~
7.72 j12.27 + 1.20 i-
~ -
Y=
_~
between October 1978 and August 1980 were calculated (Table I, Fig. 4). When these linear relations between fluorescence and dissolved organic carbon were extrapolated to the dissolved organic carbon axis there was always a positive intercent in the Ems-Dollart estuary. In the Wierbalg-Malzwin the intercept was 2 times positive but once (July 1980) a small negative intercept was found. Although the intercepts veys
mFI
0;
(
0
Fig. 4. Relation
between
2
.
,
4
dissolved organic in the Ems-Dollart
,
,
6
carbon estuary,
,
,
6 mp
,
,
IO
c. 1-1
(mg C ‘1-l) 07-12-1978.
and fluorescence
(mF1)
224
R. W.
P. M. LAANE& L. KOOLE
give non-realistic values, they can be used to distinguish the relative amount of the fluorescent matter in dissolved organic matter present in an estuary. For doing this, the constancy of the conversion factor A (equation 3) over the estuary is needed. This condition seems to be fulfilled because of the following two characteristics of the fluorescent matter: (1) conservative behaviour, and (2) similarity of the emission spectra within the estuary. The linear relations between fluorescence and dissolved organic carbon could show (1) a negative intercept (Fig. 5a), (2) no intercept (Fig. 5b), or (3) a positive intercept on the dissolved organic carbon axis (Fig. 5~). For the interpretation of these 3 examples it has to be kept in mind that dissolved organic carbon as well as fluorescent organic carbon are conservative properties in the estuary ( LAANE, 1980b, 1981). In each of the examples a line is drawn which represents the relation between fluorescence and dissolved fluorescent carbon (equation I). For determination of the ratio DOC,, : DOC, in the estuary the absolute value of A does not matter, although the minimum value is fixed by the slope of the linear relation between fluorescence and dissolved organic carbon (equation 3). In Fig. 5a, the minimum value of A has to be greater than Hsea : DOC,,, and for the relation shown in Fig. 5c, A has to be greater than Flrive, : DOC,iV,,. In the first example (Fig. 5a) the sea water entering the estuary contains relatively more fluorescent matter than the fresh water sources. In the second example, the relative amounts are the same all over the estuary. Because all natural waters contain non-fluorescent organic substances, it must be concluded that in this example a constant fraction of dissolved organic carbon has fluorescent properties over the whole estuary. In the third example (Figs 4 and 5c) the sea water contains relatively fewer fluorescent substances than the fresh water entering the estuary. This was found during all the Ems-Dollart estuary surveys and nearly all the Wierbalg-Malzwin surveys. It is obvious from
Fig. 5. Possible relations between dissolved organic carbon (DOC) and fluorescence (mF1) (0) and relation between fluorescent dissolved organic cabon (DOC,) and fluorescence (d).
DISSOLVED ORGANICCARBONIN ESTUARY
225
equation (3) that when the value of A is not known it is impossible to calculate the concentration of fluorescent matter in dissolved organic matter. There are two possible ways of quantifying the concentration of fluorescent material: (1) by calibrating the fluorescent material against a standard mixture of known fluorescent substances, and (2) by isolating the fluorescent material and calculating the conversion factor A from the fluorescence and organic carbon contents of the isolated matter. The first possibility was tested by calibrating against a standard mixture of humic acid and lignine sulphonate (Roth) as described by NYQUIST ( 1979) and also by extracting the fluorescent substances with trioctylamine (EBERLE, 1973, G~Tz, 1979). Both methods failed, the isolation of the fluorescent matter being the only possibility left. By the use of this method a conversion factor (A) of 3 1 was found for the EmsDollart estuary. To be sure that all the organic carbon isolated, was associated with the fluorescent molecules, further research is necessary. However, with the conversion factor of 31, the dissolved fluorescent carbon concentrations were calculated (equation 3) for stations 2 and 5 (Table II). For the same stations the dissolved carbohydrate carbon and amino acid carbon (after hydrolysis) as percentage of dissolved organic carbon (LAANE, unpublished results) are known (Table II). The dissolved organic carbon comprised about 1 y0 dissolved lipids (LAANE, 1980). From the present results it can be concluded that the fluorescent matter is a major constituent of dissolved organic carbon. However, the conversion factor for the fluorescent matter in the Wierbalg-Malzwin and other areas has to be calculated, and other factors influencing the fluorescence and the conversion factor, e.g. pH and water temperature have to be investigated before concentrations of the fluorescent matter can be accurately estimated.
