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Transmission spectroscopy examinations of natural waters
Estuarine
and Coastal
Marine
Science (I 977) 5, 309-3 17
Transmission Spectroscopy Examinations of Natural Waters C. Ultraviolet Spectral Characteristics of the Transition from Terrestrial Humus to Marine Yellow Substance
Murray
Brown
Institute of Physical Haraldsage 6, 2200
Oceanography, University Copenhagen N, Denmark
Received
1975
12 August
and in revised form
of Copenhagen, 25 November
1975
The author defines a spectral slope value for ultraviolet absorption spectra of natural waters, for use in a major study of spectral characteristics of Baltic waters. The results show a steepening in the spectra as total absorption decreases going from slightly saline waters of the northern Bothnian Bay to North Sea waters entering the Kattegat. This qualitative change is related to the selective loss of high molecular weight humic components of the yellow substance. The literature relating to spectral differences in molecular weight fractions of natural humus is reviewed. When spectral data are combined with salinity values, a categorization of water types is possible, giving Baltic surface, Baltic deep, and North Sea types. The various processes contributing to this differentiation are discussed, and an associated experiment examines the spectral effects of bacterial activity on marine humus.
Introduction It is especially spectra
when
difficult no clear
to attempt maximum
qualitative
may be found.
analysis of ultraviolet A well-known
example
or visible
absorption
of this is the spec-
trum of natural waters, marine as well as non-marine. In most cases the spectrum shows strong increase in absorption toward the short-wave end, with some indication of a shoulder near 280 nm. [A rare example of an absolute peak at 277 nm has been reported by Hanya & Ogura (1962) for water from the river Edogawa.] Brown (197p) has related this 280 nm shoulder to simple aromatic rings, especially those conjugated with carboxyl, carbonyl and phenolic oxygen, structures common in many models for humus. Figure I shows two typical Baltic seawater absorption spectra. The slope of the spectrum itself would appear to be the only remaining qualitative feature which might be exploited for interpretation. The best known inquiry into this matter was made by Kalle (1961, 1962, 1963, 1966), who measured an ‘extinction coefficient’ at 420 and 665 nm, and computed the ratio for these values for a number of natural water samples, as well as artificial model compounds. Kalle concluded on the basis of comparison of these ratios that ‘the mainland waters (tap water from Hamburg, water from moors, and extracts of leaves) tend to the phenol 309
310
M. Brown
humic acid type’, i.e. flatter spectra, ‘and seawater (water from the North Atlantic, the North Sea, and the Baltic) tends more to the melanoidines type’, i.e. steeper spectra. A previous study (Brown, 1974a) has examined this method, and pointed out serious objections in both interpretation and technique. Chief among these being the extremely small value which could possibly be determined with short cells in the visible wavelength region, and a systematic dilution effect which can drastically alter slope values.
II
I 250
I
I1
I
I, 300 Wavelength
I
I
I
I 350
(nm~
Figure I. Two typical absorption spectra of filtered Baltic seawater. The lower curve corresponds to the central Baltic, while the upper is typical of the Bothnian Sea.
Brown (1974a) showed that carefully prepared dilutions of one humic water sample showed an increasing slope value with decreasing concentration. Reduction by as much as one-half the calculated value can occur, for slopes in the visible as measured by Kalle. This is strong evidence that seeming qualitative changes in absorption spectra can be artifactual rather than real. The effect is greater for wider wavelength intervals, and to some degree less serious in the U.V. than in the visible for any given wavelength interval. Both problems seem to be eliminated when a short wavelength interval (< IOO nm) is specified, i.e. for reduction of dilution effect, and absorption is studied in the ultraviolet, i.e. for greater accuracy. The present study consists of a collection of absorption measurements of Baltic seawater, at two narrowly spaced short wavelengths to determine to what extent Kalle’s findings could be verified, when the ‘dilution effect’ is minimized, and a discussion of interpretation based on the humus literature. Method During the November 1974 cruise of the Swedish Fisheries Ministry research ship ‘Argos’ through the Kattegat, Danish Sound, Baltic proper, Bothnian Sea and Bay, sampfes were taken in all-plastic samplers from the surface and bottom, or in some cases from representative intermediate depths. These were left standing open for one hour to allow oxygen saturation, then filtered by glass syringe through Whatman GF/c filters into lo-cm cells.
Transmission spectroscopy of natural waters
31’
Absorbance (2 --log y. T) measurements were made in a Perkin-Elmer 139 Spectrophotometer at 380 nm and 280 nm OS.double-distilled water as reference. A cell-to-cell correction was made. As the samples are filtered, particle (> I p) scattering may be ignored. In principle some colloid scattering might occur, but this is much too small to affect measurements in a typical double-beam spectrophotometer. Scattering due to the water itself is compensated by the reference cell. The wavelength calibration of the instrument was checked several times throughout the voyage by matching the selector reading to a known peak in the source spectrum. No drift was observed for a ‘warm’ instrument which had been calibrated when cold. The repeatability of the measurements is demonstrated by the fact that a double-cruise up and down the Baltic over a period of one month consistently produced values falling within the simple pattern discussed below. Results For some time now measurements of light absorption at 380 nm have been made to determine the content of the ‘yellow substance’ (Jerlov, 1968, pp. 55-57, 164-165). For greater sensitivity, since ro-cm cells were employed, it was considered more useful to compare ultraviolet absorption values as measures of humus.
I 0,.
07 5.
5 4,p 0.5 0
02 5,
,3"
3
IO
15 Salinity
20
25
30
35
(o/oo)
Figure z. ro-cm absorbanceat 280 ( x ) and 310 nrn (+) Baltic seawater WS.salinity.
Water twice distilled
of glassfibre-filtered in glass was used as reference.
