Regions in the Amazon Basin have been associated with specific biogeochemical
processes, but a detailed chemical classification of the abundant and
ubiquitous dissolved organic matter (DOM), beyond specific indicator
compounds and bulk measurements, has not yet been established. We sampled
water from different locations in the Negro, Madeira/Jamari and Tapajós
River areas to characterize the molecular DOM composition and distribution.
Ultrahigh-resolution Fourier transform ion cyclotron resonance mass
spectrometry (FT-ICR-MS) combined with excitation emission matrix (EEM)
fluorescence spectroscopy and parallel factor analysis (PARAFAC) revealed a
large proportion of ubiquitous DOM but also unique area-specific molecular
signatures. Unique to the DOM of the Rio Negro area was the large abundance
of high molecular weight, diverse hydrogen-deficient and highly oxidized
molecular ions deviating from known lignin or tannin compositions, indicating
substantial oxidative processing of these ultimately plant-derived
polyphenols indicative of these black waters. In contrast, unique signatures
in the Madeira/Jamari area were defined by presumably labile sulfur- and
nitrogen-containing molecules in this white water river system. Waters from
the Tapajós main stem did not show any substantial unique molecular
signatures relative to those present in the Rio Madeira and Rio Negro, which
implied a lower organic molecular complexity in this clear water tributary,
even after mixing with the main stem of the Amazon River. Beside ubiquitous
DOM at average H
With an average of about 200 000 m
Amazon tributaries vary in their coloration and opacity due to their origin
and reactivity and have traditionally been classified as “black waters”,
“white waters” and “clear waters” (Sioli, 1950). These three water types
play a continuing role in the transformation of OM, due to mediating light
availability for aquatic life or photoreactivity. Black waters are
influenced by chromophoric DOM (CDOM) and have low particulate mineral
content (Sioli, 1950; Leenheer, 1980). It has been suggested that drainage
areas of black water systems are characterized by moist, acidic, hydric
soils that allow for leaching of terrestrially derived plant matter, like
lignins, tannins and other plant materials that also contribute to the CDOM
(Leenheer, 1980). White waters make up
High CDOM in black waters and suspended sediment concentrations in white waters limit light and therefore the autochthonous production of organic matter (Costa et al., 2013); accordingly, allochthonous inputs dominate the organic matter pool (Ertel et al., 1986; Hedges et al., 1994; Townsend-Small et al., 2007). In clear waters, light is abundant (Costa et al., 2013) but nutrients are limited, and as a result, OM is still expected to be influenced by allochthonous input. However, agriculture and urbanization along clear waters can supply additional nutrients and therefore increase autochthonous OM production with potential consequences for the DOM pool. These specific physicochemical properties of these three main types of waters in the Amazon Basin are expected to exhibit distinctly different organic matter signatures.
High bacteria counts (
The unique DOM environments found within the Amazon Basin, which are ultimately the drivers of aquatic life, have yet to be resolved on a fine scale. It is unclear how previous work on bulk DOM, optical properties, a few specific target compounds, isotopes or microbial processes reflects the authentic chemical diversity intrinsic to the complex OM found in black, white and clear waters. Therefore, we investigated the chemodiversity of these three water types across the Amazon Basin by employing non-target Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) interfaced with soft electrospray ionization (ESI), which has enabled the characterization of thousands of individual molecular ions in complex DOM mixtures (Gonsior et al., 2011, 2013; D'Andrilli et al., 2010; Tremblay et al., 2007; Ward et al., 2013; Hertkorn et al., 2013; Liu et al., 2011; Stubbins et al., 2010; Stenson et al., 2003). By combining this technique with advanced optical characterization, excitation emission matrix (EEM) fluorescence spectroscopy and parallel factor analysis (PARAFAC), we assess similarities and differences in DOM composition between different waters of the Amazon Basin.
Using these techniques, we attempted to answer the following questions. (a) What is the overall chemodiversity of DOM in the Amazon Basin, and do distinct differences in DOM composition exist between major Amazon tributaries and the flooded area? (b) How well do the simple optical properties represent the overall molecular composition of DOM as described by FT-ICR-MS in tropical ecosystems? Satisfactory comprehension of these relationships would have large implications for the understanding of aquatic food webs and also for predicting further transport and processing of DOM in the Amazon.
