Impact of UV254-radiation on aquatic humic substances

Chemosphere, Vol. 33, No. 5, pp. 783-790, 1996

Pergamon

PII: S0045-6535(96)00233-0

Copyright © 1996 Elsevier Science Ltd Printed in Great Britain, All rights reserved 0045-6535/96 $15.00 + 0.00

I M P A C T O F UV~54-RADIATION ON A Q U A T I C H U M I C S U B S T A N C E S

Maaret Kulovaara"l, Nina Corin, l Peter Backlund~ and Jari Tervoa

~Department of Organic Chemistry, Abo Akademi University, FIN-20500 Turku/Abo, Finland ~'I~rku Regional Institute of Occupational Health, FIN-20500 Turku/~.bo, Finland 3Defence Forces Research Centre, Department of Chemistry, P.O.Box 5, FIN-34111 Lakiala, Finland

(Received in Germany 27 April 1996; accepted 24 May 1996) Abstract

Aqueous solutions of humic substances (humic and fnlvic acids) were exposed to UV-radiation at 254 nm (UV2s4). The induced changes in the dissolved organic carbon content (DOC), in the absorbance of radiation at 272 nm (A~7~), and in the solid state lsC NMR spectral data were recorded. During the first 40-h exposure of the humic acid solution, the DOC-normalised UV~72-absorbance (A272/DOC ratio) was approximately constant, indicating a fairly high minerallsation rate as compared to the rate of the degradation of UV2n-absorbing structural subunits. During prolonged exposure, however, the minera].isation rate was clearly retarded. The corresponding normalised values for the fulvic acid solution demonstrated that the relative "rate of mineralisation" was constantly lower. Fulvic acid was more susceptibile for UV-initiated degradation than humic acid. The NMR spectra revealed that the high-molecular-weight matter in both of the exposed samples changed to a more aliphatic character and that more carboxyl and carbonyl carbon atoms appeared. Copyright ~> 1996 Elsevier Science Ltd Key words - humic substances, UV~s4-radiation, photochemical degradation, DOC, UV~72-absorbance, solid state J3C NMR

Introduction

Humic substances are ubiquitous, and are found wherever organic matter is undergoing decomposition. There are two major fractions of aquatic humie substances: humic acid and fulvic acid, which are conveniently isolated from acidified natural waters by sorption onto XAD resins [1]. In addition to

783

784 this, so called, hydrophobic acidic matter, trapped (together with some neutral matter) on the XAD8 resin at pH 2, a pool of hydrophilic organic matter and also some positively charged, originally hydrophobic, bases will pass through the resin column. Structurally still ill-defined humic substances consist of aromatic ring structures with aliphatic sidechains, mainly oxygen containing functional groups, "bridge" units consisting of oxygen or nitrogen, and associated carbohydrate or peptide fragments. The average molecular weight of humic acid is higher than that of fulvic acid [2]. Aquatic fulvic acids sometimes show slightly higher oxygen content than humic acids from the same origin [2]. Different spectrophotometric and spectroscopic methods have often been applied to study and to characterise humic substances, and the most promising nondegradative technique for humic structural analysis is NMR (nuclear magnetic resonance) spectroscopy [3l. The use of the Fourier Transform NMR technique allows rapid data accumulation which is needed for studies of isotopes, such as 13C, with low natural abundance. The cross-polarisation magic-angle spinning (CP/MAS) technique, in turn, allows acquisition of 13C NMR spectra from solid samples, which masters problems caused by the limited solubility of humic substances in NMR solvents [3]. Global solar radiation consists of ultraviolet (UV) radiation with wavelengths of approximately 290400 nm, visible (Vis) radiation of 400-700 nm, and infrared radiation > 700 nm. About 4% of the total energy in the global sunlight occurs in the UV band, but the intensity varies greatly with latitude, season, time of day, and thickness of the atmosphere and the ozone layer [4]. Shorter wavelengths imply higher energies and also a higher potential to destroy organic matter. Lately, the consequences of increased global UV-radiation which is caused by the decrease in the thickness of the ozone layer, have been of growing concern. Much attention has been focused on the effects of artificial radiation in the short-wave UV-region because of its strong ability to disinfect. Such radiant energy has been used, not only to inactivate microorganisms but also to degrade residual organic matter in drinking water [5]. The maximal germicidal effect is obtained at about 260 nm and the most efficient source for the generation of almost monochromatic radiation close to this wavelength (253.7 nm = UV2s~) is the low-pressure mercury discharge lamp [5]. The degradative power of artificial UV-radiation on aquatic humic substances is well documented (e.g. [6-9]). Various aliphatic, low-molecular-weight (LMW) carboxylic acids are known to be formed during the UV-light exposures [8,9]. According to Allart et al. [9] the acids seemed to be further degraded by continued irradiation. Photochemical aspects related to humic substances based on UV-experiments has been discussed by Frimmel [10]. Natural radiation from the sun is known to fade the colour of surface waters (reviewed in [11]), and to change the absorbance and fluorescence properties of the samples [11-13]. Natural photochemical mineralisation of the dissolved organic matter of a moderately humic lake was recently recorded by Salonen and Vahgtalo [14] as an increase in the amount of dissolved inorganic carbon. The authors emphasised that the short-wave UV range of solar radiation, UV-B, with wavelengths < 320 rim, is the most important mineraliser. LMW cleavage products, other than inorganic carbon compounds, which are formed during light-initiated degradation reactions in the environment, such as simple organic acids, sugars, and amino acids, are easily utilised by biota as a source of carbon and other elements [12,15]. Recent results by Milnster et al. [16] and Heikkinen and Miinster [17] indicate that enzymatic activities are also involved in natural degradation processes in polyhumic lake waters.

