Use of lipid tracers to estimate the composition and origin of POM
collected in the Rhône River
Sterols
Based on a literature review, cholest-5-en-3β-ol (cholesterol),
24-ethylcholest-5-en-3β-ol (sitosterol if the C-24 stereochemistry is
24α), cholesta-5,24-dien-3β-ol (desmosterol),
24-methylcholesta-5,24(28)-dien-3β-ol (24-methylenecholesterol),
24-methylcholesta-5,22-dien-3β-ol (brassicasterol and/or
epi-brassicasterol depending on C-24 stereochemistry) and
24-methylcholest-5-en-3β-ol (campesterol) have been selected as
tracers of the origin of POM. Apart from cholesterol, which can originate
from a wide number of sources, all the other quantified sterols are
relatively source-specific. Sitosterol constitutes the major sterol in
higher plants, even though it can also be found in diatoms (Volkman, 1986),
and is often used to trace terrestrial organic matter in lacustrine and
marine systems (e.g., Meyers and Ishiwatari, 1993). Desmosterol is mainly
found in algae (Volkman, 1986). 24-Methylenecholesterol is mainly found in
diatoms, more particularly in the Thalassiosira and Skeletonema genera in the marine realm (Volkman,
2003). Epi-brassicasterol is mostly found in algae (Volkman, 1986).
Percentages of sterols (relative to the sum of sterols quantified),
and sitosterol : campesterol ratio in the samples investigated.
Date
Sitosterol
Cholesterol
Desmosterol
Brassicasterol
Methylene-
Campesterol
Sitosterol :
Cholesterol
Campesterol ratio
5 April 2011
74.4
12.7
0.4
4.9
1.2
6.4
11.6
18 July 2011
21.2
45.5
2.6
16.1
3.3
11.3
1.9
4 November 2011
54.1
23.8
1.9
10.0
2.2
8.0
6.7
5 November 2011
60.2
21.4
1.2
7.7
1.7
7.9
7.6
7 November 2011
56.7
28.6
0.4
6.1
1.3
6.9
8.2
14 November 2011
34.6
45.7
0.9
10.0
1.5
7.4
4.7
19 December 2011
52.5
29.1
0.5
7.5
1.9
8.5
6.2
16 January 2012
39.2
43.6
1.3
6.7
1.8
7.4
5.3
6 March 2012
17.6
12.4
28.8
6.2
22.6
12.4
1.4
17 April 2012
36.5
39.3
4.7
8.2
2.9
8.4
4.4
2 May 2012
38.4
41.4
2.1
9.5
1.9
6.7
5.7
22 May 2012
32.5
43.6
4.4
10.3
2.6
6.5
5.0
11 June 2012
35.0
43.5
2.5
9.6
1.6
7.7
4.5
26 June 2012
24.7
54.9
2.6
10.0
1.8
6.0
4.1
25 July 2012
21.6
37.5
3.9
20.8
4.4
11.7
1.8
5 September 2012
22.6
46.6
3.4
15.4
3.3
8.6
2.6
19 September 2012
21.1
44.3
3.8
19.1
3.0
8.7
2.4
3 October 2012
27.4
46.7
2.3
11.9
1.9
9.8
2.8
16 October 2012
42.9
38.9
1.4
7.8
1.4
7.6
5.7
6 November 2012
44.9
36.9
2.0
7.3
1.8
7.1
6.3
17 December 2012
66.3
22.8
1.0
3.5
1.2
5.2
12.7
10 January 2013
46.9
34.8
2.5
7.1
1.5
7.2
6.5
22 January 2013
36.0
48.4
1.4
6.9
1.3
6.1
5.9
4 February 2013
50.0
39.2
0.8
4.0
0.8
5.1
9.8
13 February 2013
55.4
30.7
2.0
4.8
1.3
5.9
9.4
12 March 2013
35.1
27.6
14.5
9.0
7.6
6.2
5.7
21 March 2013
37.4
29.0
11.1
8.9
6.6
7.2
5.2
17 April 2013
35.6
45.6
2.6
7.0
2.5
6.7
5.3
2 May 2013
59.0
24.5
1.1
6.8
1.7
6.8
8.7
13 May 2013
49.7
38.6
0.0
4.6
1.1
6.1
8.2
Average
41.0
35.9
3.6
8.9
3.0
7.6
5.9
SD
14.6
10.7
5.6
4.2
4.0
1.8
2.8
All samples are dominated by sitosterol and cholesterol, with proportions
being on average 3 times higher than those of the other sterols, apart
from the 6 March 2012 sample (Table 1). This sample exhibits a rather
different profile, dominated by desmosterol (0.122 µg mg-1 (dry
weight)) and 24-methylenecholesterol (0.096 µg mg-1 (dry weight)),
with a strong contribution from brassicasterol (0.026 µg mg-1 (dry
weight)) compared to all other samples. All these sterols have been
considered to be planktonic markers, and have been summed to compose the
planktonic sterol fraction shown in Fig. 3a. This fraction forms the major
part of the total sterol fraction of the March 2012 sample, evidencing a
phytoplanktonic bloom event. Given the nature of the sterols involved
(desmosterol, methylene-cholesterol), diatoms seem to be major contributors
(Rampen et al., 2010). This concurs with the drops in silica observed at the
same period (MOOSE data, unpublished). As we see an increase, although less
important in the proportion of planktonic sterols in the Spring 2013 samples
(20 times less in quantity when compared to the 6 March 2012 sample, but
still constituting 31 and 27 % of the total of all sterols quantified in
the 12 and 21 March 2013 samples respectively), this type of
planktonic event is probably a yearly spring occurrence but our sampling
frequency was not adapted to study blooms that can appear and disappear in a
matter of days. Sitosterol has been previously identified in marine algae,
and more widely as the major sterol in higher terrestrial plants (Volkman,
1986, 2003). However, the clear increase observed here in March 2012
demonstrates that it is also present in potamoplankton (Fig. 3a). The
presence of cholesterol can indicate a zooplankton contribution (Volkman,
1986), but it is also often found in freshwater algae (Volkman et al., 1981;
Gagosian et al., 1983), and can also evidence human impacts (Sicre et al.,
1993). The relatively high proportions of 5β(H)-cholestan-3β-ol (coprostanol) detected in the samples investigated (see Sect. 3.3.2)
clearly show that in the Rhône River cholesterol mainly arises from waste
water inputs (Brown and Wade, 1984).