TABLE
II
Fluorescent carbon (DOC,), dissolved carbohydrate carbon (DCC) and dissolved amino acid carbon (DAC) expressed as percentages of total dissolved organic carbon at 2 stations in the Ems-Dollart estuary, 27 February 1980. Station ____
DOC,
DCC
DAC
(%/
l%i
(%I
3
66.6
5
49.3
___~.~~
9.1 5.1
1.2 2.1
226
R. W. P. M. LAANE
& L. KOOLE
IV. SUMMARY
During 4 surveys (between February and July 1980) the excitation and emission spectra of the dissolved organic matter in the Ems-Dollart estuary were recorded. During one survey (July 1980) the same spectra were recorded in the Wierbalg-Malzwin. The emission maximum and the shape of the fluorescent spectrum were similar in all samples. Given this founding it was possible to use the fluorescence as a measure for the concentration of fluorescent organic carbon in dissolved organic carbon. From the linear relation between fluorescence and dissolved organic carbon it is concluded that during nearly all the surveys there was relatively more fluorescent matter in the fresh water sources than in the sea water entering the estuary. It appeared that the fluorescent matter is the major constituent of dissolved organic carbon in the EmsDollart estuary. V. REFERENCES BROWN, M., 1974. Laboratory measurements of fluorescence spectra of Balthic waters. Kobenhavns Universitet report 29: 1-19. CHRISTMAN,R. F., 1970. Chemical structure of color producing organic substances in water. In: D. W. HOOD. Organic matter in natural water. Inst. of mar. Science occ. Publ. 1: 181-199. DAUMA,R. A., 1978. Les substances organiques complexes dissoutes dans l’eau de mer. Centre National de la Recherche Scientifique, Paris. DUURSMA,E. K., 1961. Dissolved organic carbon, nitrogen and phosphorus in the sea.-Neth. J. Sea Res. 1 (l/2): 1-147. -, 1965. The dissolved organic constituents of seawater. In: J. P. RILEY & G. SKIRROW. Chemical Oceanography. Acad. Press: 433473. DUURSMA,E. K. &J. W. ROMMETS,1961. Interpretation mathematique de la fluorescence des eaux deuces, saumatres et marines.-Neth. .J. Sea Res. 1 (3): 391405. EBERLE, S. H., 1973. Extraktion von Huminsaure aus Wasser mit trioctylamin. Kernforschungszentrum Karlsruhe, KHF-173 1: l-l 4. GIENAPP, H., 1979. Quantum corrected (“true”) fluorescence emission spectra of filtered water from the Elbe mouth.-Dt. hydrogr. 2. 32: 2044210. GC~TZ,R., 1979. Zur spektralphotometrischen Bestimmung von Ligninsulfonsaure und Huminsaure in Wassern.-Z. analyt. Chem. 296: 406407. HALL, K. J. & G. F. LEE, 1974. Molecular size and spectral characterization of‘organic matter in a meromictic lake.--Water Res. 8: 139-l 51. JOHNSSON,D. W., J. B. CALLIS & G. D. CHRISTIAN,1977. Rapid scanning fluorescence spectroscopy.-Analyt. Chem. 49: 747A-757A. KALLE, K., 1949. Fluoreszenz und Gelbstoff im Bottnischen un Finnischen MeerbusenDt. hydrogr. Z. 2: 117-124. KOOLE, L., 1980. De relatie tussen fluorescentie en opgelost organisch koolstof. Publicaties en Verslagen Biologisch Onderzoek Eems-Dollard Estuarium 1980-10: l-22. LAANE, R. W. P. M., 1980a. Some observations on the lipid concentration in the EmsDollart estuary and the western Wadden Sea.-Estuar. coast. mar. Sci. 10: 589-596.