Water samples from the main stem of the Madeira (and its small tributary Rio Jamari), Negro and Tapajós River were collected in duplicates using 1 L precombusted Pyrex glass bottles in May 2013. The bottles were filled in the main stem of the river just below the surface. In addition, 9–10 lakes, that were flooded by the individual rivers at the time of sampling, were sampled in the same manner near the cities of Santarém (confluence of Tapajós and Amazon River: Rio Tapajós area), São Carlos (Rio Madeira area) and Novo Airão (Rio Negro area) (Fig. S1 in the Supplement). The sampling sites in the Rio Tapajós area included samples from flooded clear water lakes adjacent to the main stem of the Rio Tapajós and flooded lakes that were located after the confluence of Rio Tapajós and the main stem of the Amazon River. However, the sampling sites after the confluence were still dominated by Rio Tapajós water. The collected samples were filtered and solid phase extracted either directly after collection (aboard river boats) or within 3 h after collection, when smaller boats were used for sampling.
All 1 L water samples were filtered through precombusted (500
Subsequently, the cartridges were rinsed again with acidified Milli-Q water
to remove remaining sample solution, dried and eluted with 10 mL high-purity
methanol (Chromasolv, Sigma Aldrich) into pre-cleaned 40 mL amber glass
vials. Methanolic samples were then kept on ice during the 2-week sampling
period and later kept at
Small (40 mL) aliquots of each water sample were filter-sterilized (0.2
All SPE samples were analyzed using negative-mode electrospray ionization
and a Bruker Solarix 12 Tesla FT-ICR-MS located at the Helmholtz Zentrum
Munich, Germany. Details about the FT-ICR-MS analyses used in this study
have been described previously (Gonsior et al., 2011; Hertkorn et al., 2013).
Briefly, methanolic samples were diluted 1 : 20 with methanol and then
directly injected into the electrospray at a flow rate of 120
Negative-mode ESI typically generates several
thousands of different
Van Krevelen diagrams (van Krevelen, 1950) were used to visualize the
elemental ratios of unambiguously assigned molecular formulae. Kendrick
plots (Kendrick, 1963) are also useful to determine members of homologous
series, but we used a modified Kendrick plot, where the Kendrick mass defect
(KMD) is divided by another independent parameter
CDOM was recovered almost quantitatively (> 90 %) using the
described SPE method and enabled a direct comparison of FT-ICR-MS results
and optical properties of Amazon DOM. SPE-DOM samples were prepared for
optical analyses using 100
Data mining and the application of multivariate statistics is increasingly
important to be able to analyze very complex data sets. Examples of such
multivariate approaches are hierarchical cluster analysis (HCA) and
principal component analysis (PCA) that have been recently applied to
ultrahigh-resolution mass spectrometry (Sleighter et al., 2010; Werner et
al., 2008; Stubbins et al., 2014) and EEM-PARAFAC (Chen et al., 2010). In
this study, PCA and HCA were applied to mass-spectrometry-based data sets of
all collected spectra and their exact mass lists and intensities. The
duplicate samples were first averaged and resulted in up to 16 000 variables
(
In an additional analysis, the two normalized data sets (FT-ICR-MS and
EEM-PARAFAC) were combined to be able to determine hierarchical clusters on
the variables (
Representative FT-ICR-MS spectra from each area are provided in Fig. 1.
The mass spectrum of the Rio Negro sample, in comparison to the other areas, clearly
showed much higher intensities of hydrogen-deficient
Ultrahigh-resolution FT-ICR mass spectra of SPE-DOM isolated from the
main stem of the Rio Negro, Rio Madeira and Rio Tapajós, Amazon, Brazil
and the stacked relative abundances of all ions at nominal mass 265 and 601
for all three river systems (note the differences in peak intensity for individual
Differences in SPE-DOM between the Rio Negro, Rio Tapajós and Rio Madeira identified by ESI-FT-ICR-MS.