Sitosterol and planktonic sterols (a), cuticular waxes and
betulin (b) and poly-unsaturated fatty acid (PUFA) (c)
contents of the different samples. Standard error shown was estimated to be
14 % (see Sect. 2.3). Phytoplanktonic blooms are evidenced by the spike
in planktonic sterols, and while the other tracers show the terrigenous
origin of the POM sampled, the ubiquitous nature of sitosterol is made
clear.
We also looked at the ratio sitosterol / campesterol (Table 1) as an
indicator of the terrigenous versus diatom origin of our POM. While many
plants have a sitosterol / campesterol ratio of less than 4 (Volkman,
1986), Nishimura and Koyama (1977) reported values ranging from 11.5
(Pinus densiflora) to 31 (hollyIlex pedunculosa). Dachs et
al. (1998) identified the threshold of 1 as the limit under which samples are
dominated by OM of aquatic origin, and above which sources of OM are
terrigenous. In our case, all the samples present a
sitosterol / campesterol ratio above 1, mainly between 2 and 8 (up to
12). Three samples show a ratio near 1 (July 2011 and 2012 and March 2012;
Table 1), a low value which confirms the presence of diatoms in the plankton
since freshwater diatoms exhibit sitosterol / campesterol ratios close to
1.0 (Ponomarenko et al., 2004).
Terrestrial vascular plant biomarkers
The contribution of terrestrial vascular plants to our samples is also
evidenced by the presence of the triterpenoids betulin
(lup-20(29)-ene-3β,28-diol), oleanolic (3β-hydroxyolean-12-en-28-oic) and ursolic (3β-hydroxyurs-12-en-28-oic)
acids (Razboršek et al., 2008) as well as components of cuticular waxes
(16,8-11-dihydroxyhexadecanoic, ω-hydroxyhexadecanoic, ω-hydroxyoleic and 18-hydroxyoctadec-9-enoic acids) (Kolattukudy, 1980)
(Fig. 3b). Betulin has been proposed as a tracer for paper birch (Fine et
al., 2001), a common species along the Rhône River, while oleanolic and
ursolic acids are widely distributed in terrestrial higher plants (Liu,
1995). The amount of cuticular waxes is variable amongst samples, between
0.02 and 3.8 µg mg-1(dry weight), with the highest in the
5 April 2011, 2 May 2013 and 4 November 2011 samples (3.8, 2.2 and 1.7 µg mg-1 respectively). Two of these sample dates (2 May 2013 and 4
November 2011) happen to be flood dates. It is clear that floods, during
which higher water flows are coupled to surface runoff, collect and carry
more terrestrial plant leaf debris, and hence increase the amount of
cuticular waxes found in our samples. The yearly variations in quantity are
probably due to the fact that waxes (linear compounds) are more easily
degraded by bacteria than cyclic structures such as sterols or triterpenoids
(Atlas and Bartha, 1992).
The betulin and sitosterol concentrations are significantly correlated in
most of our samples (r= 0,67 between sitosterol and betulin, on 29
samples, p-value = 3.10-5, excluding the 6 March 2012 sample due to
its out-of-range phytoplanktonic profile), thus reinforcing the idea that in
the Rhône River sitosterol mainly results from terrestrial higher plant
inputs. However, at the time of the spring bloom a significant part of this
sterol seems to derive from potamoplankton (Fig. 3b).
Another ratio commonly used to attest to the terrigenous origin of compounds
is the Terrigenous-to-Aquatic ratio (TAR, Bourbonnière and Meyers,
1996). Here we used the TAR(AL) as calculated by Van Dongen et al. (2008) for n-alkanols: (C26+C28)/(C16+C18). The
TAR(AL) in our samples is always above 1, and clearly indicates a
strong terrigenous contribution to the suspended particulate matter found in
the lower Rhône. The Average Chain Length of n-alkanols, a proxy positively
correlated to the abundance of higher plant debris (Van Dongen et al.,
2008), ranged from 26 to 22 across all samples, also attesting to the strong
contribution of terrestrial vascular plants. The long-chain even-numbered
n-alkanol profiles show a strong contribution of C22 and C28
n-alkanols. Compared with those previously described in the literature
(Diefendorf et al., 2011), this characteristic suggests a strong gymnosperm
contribution, which concurs with the low amounts of long-chain n-alkanes
detected.
