Study of the eVect of pH, salinity and DOC on fluorescence of synthetic mixtures of freshwater and marine salts Valdemar I. Esteves,* Eduarda B. H. Santos and Armando C. Duarte University of Aveiro, Department of Chemistry, 3810 Aveiro, Portugal Received 29th March 1999, Accepted 28th April 1999 In order to provide support for the discussion of the fate of organic matter in estuaries, a laboratory simulation was performed by changing freshwater ionic strength, pH and organic matter content.The change in spectroscopic characteristics caused by variations in salinity, pH and organic matter concentration in the filtered samples was observed by UV-Vis and fluorescence spectroscopy. The increase in emission fluorescence intensity of dissolved organic matter (DOM) due to increasing salinity (in the range 0 to 5 g l-1) is aVected by the pH of the samples. The emission fluorescence intensity at the three maxima observed in the fluorescence spectra, is linearly correlated with dissolved organic carbon (DOC) concentration at several salinity values in the same sample.The increase in organic matter concentration caused a shift in the emission peak wavelength at 410 nm for several salinity values.We concluded that it is necessary to take into account the influence of salinity and pH on emission fluorescence of dissolved organic matter if it is to be used as a tracer in estuarine or near shore areas.Methodology for studying the eVects of the environmental Introduction parameters The behaviour of riverine dissolved organic carbon (DOC) Adequate amounts of freeze dried sea salts were added to when entering an estuarine zone has been studied.Some aliquots of filtered freshwater in order to obtain samples with authors1,2 suggested that fractions of DOC are removed by salinity values of 0 (no adjustment), 2, 5, 10, 15, 25 and mechanisms such as flocculation, precipitation, microbial 35 g l-1. The samples were then adjusted to pH 8.0 with NaOH degradation and particulate adsorption while some others3–5 and HCl and left overnight in a darkroom.The following day, found that DOC shows an overall conservative behaviour samples were filtered through muZed (400 °C, 24 h) GF/F in estuaries. (Whatman, Maidstone, Kent, UK) filters. Filtered samples Fluorescence spectroscopy has been extremely useful as a were characterised by spectral analysis in a UV-Vis spectropho- technique for monitoring dissolved organic matter3 in freshtometer [Shimadzu (Du� sseldorf, Germany) Model UV 2101 water, estuarine or nearshore areas.Both UV-Vis and fluores- PC] and in a fluorescence spectrophotometer (JASCO, Tokyo, cence spectroscopy have also been used to quantify DOC in Japan, Model FP-770).aquatic environments.6,7 In this paper, we report the results Emission fluorescence intensities were measured at 410 nm, obtained in laboratory experiments of the eVects on UV-Vis 440 nm and 460 nm for an excitation wavelength of 360 nm, and fluorescence spectra of changing pH, salinity and DOC which corresponded to the three maxima for the freshwater concentration of freshwater mixed with prepared salt water.sample. The width of the excitation and emission lines was 5 nm. The wavelength accuracy and repeatability were Experimental ±1.5 nm and ±0.3 nm respectively (data from the manufacturer). Preparation of freeze dried salts from coastal water For each salinity value a sample of filtered water was Five litres of seawater from a non-polluted beach (Costa subsampled to obtain pH values of 7.0, 8.0 (no pH correction) Nova, Portugal ) were filtered [0.45 mm Gelman (Ann Arbor, and 8.5 with NaOH and HCl.The samples obtained were MI, USA) membrane filter]. In order to oxidize organic matter characterized by fluorescence spectroscopy. to CO2, the seawater was irradiated with UV radiation In order to study the eVect of dilution, for each salinity (1000 W; l=254 nm) over 12 h: six drops of H2O2 (30% by value, the samples at pH 8.0 were diluted with deionized water volume) were added to 200 ml quartz tubes filled with seawater, prepared at the same salinity as the samples and again and the seawater was exposed to UV radiation.After a 12 h characterized by fluorescence spectroscopy. exposure, the seawater was freeze dried to obtain the salts The fluorescence spectra were corrected for wavelength used for the preparation of solutions with diVerent salinities.dependent eVect using a dynode-feedback control system.8 The same procedure was performed with a series of blanks [Millipore (Milford, MA, USA) Milli-Q 50]. Sampling of freshwater Ten litres of freshwater were sampled in glass