DISSOLVED
ORGANIC
CARBON
IN ESTUARY
227
1980b. Conservative behaviour of dissolved organic carbon in the Ems-Dollart estuary and the western Wadden Sea.-Neth. J. Sea Res. 14 (2): 192-199. -, 198 1. The composition and distribution of dissolved fluorescent substances in the Ems-Dollart estuary-Neth. J. Sea Res. 15( 1): 88-99. NYQUIST, G., 1979. Investigation of some optical properties of seawater with spectral reference to lignin sulphonates and humic substances. Univ. Gotenberg: l-200 (thesis). SMART, P. L., B. L. FINLAYSON,W. D. RYLANDS & M. C. BALL, 1976. The relation of fluorescence to dissolved organic carbon in surface waters.-Water Res. 10: 805411. STEWART, A. J. & R. G. WETZEL, 1981. Asymmetrical relationships between absorbance, fluorescence, and dissolved organic carbon.-Limnol. Oceanogr. 26 (3) : 590-597. WEINER, E. R., 1978. Equivalence of simultaneous scanning and three-dimensional plotting of fluorescence spectra.-Analyt. Chem. 50: 158331584. WILANDER, A., H. KVARN& & T. LINDELL, 1974. A modified fluorometric method for measurement of lignin sulfonates and its in situ application in natural waters.Water Res. 8: 103771045. WILLIAMS, P. J. LEB., 1975. Biological and chemical aspects of dissolved organic material in seawater. In: J. P. RILEY & G. SKIRROW. Chemical oceanography. (2nd ed.) Acad. Press 2: 301-363.
-,
THE
RELATION BETWEEN FLUORESCENCE DISSOLVED ORGANIC CARBON IN THE EMS-DOLLART ESTUARY AND THE WESTERN WADDEN SEA*
R. Biological
W.
P. M.
LAANE
AND
and L. KOOLE
Research Ems-Dollart Estuary, Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, ‘Texel, The Netherlands
CONTENTS I. II. III.
IV. V.
Introduction ...................... Materials and Methods .................. Results and Discussion ........... I ...... A. Excitation and emission spectra ............. B. Relation between fluorescence and dissolved organic carbon Summary ....................... References .......................
217 218 220 220 222 226 226
I. INTRODUCTION
The dissolved organic matter in natural waters consists of a large number oforganic compounds (DUURSMA, 1965; WILLIAMS, 1975). This pool of organic compounds can be classified into 4 groups: carbohydrates, proteins, lipids and polyphenolic compounds. The concentrations of the first 3 groups of organic compounds are fairly easy to measure as described in the literature. Little is known about the last group. DAUMAS ( 1978) calculated that approximately 70% of the dissolved organic matter can be classified as polyphenolic compounds. In the Ems-Dollart estuary one can see thesegubstances by eye; particularly in the low salinity range the filtered water has a yellow-brown colour. Their maximum proportion can be estimated at 750/, of the dissolved organic carbon because approximately 18% carbohydrates, 5% amino acids (after hydrolysis) and 2 o/0lipids were found ( LAANE, 1980a, 198 1 and unpublished results). Physically, the polyphenolic compounds are distinguishable from most of the carbohydrates, proteins and lipids by their fluorescent characteristics and, using Lambert-Beer’s Law, fluorescence can be converted into concentrations. The fluorescent dissolved organic carbon (DOC,) can be calculated from fluorescence (FI) ex* Contribution
no. 42 from the Biological
Research
Ems-Dollart
Estuary
(BOEDE).
218
R. W. P. M. LAANE& L. KOOLE
pressed as units of millifluorescence ROMMETS 1961) by:
(mF1) (KALLE, 1949;
FL = A x DOC,
DUURSMA & (1)
in which A is a conversion factor (dimension mF1.1. mg C -‘). This equation is valid if certain requirements are met. Firstly, when fluorescence is measured at a fixed wave length, the use of equation (I) is only permitted if the fluorescence spectra of all samples have the same shape and maximum. Secondly, the percentage of carbon in the fluorescent matter has to be consistent over the whole estuary. In earlier work (LAANE, 1981) it was shown that the fluorescence behaves conservatively in the Ems-Dollart estuary and therefore it can be concluded that no major fluctuations in the organic carbon content of the fluorescent matter occur during transport in the estuary. Dissolved organic carbon (DOC) consists of fluorescent (DOC,) and non-fluorescent (DO&) carbon: DOC
:= DOC,
+ DOC,,
(2)
When the fluorescent spectra of all samples in the estuary have the same shape and maximum, the equations (1) and (2) can be combined, to the ratio:
DOG,, -= DOC,
(TX
A)-
1
In the first part of this paper the fluorescence spectra organic matter will be described. In the second part tween fluorescence and dissolved organic carbon from centration of the fluorescent matter in the Ems-Dollart be discussed.