Chlorophyll
The available data on chlorophyll a (MOOSE database, only available for 2012
and 2013) show a content variability between 0.9 (10 October and 6
November, 2012) and 14.0 (3 April 2012) mg m-3 (Fig. 4a). Chlorophyll
a is frequently used as a proxy for photosynthetic organisms and the
variation observed here is consistent with the hypothesis of a yearly
phytoplanktonic spring bloom, with a larger magnitude for the 2012 event.
Fatty and hydroxy acid (FA/HA) content of the different samples
(% of total quantified FA/HA) (PUFA: poly-unsaturated fatty
acids) * double-bond position undetermined.
2011
2012
2013
5/4
18/7
4/11
5/11
7/11
14/11
19/12
16/1
6/3
17/4
2/5
22/5
11/6
26/6
25/7
5/9
19/9
3/10
16/10
6/11
17/12
10/1
22/1
4/2
13/2
12/3
21/3
17/4
2/5
13/5
C14:0
4.4
8.4
5.4
3.5
1.6
4.2
5.1
6.1
11.4
6.8
2.9
2.2
3.8
6.0
2.2
8.2
1.6
1.2
1.5
1.2
1.1
3.9
2.0
1.3
2.1
3.5
5.3
3.6
1.6
4.6
C15:0
2.6
2.1
2.1
2.7
1.0
2.1
1.9
2.0
0.4
2.2
2.1
1.1
2.1
2.3
1.1
1.3
0.9
0.9
1.3
1.0
1.0
1.9
2.3
1.1
1.5
0.7
0.8
1.7
1.2
2.5
C:1
0.0
0.0
0.0
0.0
0.0
0.6
0.0
0.0
0.1
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
Iso-C15:0
2.1
4.3
2.8
1.6
0.2
3.2
2.3
3.1
0.1
1.5
1.2
0.4
1.4
1.1
0.7
2.9
0.5
0.9
0.9
0.3
0.2
1.5
0.3
0.2
0.4
0.2
0.5
1.2
0.7
0.5
Anté-iso-C15:0
1.4
2.1
2.1
1.3
0.2
2.1
2.3
2.9
0.1
1.3
1.0
0.5
1.2
0.9
0.4
1.4
0.4
0.7
0.9
0.4
0.2
1.5
0.4
0.2
0.5
0.2
0.5
1.1
0.6
0.7
C16:0
40.7
42.9
42.6
57.3
40.4
47.2
42.2
46.0
17.9
46.5
58.7
45.3
56.5
64.3
46.2
46.5
46.1
4.8
5.1
50.3
51.8
45.3
63.9
53.7
51.9
25.5
27.2
40.5
54.8
71.0
C16:1ω7
5.5
14.6
12.3
5.7
1.2
14.0
7.6
10.7
23.2
11.9
7.0
6.3
5.2
7.0
11.4
12.9
8.5
53.6
6.6
2.4
1.0
6.9
3.8
2.6
3.9
14.1
13.6
8.8
4.1
2.3
C16:2*
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
6.1
0.0
0.0
0.0
0.0
0.0
2.4
2.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
3.2
0.8
0.0
0.0
C16:3*
0.0
0.4
0.0
0.0
0.0
0.0
0.0
0.0
11.7
0.0
0.0
0.0
0.0
0.0
0.0
2.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.9
6.0
1.2
0.0
0.0
C16:4*
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4.1
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4.5
3.0
0.0
0.0
0.0
C17:0
0.9
1.0
0.8
0.9
0.9
1.0
0.7
0.7
0.0
0.6
0.6
0.7
0.7
0.8
0.8
0.6
0.7
1.4
1.2
0.9
0.7
0.8
0.8
0.8
0.6
0.3
0.3
0.9
0.0
0.0
C18:0
9.3
9.1
7.5
12.4
20.6
11.0
10.2
12.2
1.2
10.5
13.5
20.3
14.6
13.5
10.7
6.9
12.8
15.3
21.1
24.3
20.5
16.5
16.7
21.3
17.7
5.6
7.3
13.2
18.6
13.2
C18:1ω9 (Oleic)
8.1
6.6
10.6
0.8
6.9
6.3
9.1
8.2
0.9
7.9
6.2
9.5
5.2
0.7
11.8
6.5
14.0
10.0
11.7
9.8
6.4
2.5
4.9
12.3
13.1
7.5
9.0
11.9
10.8
3.5
C18:1ω7 (Vaccenic)
3.3
4.9
4.6
3.7
5.1
5.3
5.7
4.2
0.0
3.6
3.5
6.2
3.5
2.1
6.4
3.2
6.5
7.2
10.1
5.1
2.3
10.5
1.8
3.4
3.9
2.0
3.2
4.8
3.4
0.9
C18:2ω6
3.3
1.2
4.0
0.1
1.5
1.2
3.4
1.7
0.0
2.2
0.9
2.1
0.9
0.0
2.2
1.0
2.8
2.1
2.3
1.9
11.4
4.5
0.9
1.6
2.4
1.9
2.7
2.5
0.0
0.0
C18:3ω3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
C18:4ω3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.4
3.6
0.0
0.0
0.0
C20:0
2.2
0.9
2.0
1.4
3.6
0.9
1.6
1.8
0.1
0.9
1.1
1.6
1.2
0.0
0.8
0.5
1.1
1.3
33.1
1.7
1.7
1.6
0.9
0.7
1.0
0.4
0.7
1.6
1.6
0.4
C20:5ω3
12.2
0.6
0.8
7.0
11.7
0.1
4.4
0.0
17.3
0.9
0.0
2.0
1.7
0.4
2.1
1.3
2.8
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
20.0
12.1
3.9
0.0
0.0
C22:0
3.0
0.8
2.0
1.3
3.9
0.8
2.1
0.2
0.0
0.6
1.0
1.4
1.3
0.7
0.6
1.3
1.0
0.6
3.2
0.0
1.6
1.9
0.9
0.5
0.8
0.2
0.6
1.8
2.3
0.3