785 In this work we have utilised tsC NMR spectroscopy to study the structural changes of dissolved humie substances (isolated humie and fulvie acids), caused by the UV-light energy. In addition, the ehange.,~in the UV-absorbanee (272 nm) and the dissolved organic carbon (DOC) contents were recorded.

Materials and m e t h o d s

Water sam~ples. Two samples each of Nordic reference fulvie acid (NoFA) and humic acid (NoHA) were dissolved in water. Bottled high-performance liquid chromatography-grade water, "Aqua sterifisata" (Kabi Pharmacia), was used as a solvent. The concentration of the NoFA samples were 0.30 mg/ml (150 rag/500 mill, whereas the two NoHA solutions were more diluted, with concentrations of 0.125 mg/ml (100 rag/800 ml), because of solubility problems. Furthermore, the NoHA samples had to be filtered (0.45 t~m membranes, Millipore) in order to get clear solutions without suspended material. The pH of the NoFA solution was 2.6 and of the NoHA solution 3.6.

Irradiation of water samples. The aquatic humic samples were irradiated at room temperature in two parallel quartz glass cylinders, 5 em (o.d.) x 40 em each, using six parallel UV lamps (Philips TUV, 15 W). The lamps were installed in a reflector and placed at 20 cm distance from the sample. According to the manufacturer, the radiation intensity of a 15-W lamp, at a distance of 1 m, is 42 uW/em 2. The solutions were stirred by a magnet during the exposure. The irradiation times were 40 h and 80 h, respectively. Separate aliquots of a third NoFA solution were sampled after 8, 16, 24, and 32 h of irradiation and used for A272 and DOC measurements.

Determinaiion of DOC. The DOC contents of the aquatic samples were determined on a Shimadzu Total Organic Carbon 5050 Analyzer equipped with an automatic sample injector, Shimadzu ASI 5000.

Measurement of light absorbances. Absorbances of the humic solutions at 272 nm were recorded using a Shimadzu UV-160 A UV-Vis spectrofotometer.

Extraction and concentration of samples. In order to remove LMW degradation products formed during the fight exposures, the NoFA solutions were extracted by four portions of redistilled diethyl ether (70 + 20 + 20 + 20 ml). The NoHA samples, being of larger volumes, were correspondingly extracted with 120 + 40 + 40 + 40 ml. The water phas~ containing the remaining high-molecular-weight (HMW) material were then concentrated by rotm'y evaporation (40°C) to 15-20 ml and the residual water was subsequently removed from the sample:~ by a vacuum freeze-dryer for three days. Unexposed solutions of NoFA and NoHA were treated in a similiar manner. Analysis of the etheral extracts has been published earlier [18].

