Essential characteristics of natural dissolved organic matter affecting ...

Aquat. Sci. 66 (2004) 171– 177 1015-1621/04/020171-07 DOI 10.1007/s00027-004-0705-x © EAWAG, Dübendorf, 2004

Aquatic Sciences

Research Article

Essential characteristics of natural dissolved organic matter affecting the sorption of hydrophobic organic contaminants Jarkko Akkanen 1, *, Rolf D. Vogt 2 and Jussi V. K. Kukkonen 1 1

2

Laboratory of Aquatic Ecology and Ecotoxicology, Department of Biology, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland Department of Chemistry, University of Oslo, P.O. Box 1033, N-0315 Oslo, Norway

Received: 26 June 2003; revised manuscript accepted: 27 October 2003

Abstract. Association of benzo[a]pyrene (BaP), pyrene, 3,3¢,4,4¢-tetrachlorobiphenyl (TCB) and 2,2¢,4,4¢-tetrabromo diphenyl ether (TBDE) with natural dissolved organic matter (DOM) was studied. The DOM samples were previously collected from natural waters at five Nordic sites during fall 1999 and spring 2000, isolated by the reverse osmosis method, and thoroughly characterized. The purpose was to determine the essential characteristics that predict the sorption capacity of DOM for hydrophobic contaminants. DOM isolates were dissolved in artificial freshwater to give a dissolved organic carbon concentration of 15 mg L–1. Partition coefficients (KDOC) of the model compounds between water and DOM were measured by the equilibrium dialysis method. Further, the bioavailability of BaP and pyrene to Daphnia magna

was tested in the presence of DOM with various qualities. A strong negative correlation was found for the KDOC values of BaP (R = –0.922) and pyrene (R = –0.929) with spectral absorbency ratio (A254 /A400), while the correlation (R = –0.760) was weaker for the KDOC values of TCB. The KDOC values for TCB correlated (R = 0.849) strongly with specific visible absorbency (A600 /TOC), while the KDOC values for TBDE correlated (R = –0.739) with relative fluorescence emission. Generally, bioavailability of BaP and pyrene to D. magna reflected the measured association of the compounds with DOM. The data emphasize the importance of aromaticity of DOM, estimated by simple spectroscopic methods, in predicting sorption capacity for polycyclic aromatic hydrocarbons. The situation with halogenated compounds still remains unclear.

Key words. Polychlorinated biphenyl; polycyclic aromatic hydrocarbon; polybrominated diphenyl ether; partitioning.

Introduction Dissolved organic matter (DOM) is present in all natural waters, but DOM concentrations and properties may vary greatly among locations. For many ecological functions, the importance of DOM in the environmental fate of many contaminants has been shown in a large number of studies. Processes, such as transport, solubility, degradation, volatilization, and bioavailability of hydrophobic organic compounds, can be greatly affected by DOM (Piccolo, * Corresponding author phone: +358-13-251 3549; fax: +358-13-251 3590; e-mail: [email protected] Published on Web: June 16, 2004

1994). Therefore, it is essential to identify the properties that control the capacity of various DOM to sorb contaminants. A project called Natural Organic Matter in the Nordic Countries (NOMiNiC) (Vogt et al., 2001; 2004) was established to study natural organic matter in surface waters from five Nordic sites. One of the primary tasks was to thoroughly study the properties of DOM in spring and fall samples from these sites, which differed mainly by climate and sulphur deposition. The outcome was 10 fully characterized reverse osmosis isolates of natural organic matter. The model compounds for this study were chosen to represent a variety of different classes of organic chemi-