C22:1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
C23:0
0.0
0.0
0.0
0.0
0.0
0.0
0.3
0.0
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
C24:0
1.1
0.1
0.4
0.3
1.3
0.1
0.9
0.1
0.0
0.2
0.4
0.4
0.3
0.3
0.2
0.1
0.3
0.1
1.0
0.5
0.3
0.6
0.2
0.2
0.3
0.1
0.1
0.5
0.4
0.1
C26:0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
% PUFA
15.5
2.3
4.9
7.1
13.2
1.3
7.8
1.7
44.6
5.4
0.9
4.1
2.6
0.4
6.8
7.7
5.6
2.1
2.3
1.9
11.4
4.5
0.9
1.6
2.4
39.7
30.7
8.4
0.0
0.0
ω-hydroxy-C16:0
1.0
0.3
1.1
1.3
0.4
0.1
0.4
0.4
15.8
1.3
0.1
0.4
0.1
0.1
0.4
0.2
0.2
1.3
0.2
0.5
0.3
0.3
0.1
0.3
0.4
0.8
0.4
0.5
2.7
0.6
ω-hydroxy-C18:1ω9
0.8
0.0
0.5
0.5
0.2
0.0
0.4
0.0
0.0
0.4
0.0
0.1
0.0
0.0
0.2
0.1
0.1
0.2
0.1
0.1
0.2
0.2
0.0
0.1
0.3
1.1
0.4
0.2
0.7
0.2
18.(8-11)-dihydroxy-C16:0
5.0
1.0
9.3
7.2
1.5
0.2
2.3
1.9
7.8
7.2
0.1
2.9
0.9
0.2
1.1
0.6
0.8
3.0
1.7
3.7
2.3
1.9
0.0
1.3
2.5
2.5
2.5
1.7
3.1
0.9
ω-C16:0 diacid
0.3
0.0
0.3
0.2
0.0
0.0
0.1
0.0
0.0
0.2
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.1
0.0
0.1
0.0
0.0
0.1
0.0
0.0
0.0
0.1
0.0
ω-hydroxy-C22:0
92.9
98.6
88.8
90.8
97.9
99.7
96.9
97.6
76.4
91.0
99.8
96.5
98.9
99.7
98.3
99.1
98.8
95.5
97.9
95.6
97.2
97.5
99.9
98.3
96.7
95.6
96.6
97.5
93.5
98.3
Fatty acids
A number of saturated linear fatty acids have been found in our samples, with
C16:0 and C18:0 being the most abundant (Table 2). These fatty
acids are not specific, and can stem from a number of sources including
terrestrial vascular plants, algae and bacteria (Volkman et al., 1981). More
recently. C16:1ω7 and C20:5ω3 (eicosapentaenoic acid)
were identified as two of the main fatty acids in
Bacillariophyceae
(Taipale et al., 2013). These two markers are present in our samples, and
together form 40.5 and 34.1 % of the 6 March 2012 and 12 March 2013
samples (versus an average of 13.2 % across all samples), which, when
coupled with our sterol analysis, concurs with our hypothesis that diatoms
are major contributors in the algal blooms identified. It is worth noting
that in the 3 October 2012 sample, C16:1ω7 forms 53.6 % of all
quantified fatty acids, while C20:5ω3 is completely absent
(0 %), and therefore these markers alone cannot be considered to be
specific enough in natural river water samples which can contain a number of
fatty acids from various sources.
Chlorophyll a levels (MOOSE data) and percentage of photodegradation
of chlorophyll, calculated using CPPI (Cuny et al., 1999) in the Rhône waters
on and around sample dates.
Longer-chain saturated fatty acids (between C20 and C28) with a
strong even-chain predominance, which are specific to the epicuticular waxes
of vascular plants (Kunst and Samuels, 2003), are scarcely present in our
samples, with C20 and C22 being the most abundant relative to the
others: over 98 % (on average) of the total of long-chain
(C20–C28) saturated fatty acids across all samples. Due to the high
degradability of fatty acids, a number of them could not be quantified,
potentially leading to an underestimation of higher plant contribution.
Polyunsaturated fatty acids are present in very low proportions in our
samples, apart from the 6 March 2012 and 12 March 2013 samples where they
contributed to 44 and 40 % of total fatty acids (Fig. 3c – quantified
using the total of all PUFA quantified between C14 and C26). These
high contributions support the presence of a high proportion of fresh algal
material in these samples.