Note:
Detailed molecular formula assignments of duplicate SPE-DOM samples from the
Rio Negro area (black water), Rio Tapajós area (clear water) and Rio
Madeira area (white water) in both the main stem of the river as well as
flooded adjacent lakes revealed an overall molecular composition of Amazon
DOM that was remarkably diverse (Fig. 2). CHO formulae covered almost the
entire area of chemically reasonable O
Van Krevelen diagrams of the total and the ubiquitous
A simple computation of unique signatures (
Unique
A removal of these unique high molecular weight poly-aromatic DOM molecules by means of mineral adsorption or flocculation with iron (Philippe and Schaumann, 2014) or aluminum oxides (Galindo and Del Nero, 2014) after mixing with the high-sediment-load Solimões River is conceivable and corresponded to previous reports that between 4 % (Aucour et al., 2003) and 40 % (Moreira-Turcq et al., 2003) of the DOC was removed at the confluence of the Rio Negro and Solimões. We were not able to sample the Solimões River directly, and hence a direct comparison between FT-ICR-MS results from the Rio Negro and Solimões were not achieved. However, investigations of Amazon wetland hydrogeochemistry also found analogous preferential sorption of higher molecular weight DOM to sediments, resulting in an enriched low molecular weight aliphatic DOM pool under high suspended solids conditions (Maurice et al., 2002).
In contrast, the distinct signatures indicative of the Rio Madeira area were
comprised of diverse CHNO and CHOS compounds that may define the readily
bioavailable and labile DOM pool, in part because of its low molecular
weight, in particular for the sulfur-bearing
The Tapajós area showed indicative aliphatic CHO signatures (high H
EEM spectra of SPE-DOM also showed distinct differences between each area
(Fig. S4), and a highly intense long-wavelength fluorescent peak in Rio
Negro water samples was apparent (Fig. S4). A five-PARAFAC-component (Fmax 1–5) model (Fig. S2) was most adequate to explain the differences in
the fluorescence data set and also captured the indicative fluorescence
signal of the Rio Negro (Fmax 3 and 4) at high emission wavelengths (Fig. S5).
These high emission wavelengths can either be explained by large
complex conjugated
Additional multivariate analysis, such as HCA and PCA, of the FT-ICR-MS and EEM-PARAFAC data of all samples also produced distinct separation between sampling areas (Fig. 4). It was gratifying that the EEM-PARAFAC PCA and HCA clusters matched remarkably close to the results of the FT-ICR-MS data. These statistical results suggested that many of the molecular changes associated with each sampling area might be associated with certain changes in the fluorescent DOM (FDOM). This at least should apply for the Rio Negro area because FT-ICR-MS-derived molecular signatures showed high hydrogen deficient molecules indicative of aromatic structures. However, correlations between optical properties (EEMs) and FT-ICR-MS data are not a certain proof of a causal relationship.
Traditionally, the classification of Amazon rivers was based on appearance and color of the water, and largely defined by CDOM (e.g., black water – Rio Negro vs. clear water – Rio Tapajós) and its sediment load (e.g., white water – Rio Madeira, Solimões). Hence, optical properties were important indicators, at least for black and clear water systems. Our results suggested that this classification based on optical properties might expand to include specific molecular characteristics of waters from various Amazon Basin areas, but presumably non-fluorescent aliphatic CHOS and CHNO compounds were indicative of the Madeira sampling area and were outside the analytical window of the EEM-PARAFAC approach (Fig. 4). Aromatic CHNO compounds, which were present in the Madeira area samples, presumably carry the ability to show long wavelengths' absorbance and fluorescence, even at relatively low molecular weight. At present, it remains unknown whether aromatic nitrogen heterocyclic compounds play an important role in FDOM.
Averaged nutrients, chlorophyll, DOC and TDN concentrations of all water samples collected in the main stem of the Rio Negro, Rio Madeira, Rio Jamari and Rio Tapajós as well as in 8–10 flooded lakes within the Rio Negro area, Rio Madeira area as well as within the Rio Tapajós and after its confluence with the Amazon River (Tapajós area) in May 2013.
Note: DOC
Principal component analysis and hierarchical cluster
analysis of the normalized data of all
Our results indicated that classified Amazon water systems (black, white and clear water) were associated with the presence or absence of many regionally unique compounds. It remains an unanswered question as to what happens with the FDOM that is presumably adsorbed to the mineral phase or coagulated and become part of the particulate fraction, and whether or not it is respired or transported to the Atlantic Ocean and eventually desorbed, or if it is added to the downstream sediments or to the seasonally flooded forest floor.