NMR analyses. The NMR spectra were recorded on a Bruker AC 300 P Fourier Transform NMR spectrometer operating at 75.48 MHz. The following instrumental conditions were used: contact time 1 ms, repetition

786 delay 2.5 s, spectral width 41.7 kHz, exponential window function 50 Hz, 13C 90° (Hartmann-Hahn) pulse 5.3 us, number of datapoints 16 k, and a number of transients which varied between 8800 and 14000. The freeze-dried samples (50 - 80 rag) were packed into a 4 mm o.d. zirkonia rotor and, in case of a too small sample, the specimen holder was filled with a required amount of A1203. Crosspolarization magic angle spinning at 6 kHz was utilised. The carbonyl carbon of glycine (~¢ = 176.1 ppm from tetramethylsilane reference) was used as an external reference for chemical shifts. Signal areas of various distinct groups of carbon atoms were integrated instrumentally.

R e s u l t s and discussion

Light exposure of the samples initiated a degradation of the dissolved humie matter. Following exposure of the NoFA samples for 40 h and 80 h, the DOC values had decreased by 11.2% and 23.9%, respectively. The corresponding values for the NoHA samples were 27.1% and 34.8%, respectively. The NoFA seemed to mineralise at a relatively constant rate throughout the 80-h exposure period, whereas the mineralisation of NoHA proceeded more rapidly during the first 40 h and was then somewhat retarded. It should be noted that the structural changes in the NoFA and the NoHA versus time can not be directly compared because of different concentrations at the beginning of the exposure experiments. The absorption of light by humic substances in the UV and Vis wavelength ranges is due to conjugated unsaturated bonds and also to the presence of free electron pairs on heteroatoms. The absorbance of visible light near 400 nm is related to the intensity of the yellow colour caused by extended chromophoric systems in conjugated structures. The wavelength of 272 nm is the region of ~r-~* transitions in substituted benzenes and most polyenes but, as Gauthier et al. [19] pointed out, alternatively, the adjacent wavelengths such as 254 nm should give an equally useful correlation. In this work the degradation of olefinic/aromatic parts of the humic substances was recorded by measuring the absorbance at 272 nm. The overall course of the photolyses studied by spectrophotometric means simply reveal that the light exposure of the samples had brought about changes in the UV2~2-absorbing olefinic/aromatic units of the macromolecules. The A~2 decreased during irradiation. For the NoFA the decrease was somewhat faster during the first 40 h of exposure, but, for the NoHA the decrease proceeded at a relatively constant rate. In spite of the fact that the NoFA solution was more concentrated at the start of the experiment, the relative A~r2 of NoFA decreased more extensively (40 h: to 0.54; 80 h: to 0.34 relative absorbance units, AU) than that of NoHA (40 h: to 0.77; 80 h: 0.51 relative AU). This means that the degradation of the unsaturated structures of NoFA proceeds easier than that of the corresponding stuetures of NoHA. The "rates" of degradation were also evaluated by normalising the absorbance readings (A272) by the values of DOC in the corresponding samples (cf. Fig. 1). The starting A27~/DOC values are, in a way, a measure of the "quality" of the dissolved material. Natural waters with relatively high DOC contents might consist of numerous small-molecular and less UV-light-absorbing compounds, which results in low A2r~/DOC values. A high A2T2/DOC ratio, on the other hand, indicates a large proportion of UV-absorbing material.

787 As shown in Fig. 1, the DOC normalised graph of A~72 (A2n/DOC) versus time obtained for the NoFA differs from that of the NoHA during the first 40 h of the light exposure. The A272/DOC ratio for NoFA versus time resulted in a continuously descending line whereas the ratio for the NoHA gave a more or less straight horizontal llne. This indicates that, as compared to the rate of alteration of olelinic/aromatic units to intermediate, non-absorbing (272 nm) structures, the mineraiisation of NoHA was faster than that of NoFA during the first 40 h. Formation of simple aliphatic carboxylic acids (which do not absorb much UV-light _> 260 nm) upon UV-exposure of natural humic waters has been w.~rifiede.g. by Bscklund [8]. 0.06

O.O5 |

0.04

# NOFA NoHA

0.03 e~

.l .~

0.02 0.01

0

I 8

t 16

J 24

I 32

I 40

I 48

I 56

I 64.