If we compare the average chain length (ACL) of fatty acids in our samples
with that of n-alkanols, it appears clearly that the ACL of fatty acids is
lower, with an average of 16.7 across samples, against 23.9 for alcohols. It
is widely accepted that fatty acids are more prone to bacterial degradation
than other lipids (Wakeham, 1995), and long-chain fatty acids tend to be
degraded more efficiently by a number of bacteria (Novak and Carlson, 1970).
Such a bacterial degradation could explain the ACL difference between
n-alkanols and fatty acids, and the lack of terrestrial higher-plant fatty
acids in our samples, while other markers for higher plants (such as waxes
or betulin) are present in large quantities. This is reinforced by the fact
that we also find a relatively high proportion of vaccenic acid in our
samples, a specific marker for bacterial activity (Sicre et al., 1988)
Hydroxyacids
Hydroxyacid contents were low during the period studied but some samples (18
July 2011, 4 November 2011 and 16 January 2012) exhibited high amounts of
C22 ω-hydroxyacid and small quantities of C20 and C24
homologues (Table 2). These compounds are generally considered to be suberin
markers when found in soil (Nierop, 1998; Otto et al., 2005), even though
they have been found in leaves and stems of higher plants as well
(Mendez-Millan et al., 2010). Suberin is a cell wall component of cork cells,
and is mainly found in bark, woody stems and roots (Kolattukudy, 1980).
Given the geographical location of our sampling point, we can consider this
compound to be a marker of industrial activity, probably associated with a
paper paste mill less than 3 km upstream. The mill uses
mainly local conifers (collected within a 250 km radius according to the
company, Paper Excellence, 2014), more particularly Pinus halepensis, Pinus nigra, and Pinus sylvestris (Etude
AGRESTE, 2011) and is authorized to reject a certain amount of wastewater in
the river. This is consistent with our findings that the POM is dominated by
gymnosperms. Pinus species also display a
sitosterol / campesterol ratio of between 5 and 10 (Conner et al.,
1980) in line with most of our samples as well. This industrial contamination
could explain the large contribution of gymnosperms to our POM, and implies
that we consider the extra input of terrestrial plant matter that will be
released, and degraded, at sea.
As a summary, the overall lipid composition of the Rhône River SPM is
characterized by major terrestrial higher plant components (mainly derived
from gymnosperms) with episodic, but significant, contributions from
freshwater algal material (probably dominated by diatoms) in the spring.
Despite the strong concentration of industries along this river, SPM appears
to be very weakly contaminated by petroleum hydrocarbons, but is strongly
impacted by the local paper mill and wastewater discharges (see Sect. 3.2.2).
Use of lipid tracers to estimate the degradation state of POM from
the Rhône River
If they can inform us on the origin of organic matter in natural
environments, lipid biomarkers are also invaluable in helping us estimate
biotic and abiotic alterations of organic matter and determine what are the
main processes involved in its degradation. Products resulting from the
degradation of sterols, chlorophyll, monounsaturated fatty acids and
hydroxyacids are among the most useful and specific tracers.
Chlorophyll
The absorption of light by some compounds, called photosensitizers, in the
presence of oxygen (regardless of these compounds being endogenous or not)
causes an oxidation whose effects, chemical or biological, are mostly
adverse (Spikes and Straight, 1967). Photosensitizers induce chemical
reactions via the absorption of light that would not occur in their absence.
Photosensitizers (Sens) are involved in indirect photo-oxidative processes:
they have two systems of electronically excited states, 1Sens and
3Sens. The triplet state is much longer lived than the singlet state,
which is the initial product issued from light absorption. Indirect
photo-oxidation (photo-sensitized oxidation) can be intense during the
senescence of phototrophic organisms (Rontani, 2012) due to the presence of
chlorophyll, which is a very efficient photosensitizer (Foote, 1976) capable
of generating singlet oxygen particularly reactive towards unsaturated
cellular components (Type II photoprocesses). Chlorophyll may be also
directly photodegraded by solar light (Nelson, 1993). Direct
photodegradation of chlorophyll and Type II photo-oxidation of unsaturated
cellular components can be thus considered two competitive photo-processes.
In the photic layer of aquatic environments, photo-oxidation has long been
considered a major degradation process for phytoplankton chlorophyll
pigments (Lorenzen, 1967; Vernet, 1991). Since we have no marker stable and
specific enough for chlorophyll tetrapyrrolic ring photodegradation, we used
the CPPI (Chlorophyll Phytyl side chain Photodegradation Index) for the
in situ determination of the rate of photodegradation of chlorophyll (Cuny
et al., 1999). Indeed, the photodegradation of the chlorophyll phytyl side
chain produces 3-methylidene-7,11,15-trimethylhexadecan-1,2-diol
(phytyldiol), specific of Type II chlorophyll photodegradation and
widespread in the environment (Cuny and Rontani, 1999). The CPPI,
(phytyldiol:phytol molar ratio) can be linked, through a mathematical model,
to the global quantity of photodegraded chlorophyll (Cuny et al., 1999),
The photodegradation rate of chlorophyll fluctuates greatly (Fig. 4b). The
6 March 2012 and 12 March 2013 samples display very low rates (2.6 and 9.8 %
respectively), in line with our identification of planktonic blooms in March
2012 and 2013. Such blooms result in an increase of fresh chlorophyll
inputs, with intact phytyl side chains, and thus in a decrease of CPPI. The
dips in chlorophyll photodegradation rates can help us identify blooms, or
at least periods when the input of chlorophyll is higher. The summer 2012
samples (July and September) also display a low photodegradation rate
ranging from 8.8 to 13.9 % while their amount of planktonic sterols
increases slightly.