DOC concentrations in the Rio Negro (10.8 mg L
High Rio Negro DOC concentrations may be indicative of watershed characteristics (Leenheer, 1980) or a lower mineral content (Moreira-Turcq et al., 2003; Maurice et al., 2002; Aucour et al., 2003) compared to the other tributaries. The DOC data were in agreement with the observations resulting from FT-ICR-MS (Figs. 1 and 3) and EEM-PARAFAC analyses (Fig. S5) that indicated a removal of high molecular weight polyphenolic-like compounds from the Rio Negro waters, when compared to samples collected downstream or in other catchments. Total dissolved nitrogen (TDN) and total dissolved phosphorus (TDP) were low in all waters (Table 2), contributing to the observed low primary productivity despite the differences in light availability between the systems. A high DOM C : N ratio was observed in all waters consistent with previous findings (Ertel et al., 1986; McClain et al., 1995).
Heat maps of specific Spearman's rank-correlated
hierarchical clusters (distance threshold 0.6) that showed correlations
between
To address the question of which
In contrast, Fmax 1, 2 and 5 only correlated with a very few aliphatic molecular ions (Figs. 5 and S6), which were improbable fluorescent compounds itself, indicating an indirect correlation. Accordingly, correlations of Fmax 1, 2 and 5 were found within a distinct CHNO cluster, indicating that the fluorophores responsible for these PARAFAC components might have derived from heterocyclic or other aromatic nitrogen-containing molecules. However, components Fmax 3 and 4 did not show any correlation with molecular ions in the CHNO pool, nor in the CHOS pool, supporting the supposition that these PARAFAC components were only derived from CHO molecules.
The heat map and correlations of CHOS molecular ions (Fig. S6) reflected the
already mentioned enrichment of CHOS compounds in the Madeira and
Tapajós area samples and the absence of these signatures in the Rio
Negro area. The unique highly hydrogen-deficient CHOS pool only found in the
Madeira sampling area also manifested in a distinct cluster in this
analysis. The CHOS-based cluster that co-varied with PARAFAC components
Fmax 1, 2 and 5 showed mostly molecular ions with high O
Here, we present direct evidence that Rio Negro waters contained a unique, high molecular weight and highly fluorescent DOM component that is neither present in the Rio Madeira nor in the Rio Tapajós/Amazon confluence. This Rio Negro FDOM was confirmed by FT-ICR-MS to be indicative of high molecular weight polyphenolic-like compounds. DOC concentrations were also the highest in the Rio Negro. There is no reason to suspect widely different DOC produced by plants among the regions, but differences in geology is expected to influence the DOC pool, which is in part reflected in the large differences in pH and it is likely that this specific Rio Negro DOM fraction is sensitive to adsorption to mineral particles or coagulation with metals and that this specific DOM can be rapidly removed from the water during mixing of major Amazon tributaries as suggested in previous studies (Moreira-Turcq et al., 2003; Aucour et al., 2003). Such a removal would also substantially reduce the molecular complexity of Amazon DOM as observed in this study.
The similarities in DOM quality within regions between lakes and rivers (Table 2) indicated that the DOM composition is relatively stable under certain sets of environmental conditions. On the other hand, as discussed above, changing conditions may rapidly change DOM composition, in turn affecting conditions for aquatic life. While the commonly measured spectral properties of the DOM indicated these overall patterns, some important details were missed by solely measuring optical properties. Distinction between unique types of DOM with different origin, functions and turnover times, such as the previously discussed CHNO and CHOS compounds, would not have been possible without FT-ICR-MS and multivariate statistical analyses. Thus, for improved capacity to understand and predict the fates and functions of DOM, new methods, such as ultrahigh-resolution FT-ICR-MS, allowed detailed DOM characterization and emphasized the importance of non-target high-resolution techniques. To stimulate additional comparisons of simultaneously collected FT-ICR-MS and EEM-PARAFAC data, we encourage researchers to freely access the data sets used in this study (Gonsior et al. 2016).
All FT-ICR-MS and EEM-PARAFAC data are freely available online (Gonsior et al., 2016).
This research was supported by the Brazilian research agencies FAPERJ, CAPES and CNPq, by the German research agencies Alexander von Humboldt and DAAD and the Swedish Research agencies STINT and VR. Alex Enrich-Prast has additional support as a research fellow from CNPq and a Cientista do Nosso Estado from FAPERJ. This is contribution 5207 of the University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory. Edited by: M. Tzortziou Reviewed by: two anonymous referees