I 72

80

Irradiation T i m e (b)

Fit~re 1. The DOC-normalised absorbance of UV~72-1ight versus exposure time for aqueous NoFA (0.30 mg/ml) and NoHA (0.125 mg/ml) solutions. The NoHA solution was more diluted than the NoFA solution because of the earlier mentioned solubility problems. But in spite of this, the relative losses of HMW material during the first 40 h of exposure was approximately equal in the NoHA (76.4%) and the NoFA solutions (80.6%). This also indicates that humie acids posess a greater resistance towards light-initiated degradation. During prolonged treatment, the relative decrease in the HMW NoFA material was even higher (54.5~) than that of the l~umic acid solution (65.2%). The CP/MAS 13C NMR spectra displayed six - in some spectra seven - distinct groups of carbon signals: aliphatic carbons and methoxy carbons, carbohydrate carbons, olefinic and aromatic tertiary carbons, aromatic quaternary carbons and oxygen-, nitrogen-bonded aromatic carbons, carboxylic carbons and. amide carbonyl carbons, as well as aldehyde and ketone carbonyl carbons (el. Fig. 2). Methoxy groups at 55.7 ppm could most clearly be distinguished in the starting spectrum of NoHA. The areas of the signal groups were integrated, whereby the aliphatic (methyl, methylene, and methine) signals and the possible methoxy signals were integrated together. The normalised relative intensities of the groups are presented in Table 1. The signal intensities of the olefinic/aromatic, the aromatic quaternary, the phenolic and the nitrogen-bonded aromatic carbon groups at 90-157 ppm are combined. The lsC NMR spectra in Fig. 2 reveal that, during the UV2s4-exposure the olefmic/aromatic part of the samples underwent a more rapid degradation than the aliphatic one. The same trend can be

788 seen when the spectra are expressed as relative signal intensities (Table 1). During the irradiation, the unsaturated carbon-carbon bonds with chemical shifts of 90-157 ppm in the remaining HMW material changed in favour of aliphatic structures at 0-60 ppm. This change was more obvious for the NoHA reference substance which had a pronouncedly aromatic character at the start of the exposure (Table 1). At the end of the experiment the NoHA 13C NMR spectrum appearing at 0-185 ppm resembles the spectrum of unexposed NoFA. The amount of carboxylic (157-185 ppm) and carbonyl (185-222 ppm) functional groups increased in both samples during the experiment.

NoHA

NoFA

•

Oh

i

i

200

i

100

I

0

I

i

i

200

i

100

i

0

ppm

40 h

40 h

i

i

200

i

100

i

0

"5

i

i

i

200

ppm

80 h

i

i

l~m

i

IO0

I

0

ppm

80 h

i

200

I

I O0

Fieure 2. Solid state ( C P / M A S ) IsC N M R after different times of UV2~-exposure.

i

0

I

ppm

;

a

200

i

100

l

0

i

ppm

spectra of humic reference substances N o H A

and N o F A

Visser [20] studied the effects of the germicidal UV-radiation on solutions of humic matter by means of calculated H:C and O:C atomic ratios based on the results from elemental analyses. He found that the studied material was partially oxidised and had lost part of its aromaticity which resulted in the formation of highly oxygenated aliphatic compounds with high H:C and O:C atomic ratio values. In the same work, fulvic acid was shown to be more susceptibile to degradation by UV-irradiation than humic acid. This observation has also been made by Allart et al. [9].

789 Table 1. Relative signal intensities of different types of carbon atoms in ~aC NMR spectra of humic reference substance samples irradiated with UV~54-radiation.

*I= II = III= IV = V= VI=

Irradiation Sample time (h)

I*

II

III + IV

V

VI

0 4:0 80

NoFA NoFA NoFA

34.7 32.5 36.8

18.8 18.3 17.0

28.5 29.3 23.4

15.2 16.3 18.8

2.8 3.6 4.0

0 40 ~;0

NoHA NoHA NoHA

27.5 30.9 31.9

17.7 16.3 16.1

36.7 32.2 26.8

13.3 15.8 16.4

4.7 4.9 8.8

0-60 ppm, aliphatic carbons + methoxy carbons (at 55.7 ppm) 60-90 ppm, carbohydrate carbons 90-141 ppm, olefinic + aromatic carbons 141-157 ppm, aromatic quaternary carbons + oxygen, nitrogen bonded aromatic carbons 157-185 ppm, carboxylie carbons + amide carbonyl carbons 185-222 ppm, carbonyl carbons (aldehydes + ketones)