containers at Results and discussion Frei-Gil on the Mira channel of the Aveiro lagoon, Portugal.Conductivity, pH and temperature were measured in the field. The eVect of salinity on the UV-Vis spectra The sample was filtered (Gelman 0.45 mm) in the laboratory. Dissolved organic carbon (DOC) of this filtered sample was The variations in absorbance with salinity of a freshwater sample are shown in Fig. 1 where the UV-Vis absorbance of measured using a Dohrmann (Santa Clara, CA, USA) DC-180 Carbon analyser. filtered samples is represented as a function of salinity for two J. Environ. Monit., 1999, 1, 251–254 251Fig. 1 EVects of salinity on freshwater absorbance (250 and 350 nm). Fig. 2 Emission fluorescence spectra of a freshwater sample for an excitation wavelength of 360 nm.wavelengths. A general trend is observed for the wavelengths studied: the UV-Vis absorbance decreases as the salinity increases. The DOC value of the samples was 3.5 mg l-1 of C, before increasing salinity. As the salinity was increased there was a visible increase (not measured) of particulate matter content retained in the GF/F filters associated with the decrease in UV-Vis absorbance of filtered samples.This suggests flocculation of dissolved organic matter (DOM). The extent of decrease in the UV-Vis absorbance due to the salinity is independent of wavelength: for instance, for wavelengths of 365 nm and 250 nm the UV-Vis absorbance decrease was 11% and 5%, respectively, calculated for a salinity value of 35 g l-1. Assuming a strong correlation between DOC and UV-Vis absorbance at 250 nm,7,9 the results confirm the occurrence of flocculation of DOM during mixing of freshwater and seawater.According to Sholkovitz1 3% to 11% of the river DOM is flocculated when in contact with seawater, which is in concordance with our results. The eVect of salinity and pH on emission fluorescence spectra A typical emission fluorescence spectrum of river water is shown in Fig. 2; three peaks are easily identified at wavelengths of 410, 440, and 460 nm. The change in salinity and pH of Fig. 3 Emission peak wavelength (lexc=360 nm), as a function of freshwater did not cause peak wavelength displacement in DOC concentration. Each point is the data average for salinities 0, 5, 15, and 35 g l-1. Confidence interval has a 95% confidence level.fluorescence emission spectra. However, an increase in DOC content for several salinity values caused a small but consistent shift in the first peak wavelength (attributed to Raman scattering or superimposed with the Raman peak) to higher in order to obtain samples with diVerent salinities and the same amount of organic matter. As shown in Fig. 4, the wavelengths as shown in Fig. 3.Correlating the DOC concentration with the wavelength of the maximum of the first peak increase in fluorescence intensity due to the increase in salinity is clear in the range of 2 to 5 g l-1 and nearly constant for of 32 solutions in a range of salinities between 0 and 35 g l-1 gave the equation y=(4.07×102)+0.75x, and r2=0.926, salinities greater than 5 g l-1.Willey10 attributed this fluorescence increase to the presence in seawater of magnesium which where y is the peak wavelength and x is DOC concentration (mg l-1 of C). The 410 and 440 nm peaks must not be used complexes with fluorescent fulvic and humic acids, enhancing fluorescence by crosslinking the structure between internal to evaluate the organic matter content of solutions with high concentrations, to prevent overlap of the two peaks.oxygens. This crosslinking aVec fluorescence by changing the positions and energy level of p electrons. Furthermore, removal The other two peaks did not produce any displacement with changes in organic matter content. of the quenching eVect of metals such as copper or iron by magnesium could enhance fluorescence.Although fluorescence intensity was aVected by salinity, the largest changes in fluorescence intensity were observed when The eVect of salinity on emission fluorescence intensity becomes more accentuated when the pH is higher. For pH= the organic matter content of the samples was varied. The increase in natural fluorescence intensity observed by 7.0 the increase in emission fluorescence intensity due to the increase in salinity from 0 to 35 g l-1 is 3% (lem=440 nm) and Willey10 during the mixture of freshwater and saltwater in estuaries, was reproduced in this laboratory in terms of salinity, 9% when the pH is 8.5 (lem=440 nm).The results obtained show that the increase in fluorescence due to the change of adding adequate amounts of freeze dried