Figure z shows IO-cm absorbance (2 -log o/oT) at 280 nm and 3 IO nm vs. salinity. 280 nm was chosen as it lies directly on the above-mentioned ‘shoulder’ in the transmission spectrum, while 310 nm has been shown to occupy a similar ‘shoulder’ position on the corrected fluorescence excitation spectrum of Baltic seawater (Brown, 19743). Both absorption and fluorescence excitation spectra are remarkably similar-excepting the exact position of
312
M. Brozon
these shoulders-with strong end rise below 250 nm. The figure shows that a close relationship between the salinity and absorption intensity is maintained. As many hundreds of separate streams feed the Baltic, and sampling positions were by no means confined to one area, then the salinity must be a strong factor in controlling the amount of organic materials remaining in solution. The convex shape of the 380 nm absorption as. salinity plot has been explained (Jerlov, 1955) by a precipitation of the humus; the same undoubtedly holds for the evidence of the present discussion. The yellow substance, then, is not truly conservative, but, as shown by the figure, functionally related to a conservative property-the salinity. Quantitative comparison of the values obtained in this study with those of many published studies is impossible, as absorption values at 380 nm were taken only at a few stations. It is possible, however, to calculate dissolved organic material, DOM, from total organic carbon, TOC, measurements made during the September 197-J cruise of the Finnish research ship Aranda, together with absorption measurements at 280 nm made by this author. Assuming DOM = z x TOC, a specific absorption coefficient, k, may be calculated as shown by Brown (1974~) equal to 19.0 (IO-cm cell, concentration in 81-l). For each sample a spectral slope, CD,has been calculated as:
These ratio values have been plotted in Figure 3 ws. absorbance at 310 nm. Plotting CDvs. absorbance at 280 nm gives a qualitatively identical figure. As the figure shows, a clear steepening in the absorption curves of natural humic waters occurs in the transition zone from the rivers to the open sea-the estuary. At the same time a great deal of dissolved material is lost, presumably to precipitation, as evidenced by the non-linearity of Figure 2. That there is a precipitation of humic materials is clear, as first demonstrated by Jerlov (1955) in experiments supporting his measurements of yellow substance in the Baltic. The theoretical possibility remains, however, that a part, at least, of the decrease in absorption could be due to hypochromicity of the humus macromolecules and colloids when condensing under saline conditions. Brown (197p) has discussed this possibility for natural water humus. A first examination of Figure 3 shows three separate categories of water types. A stylized representation of this classification is given in Figure 4. Here Type I refers to the high salinity (>Is%,,), low yellow-substance-containing waters of the North Sea entering the Baltic area via the Kattegat and Skagerak. Type II refers to waters of intermediate salinity (15%~ to 7.5x0), usually below the 50-m horizon in the Baltic proper; while Type III is assigned to the surface waters of low salinity (8x, to ozO). There is some overlap in salinity values between Types II and III. The approximate salinity ‘gradient’ has been supplied by inspection from Figure z. Bearing in mind that it is probably only North Sea water of a certain depth which can enter the Baltic over its 17-m sill, owing to density stratification, then an understanding of the relationships between the various masses and their mixing, requires that only a portion, not the whole, of Type I be considered as contiguous with Types II and III within the inland sea. This is important, as a wide range of values is found in these true marine waters. Figure 5 shows @ values US.depth for many stations taken in the Danish Belts and Sound and in the southern Kattegat during cruises of the Martin Knudsen, the research ship operated by the Danish government’s Environmental Ministry. A decrease in CDis apparent with depth. A typical value at the sill depth would be 2.10.
Transmission spectroscopy of natural waters
3’3
2-5
B
2c
.
If o-2
0
IO-cm A S,O-nm
Figure 3. Spectral slope values, CD,OS. lo-cm
I ’ %o
I
I
04
0.2
.
.
06
04
absorbance
at 310 nm.
I
06
AIO-cm
310-nm
Figure 4. Simplified schematic salinity values are approximate,
of the data presented in Figure 3. The indicated having been supplied by inspection of Figure 2.
The classical model for renewal of Baltic deep waters hypothesizes an inflow over the sill which entrains and reintroduces approximately one third of the overlying low salinity water which is flowing outward. This model is strongly supported by the fact that Type II lies at exactly the expected position in the present figures which would be called for by mixing between I (a = 2.1) and III (Q, = 225), on the basis of salinities. Although Ogura & Hanya (1966) have shown that nitrate and bromide contribute significantly to the total absorption in the wavelength interval 21~~230 nm, the possibility that variations in these species could give rise to the observed variations is slight, as they do not absorb appreciably above 230 nm, and the observed effects are the opposite of those which could be expected. A strong bromide interference would be characterized by systematic increase in slope values through the entire salinity range to a maximum at the highest values (not seen). A nitrate effect would cause deep water values (Type II) to be higher than those for surface water (also not seen).
314
M. Brown
.-..--
0
/’
.
I.
.
’ 0’ .
: .:*
If 0,‘.
l l . . .
.*m/ I I’
.
IO -
,.’
.
4
.
. . 5OL
’ I ,75
I 2.00
I 2.25
; !Cio
0
Figure 5. @ values vs. depth for water samples taken at stations in the Danish Belts and Sound, and in the southern Kattegat.