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cals of environmental concern. Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene and pyrene, are formed during the combustion of fossil fuels and other organic matter and are also produced by natural processes. However, the major proportion of PAHs released into the environment is from human activities (Neff, 1985). Polychlorinated biphenyls (PCBs), such as 3,3¢,4,4¢-tetrachlorobiphenyl, have been produced for industrial purposes. PCBs are non-flammable, chemically stabile and have low conductivity. Therefore, they have been used as cooling and insulating fluids in transformers and capacitors, hydraulic fluids, surface coatings of copy papers, printing inks, rubbers, paints, waxes, asphalt and pesticides as well as in flame retardants in lubricating oils (Danse et al., 1997). Environmental concern of polybrominated diphenyl ethers (PBDEs), such as 2,2¢,4,4¢-tetrabromo diphenyl ether, is increasing. Due to their broad use as flame retardants in many resins and polymers, PBDEs can be found in many products (textiles, furniture, TV sets, computers and many other types of electrical equipment) used in every day life (Rahman et al., 2001). The increase in their use has lead to increases in the PBDE concentrations in environmental samples over the past two decades (Ikonomou et al., 2002; de Wit, 2002). Sorption of PAHs to DOM has been shown to correlate with the aromaticity (measured by UV-spectroscopy) of DOM. This relationship has been shown to apply to isolated humic and fulvic acids (Gauthier et al., 1987; Chin et al., 1997; Haitzer et al., 1999; Perminova et al., 1999) and, in most cases, for natural water samples (McCarthy et al., 1989; Kukkonen and Oikari, 1991; Akkanen et al., 2001). In addition, sorption of PAHs has been shown to correlate with the proportion of hydrophobic acids (Kukkonen and Oikari, 1991; Akkanen et al., 2001). PCBs have been shown to associate with a different fraction of DOM than the PAHs. Kukkonen et al. (1990) showed that PCBs associate mainly with the hydrophobic neutral fraction (XAD-8 fractioned), which is characterized by low aromaticity. This association was also evident in a series of 20 freshwaters, where the sorption of TCB to DOM tended to be higher in samples where the hydrophobic neutral fraction was larger (Kukkonen and Oikari, 1991). Further, the sorption of TCB to Nordic reference fulvic acid (high aromaticity) was shown to be extremely low (Akkanen and Kukkonen, 2001). On the other hand, Uhle et al. (1999) pointed out that the sorption of PCBs is also driven by the aromaticity of DOM, but only two different types of DOM were used in that particular study. The association of PBDEs with DOM is still largely unknown. The interaction between PAHs and DOM has been widely studied. However, in most studies isolated fractions, mainly humic and fulvic acids, of DOM have been used and therefore studies of bulk DOM are necessary. Other organic compounds, such as PCBs have received

Association of organic contaminants with DOM

less attention. Furthermore, the environmental concern of “new” contaminants such as PBDEs is rising, and there is a need to determine their behaviour in the environment. In this study, the association of four model compounds with DOM present in redissolved reverse osmosis isolates was measured. Correlation analysis was used to distinguish the parameters that predict the potential sorption capacity of DOM for organic contaminants. Further, bioaccumulation studies were conducted for two PAHs to see if the measured association with DOM was related to the apparent bioavailability to the water flea (Daphnia magna).