of the dissolved the relation bewhich the conis derived, will
Acknowledgements.-Thanks are due to our colleagues H. Postma, P. de Wolf, W. Helder, E. Duursma, L. van Geldermalsen and J. Burrough for their suggestions and critical reading of the manuscript. II. MATERIALS
AND METHODS
The methods of investigating fluorescence and dissolved organic carbon have been described (LAANE, 1980b, 1981). Fluorescence spectra of water samples were recorded with an Aminco-Bowman SPF-500 fluorometer (excitation and emission band path 8 nm). All emission spectra were corrected for Raman scattering. The maximum excitation
DISSOLVED
ORGANIC
CARBON
219
IN ESTUARY
30’
20’
53°1d
IJSSELMEER
Fig.
1. Map of the Ems-Dollart
estuary sampling
(a) and the Wierbalg-Malzwin stations (0).
(b) showing
R. w. P. M. LAANE& I.. KooLE
220
and maximum emission wave length were determined by combining the excitation and emission spectra into a single graph (JOHNSSON, CALLIS & CHRISTIAN, 1977; WEINER, 1978; NYQUIST, 1979), and published elsewhere (KOOLE, 1980). Fluorescent matter from peat was isolated by 2 methods: In the first method 150 g dried (80” C) peat was suspended in 800 ml 0.1 N NaOH and stirred for 12 hours. After filtration, the filtrate was brought to pH 2 with 6 N HCl and the humic-like substances precipitated. Humic-like and fulvic-like substances were separated by centrifugation (20 min, 3000 g) . The second method was performed with 50 g dried (80” C) peat and 250 ml aqua-bidest. After a working-up as described above, the pH was adjusted to 7.4 with 6 N NaOH and HCl. No separation was made between humic-like and fulvic-like substances. Fluorescent matter from water was isolated by filtering a 25 litres sample (station 2, Fig. l), adjusting to pH 2 with 6 N HCl and extracting with chloroform to remove the lipid fraction. The water was passed over Amberlite XAD-8 resin (1 ml. min -l), then the column was eluted with 250 ml 0.1 N NaOH. Absorption and emission spectra of the isolated material appeared to be identical to the spectra of the starting material. Humic-like and fulvic-like substances were separated by acidification to pH 2 (6 N HCl) and centrifugation (20 min, 3000 g) . Emission spectra of the matter isolated from the peat and estuarine water were recorded on a Zeiss fluorometer (excitation 365 nm, Hg lamp). The sampling stations in the Ems-Dollart estuary and the WierbalgMalzwin are shown in Fig. 1. III.
RESULTS
A. EXCITATION
AND DISCUSSION AND EMISSION SPECTRA
Four surveys were carried out in the Ems-Dollart estuary between February and July 1980, and one survey in the Wierbalg-Malzwin in July 1980 (Fig. 1). E mission spectra are plotted for different sampling stations in the Ems-Dollart (Fig. 2a) and in the Wierbalg-Malzwin (Fig. 2b), both during one survey. The variation in the emission spectra at sampling station 3, as recorded during 4 surveys are also plotted (Fig. 2~). It can be concluded that for all samples taken the emission maximum (excitation 350 nm) of the organic matter in the Ems-Dollart and the Wierbalg-Malzwin does not deviate much outside 420 to 440 nm in time and space. It is difficult to compare the maximum excitation and emission wave
DISSOLVEDORGANICCARBONIN ESTUARI
221
lengths with those previously published (BROWN, 1974; HALL & LEE, 1974; WILANDER, KVARN~ & LINDELL, 1974; SMART et al. 1976; NYQUIST, 1979) because all these spectra were not corrected for the spectral sensivity factor of the equipment used. GIENAPP (1979) described “true” emission spectra of which the maximum wave lengths were around 450 to 460 nm (excitation 367 nm). CHRISTMAN(1970) and GIENAPP (1979) observed that the emission spectra of different natural waters are indeed very similar. The results of the emission spectra in the Ems-Dollart and Wierbalg-Malzwin agree with these
Fig. 2. Emission spectra (relative intensity) of fluorescent material in the Ems-Dollart estuary and the Wierbalg-Malzwin (excitation 350 nm). a. Stations 1 to 6, 21-04-1980. b. Station 3, 4 surveys. c. Stations 7 to 12, 09-07-1980.