Δ5-sterols
Δ5-sterols possess structural features that can be restricted to a
limited number of organisms (Volkman, 1986, 2003). Moreover, biotic and
abiotic degradation processes result to specific functionalizations of their
cyclic skeleton (De Leeuw and Baas, 1986), which are very useful to estimate
the relative importance of these processes (Christodoulou et al., 2009;
Rontani et al., 2009). Consequently, degradation products of
Δ5-sterols constitute excellent biomarkers for tracing diagenetic
transformations of specific organisms (Mackenzie et al., 1982). Using these
tracers to evaluate the relative influence of different degradation processes
requires that their removal rate (by further degradation) is comparable to
that of the parent Δ5-sterol. Although each sterol and its
degradation products may be potentially totally mineralized by marine
bacteria, we assume that they should exhibit similar reactivity towards
bacterial degradation. This assumption is based on the fact that aerobic
biodegradation of sterols generally involves initial attack on the side
chain, which is similar in all the degradation tracers selected to that of
the corresponding parent Δ5-sterol. Moreover, it may be noted that
3β,5α,6β-steratriols, employed for autoxidation estimates are
weakly affected by abiotic degradation processes. This is also the case for
Δ4-6α/β -hydroperoxysterols (photo-oxidation
tracers), which are much more stable than Δ5-7α/β-
and Δ6-5α-hydroperoxysterols (Christodoulou et al., 2009). Indeed, β-scission
of the alkoxyl radicals resulting from homolytic cleavage of
Δ5-7-hydroperoxysterols and Δ6-5-hydroperoxysterols
affords secondary and tertiary radicals, respectively, more stable than the
primary radical resulting from the cleavage of
Δ4-6-hydroperoxysterols (Christodoulou et al., 2009). Moreover,
proton driven cleavage (Hock cleavage) of Δ5-7-hydroperoxysterols
and Δ6-5-hydroperoxysterols involves a highly favored migration of
vinyl group (Frimer, 1979), while only an unfavored migration of alkyl group
is possible in the case of Δ6-5-hydroperoxysterols (Rontani et al.,
2014a).
Aerobic bacterial hydrogenation may convert Δ5-sterols to 5α(H)-stanols, 5α(H)-stanones and ster-4-en-3-ones (Gagosian et al.,
1982; De Leeuw and Baas, 1986; Wakeham, 1989). During the treatment undergone
by our samples (NaBH4 reduction), 5α(H)-stanones and
ster-4-en-3-ones are respectively converted in 5α(H)-stanols and
ster-4-en-3-ols; these compounds hence constitute useful markers of bacterial
degradation of sterols. To evaluate the proportion of biological degradation
of cholesterol, and to better trace human impacts on the OM found in the
Rhône, we also included coprostanol and epicoprostanol in its biodegradation
products. Sterol biodegradation percentages were estimated using Eq. (1a) and
(1b). Coprostanol (5β(H)-cholestan-3β-ol) is a stanol that
arises from the anaerobic microbial degradation of cholesterol in the
digestive tracts of higher land mammals, including man (Martin et al., 1973).
Epicoprostanol (5β(H)-cholestan-3α-ol) is not a major sterol
in human faeces, but it is often used as an indicator of sewage treatment
(McCalley et al., 1981):
sitosterolbiodegradation%=[sitostanol]/[sitosterol]×100
cholesterolbiodegradation%=[cholestanol+coprostanol+epicoprostanol]/[cholesterol]×100.
Free radical autoxidation yields mainly Δ5-3β, 7α/β-hydroperoxides and in smaller quantities 5,6-epoxysterols and
3β,5α,6β-trihydroxysterols. 3β,5α,6β-trihydroxysterols were chosen as tracers of autoxidation
(Christodoulou et al., 2009; Rontani et al., 2009) and the sterol
autoxidation percentage was estimated using Eq. (2) based on autoxidation
rate constants calculated by Morrissey and Kiely (2006):
sterolautoxidation%=([3β,5α,6β-trihydroxysterols]×2.4)/[sitosterolorcholesterol]×100.
1O2-mediated photo-oxidation (Type II photoprocesses) yields mainly
Δ6-5α-hydroperoxides and to a lower extent
Δ4-3β, 6α/β-hydroperoxides. Δ6-5α-hydroperoxides are unstable and are converted very easily to
the non-specific 7-hydroperoxides, so they were discarded as markers of
photo-oxidation. Although produced in lesser amounts, Δ4-3β, 6α/β-hydroperoxides, which are relatively stable and highly
specific, have been chosen as tracers of photo-oxidation processes and
quantified after NaBH4 reduction to the corresponding diols. The
percentage of sterol photo-oxidation was estimated using Eq. (3)
(Christodoulou et al., 2009), based on the ratio Δ4-6α/β-hydroperoxides/Δ6-5α-hydroperoxides found in
biological membranes (0.30) (Korytowski et al., 1992):
sterolphoto-oxidation%=([Δ4-3β,6α/β-dihydroxysterol]×(1+0.3)/0.3)/[sitosterolorcholesterol]×100.
Here, values are expressed in proportions relative to the amount of
remaining parent sterol in the sample. A total percentage of over 100 %
hence only means that degradation products were present in larger quantities
than their associated parent sterol.