Discussion Several processes which could cause the indicated spectral changes suggest themselves: (I) biological production of a new dissolved component; (2) photo-activated oxidation introducing more oxygen-containing functional groups into existing molecules ; (3) bacterial oxidation and hydrolysis; (4) selective loss of a portion of the terrestrial humus which absorbs relatively more strongly toward the longer wavelengths than the ‘residual’ component. The possibility of the in situ production of a Baltic humic component peculiar to these waters, and responsible for absorption increase in the short wave end, has been discarded as there is no reason to believe that a special biochemical regime exists here. The spectral characteristics of the Baltic water finally produced by one or a combination of these processes is very different from open-sea marine yellow substance which is mainly the result of in situ biological processes. In addition, Figures 3 and 4 show that the alteration in the shape of the spectrum of Type III is very closely related to a loss in material rather than the opposite. Oxidation in the natural environment has been little studied, owing to obvious problems in microtechnique and determination. Gjessing & Gjerdahl(1970) have made the only study known to this author on the reduction of natural aquatic humus by solar radiation. They showed that ‘about 207$ of the reduction of colour between the inlet and outlet of lake Byvatn (in Norway) is due to natural U.V. radiation’ during a 75-day retention in the lake. Retention time for the whole Baltic system has been roughly estimated by Fonselius (1972) to be about 22 years; and, during the winter at least, the upper 50 m or so, the least saline, are well mixed. For these reasons we can assume that the total complement of humic materials is affected to some degree by photo-oxidation. That this process could induce the indicated spectral steepening is possible, but unsubstantiated.
Transmission
spectroscopy
of natural
waters
315
Bacterial oxidation and hydrolysis of macromolecular substances in solution has only recently become an area of active inquiry. Naturally, the surface areas provided by suspended particles are important in this process; Khailov & Finenko (1969) studied various bacterialsolute interactions at this interface, and gave approximate rate constants for organic transformations. However, Postma (1969), Jannasch (1967) and, originally, Zobell (1946) have pointed out that due to (a) the extremely low substrate concentration, and (b) sparse available surface area in situ, bacterial utilization of dissolved organics in seawater is very small. As discussed by Postma (1969), storage of seawater in bottles should hasten bacterial processes. A small experiment was performed to see what spectral change occurred in bottle-stored samples over a long period of time. (I) Samples taken in the Bothnian Sea and Bay during the September 1973 cruise of the Finnish research vessel Aranda, were stored unfiltered for 14 months in Ioo-ml brown glass bottles with glass stoppers. The bottles had been cleaned in acid, then base, and rinsed in alcoholic KOH to remove contaminants, especially surfactants, before use. (2) Transmission spectra of filtered aliquots were run immediately upon return to the laboratory (1-2 weeks after sampling) and again after 14 months. IO-cm cells were employed, and the reference water was prepared in exactly the same manner both times, i.e. passage through activated carbon and membrane filtration. The results of this study, given in Table I, show that storage in this manner, presumably accelerating bacterial processes while discriminating against photo-oxidation, consistently gives a reduction in CDvalues, that is-a flattening of the curve. In one case there was even an increase in absorption at the longer wavelength, which might be accounted for by particle degradation and dissolution. It is likely, therefore, that bacterial activity plays a minor role in the observed spectral transformation. TABLE
I.
Transmission spectral changesafter
Sample” I-3
Initial values /p-m @ 110nnl
II-Z
0.980 0.497
I.715 1.780
III-I RR-8 KOK-A
0.387 0’340 0'301
I ,869 “9’4
VII-2 VII-4
0.266
Spec-1
0.238 0’212
IX-I
0'252
I .g8z
14
months storage
Final values /p-cm Q 310nm 0.740 0.434 0.366 0.338 0.265
1.643 1.738
0.248 0.228
“994
2.068 2.063 2.141
0'221
2’143
0.228
I.796 1.807 1.845 2.017
“995 1-d
% ;to~c in 310nm
% $gge
-22.5
-42
-12.6 - 5’4 - 0.6
-2.4
- 6.9
-12.0
+
-3’9 -- 5’9
6.8 9’5 7” 7.6
-3.6 -2.7 -6.8 -8.7
‘Sample designationsrefer to sampling stations annually occupied by the Finnish Marine ResearchInstitute, Helsinki, with the exception of Spec-x near regular station VII-4. From a thermodynamic standpoint, greater molecular or particle size is favorable for coagulation in an ionic medium (Stumm & Morgan, 1970, p. 498). It is suggested therefore that removal of total color from terrestrial waters entering the marine environment will proceed in the order colloids > macromolecules > smaller molecules. Brown (1975a) has shown that a decrease in high molecular weight material (> IO ooo) with increasing salinity occurs in the northern Baltic. The possibility exists, therefore, that the observed spectral changes could partially be explained by a selective coagulation process,
316
M. Brown
if evidence could be produced that the higher molecular weight is indeed different in its spectral properties from the lower weight material. Butler & Ladd (1969) h ave studied the spectra of humus and report a close correlation between carboxyl groups and absorption at 260 nm. With increasing molecular weight, the relative abundance of the carboxyl groups-and therefore absorption at 260 nm-decreases. Presumably, they refer to carboxyl groups conjugated with aromatic or other oxygencontaining moeties, since aliphatic carboxylic acids show absorption maxima near 200210 nm; benzoic acid, on the other hand (C,H,COOH) has absorption bands at 228 and 279 nm. This relates well to Brown’s (1974~) model hypothesizing substituted aromatics in general to explain the broad shoulder near 280 nm in absorption spectra of natural waters. Kononova & Bel’chikova (1960) found that ratios of extinction at 230 nm to 310 nm, or 230 to 320 nm are low for ‘complex particles’, that is high molecular weight humus, and high for smaller molecules. They go on to quote several other studies as showing that higher weight material has a less steep spectrum than low weight material. Swift, Thornton & Posner (1970) state, after spectral examination of humus fractions, ‘(the) visible portion of the spectrum shows that the lower molecular weight fractions exhibit a greater slope through the visible region than the high molecular weight fractions’. Finally, Soukup (1964) found that ratios of absorption at 465 nm to 619 nm increase with decreasing molecular weight. These results lend support to the contention that a gradual reduction of the colloidal and high molecular weight fractions due to salinity flocculation should assist, at least, in the observed changes. That this is not the whole picture is, however, quite clear as shown in Figure 4 where Type II, the Baltic deep water, has slope values lower than the overlying less saline waters. It would appear that the ‘ripening’ process occurs only appreciably in the surface waters up to the point where a certain fraction of the outflowing surface waters are entrained by the Type I inflow over the Baltic sill. The new mixture, Type II, retains its spectral characteristics from that point with little change. This does not, as it would seem at first, weigh the evidence more heavily for photo-oxidation-which certainly could not occur appreciably below 50 m-as it is also known that coagulation processes due to salinity are repressed at high salinities due to formation of a charge anti-layer. Brown (19;~~~ has found a reduction in flocculation rate of humus for high salinities in laboratory
Conclusions Very certainly there is a change in the optical properties of terrestrial humic material which enters the Baltic by the time it reaches the deep sea. Whether this is due to photo-oxidative alteration, or selective removal of molecular weight fractions, or a combination of the two together with some bacterial activity is not yet known. However, the optical classification system (Figure 4) afforded by these changes could be of great value in studies of water exchange and mixing at the mouth of the Baltic. There are some preliminary indications that departures from the model for spectral characteristics combined with salinity, as shown in Figure 4, constitutes a reliable index of unusual chemical conditions. In a recent study concentrated organic matter taken from various locations, Kerr & Quinn (1975), found that humic substances from the soil display an inverse relation between absorption intensity and spectral slope-corresponding to the right hand side of Figure 3-while open marine material shows a direct relation-as on the left side of the figure. Their con-
Transmission
spectroscopy
of natural
waters
317
elusion, that this difference constitutes good evidence for different sources and/or diagenetic processes (Kerr & Quinn, 1975, p. 114) is emphatically supported by the present results. Acknowledgements The author is grateful to the governments of Finland, Sweden and Denmark for permission to participate in various cruises, and many acts of courtesy. Special personal thanks are due to Dr Aarno Voipio, Helsinki, Docent Stig Fonselius, Gothenburg and Professor Niels Jerlov, Copenhagen. References Brown, M. 197qz Transmission spectroscopy examinations of natural waters; A. On standardization of optical terminology in oceanographic investigations; B. Interpretation of spectra. Reports of the Institute of Physical Oceanography, University of Copenhagen, Denmark 28, 26 pp. Laboratory measurements of fluorescence spectra of Baltic waters. Reports of the Brown, M. 1974 Institute of Physical Oceanography, University of Copenhagen, Denmark 29, 19 pp. Brown, M. I975a High molecular weight material in Baltic waters. Marine Chemistry, 3, 253-258. PhD Dissertation, University of Brown, M. 1973b Humic particle formation in saline admixtures. Copenhagen. Butler, J. H. A. & Ladd, J. N. 1969 Effect of extractant and molecular size on the optical and chemical properties of soil humic acids. Australian Journal of Soil Research 7, 229. Fonselius, S. 1972 On eutrophication and pollution in the Baltic Sea. In Marine Pollution and Sea Life. (Ruivo, M., ed.) FAO, Fishing News (Books) Ltd. Gjessing, E. T. & Gjerdahl, T. 1970 Influence of ultra-violet radiation on aquatic humus. Ihtten 29 144. Hanya, T. &-Ogura, N. r962 Application of ultra-violet spectroscopy to the examination of dissolved organic submstances in water. In Advances in Organic Geochemistry. Proceedings of the International Meeting in Organic Chemistry, Milano (Colombo & Hodson, eds) 447 pp. Jannasch, H. W. 1967 Growth of marine bacteria at limiting concentrations of organic carbon in seawater. Limnology &Y Oceanography 12, 264. Jerlov, N. 1955 Factors influencing the transparency of the Baltic waters. Reports from the Oceanographic Institute in Gothenburg 25, 18 pp. Jerlov, N. 1968 Optical Oceanography. Elsevier Oceanography Series, Amsterdam. Union Geodesquie et Geophysique International, Kalle, K. 1961 What do we know about Gelbstoff? Monographie IO, 59. Kalle, K. 1962 uber die gel&ten organisehen komponenten im Meerwasser. Kieler Meeresforschungen 18, 128.
Kalle,
K. 1963 Uber das Verhalten und die Kerkunft in den Gewassern und in der Atmosphlre vorhandenen himmelblauen Fluoreszenz. Deutsche Hydrographische Zeitschrift 16, I 53. Kalle, K. 1966 The problem of the Gelfstoff in the sea. Oceanography and Marine Biology, Annual Review 4, 91. Kerr, R. A. & Quinn, J. G. 1975 Chemical studies on the dissolved organic matter in sea water. Isolation and fractionation. Deep-Sea Research 22, 107. Khailov, K. M. & Finenko, 2. Z. 1969 Interaction of detritus with high-molecular weight components of dissolved organic matter in sea water. Oceanology 8, 776 (English). Kononova. M. M. & Bel’chikova. N. P. 1060 Investigation of the nature of soil humus substances bv fractionation. Soviet Soil Science (196b), p. I r49: Ogura, N. & Hanya, T. 1966 Nature of ultraviolet absorption of sea water. Nature 212, 758. Postma, H. 1969 Dissolved organic matter in the oceans. Advances in Organic Geochemistry 1968, International Series of Monographs in Earth Sciences, vol. 3r (Schenck & Havenaar, ed.). Soukup, M. 1964 Separation of humic substances by gel filtration on Sephadex. Collection of Czechoslovakian Chemical Communications (1964) p. 3182. Stumm, W. & Morgan, J. J. 1970 Aquatic Chemistry. Wiley-Interscience. Swift, R. S., Thornton, B. K. & Posner, A. M. 1970 Spectral characteristics of a humic acid fractionated with respect to molecular weight using an agar gel. Soil Science 1x0, 93. Zobell, C. E. 1946 Marine Microbiology. Chronica Botanica, Waltham, Massachussetts (Quoted by Jerlov, 195:;).