222
R. W. P. M. LAANE & L. KOOLE
observations. When the wave length of the maximum emission does not vary, it is permissible to use the unit millifluorescence as a measure of the concentration of fluorescent substances. But it is not true that the spectra in a series of investigations will not vary in wave length; every spectrum has to be checked individually. This is illustrated by the following example in which emission spectra of the estuarine water of the Ems-Dollart and of the organic matter obtained from peat from Wadden Sea sediment are compared (Fig. 3). Different spectra were obtained showing that in this case it is not permissible to express fluorescence in the unit millifluorescence. B. RELATION
BETWEEN FLUORESCENCE ORGANIC CARBON
AND
DISSOLVED
An asymmetrical relationship between fluorescence and dissolved organic carbon in leachates from leaves and in lakes is described by STEWART & WETZEL (1981). SMART et al. (1976) described a linear relation for various surface waters, all showing a small (positive) intercept on the dissolved organic carbon axis. They concluded that some non-fluorescent carbon was present in these waters. Because the fluorescence and the dissolved organic carbon in the Ems-Dollart estuary and the Wierbalg-Malzwin behave conservatively, they also must be linearly related (LAANE, 1980b, 198 1). The regressions for all 14 sur-
Fig. 3. Emission spectra (excitation 350 nm) of aqua-bidest peat extract (Aj, fluorescent substances in the Ems-Dollart estuary, pH 7.0 (B), in the humic fraction, pH 7.3 (C), and in the fulvic fraction, pH 7.3 (D).
DISSOLVED
ORGANIC
CARBON
TABLE
223
IN ESTUARY
I
Linear relation between fluorescence Cy) and dissolved organic carbon (x) in the EmsDollart estuary and the Wierbalg-Malzwin, with number of samples taken (n) and correlation coefficient (r’), The Wierblag-Malzwin relation of 1957 calculated from results published by DUURSMA (196 1). Date
y=a-bx
of suruty
Ems-Dollart 22-10-1978 7-12-1978 26- 3-1979 14- 5-1979 9- 7-1979 17-10-1979 29-l 1-1979 27- 2-1980 21- 4-1980 3- 6-1980 7- 7-1980 14- 8-1980 Wierbalg-Malzwin 27- 8-1957 27-10-1978 9- 7-1980
Y Y Y Y Y Y Y Y Y y y Y
= = = = = = = = = = = =
+ + + + + + + $ $$~ $$-
- 30.08 - 20.08 5.68 - 20.00 - 22.83 5.32 17.74 - 26.13 - 50.50 -156.61 11.25 18.00
Y = Y =
n
r2
19.96x 20.13x 12.96x 13.75x 12.83.x 9.29x 12.93x 23.18x 21.63.x 38.46x 8.68x 15.89x
13 11 7 4 6 8 5 11 11 6 9 8
0.75 0.98 0.98 0.89 0.96 0.97 0.98 0.91 0.98 0.99 0.98 0.99
8.75x 10.10x 6.07x
12 8 14 __~_____
0.89 0.85 0.94 ~
7.72 j12.27 + 1.20 i-
~ -
Y=
_~
between October 1978 and August 1980 were calculated (Table I, Fig. 4). When these linear relations between fluorescence and dissolved organic carbon were extrapolated to the dissolved organic carbon axis there was always a positive intercent in the Ems-Dollart estuary. In the Wierbalg-Malzwin the intercept was 2 times positive but once (July 1980) a small negative intercept was found. Although the intercepts veys
mFI
0;
(
0
Fig. 4. Relation
between
2
.
,
4
dissolved organic in the Ems-Dollart
,
,
6
carbon estuary,
,
,
6 mp
,
,
IO
c. 1-1
(mg C ‘1-l) 07-12-1978.