Biotic and abiotic degradation of sitosterol (a) and
cholesterol (b) in the different samples. Full error shown here
incorporates the 14 % analytical standard error estimated for lipid
quantification for all terms of the equations used. Sitosterol and
cholesterol clearly have very different degradation patterns.
The results of the evaluation of sitosterol and cholesterol degradation
processes are shown in Fig. 5. The most highly degraded samples
sitosterol-wise were the ones from 18 July 2011, 26 June 2012 and 22 January
2013. Interestingly, cholesterol degradation shows a completely different
trend. When looking at the type of degradation undergone by
Δ5-sterols, it also appears clearly that, if auto- and
photo-oxidation processes are the major drivers of sitosterol oxidation,
biodegradation is the major player in cholesterol degradation. Hedges and
Keil (1995) hinted that sterols associated with waxy higher plant material
might not be as prone to enzymatic degradation as other sterols, which would
explain why sitosterol is only weakly biodegraded in our samples. Indeed,
even though we showed that sitosterol is also produced during spring
phytoplanktonic blooms, it is mainly derived from higher plants in most of the
samples investigated. The dominance of coprostanol relative to cholestanol
attests to a strong contribution of human or animal faeces to the cholesterol
present in the Rhône River. Here we used two different ratios in order to
better understand how human activities and wastewaters affect the Rhône
waters. The first ratio, used to determine if waters are affected by waste
water inputs and runoff has been previously used by Writer et al. (1995), and
is calculated as follows: coprostanol / (cholesterol + cholestanol).
These authors have defined a threshold of 0.06 above which samples are
considered affected by wastewater inputs and runoff from pastures and
feedlots, and below which they can be considered pristine. The second ratio
is the epicoprostanol : coprostanol ratio proposed by Mudge and
Seguel (1999), in order to illustrate the level of treatment undergone by
wastewaters. The higher the ratio, the more treated the waters are, as
epicoprostanol is only present in traces in primary sludge, while it is
present in much higher quantities in treated sludge (McCalley et al., 1981).
Both ratios are represented in Fig. 6 and they show that the Rhône is
undeniably affected by wastewaters and, to a much lower extent given the
historical land use around the Rhône, pasture runoff. However, we noted that
both the ratio and the threshold used are unofficial and in no way are proof
of a large-scale contamination or pollution. We only highlight here the
non-pristine state of the Rhône waters, which is logical given the level of
urbanization along the river. The epicoprostanol : coprostanol ratio also shows
fluctuations in the level of treatment of wastewaters, and fluctuates with
flow rates and precipitation levels.
Coprostanol:(cholesterol+cholestanol) and
epicoprostanol : coprostanol ratios of the different samples. Full error
shown here incorporates the 14 % analytical standard error estimated for
lipid quantification for all terms of the ratios. Contamination threshold is
0.06 (See Sect. 3.3.2).
During senescence, unsaturated higher plant lipids (and notably
Δ5-sterols) may be photodegraded (type II photo-oxidation), with
chlorophyll acting as a sensitizer (Rontani et al., 1996). Sitosterol present
in higher plant phytodetritus should thus have been intensely photodegraded
on land. However, the photo-oxidation rate estimates appeared to be
relatively low compared to that of autoxidation and biodegradation (Fig. 5a).
This is probably due to an intense free-radical-driven breakdown of
hydroperoxides produced during photo-oxidation (Rontani et al., 2003). The
photo-oxidation percentages displayed here are thus certainly underestimated.
The presence of large amounts of 24-ethylcholestan-3β,5α,6β-triol in most of the samples indicates that autoxidation plays an
important role in the degradation of sitosterol (Fig. 5a). Autoxidation
(spontaneous free radical reaction of organic compounds with O2), which
has been largely ignored until now in the environment, seems to play a key
role in the degradation of sitosterol (Fig. 5a) and thus of higher plant
material carried by the Rhône River. This assumption was well supported by
the detection of significant proportions of compounds deriving from betulin
autoxidation (data not shown). Recently, it has been demonstrated that
autoxidation plays a key role in the degradation of terrestrial (Rontani et
al., 2014b) and marine (Rontani et al., 2014a) vascular plant debris in
seawater. There is clearly a growing body of evidence suggesting that
autoxidation reactions can strongly impact the preservation of particulate
organic matter in the environment and should be considered carefully
alongside other removal processes such as biodegradation when constructing
carbon cycles and evaluating carbon budgets. The lowest autoxidation rates
observed in samples from 6 March 2012 and 12 March 2013 may be attributed to
the phytoplanktonic bloom events, with high inputs of fresh material. While
there is variability in the amount and type of degradation undergone by
sterols in the sampled particulate matter, it is evident that sitosterol and
cholesterol behave very differently when being degraded.
Unsaturated fatty acids and cuticular waxes
Fatty-acid sensitivity to photo- and autoxidation is intrinsically linked to
their number of double bonds (Frankel, 1998), and we will only be looking at
unsaturated fatty acids here. Unfortunately, oxidation products of
polyunsaturated fatty acids (PUFA) are not stable enough to be used to
monitor PUFA degradation. In contrast, photo- and autoxidation products of
mono-unsaturated fatty acids (allylic hydroperoxyacids) are much more stable,
and can be used (after NaBH4-reduction to the corresponding
hydroxyacids) as tracers of the abiotic oxidation processes affecting POM
(Marchand and Rontani, 2001). Free-radical-mediated oxidation (autoxidation)
processes can be easily discriminated against photo-oxidation processes
thanks to the specific allylic hydroperoxyacids specifically produced by
autoxidative processes (Marchand and Rontani, 2001).