and Coastal
Marine
Science (I 977) 5, 309-3 17
Transmission Spectroscopy Examinations of Natural Waters C. Ultraviolet Spectral Characteristics of the Transition from Terrestrial Humus to Marine Yellow Substance
Murray
Brown
Institute of Physical Haraldsage 6, 2200
Oceanography, University Copenhagen N, Denmark
Received
1975
12 August
and in revised form
of Copenhagen, 25 November
1975
The author defines a spectral slope value for ultraviolet absorption spectra of natural waters, for use in a major study of spectral characteristics of Baltic waters. The results show a steepening in the spectra as total absorption decreases going from slightly saline waters of the northern Bothnian Bay to North Sea waters entering the Kattegat. This qualitative change is related to the selective loss of high molecular weight humic components of the yellow substance. The literature relating to spectral differences in molecular weight fractions of natural humus is reviewed. When spectral data are combined with salinity values, a categorization of water types is possible, giving Baltic surface, Baltic deep, and North Sea types. The various processes contributing to this differentiation are discussed, and an associated experiment examines the spectral effects of bacterial activity on marine humus.
Introduction It is especially spectra
when
difficult no clear
to attempt maximum
qualitative
may be found.
analysis of ultraviolet A well-known
example
or visible
absorption
of this is the spec-
trum of natural waters, marine as well as non-marine. In most cases the spectrum shows strong increase in absorption toward the short-wave end, with some indication of a shoulder near 280 nm. [A rare example of an absolute peak at 277 nm has been reported by Hanya & Ogura (1962) for water from the river Edogawa.] Brown (197p) has related this 280 nm shoulder to simple aromatic rings, especially those conjugated with carboxyl, carbonyl and phenolic oxygen, structures common in many models for humus. Figure I shows two typical Baltic seawater absorption spectra. The slope of the spectrum itself would appear to be the only remaining qualitative feature which might be exploited for interpretation. The best known inquiry into this matter was made by Kalle (1961, 1962, 1963, 1966), who measured an ‘extinction coefficient’ at 420 and 665 nm, and computed the ratio for these values for a number of natural water samples, as well as artificial model compounds. Kalle concluded on the basis of comparison of these ratios that ‘the mainland waters (tap water from Hamburg, water from moors, and extracts of leaves) tend to the phenol 309
310
M. Brown
humic acid type’, i.e. flatter spectra, ‘and seawater (water from the North Atlantic, the North Sea, and the Baltic) tends more to the melanoidines type’, i.e. steeper spectra. A previous study (Brown, 1974a) has examined this method, and pointed out serious objections in both interpretation and technique. Chief among these being the extremely small value which could possibly be determined with short cells in the visible wavelength region, and a systematic dilution effect which can drastically alter slope values.
II
I 250
I
I1
I
I, 300 Wavelength
I
I
I
I 350
(nm~
Figure I. Two typical absorption spectra of filtered Baltic seawater. The lower curve corresponds to the central Baltic, while the upper is typical of the Bothnian Sea.
Brown (1974a) showed that carefully prepared dilutions of one humic water sample showed an increasing slope value with decreasing concentration. Reduction by as much as one-half the calculated value can occur, for slopes in the visible as measured by Kalle. This is strong evidence that seeming qualitative changes in absorption spectra can be artifactual rather than real. The effect is greater for wider wavelength intervals, and to some degree less serious in the U.V. than in the visible for any given wavelength interval. Both problems seem to be eliminated when a short wavelength interval (< IOO nm) is specified, i.e. for reduction of dilution effect, and absorption is studied in the ultraviolet, i.e. for greater accuracy. The present study consists of a collection of absorption measurements of Baltic seawater, at two narrowly spaced short wavelengths to determine to what extent Kalle’s findings could be verified, when the ‘dilution effect’ is minimized, and a discussion of interpretation based on the humus literature. Method During the November 1974 cruise of the Swedish Fisheries Ministry research ship ‘Argos’ through the Kattegat, Danish Sound, Baltic proper, Bothnian Sea and Bay, sampfes were taken in all-plastic samplers from the surface and bottom, or in some cases from representative intermediate depths. These were left standing open for one hour to allow oxygen saturation, then filtered by glass syringe through Whatman GF/c filters into lo-cm cells.
Transmission spectroscopy of natural waters
31’
Absorbance (2 --log y. T) measurements were made in a Perkin-Elmer 139 Spectrophotometer at 380 nm and 280 nm OS.double-distilled water as reference. A cell-to-cell correction was made. As the samples are filtered, particle (> I p) scattering may be ignored. In principle some colloid scattering might occur, but this is much too small to affect measurements in a typical double-beam spectrophotometer. Scattering due to the water itself is compensated by the reference cell. The wavelength calibration of the instrument was checked several times throughout the voyage by matching the selector reading to a known peak in the source spectrum. No drift was observed for a ‘warm’ instrument which had been calibrated when cold. The repeatability of the measurements is demonstrated by the fact that a double-cruise up and down the Baltic over a period of one month consistently produced values falling within the simple pattern discussed below. Results For some time now measurements of light absorption at 380 nm have been made to determine the content of the ‘yellow substance’ (Jerlov, 1968, pp. 55-57, 164-165). For greater sensitivity, since ro-cm cells were employed, it was considered more useful to compare ultraviolet absorption values as measures of humus.
I 0,.
07 5.
5 4,p 0.5 0
02 5,
,3"
3
IO
15 Salinity
20
25
30
35
(o/oo)
Figure z. ro-cm absorbanceat 280 ( x ) and 310 nrn (+) Baltic seawater WS.salinity.
Water twice distilled
of glassfibre-filtered in glass was used as reference.