Materials and methods DOM samples were collected from five sites: Hietajärvi (Finland), Valkea-Kotinen (Finland), Svartberget (Sweden), Birkenes (Norway) and Skjervatjern (Norway) during fall 1999 (F) and spring 2000 (S). All were stream samples taken near the lakes except for Valkea-Kotinen, which was the only lake sample. DOM was isolated by the reverse osmosis method and then freeze dried. For more detailed description of the sites and samples, see Vogt et al. (2001; 2004). To compare functional properties of the samples under similar conditions, the partition and bioavailability experiments were conducted at the same DOM concentration for all samples. For example, carbon normalized partition coefficients and bioconcentration of organic contaminants can vary with the concentration of DOM (Akkanen and Kukkonen, 2003b). Therefore, DOM isolates were dissolved in soft artificial freshwater to achieve a dissolved organic carbon (DOC) concentration of 15 mg L–1. Artificial freshwater was prepared in Milli-Q grade water by dissolving the following salts: CaCl2 ¥ 2H2O 58.8 mg L–1, MgSO4 ¥ 2H2O 24.7 mg L–1, NaHCO3 13.0 mg L–1, and KCl 1.2 mg L–1 (hardness [Ca2+] + [Mg2+] = 0.5 mM). The redissolved samples were filtered through 0.45-mm membrane filters (Schleicher & Schuell, Dassel, Germany). Radiolabeled model compounds were: [3H]-benzo[a]pyrene (BaP; Amersham, Little Chalfront, UK; specific activity 81 Ci mmol–1), [14C]-pyrene (Sigma, St. Louis, MO, USA; 58.7 mCi mmol–1), [14C]-3,3¢,4,4¢-tetrachlorobiphenyl (TCB; Sigma; 104.0 mCi mmol–1), and [14C]2,2¢,4,4¢-tetrabromo diphenyl ether (TBDE; Stockholm University, Department of Environmental Chemistry, Sweden; 73.8 mCi mmol–1). Due to rapid autoradiolysis of tritiated BaP, this compound was purified just before the experiments by solvent extraction and thin layer chromatography (TLC). The TLC plates (Silica Gel 60, Merck, Darmstadt, Germany) were developed in pentane: diethyl ether (9:1 v/v). The log Kow values for the model compounds were 6.0 for BaP (De Maagd et al., 1998), 5.3 for pyrene (Helweg et al., 1997), 6.6 for TCB (Sabljic et al., 1993), and 6.6 for TBDE (Tittlemier et al., 2002). Ethanol

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was used as the solvent carrier for the model compounds. In the experiments, the volume of the carrier never exceeded 0.01% of the total solution volume. Nominal aqueous concentrations of the model compounds were 0.01 mg L–1 for BaP and 1.0 mg L–1 for the others. Partition coefficients were determined by the equilibrium dialysis method (Carter and Suffet, 1982; McCarthy and Jimenez, 1985). A detailed description of the method can be found in Akkanen and Kukkonen (2003b). Briefly, a 6-ml DOM sample in a dialysis tube was placed in 210-mL glass jar with screw cap (four replicates/ sample) containing one of the model compounds dissolved in 200 ml of artificial freshwater. Parallel control samples were made with artificial freshwater inside the tubes to verify free diffusion of the model compounds through the dialysis membrane. The jars were incubated four days on a shaker (45 rpm) in the dark at room temperature (20 ± 1°C). Hydrophobic chemicals can adsorb to the dialysis membranes and glassware. However, the method is based on the fact that the whole system is at equilibrium, i.e., chemical adsorbed to the membranes or glassware does not interfere with the equilibrium between DOM-associated and the freely dissolved fraction of the chemical. The partition coefficients were calculated as KDOC = Cb/(Cf*DOC), where Cb is the concentration of DOM-associated chemical (i.e. the difference between concentrations inside and outside the dialysis tube), Cf is the concentration of freely dissolved compound (i.e. the concentration outside the dialysis tube), and DOC is the concentration of dissolved organic carbon (kg L–1) in the DOM sample. The compounds were quantified by liquid scintillation counting. The effect of DOM on the bioavailability of BaP and pyrene to Daphnia magna was also tested. Bioavailability of the model compounds was determined in 120-mL glass jars with screw caps containing 100 ml of test media containing DOM (three replicates per sample). Control samples contained artificial freshwater without DOM (DOC < 0.2 mg L–1). The solutions were spiked with one model compound at a time. The model compounds used in this study tended to sorb to glassware depending mostly on their hydrophobicity, leading to lower aqueous concentrations. Therefore, to ensure steady exposure concentration, the spiked solutions were kept overnight in the dark at room temperature before exposure. Five subadult (no eggs in the brood chamber) D. magna were added to each jar. After 24 h exposure in the dark at 20 ± 1°C, radioactivity in the water and water fleas was analyzed by liquid scintillation to quantify the model compounds. Bioconcentration factors after 24 h of exposure (BCF24) were calculated as the ratio between the concentration of the model compound in the water fleas (ng g–1 wet wt) and the concentration of model compound in water after exposure. The proportion of DOM-associated compound was calculated as the ratio between the BCF values in the

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presence and absence of DOM. A more detailed description of the method can be found in Akkanen and Kukkonen (2003b).