Samples only displayed small amounts of oxidation products of oleic acid (not
quantified), probably due to the fact that unsaturated fatty acids and their
degradation products are very labile and easily metabolized by bacteria
(Marchand et al., 2005). Despite this degradation, the profiles obtained by
GC-MS (exhibiting relatively high proportions of cis oxidation
products) allowed us to confirm the important role played by autoxidation in
the degradation of POM in the Rhône River. Cutin is present in cuticles
covering all aerial parts of higher plants. It is constituted of
biopolyesters mainly composed of hydroxy fatty acids. Long-chain
n-alkanoic, ω-hydroxy, dihydroxy, trihydroxy and epoxy-hydroxy
acids constitute the major aliphatic monomers (Kolattukudy, 1980). It was
previously demonstrated that Type II photo-oxidation processes act on some
unsaturated cutin monomers such as ω-hydroxyoctadec-9-enoic acid
(ω-hydroxyoleic acid) during the senescence of higher plants (Rontani
et al., 2005). 1O2 reacts with the carbon–carbon double bond, and
leads to the formation of a hydroperoxide at each unsaturated carbon. Due to
the involvement of allylic rearrangements, Type II photosensitized oxidation
of ω-hydroxyoleic acid results (after NaBH4-reduction of
hydroperoxides to the corresponding alcohols) in the formation of isomeric
allylic 18,(8–11)-dihydroxyoctadecanoic acids, with a trans double
bond. These compounds constitute interesting specific tracers of higher plant
material photo-oxidation. Autoxidation of this compound was never studied,
but by analogy with oleic acid oxidation the autoxidative formation of
specific cis allylic hydroperoxyacids was expected.
Significant amounts of allylic 18,(8–11)-dihydroxyoleic acid, with
cis and trans double bonds have been effectively detected
in some (not all) samples analyzed attesting to the involvement of auto- and
photo-oxidation of higher plant material (Galeron and Rontani, unpublished
data). The high proportions of cis isomers observed confirmed the
dominance of autoxidation processes. Some samples (16 January 2012, 26 June
2012 and 18 July 2011) even displayed larger amounts of oxidation products
than ω-hydroxyoleic acid, which evidences the importance of
degradative processes on this compound. The previously discussed yearly
variability in cuticular wax content in our samples (see Sect. 3.2.2 and
Fig. 3b) explains some of these results.
Hydroperoxide stability in SPM
It was previously proposed that photochemically produced hydroperoxides could
induce intense autoxidation processes in the marine environment (Rontani et
al., 2014a). Hydroperoxides resulting from photo-oxidation processes may
undergo: (1) heterolytic cleavage catalyzed by protons (Frimer, 1979) and (2)
homolytic cleavage induced by transition metal ions (Pokorny, 1987) or UVR
(Horspool and Armesto, 1992). Homolytic cleavage of hydroperoxides would lead
to the formation of alkoxyl radicals, which can then: (1) abstract a hydrogen
atom from another molecule to give alcohols, (2) lose a hydrogen atom to
yield ketones, or (3) undergo β-cleavage reactions affording volatile
products. During the NaBH4-reduction, hydroperoxides and ketones were
reduced to the corresponding alcohols. The sum of the corresponding
hydroperoxides, ketones and alcohols was evaluated under the form of
alcohols. Application of a different treatment allowed us to specifically
quantify hydroperoxides, alcohols and ketones (remaining in cuticular waxes,
phytol, oleic acid, sitosterol and cholesterol oxidation products) (Fig. 7).
Clearly, the proportion of remaining hydroperoxides was highest in the case
of sterols, with 49.4 and 31.3 % respectively for 3,6- and 3,7-diols of
sitosterol, and 51.5 and 33.5 % for 3,6- and 3,7-diols of cholesterol,
against less than 20 % (17.3 %) for cutins, 12.0 % for oleic acid, and
6.6 % for phytol. Standard error was calculated based on all the results
obtained (standard error = standard deviation / √(n) for n
samples). These results clearly indicate that despite the involvement of an
intense free radical oxidation (autoxidation) inducing homolytic cleavage of
peroxy bonds, a significant proportion of hydroperoxides is still intact in
POM of the Rhône River. This proportion reaches 10 % of the parent
residual compound in the case of sitosterol and 5 % in the case of
cholesterol. Probably due to high compartmentalization effects, preservation
of these compounds seems to be enhanced in higher plant debris. It was
recently proposed that homolytic cleavage of photochemically produced
hydroperoxides in riverine POM could be catalyzed by some redox-active metal
ions released from SPM in the mixing zone of riverine and marine waters
(Rontani et al., 2014b). Due to the presence of significant amounts of
hydroperoxides in higher plant residues, the involvement of intensive
autoxidation of this material in the Rhône estuary is thus likely.
Relative percentages of intact hydroperoxides and their ketonic and
alcoholic degradation products measured in the case of ω-hydroxyoleic
(cuticular waxes) and oleic acids, phytol, sitosterol and cholesterol
oxidation products. Standard error was calculated based on the results
obtained for all samples.