Natural abundance experiments

Natural abundance methods provide an important tool to study the circulation and interactions of elements in the different ecosystems (Lampert and Sommer 2007). Measurements of the natural abundance of stable isotopes can be used to differentiate trophic levels and sources, and also to detect energy and organic matter flows in food webs (Middelburg 2014). Natural abundance methods are

usually more suitable for studies of slow-turnover pools than short-term labelling experiments, and these methods can be used to recognize naturally occurring seasonal and spatial variations of stable isotopes (Fry 2006).

The differences in isotopic composition of the inorganic carbon compounds and the photosynthetic pathway (C3 or C4) cause variation to stable isotope ratios (δ¹³C) of plants, and these values are further reflected in the consumers of aquatic food webs (Middelburg 2014). δ¹³C of terrestrial C3 plants usually varies from -23 to -30 ‰, while δ¹³C of terrestrial C4 plants is about -10 to -14 ‰ (Fry and Sherr 1984). The basic principle of natural abundance studies is that the isotope ratio is transferred between different trophic levels (Middelburg 2014). The isotopic fractionation of carbon during trophic transfers is usually small (about 0.4 ‰ per trophic level) compared to other elements (e.g. 3.4

‰ for δ¹⁵N) (Lampert and Sommer 2007), and the fractional contributions of carbon derived from resource can be calculated by using a simple two-source mixing model (formula 11) (Fry 2006;

Middelburg 2014):

𝑓1 = 𝛿𝑠𝑎𝑚𝑝𝑙𝑒−𝛿𝑠𝑜𝑢𝑟𝑐𝑒 2

𝛿𝑠𝑜𝑢𝑟𝑐𝑒1− 𝛿𝑠𝑜𝑢𝑟𝑐𝑒2 𝑎𝑛𝑑 𝑓2 = 1 − 𝑓1 (11)

where f1 and f2 are the fractions for source 1 and 2, and δsample, δsource1 and δsource2 are the isotope values of the sample and two sources. There can also be several different resources in aquatic ecosystems, and thus, the proportions of each source have to be calculated by using multisource mixing models (Fry 2006).

Most δ¹³C values of natural abundance samples generally vary between −100 and +50 ‰ (Fry 2006).

Stable carbon isotope signatures in freshwater ecosystems depend on the source of carbon (e.g.

autochthonous phytoplankton vs. allochthonous terrestrial plant detritus) (Grey 2016). In freshwaters with strong respiration inputs, δ¹³C of DIC can approach -20 ‰ (Peterson and Fry 1987). In eutrophic and poorly oxygenated environments δ¹³C of DIC may decrease significantly because of increased production and oxidation of CH4 in these conditions (Michener and Lajtha 2007).

Stable isotopes can be used in the identification and quantification of methanogenic pathways (Conrad 2005). The δ¹³C values of CH4 are varying according the production pathway: thermogenic CH4 is enriched in ¹³C compared to bacterially produced CH4. δ¹³C of thermogenic CH4 is typically between -40 ‰ and -45 ‰, whereas biogenic CH4 can have a δ¹³C value of -45 ‰ to -100 ‰ (Grey 2016). In addition, CH4 derived from H2/CO2 is typically more depleted (-60 ‰ to -100 ‰) than CH4

derived from acetate (-50 ‰ to -65 ‰) (Schlesinger and Bernhardt 2013). Furthermore, natural abundance methods can be used to detect methanotrophic pathways (Conrad 2005). For example, it

is possible to estimate water column CH4 oxidation by using the δ¹³C-CH4 values, since CH4

oxidation fractionates against ¹³CH4. Thus, CH4 oxidation of ¹³C-depleted biogenic CH4 leads to enrichment of ¹³C in the residual CH4 pool (Bastviken et al. 2008).

13C analysis can also detect important carbon pathways from CH4 to other organisms (Lampert and Sommer 2007). Several stable isotope studies have suggested methanotrophic bacteria to be an important food source for consumers such as chironomid larvae and zooplankton (Grey et al. 2004;

Deines and Grey 2006; Jones et al. 1999; Kankaala et al. 2006b). Low δ¹³C ratios of consumer may indicate utilization of CH4-derived carbon: δ¹³C ratios of CH4 from biogenic sources are usually very negative, and thus, for example extraordinary low δ¹³C ratios of chironomids may be a consequence of feeding on methanotrophs at the oxic-anoxic interface of sediments (Deines and Grey 2006).

2.3.5 ¹³C labelling experiments

In many studies additional tracers are needed to completely understand the isotopic composition of aquatic environments (Michener and Lajtha 2007). It is possible to study gross CH4 production and consumption by using labelling experiments (Fischer and Hedin 2002). Natural abundance approaches are suitable for excluding important sources, but when identifying the importance of sources, labelling methods can provide strong signals and good source targeting (Fry 2006). Labelling experiments describe how the ratio of labelled to unlabelled CH4 changes over time (Fischer and Hedin 2002).

The advantage of labelling experiments is that they are more conclusive compared to natural abundance studies, and under experimental control it is possible to attain larger differences between isotopic ratios. When planning labelling experiment, it is important to decide the suitable tracer, the form of addition to the system (organic/inorganic, dissolved/particulate, reduced/oxidized), and the duration of the study. Tracer additions can be carried out continuously, or there can be only a single addition (Middelburg 2014).

Labelling experiments with different substrate levels are suitable for identifying distinct methanotrophic responses in different water bodies (Mau et al. 2013). These experiments can be accomplished in enclosures (e.g. mesocosms) or in situ (Middelburg 2014). Also some methods are based on incubation of water samples in containers: for example ¹³C-labeled CH4 is added to the bottle or vial containing the water sample, and after incubation the transformation to ¹³CO2 or ¹³C-labeled biomass is measured (Bastviken et al. 2002).

Another possibility is to add ¹³C directly to the water column. These whole-lake approaches can be used especially in studies focusing whole system carbon metabolism (Bastviken et al. 2002). In the whole-lake approach of Cole et al. (2002), an inorganic carbon tracer (NaH¹³CO3) was added in a single pulse to the epilimnion of the small, humic study lake. Analysis of the collected water samples showed an immediate increase in the δ¹³C-DIC at the time of the tracer addition, but 10 days after the addition δ¹³C-DIC was only slightly increased compared to natural abundance levels. They also concluded that allochthonous carbon (DOC) was respired by bacteria, but very little of this carbon was transferred to higher trophic levels (Cole et al. 2002). Overall, labelling experiments are able to trace contemporary production in enriched areas, but complex substrates such as detritus may not be reproduced very well (Middelburg 2014). Therefore, labelling experiments are always the most useful used alongside with natural abundance studies and other supplementary techniques (Fry 2006;

Michener and Lajtha 2007). The advantages and disadvantages of both techniques are summarized in table 3.

Table 3. Comparison between natural abundance and labelling experiments (Middelburg 2014) Natural abundance experiments Labelling experiments

+Tracer additions can be done in different forms and combinations