Results The KDOC values were substantially higher for BaP than for the other model compounds (Fig. 1). Sorption of pyrene was an order of a magnitude lower, but the KDOC values correlated highly with those of BaP. For TCB, the KDOC values were similar to those of pyrene, while the values for TBDE were slightly greater. The KDOC values for BaP and pyrene differed significantly (p < 0.05) among sites, and in some cases, between seasons at a particular site (Fig. 1). The KDOC values for TCB were lowest in three samples (Svartberget S, Birkenes F and S), otherwise the values were quite uniform. The values for TBDE were quite stable throughout the sample series and no significant differences (p > 0.05) were found (Fig. 1). The KDOC values ranged from 38 000 (Birkenes S) to 360 000 (Svartberget F) for BaP, from 3 500 (Birkenes S) to 45 000 (Svartberget F) for pyrene, from 15 000 (Svartberget S) to 51 000 (Svartberget F) for TCB, and from 37 000 (Hietajärvi S) to 80 000 (Hietajärvi F) for TBDE. More detailed information on the parameters presented in Table 1 can be found in Vogt et al. (2001; 2004). The KDOC values for the two PAHs correlated strongly with spectroscopic parameters such as specific UV absorbance (sUVa, positive correlation), specific visible absorbance (sVISa, positive correlation) and the spectral absorbency ratio (SAR, negative correlation) (Table 1). The KDOC values for TCB also correlated with parameters like SUVA and SAR, but the correlation was weaker than for the two PAHs. The strongest correlation for the KDOC

Figure 1. Association of the model compounds with dissolved organic matter in samples taken in the fall (F) and spring (S). Log KDOC = partition coefficient determined by the equilibrium dialysis technique, V-K = Valkea-Kotinen, Hi = Hietajärvi, Sv = Svartberget, Bi = Birkenes and Sk = Skjervatjern.

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Table 1. Significant (* = p < 0.05, ** = p < 0.01) correlations between KDOC values of the model compounds and selected characteristics of dissolved organic matter in the samples. For more detailed description of the parameters see Vogt et al. (2001, 2004). Parameter A600 SAR sUVa sVISa RkSF RkL SFE Alkyl-C EM 1 RFI-A EM 2 KDOC for BaP KDOC for Pyrene KDOC for TCB KDOC for TBDE

KDOC for BaP

KDOC for Pyrene

KDOC for TCB

KDOC for TBDE

0.706* –0.922** 0.771** 0.849** –0.112 0.128 –0.714* –0.033 0.732* –0.661* 0.721* – 0.971** 0.731* 0.023

0.654* –0.929** 0.726* 0.802** –0.025 0.288 –0.712* –0.071 0.768** –0.761* 0.631* 0.971** – 0.724* –0.051

0.779** –0.760* 0.769** 0.849** –0.325 0.160 –0.338 0.194 0.254 –0.437 0.418 0.731* 0.724* – 0.056

0.17 –0.208 0.031 0.183 –0.739 * –0.728 * –0.384 0.696 * 0.160 –0.269 –0.257 0.023 –0.051 0.056 –

A600 = absorbance at 600 nm; SAR = A254 /A400 ; sUVa = A254 /DOC (mg C L–1); sVISa = A600/DOC; RkSF = relative fluorescence emission (AU*L mg–1); RkL = relative fluorescence emission in blue region (330–345 nm); SFE = fluorescence efficiency (AU cm); Alkyl-C = alkylic carbon (13C-CPMAS NMR); EM 1 = emission wavelength of phenolic peak (Total luminescence spectra, TLS); RFI-A = relative fluorescence intensity of phenolic peak (TLS); EM 2 = emission wavelength of phenolic peak (TLS).