sea salt to freshwater 252 J.Environ. Monit., 1999, 1, 251–254Fig. 5 Fluorescence intensity as a function of DOC, for salinities (Sal.) of 0, 5, 15 and 35 g l-1; pH 8.0. The eVect of dissolved organic carbon content on the emission fluorescence intensity A series of samples with salinities of 0, 5, 15 and 35 g l-1 was used to study the relationship between DOC content and fluorescence intensity (Fig. 5). For each salinity, seven solutions were prepared with diVerent organic matter content, by diluting each solution with Milli-Q water with the same salinity. The fluorescence intensity was measured at three wavelengths: 410, 440, and 460 nm (lex=360 nm). For a given salinity, the fluorescence intensity is linearly correlated with Fig. 4 Variation of emission fluorescence intensity (at pH 7.0, 8.0 and the DOC concentration. The results for all salinity values in 8.5) with salinity (lem=$, 410 nm;&, 440 nm; and +, 460 nm; lexc= this experiment fitted straight lines with correlation coeYcients 360 nm).The Raman peak of Milli-Q water was subtracted from the better than 0.997. Any of the three emission wavelengths (410, fluorescence at 410 nm. 440 and 460 nm) could be used to quantify the content of organic matter, but 440 and 460 nm give higher sensitivity ( larger slope) than 410 nm. The good correlation between fluorescence intensity and pH from 7.0 to 8.5 is remarkable for salinities greater than 2 where this increase is around 10%. DOC concentration shows that in the range of 0 to 35 g l-1 salinity there are no inner filter eVects, hence this is not the Several authors3,5,11 have not observed the increase in fluorescence intensity during the mixture of freshwater and reason for the anomalous increase in fluorescence intensity with salinity. seawater, while others12 have noted a net influence of the salinity in the range 0 to 5, which can be explained by diVerent pH changes.Conclusions Emission spectra of 21 samples with salinities ranging from 0 to 35 g l-1 and pH values of 7.0, 8.0 and 8.5 were recorded The mixing of freshwater and seawater, in laboratory studies, removes 5% to 10% of dissolved organic matter as particulate, and no shift was observed in the three wavelengths corresponding to emission maxima.This fact indicates that the increase measured as absorbance decrease.Changes in ionic strength increase fluorescence intensity for salinities ranging between 0 in fluorescence due to the salinity or change in pH is not a result of a change in the chemical structure of the fluorophores. to 5 g l-1. The good correlation between fluorescence intensity and DOC concentration shows that, in the range of 0 to Therefore, it is possible to compare spectral intensities of samples with diVerent salinities and pH values. 35 g l-1 salinity, there are no inner filter eVects, hence this is J. Environ. Monit., 1999, 1, 251–254 2533 J. E. Dorsh and T. F. Bidleman, Estuarine Coastal Shelf Sci., 1982, not the reason for the anomalous increase in fluorescence 15, 701. intensity with salinity. 4 R. F. C.Mantoura and E. M.SWoodward, Geochim. Cosmochim. The increase (%) of emission fluorescence intensity (lexc= Acta, 1983, 47, 1293. 360 nm; lem=440 nm) due to salinity is aVected by pH and is 5 P. Berger, R. W. P. M. Laane, A. G. Ilahude, M. Ewald and higher for pH=8.5 (9%) than for pH=7.0 (3%). Increased P. Courtot, Oceanolog. Acta, 1984, 7, 309. concentration of organic matter, for several salinity values, 6 P.L. Smart, B. L. Finlayson, W. T. Rylands and C.MBall,Water Res., 1976, 10, 805. caused a small but consistent shift in the emission peak 7 H. De Haan and T. De Boer, Water Res., 1987, 21, 731. wavelength at 410 nm while for the other two peaks (440 and 8 H. Okahana and M. Arita, Model FP-770 Spectrofluorometer 460 nm) no displacement was produced. product description Jasco. Jasco International Co., Ltd. (4–21, To use emission fluorescence of dissolved organic matter as Sennin-cho 2-chome, Hachioji, Tokyo 193, Japan), 1987. a tracer in estuarine or nearshore areas it is necessary to take 9 R. S. Summers, P. K. Cornel and P. V. Roberts, Sci. Total into account the influence of salinity and pH on fluorescence Environ., 1987, 62, 27. intensity. 10 J. D. Willey, Marine Chem., 1984, 15, 19. 11 J. D. Willey and L. P. Atkinson, Estuarine Coastal Shelf Sci., 1982, 14, 49. References 12 S. E. Cabaniss and M. S. Shuman, Mar. Chem., 1987, 21, 37–50. 1 E. R. Sholkovitz, Geochim. Cosmochim. Acta, 1976, 40, 831. 2 E. R. Sholkovitz and D. Copland, Geochim. Cosmochim. Acta, 1981, 45, 181. Paper 9/02529D 254 J. Environ. Monit., 1999, 1, 251–254