1H NMR spectroscopy has been used to study the changes in the natural organic solute fractions of lake water in the HUMEX project to evaluate the effect of acid rain [21]. In the present work, the light-induced degradation processes of isolated humie reference substances were initiated by artificial UV-radiation and studied by lzC NMR. The next interesting task would be to use the same technique to study the corresponding effects of natural radiation on the HMW part of the dissolved organic matter in natural humic surface waters. A better understanding of the photolyses of the aquatic humic substances is needed considering the fact that increased global UV-radiation causes changes also in the aquatic ecosystem.

Acknowledgements - This study was partly financed by the Ella and George Ehrnrooth Foundation and the Oskar Oflund Foundation, which is gratefully acknowledged.

References

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790 4. R.A. Larson and M.R. Berenbaum, Environmental phototoxicity, Environ. Sci. Technol. 22, 354-360 (1988). 5. W.J. Masschelein, Behandeling van water door middel van ultra-violet licht, H~O 19, 348-359 (1986). 6. F.H. Frimmel and S.A. Huber, Liquid chromatography with three dimensional detection of humic substances and oxidation products, Finnish Humus News 3 (3) 127-132 (1991). 7. E.T. Gjessing and T. Kfillqvist, Algicidal and chemical effect of u.v.-radiation of water containing humic substances, War. Res. 25,491-494 (1991). 8. P. Backlund, Degradation of organic humic material by ultraviolet light, Chemosphere 25, 1869-1878 (1992). 9. B. Allart, H. BorOn, C. Pettersson and G. Zhang, Degradation of humic substances by UV irradiation, Environ. Int. 20, 97-101 (1994). 10. F.H. Frimmel, Photochemical aspects related to humic substances, Environ. Int. 20, 373-385 (1994). 11. D.J. Strome and M.C. Miller, Photolytic changes in dissolved humic substances, Verb. Int. Verein. Limnal. 20, 1248-1254 (1978). 12. A.J. Stewart and R.G. Wetzel, Dissolved humic materials: Photodegradation, sediment effects, and reactivity with phosphate and calcium carbonate precipitation, Arch. Hydrobioi. 92, 265286 (1981). 13. H. De Haa~a and T. De Boer, UV-degradation of aquatic humic substances, Finn~h Humus News 3 (3) 177-182 (1991). 14. K. Salonen and A. Vahatalo, Photochemical mineralisation of dissolved organic matter in Lake Skjervatjern, Environ. Int. 20, 307-312 (1994). 15. C. Steinberg and U. Mfinster, Geochemistry and ecological role of humic substances in lakewater. In Humic Substances in Soil, Sediment, and Water(Edited by G.R. Aiken, D.M. McKnight, R.L. Wershaw, and P. MacCarthy) pp. 105-145. Wiley-Interscience, New York (1985). 16. U. Miinster, E. Heikkinen, K. Salonen and H. De Haan, Significance of Ligninperoxidase (LIP) activities in polyhumic lakes. Abstract of the Fifth Nordic Symposium on Humic Substances in Soil and Water, p. 16. University of Lund, Lund, Sweden (1995). 17. E. Heikkinen and U. Mfinster, Studies on microbial lignin peroxidase (LiP)-activity in polyhumic lakes - A methodological approach. Abstract of the Fifth Nordic Symposium on Humic Substances in Soil and Water, p. 49. University of Lund, Lund, Sweden (1995). 18. N. Corin, P. Backlund and M. Kulovaara, Degradation products formed during UV-irradiation of humic waters, Chemosphere, 33, 245-255 (1966). 19. T.D. Ganthier, W.R. Seitz and C.L. Grant, Compositional variations of dissolved humic materials on pyrene Ko¢ values, Environ. Sci. Technol. 21,243-248 (1987). 20. S.A. Visser, Application of Van Krevelen's graphical-statistical method for the study of aquatic humic material, Environ. Sci. Technol. 17, 412-417 (1983). 21. R.L. Malcolm and T. Hayes, Organic solute changes with acidification in Lake Skjervatjern as shown by XH NMR spectroscopy, Environ. Int. 20, 299-305 (1994).