Figure 2. The relationship between the proportion of DOM-associated chemicals evaluated on the basis of a decrease in bioavailability to D. magna (BCF). Partition coefficients were determined by equilibrium dialysis (KDOC).

values of TCB was with sVISa (Table 1). The KDOC values for TBDE correlated with relative fluorescence emission (RkSF, positive correlation) and the proportion of alkylgroups (Table 1). The measured BCFs reflected, in most cases, the measured partition of PAHs between water and DOM indicating that DOM-associated compound was not bioavailable to Daphnia magna (Fig. 2). However, pyrene exhibited

especially anomalous behavior in some cases. In the cases of Valkea-Kotinen and Skjervatjern spring samples, the effect of DOM on BCF was much lower than expected on the basis of measured partitioning. In contrast, the effect of DOM on BCF in the Birkenes fall sample was much higher than expected.

Discussion The molecular size and weight of humic substances has been suggested to be the most important characteristic affecting the sorption capacity for organic contaminants (Chiou et al., 1986; Engebretson et al., 1996). It appears that smaller molecules do not have the size and flexibility to form hydrophobic domains considered to be essential for sorption of hydrophobic chemicals (Engebretson and von Wandruszka, 1997). In addition, the relative number of these domains increases with increasing molecular weight of humic substances (Chin et al., 1994). Lower SAR and higher sUVa values have been shown to indicate higher molecular weight of DOM (Peuravuori and Pihlaja, 1997; Vogt et al., 2004). However, the relationship between spectroscopic parameters and structure of DOM can vary with the source of DOM. Furthermore, inorganic constituents in the samples, such as nitrate and iron, can also contribute to UV-absorbance (Dilling and Kaiser, 2002). In this study, however, lower SAR and higher sUVa produced higher sorption of BaP, pyrene and TCB to DOM. These findings support the importance of molecular weight of DOM for sorption capacity. In contrast, Perminova et al. (1999) found a strong correlation between the sorption of PAH and several parameters describing the aromaticity of humic substances, but they did not find a correlation between sorption and molecular weight. In this study, total luminescence spectra supported the importance of aromaticity in the sorption of PAHs. Sorption of PAHs followed the aromaticity of DOM measured by UV-spectroscopy (sUVa). A similar correlation was not found with aromatic C% measured by NMR from solid RO isolates (Vogt et al., 2004). However, this finding is mainly due to the results from the Birkenes samples that had relatively high aromatic C% but low sorption capacity. It appears, however, that UV-absorption, which indicates aromatic and carbonylic structures, characterizes the functionality of DOM better than NMR in this case. On the other hand, aromaticity of DOM determined by solution-state NMR has been found to correlate with the sorption capacity for PAHs (Perminova et al., 1999). As shown, PAHs exhibited higher affinity to DOM than the halogenated compounds relative to their lipophilicity. For example, PCB congeners were shown to have lower KDOC values than PAHs in both natural water sam-