Figure z shows IO-cm absorbance (2 -log o/oT) at 280 nm and 3 IO nm vs. salinity. 280 nm was chosen as it lies directly on the above-mentioned ‘shoulder’ in the transmission spectrum, while 310 nm has been shown to occupy a similar ‘shoulder’ position on the corrected fluorescence excitation spectrum of Baltic seawater (Brown, 19743). Both absorption and fluorescence excitation spectra are remarkably similar-excepting the exact position of
312
M. Brozon
these shoulders-with strong end rise below 250 nm. The figure shows that a close relationship between the salinity and absorption intensity is maintained. As many hundreds of separate streams feed the Baltic, and sampling positions were by no means confined to one area, then the salinity must be a strong factor in controlling the amount of organic materials remaining in solution. The convex shape of the 380 nm absorption as. salinity plot has been explained (Jerlov, 1955) by a precipitation of the humus; the same undoubtedly holds for the evidence of the present discussion. The yellow substance, then, is not truly conservative, but, as shown by the figure, functionally related to a conservative property-the salinity. Quantitative comparison of the values obtained in this study with those of many published studies is impossible, as absorption values at 380 nm were taken only at a few stations. It is possible, however, to calculate dissolved organic material, DOM, from total organic carbon, TOC, measurements made during the September 197-J cruise of the Finnish research ship Aranda, together with absorption measurements at 280 nm made by this author. Assuming DOM = z x TOC, a specific absorption coefficient, k, may be calculated as shown by Brown (1974~) equal to 19.0 (IO-cm cell, concentration in 81-l). For each sample a spectral slope, CD,has been calculated as:
These ratio values have been plotted in Figure 3 ws. absorbance at 310 nm. Plotting CDvs. absorbance at 280 nm gives a qualitatively identical figure. As the figure shows, a clear steepening in the absorption curves of natural humic waters occurs in the transition zone from the rivers to the open sea-the estuary. At the same time a great deal of dissolved material is lost, presumably to precipitation, as evidenced by the non-linearity of Figure 2. That there is a precipitation of humic materials is clear, as first demonstrated by Jerlov (1955) in experiments supporting his measurements of yellow substance in the Baltic. The theoretical possibility remains, however, that a part, at least, of the decrease in absorption could be due to hypochromicity of the humus macromolecules and colloids when condensing under saline conditions. Brown (197p) has discussed this possibility for natural water humus. A first examination of Figure 3 shows three separate categories of water types. A stylized representation of this classification is given in Figure 4. Here Type I refers to the high salinity (>Is%,,), low yellow-substance-containing waters of the North Sea entering the Baltic area via the Kattegat and Skagerak. Type II refers to waters of intermediate salinity (15%~ to 7.5x0), usually below the 50-m horizon in the Baltic proper; while Type III is assigned to the surface waters of low salinity (8x, to ozO). There is some overlap in salinity values between Types II and III. The approximate salinity ‘gradient’ has been supplied by inspection from Figure z. Bearing in mind that it is probably only North Sea water of a certain depth which can enter the Baltic over its 17-m sill, owing to density stratification, then an understanding of the relationships between the various masses and their mixing, requires that only a portion, not the whole, of Type I be considered as contiguous with Types II and III within the inland sea. This is important, as a wide range of values is found in these true marine waters. Figure 5 shows @ values US.depth for many stations taken in the Danish Belts and Sound and in the southern Kattegat during cruises of the Martin Knudsen, the research ship operated by the Danish government’s Environmental Ministry. A decrease in CDis apparent with depth. A typical value at the sill depth would be 2.10.
Transmission spectroscopy of natural waters
3’3
2-5
B
2c
.
If o-2
0
IO-cm A S,O-nm
Figure 3. Spectral slope values, CD,OS. lo-cm
I ’ %o
I
I
04
0.2
.
.
06
04
absorbance
at 310 nm.
I
06
AIO-cm
310-nm
Figure 4. Simplified schematic salinity values are approximate,
of the data presented in Figure 3. The indicated having been supplied by inspection of Figure 2.
The classical model for renewal of Baltic deep waters hypothesizes an inflow over the sill which entrains and reintroduces approximately one third of the overlying low salinity water which is flowing outward. This model is strongly supported by the fact that Type II lies at exactly the expected position in the present figures which would be called for by mixing between I (a = 2.1) and III (Q, = 225), on the basis of salinities. Although Ogura & Hanya (1966) have shown that nitrate and bromide contribute significantly to the total absorption in the wavelength interval 21~~230 nm, the possibility that variations in these species could give rise to the observed variations is slight, as they do not absorb appreciably above 230 nm, and the observed effects are the opposite of those which could be expected. A strong bromide interference would be characterized by systematic increase in slope values through the entire salinity range to a maximum at the highest values (not seen). A nitrate effect would cause deep water values (Type II) to be higher than those for surface water (also not seen).
314
M. Brown
.-..--
0
/’
.
I.
.
’ 0’ .
: .:*
If 0,‘.
l l . . .
.*m/ I I’
.
IO -
,.’
.
4
.
. . 5OL
’ I ,75
I 2.00
I 2.25
; !Cio
0
Figure 5. @ values vs. depth for water samples taken at stations in the Danish Belts and Sound, and in the southern Kattegat.