and fluorescence
(mF1)
224
R. W.
P. M. LAANE& L. KOOLE
give non-realistic values, they can be used to distinguish the relative amount of the fluorescent matter in dissolved organic matter present in an estuary. For doing this, the constancy of the conversion factor A (equation 3) over the estuary is needed. This condition seems to be fulfilled because of the following two characteristics of the fluorescent matter: (1) conservative behaviour, and (2) similarity of the emission spectra within the estuary. The linear relations between fluorescence and dissolved organic carbon could show (1) a negative intercept (Fig. 5a), (2) no intercept (Fig. 5b), or (3) a positive intercept on the dissolved organic carbon axis (Fig. 5~). For the interpretation of these 3 examples it has to be kept in mind that dissolved organic carbon as well as fluorescent organic carbon are conservative properties in the estuary ( LAANE, 1980b, 1981). In each of the examples a line is drawn which represents the relation between fluorescence and dissolved fluorescent carbon (equation I). For determination of the ratio DOC,, : DOC, in the estuary the absolute value of A does not matter, although the minimum value is fixed by the slope of the linear relation between fluorescence and dissolved organic carbon (equation 3). In Fig. 5a, the minimum value of A has to be greater than Hsea : DOC,,, and for the relation shown in Fig. 5c, A has to be greater than Flrive, : DOC,iV,,. In the first example (Fig. 5a) the sea water entering the estuary contains relatively more fluorescent matter than the fresh water sources. In the second example, the relative amounts are the same all over the estuary. Because all natural waters contain non-fluorescent organic substances, it must be concluded that in this example a constant fraction of dissolved organic carbon has fluorescent properties over the whole estuary. In the third example (Figs 4 and 5c) the sea water contains relatively fewer fluorescent substances than the fresh water entering the estuary. This was found during all the Ems-Dollart estuary surveys and nearly all the Wierbalg-Malzwin surveys. It is obvious from
Fig. 5. Possible relations between dissolved organic carbon (DOC) and fluorescence (mF1) (0) and relation between fluorescent dissolved organic cabon (DOC,) and fluorescence (d).
DISSOLVED ORGANICCARBONIN ESTUARY
225
equation (3) that when the value of A is not known it is impossible to calculate the concentration of fluorescent matter in dissolved organic matter. There are two possible ways of quantifying the concentration of fluorescent material: (1) by calibrating the fluorescent material against a standard mixture of known fluorescent substances, and (2) by isolating the fluorescent material and calculating the conversion factor A from the fluorescence and organic carbon contents of the isolated matter. The first possibility was tested by calibrating against a standard mixture of humic acid and lignine sulphonate (Roth) as described by NYQUIST ( 1979) and also by extracting the fluorescent substances with trioctylamine (EBERLE, 1973, G~Tz, 1979). Both methods failed, the isolation of the fluorescent matter being the only possibility left. By the use of this method a conversion factor (A) of 3 1 was found for the EmsDollart estuary. To be sure that all the organic carbon isolated, was associated with the fluorescent molecules, further research is necessary. However, with the conversion factor of 31, the dissolved fluorescent carbon concentrations were calculated (equation 3) for stations 2 and 5 (Table II). For the same stations the dissolved carbohydrate carbon and amino acid carbon (after hydrolysis) as percentage of dissolved organic carbon (LAANE, unpublished results) are known (Table II). The dissolved organic carbon comprised about 1 y0 dissolved lipids (LAANE, 1980). From the present results it can be concluded that the fluorescent matter is a major constituent of dissolved organic carbon. However, the conversion factor for the fluorescent matter in the Wierbalg-Malzwin and other areas has to be calculated, and other factors influencing the fluorescence and the conversion factor, e.g. pH and water temperature have to be investigated before concentrations of the fluorescent matter can be accurately estimated.