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ples and isolated humic fractions (Landrum et al., 1984; Kukkonen et al., 1990; Kukkonen and Oikari, 1991; Akkanen and Kukkonen, 2001). Similarly, the sorption of DDT to DOM was shown to be lower than that of BaP (Kulovaara et al., 1992; Kulovaara, 1993; Cho et al., 2002). This result was evident also in this study, as the KDOC values of TCB were similar to the values for pyrene, slightly higher for TBDE, but still lower than those of BaP. TCB has been shown to associate with a different fraction of DOM than PAHs. Sorption of TCB, and other PCB congeners, is rather low to DOM fractions (XAD-8 fractioned) other than the hydrophobic neutral fraction, while BaP has high affinity for both the hydrophobic acid and hydrophobic neutral fraction (Kukkonen et al., 1990). Difference in the electron density of contaminants and DOM fractions has been suggested to be the underlying reason for dissimilar sorption. Electrophilic PCBs seem to have a greater affinity to the hydrophobic neutral fraction, which has a high electron density (Kukkonen et al., 1990; McCarthy et al., 1994). However, this relationship does not explain the behavior of electron-rich BaP. In addition, the sorption of TCB to Nordic reference fulvic acid (high aromaticity) was shown to be extremely low (Akkanen and Kukkonen, 2001). Fulvic acids are a major component of hydrophobic acids. Furthermore, the interaction between DOM and TCB seems to be weaker than that of DOM and BaP. This observation is based on the fact that TCB can be more easily extracted from DOMrich water (Akkanen and Kukkonen, 2003b). In this study, however, the sorption of TCB followed, at least loosely, a similar pattern as the sorption of PAHs, although the correlation with SAR was weaker (Table 1). TBDE appeared to behave differently than the other model compounds. The KDOC values for TBDE varied only slightly among samples, and the deviation between replicates was relatively large with some samples with some samples. However, some significant correlations between KDOC values for TBDE and the characteristics of DOM were found. For instance, KDOC values for TBDE correlated negatively with fluorescence emission parameters such as relative fluorescence emission (RkSF) and relative fluorescence emission in blue region (RkL) (Table 1). These parameters apparently indicate the degree of condensation of the organic matter (RkL) and molecular weight of DOM (RkSF) (Vogt et al., 2004). On the other hand, a weak correlation was found with DOM and alkyl-C (Table 1). This suggests that the association of TBDE is occurring through different mechanisms or with a different fraction of DOM than that of PAHs. Since this chemical class is still little studied, further studies on sorption of PBDEs are needed. The magnitude of the sorption of PCBs to DOM also depends on their substitution pattern. Chlorines in the ortho position hinder free rotation around the 1,1¢-carbon. Because of this free rotation, non-ortho substituted (cop-

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lanar) congeners, such as the PCB congener used in this study, have higher KDOC values than the ortho substituted ones (Kukkonen et al., 1990; Uhle et al., 1999). In other words, molecules that have the possibility to exhibit planar configuration also exhibit higher sorption. Because of the bend in the ether linkage, PBDEs can not be planar (Wong et al., 2001). Therefore, planarity cannot explain why the sorption of TBDE is higher than that of TCB in the present study. There may be other steric factors controlling the sorption of halogenated organic compounds. The bioavailability of BaP and pyrene to Daphnia magna followed, in most cases, the amount of freely dissolved fraction of the compounds based on the measured KDOC values. In some cases, however, the bioavailability of pyrene diverged substantially from the general trend. This results may be, at least partly, due to the direct effects of DOM. Few studies have shown that DOM may affect the physiology of different organisms (Pflugmacher et al., 2003; Wiegand et al., 2003). For example, it has been shown that DOM may alter the function of biotransformation enzymes. In addition, pyrene is extensively metabolized in D. magna (Akkanen and Kukkonen, 2003a) and, therefore, DOM may directly affect the physiology of the organism and, in this way, also the accumulation of pyrene. However, it was not possible to relate the anomalous behavior of pyrene to any of the characteristics of DOM.

Conclusions Overall, the association of DOM with PAHs is largely controlled by aromaticity of DOM that can be measured by simple spectroscopic methods. Halogenated organic compounds are shown to behave differently from PAHs when their association with DOM is concerned and, furthermore, there are still some contradictions in the data. Therefore, it is difficult to make any definitive conclusions about the main characteristics of DOM, which control the association of halogenated compounds. The mechanisms and stability of the interaction between DOM and different contaminants are also unclear. This emphasizes the need for additional studies to establish the main properties of DOM that control the environmental fate of organic contaminants in freshwaters.

Acknowledgments The authors wish to thank Ms. Anita Mustonen, Ms. Heidi Arponen, Dr. Jörg Luster, Dr. Ádám Zsolnay and Prof. Ismo J. Holopainen. This study was financed by the Academy of Finland (projects 63017 and 73166) and the European Union (contract EVK1-CT-2001-00094).

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