Discussion Several processes which could cause the indicated spectral changes suggest themselves: (I) biological production of a new dissolved component; (2) photo-activated oxidation introducing more oxygen-containing functional groups into existing molecules ; (3) bacterial oxidation and hydrolysis; (4) selective loss of a portion of the terrestrial humus which absorbs relatively more strongly toward the longer wavelengths than the ‘residual’ component. The possibility of the in situ production of a Baltic humic component peculiar to these waters, and responsible for absorption increase in the short wave end, has been discarded as there is no reason to believe that a special biochemical regime exists here. The spectral characteristics of the Baltic water finally produced by one or a combination of these processes is very different from open-sea marine yellow substance which is mainly the result of in situ biological processes. In addition, Figures 3 and 4 show that the alteration in the shape of the spectrum of Type III is very closely related to a loss in material rather than the opposite. Oxidation in the natural environment has been little studied, owing to obvious problems in microtechnique and determination. Gjessing & Gjerdahl(1970) have made the only study known to this author on the reduction of natural aquatic humus by solar radiation. They showed that ‘about 207$ of the reduction of colour between the inlet and outlet of lake Byvatn (in Norway) is due to natural U.V. radiation’ during a 75-day retention in the lake. Retention time for the whole Baltic system has been roughly estimated by Fonselius (1972) to be about 22 years; and, during the winter at least, the upper 50 m or so, the least saline, are well mixed. For these reasons we can assume that the total complement of humic materials is affected to some degree by photo-oxidation. That this process could induce the indicated spectral steepening is possible, but unsubstantiated.
Transmission
spectroscopy
of natural
waters
315
Bacterial oxidation and hydrolysis of macromolecular substances in solution has only recently become an area of active inquiry. Naturally, the surface areas provided by suspended particles are important in this process; Khailov & Finenko (1969) studied various bacterialsolute interactions at this interface, and gave approximate rate constants for organic transformations. However, Postma (1969), Jannasch (1967) and, originally, Zobell (1946) have pointed out that due to (a) the extremely low substrate concentration, and (b) sparse available surface area in situ, bacterial utilization of dissolved organics in seawater is very small. As discussed by Postma (1969), storage of seawater in bottles should hasten bacterial processes. A small experiment was performed to see what spectral change occurred in bottle-stored samples over a long period of time. (I) Samples taken in the Bothnian Sea and Bay during the September 1973 cruise of the Finnish research vessel Aranda, were stored unfiltered for 14 months in Ioo-ml brown glass bottles with glass stoppers. The bottles had been cleaned in acid, then base, and rinsed in alcoholic KOH to remove contaminants, especially surfactants, before use. (2) Transmission spectra of filtered aliquots were run immediately upon return to the laboratory (1-2 weeks after sampling) and again after 14 months. IO-cm cells were employed, and the reference water was prepared in exactly the same manner both times, i.e. passage through activated carbon and membrane filtration. The results of this study, given in Table I, show that storage in this manner, presumably accelerating bacterial processes while discriminating against photo-oxidation, consistently gives a reduction in CDvalues, that is-a flattening of the curve. In one case there was even an increase in absorption at the longer wavelength, which might be accounted for by particle degradation and dissolution. It is likely, therefore, that bacterial activity plays a minor role in the observed spectral transformation. TABLE
I.
Transmission spectral changesafter
Sample” I-3
Initial values /p-m @ 110nnl
II-Z
0.980 0.497
I.715 1.780
III-I RR-8 KOK-A
0.387 0’340 0'301
I ,869 “9’4
VII-2 VII-4
0.266
Spec-1
0.238 0’212
IX-I
0'252
I .g8z
14
months storage
Final values /p-cm Q 310nm 0.740 0.434 0.366 0.338 0.265
1.643 1.738
0.248 0.228
“994
2.068 2.063 2.141
0'221
2’143
0.228
I.796 1.807 1.845 2.017
“995 1-d
% ;to~c in 310nm
% $gge
-22.5
-42
-12.6 - 5’4 - 0.6
-2.4
- 6.9
-12.0
+
-3’9 -- 5’9
6.8 9’5 7” 7.6
-3.6 -2.7 -6.8 -8.7
‘Sample designationsrefer to sampling stations annually occupied by the Finnish Marine ResearchInstitute, Helsinki, with the exception of Spec-x near regular station VII-4. From a thermodynamic standpoint, greater molecular or particle size is favorable for coagulation in an ionic medium (Stumm & Morgan, 1970, p. 498). It is suggested therefore that removal of total color from terrestrial waters entering the marine environment will proceed in the order colloids > macromolecules > smaller molecules. Brown (1975a) has shown that a decrease in high molecular weight material (> IO ooo) with increasing salinity occurs in the northern Baltic. The possibility exists, therefore, that the observed spectral changes could partially be explained by a selective coagulation process,
316
M. Brown
if evidence could be produced that the higher molecular weight is indeed different in its spectral properties from the lower weight material. Butler & Ladd (1969) h ave studied the spectra of humus and report a close correlation between carboxyl groups and absorption at 260 nm. With increasing molecular weight, the relative abundance of the carboxyl groups-and therefore absorption at 260 nm-decreases. Presumably, they refer to carboxyl groups conjugated with aromatic or other oxygencontaining moeties, since aliphatic carboxylic acids show absorption maxima near 200210 nm; benzoic acid, on the other hand (C,H,COOH) has absorption bands at 228 and 279 nm. This relates well to Brown’s (1974~) model hypothesizing substituted aromatics in general to explain the broad shoulder near 280 nm in absorption spectra of natural waters. Kononova & Bel’chikova (1960) found that ratios of extinction at 230 nm to 310 nm, or 230 to 320 nm are low for ‘complex particles’, that is high molecular weight humus, and high for smaller molecules. They go on to quote several other studies as showing that higher weight material has a less steep spectrum than low weight material. Swift, Thornton & Posner (1970) state, after spectral examination of humus fractions, ‘(the) visible portion of the spectrum shows that the lower molecular weight fractions exhibit a greater slope through the visible region than the high molecular weight fractions’. Finally, Soukup (1964) found that ratios of absorption at 465 nm to 619 nm increase with decreasing molecular weight. These results lend support to the contention that a gradual reduction of the colloidal and high molecular weight fractions due to salinity flocculation should assist, at least, in the observed changes. That this is not the whole picture is, however, quite clear as shown in Figure 4 where Type II, the Baltic deep water, has slope values lower than the overlying less saline waters. It would appear that the ‘ripening’ process occurs only appreciably in the surface waters up to the point where a certain fraction of the outflowing surface waters are entrained by the Type I inflow over the Baltic sill. The new mixture, Type II, retains its spectral characteristics from that point with little change. This does not, as it would seem at first, weigh the evidence more heavily for photo-oxidation-which certainly could not occur appreciably below 50 m-as it is also known that coagulation processes due to salinity are repressed at high salinities due to formation of a charge anti-layer. Brown (19;~~~ has found a reduction in flocculation rate of humus for high salinities in laboratory
Conclusions Very certainly there is a change in the optical properties of terrestrial humic material which enters the Baltic by the time it reaches the deep sea. Whether this is due to photo-oxidative alteration, or selective removal of molecular weight fractions, or a combination of the two together with some bacterial activity is not yet known. However, the optical classification system (Figure 4) afforded by these changes could be of great value in studies of water exchange and mixing at the mouth of the Baltic. There are some preliminary indications that departures from the model for spectral characteristics combined with salinity, as shown in Figure 4, constitutes a reliable index of unusual chemical conditions. In a recent study concentrated organic matter taken from various locations, Kerr & Quinn (1975), found that humic substances from the soil display an inverse relation between absorption intensity and spectral slope-corresponding to the right hand side of Figure 3-while open marine material shows a direct relation-as on the left side of the figure. Their con-
Transmission
spectroscopy
of natural
waters
317
elusion, that this difference constitutes good evidence for different sources and/or diagenetic processes (Kerr & Quinn, 1975, p. 114) is emphatically supported by the present results. Acknowledgements The author is grateful to the governments of Finland, Sweden and Denmark for permission to participate in various cruises, and many acts of courtesy. Special personal thanks are due to Dr Aarno Voipio, Helsinki, Docent Stig Fonselius, Gothenburg and Professor Niels Jerlov, Copenhagen. References Brown, M. 197qz Transmission spectroscopy examinations of natural waters; A. On standardization of optical terminology in oceanographic investigations; B. Interpretation of spectra. Reports of the Institute of Physical Oceanography, University of Copenhagen, Denmark 28, 26 pp. Laboratory measurements of fluorescence spectra of Baltic waters. Reports of the Brown, M. 1974 Institute of Physical Oceanography, University of Copenhagen, Denmark 29, 19 pp. Brown, M. I975a High molecular weight material in Baltic waters. Marine Chemistry, 3, 253-258. PhD Dissertation, University of Brown, M. 1973b Humic particle formation in saline admixtures. Copenhagen. Butler, J. H. A. & Ladd, J. N. 1969 Effect of extractant and molecular size on the optical and chemical properties of soil humic acids. Australian Journal of Soil Research 7, 229. Fonselius, S. 1972 On eutrophication and pollution in the Baltic Sea. In Marine Pollution and Sea Life. (Ruivo, M., ed.) FAO, Fishing News (Books) Ltd. Gjessing, E. T. & Gjerdahl, T. 1970 Influence of ultra-violet radiation on aquatic humus. Ihtten 29 144. Hanya, T. &-Ogura, N. r962 Application of ultra-violet spectroscopy to the examination of dissolved organic submstances in water. In Advances in Organic Geochemistry. Proceedings of the International Meeting in Organic Chemistry, Milano (Colombo & Hodson, eds) 447 pp. Jannasch, H. W. 1967 Growth of marine bacteria at limiting concentrations of organic carbon in seawater. Limnology &Y Oceanography 12, 264. Jerlov, N. 1955 Factors influencing the transparency of the Baltic waters. Reports from the Oceanographic Institute in Gothenburg 25, 18 pp. Jerlov, N. 1968 Optical Oceanography. Elsevier Oceanography Series, Amsterdam. Union Geodesquie et Geophysique International, Kalle, K. 1961 What do we know about Gelbstoff? Monographie IO, 59. Kalle, K. 1962 uber die gel&ten organisehen komponenten im Meerwasser. Kieler Meeresforschungen 18, 128.
Kalle,
K. 1963 Uber das Verhalten und die Kerkunft in den Gewassern und in der Atmosphlre vorhandenen himmelblauen Fluoreszenz. Deutsche Hydrographische Zeitschrift 16, I 53. Kalle, K. 1966 The problem of the Gelfstoff in the sea. Oceanography and Marine Biology, Annual Review 4, 91. Kerr, R. A. & Quinn, J. G. 1975 Chemical studies on the dissolved organic matter in sea water. Isolation and fractionation. Deep-Sea Research 22, 107. Khailov, K. M. & Finenko, 2. Z. 1969 Interaction of detritus with high-molecular weight components of dissolved organic matter in sea water. Oceanology 8, 776 (English). Kononova. M. M. & Bel’chikova. N. P. 1060 Investigation of the nature of soil humus substances bv fractionation. Soviet Soil Science (196b), p. I r49: Ogura, N. & Hanya, T. 1966 Nature of ultraviolet absorption of sea water. Nature 212, 758. Postma, H. 1969 Dissolved organic matter in the oceans. Advances in Organic Geochemistry 1968, International Series of Monographs in Earth Sciences, vol. 3r (Schenck & Havenaar, ed.). Soukup, M. 1964 Separation of humic substances by gel filtration on Sephadex. Collection of Czechoslovakian Chemical Communications (1964) p. 3182. Stumm, W. & Morgan, J. J. 1970 Aquatic Chemistry. Wiley-Interscience. Swift, R. S., Thornton, B. K. & Posner, A. M. 1970 Spectral characteristics of a humic acid fractionated with respect to molecular weight using an agar gel. Soil Science 1x0, 93. Zobell, C. E. 1946 Marine Microbiology. Chronica Botanica, Waltham, Massachussetts (Quoted by Jerlov, 195:;).