TABLE
II
Fluorescent carbon (DOC,), dissolved carbohydrate carbon (DCC) and dissolved amino acid carbon (DAC) expressed as percentages of total dissolved organic carbon at 2 stations in the Ems-Dollart estuary, 27 February 1980. Station ____
DOC,
DCC
DAC
(%/
l%i
(%I
3
66.6
5
49.3
___~.~~
9.1 5.1
1.2 2.1
226
R. W. P. M. LAANE
& L. KOOLE
IV. SUMMARY
During 4 surveys (between February and July 1980) the excitation and emission spectra of the dissolved organic matter in the Ems-Dollart estuary were recorded. During one survey (July 1980) the same spectra were recorded in the Wierbalg-Malzwin. The emission maximum and the shape of the fluorescent spectrum were similar in all samples. Given this founding it was possible to use the fluorescence as a measure for the concentration of fluorescent organic carbon in dissolved organic carbon. From the linear relation between fluorescence and dissolved organic carbon it is concluded that during nearly all the surveys there was relatively more fluorescent matter in the fresh water sources than in the sea water entering the estuary. It appeared that the fluorescent matter is the major constituent of dissolved organic carbon in the EmsDollart estuary. V. REFERENCES BROWN, M., 1974. Laboratory measurements of fluorescence spectra of Balthic waters. Kobenhavns Universitet report 29: 1-19. CHRISTMAN,R. F., 1970. Chemical structure of color producing organic substances in water. In: D. W. HOOD. Organic matter in natural water. Inst. of mar. Science occ. Publ. 1: 181-199. DAUMA,R. A., 1978. Les substances organiques complexes dissoutes dans l’eau de mer. Centre National de la Recherche Scientifique, Paris. DUURSMA,E. K., 1961. Dissolved organic carbon, nitrogen and phosphorus in the sea.-Neth. J. Sea Res. 1 (l/2): 1-147. -, 1965. The dissolved organic constituents of seawater. In: J. P. RILEY & G. SKIRROW. Chemical Oceanography. Acad. Press: 433473. DUURSMA,E. K. &J. W. ROMMETS,1961. Interpretation mathematique de la fluorescence des eaux deuces, saumatres et marines.-Neth. .J. Sea Res. 1 (3): 391405. EBERLE, S. H., 1973. Extraktion von Huminsaure aus Wasser mit trioctylamin. Kernforschungszentrum Karlsruhe, KHF-173 1: l-l 4. GIENAPP, H., 1979. Quantum corrected (“true”) fluorescence emission spectra of filtered water from the Elbe mouth.-Dt. hydrogr. 2. 32: 2044210. GC~TZ,R., 1979. Zur spektralphotometrischen Bestimmung von Ligninsulfonsaure und Huminsaure in Wassern.-Z. analyt. Chem. 296: 406407. HALL, K. J. & G. F. LEE, 1974. Molecular size and spectral characterization of‘organic matter in a meromictic lake.--Water Res. 8: 139-l 51. JOHNSSON,D. W., J. B. CALLIS & G. D. CHRISTIAN,1977. Rapid scanning fluorescence spectroscopy.-Analyt. Chem. 49: 747A-757A. KALLE, K., 1949. Fluoreszenz und Gelbstoff im Bottnischen un Finnischen MeerbusenDt. hydrogr. Z. 2: 117-124. KOOLE, L., 1980. De relatie tussen fluorescentie en opgelost organisch koolstof. Publicaties en Verslagen Biologisch Onderzoek Eems-Dollard Estuarium 1980-10: l-22. LAANE, R. W. P. M., 1980a. Some observations on the lipid concentration in the EmsDollart estuary and the western Wadden Sea.-Estuar. coast. mar. Sci. 10: 589-596.
DISSOLVED
ORGANIC
CARBON
IN ESTUARY
227
1980b. Conservative behaviour of dissolved organic carbon in the Ems-Dollart estuary and the western Wadden Sea.-Neth. J. Sea Res. 14 (2): 192-199. -, 198 1. The composition and distribution of dissolved fluorescent substances in the Ems-Dollart estuary-Neth. J. Sea Res. 15( 1): 88-99. NYQUIST, G., 1979. Investigation of some optical properties of seawater with spectral reference to lignin sulphonates and humic substances. Univ. Gotenberg: l-200 (thesis). SMART, P. L., B. L. FINLAYSON,W. D. RYLANDS & M. C. BALL, 1976. The relation of fluorescence to dissolved organic carbon in surface waters.-Water Res. 10: 805411. STEWART, A. J. & R. G. WETZEL, 1981. Asymmetrical relationships between absorbance, fluorescence, and dissolved organic carbon.-Limnol. Oceanogr. 26 (3) : 590-597. WEINER, E. R., 1978. Equivalence of simultaneous scanning and three-dimensional plotting of fluorescence spectra.-Analyt. Chem. 50: 158331584. WILANDER, A., H. KVARN& & T. LINDELL, 1974. A modified fluorometric method for measurement of lignin sulfonates and its in situ application in natural waters.Water Res. 8: 103771045. WILLIAMS, P. J. LEB., 1975. Biological and chemical aspects of dissolved organic material in seawater. In: J. P. RILEY & G. SKIRROW. Chemical oceanography. (2nd ed.) Acad. Press 2: 301-363.
-,