1
ACID WASHING EFFECT ON ELEMENTAL AND ISOTOPIC COMPOSITION OF
WHOLE BEACH ARTHROPODS: IMPLICATIONS FOR FOOD WEB STUDIES
USING STABLE ISOTOPES
O Serrano1, L Serrano1, MA Mateo1*, I Colombini2, L Chelazzi2, E Gagnarli2, M
Fallaci2
1
Centro de Estudios Avanzados de Blanes, Consejo Superior de Investigaciones Científicas.
Acceso a la Cala St. Francesc, 14. 17300 Blanes, Girona, Spain. Fax number: (+34) 972
337806.
2
Istituto per lo Studio degli Ecosistemi del CNR Sezione di Firenze, Via Madonna del Piano,
Sesto Fiorentino, Florence, Italy.
*Corresponding author: mateo@ceab.csic.es
2
ABSTRACT
Inorganic carbon removal through acidification is a common practice prior to isotopic
analysis of macroinvertebrate samples. We have experimentally tested the effect of
acidification on the elemental and isotopic composition of a range of beach arthropod
species. Acidification resulted in a significant depletion of 7.7 % and 1.2 % in average for
carbon and nitrogen, respectively. This highlights that acid washing affects other body
compounds apart from carbonates. With a few exceptions, 13C and 15N showed no changes
following the 1N HCl attack. Our results strongly suggest that samples with a presumable
high CaCO3 content, result impoverished in
13
C as a consequence of acidification, while
those suspected to be carbonate-free are not significantly affected. Regarding 15N values, it
seemed that only high carbonate species may be 15N impoverished when treated with HCl. It
is recommended to acidify only carbonate-rich samples prior 13C analyses as a standard
protocol.
KEYWORDS: acidification, carbon, nitrogen, carbonates, isotopic ratios, macroinvertebrates
3
INTRODUCTION
Carbon and nitrogen isotopic ratios are very useful tools for the study of food web structures
in aquatic ecosystems (e.g., Jennings et al., 1997; Pinnegar and Polunin, 2000; Vizzini and
Mazzola, 2003). This approach helps to elucidate the origin of the ingested organic matter
(Fry and Sherr, 1984; Owens, 1987; Preston, 1992) and to characterize flows of mass and
energy through ecosystems (Fry, 1988; Owens 1988; Hobson and Welch, 1992; Rau et al.,
1992; Hesslein et al., 1993).
Most arthropod species contain carbonates which contribute to the global animal isotopic
signature. It is common that samples are acidified before analysis in order to avoid the
alteration of the 13C values by the high 13C content of this non-dietary carbon fraction (e.g.,
Nieuwenhuize et al., 1994; Adin and Riera, 2003). The chemical reaction that follows the
acidification treatment is:

CaCO3  2HCl  CO2  CaCl2  H 2O
[eq. 1]
However, the need and the convenience of using HCl as part of the sample preparation for
duel isotopic analysis is still a matter of controversy that only recently begins to be directly
addressed (e.g., Soreide et al. 2006; Mateo et al., submitted for publication). A few
observations have reported that acidification may also alter 15N values (Pinnegar and
Polunin, 1999), supposedly as a consequence of partial loss of compounds containing
nitrogen (Bunn et al., 1995; Jacob et al., 2005) such as chitin, proteins, and glycoprotein
(Goering et al., 1990; Shafer et al., 1994). Based on this, some authors prefer to divide the
sample into two aliquots, one acidified for the 13C analysis and another one not acidified for
the 15N analysis (Polunin et al., 2001; Bouillon et al., 2002; Nyssen et al., 2002). Other
studies have reported no significant effect by HCl attack on the isotopic signatures of marine
4
animals, concluding that acidification of the samples was unnecessary (e.g., Chanton and
Lewis, 1999; Grey et al., 2001; Nyssen et al., 2005). Some authors have tried to limit the
extent of the eventual effect of acidification by acidifying samples before drying and
grinding (Bunn et al., 1995) or using more gentle carbonate removal methods such as wetting
samples in weak acid (0.1 N HCl; e.g., Hobson et al., 2002). However, only few studies
presented data of the elemental composition (C and N) and discussed the effects of
acidification on the elemental composition (Bunn et al., 1995). To contribute to the
assessment of these methodological effects in beach macroinvertebrates, we evaluated the
effects of acidification on C and N elemental composition and on the 13C and 15N values of
some representative beach arthropods. The effect of acid washing in the carbon and nitrogen
composition of these species has never been reported. To this end, the isotopic and elemental
compositions of acidified and non-acidified aliquots have been compared.
Recently, Jacob et al. (2005) reported a positive linear relation between the effect of
acidification on invertebrate 13C values and an estimate of its carbonate content. Since then,
however, the sample’s CaCO3 content related to acid washing effect on isotopic values has
received little attention. An exception is a study by Ng et al. (2007), where they calculated a
carbonate proxy (as in Jacob et al., 2005) and recommended acid rinsing only for carbonaterich algae. We hypothesise that acidification of invertebrate samples prior to analytical
procedures involves changes both in the elemental and in the isotopic composition that can
seriously confound food web analysis based on isotopic trophic scenarios. More specifically,
we hypothesise that the carbonate content of the invertebrate is the most important factor
causing isotopic shifts during acidification. To assess this problem, we have selected various
beach and semi-terrestrial macroinvertebrate species and compared the elemental and
isotopic compositions of acid-washed to raw aliquots and their relation with a carbonate
5
proxy. In the light of the results, simple criteria for sample pre-treatment are proposed for
standardization.
MATERIALS AND METHODS
Sample collection was carried out in May 2005 on the beach of Burano (N 42°23’51”; E
11°22’ 40”, Grosseto, Italy) using pitfall cross traps that intercepted macroinvertebrates. Six
pitfall cross traps were deployed along the beach at 3 m-intervals from the shoreline to the
base of the dune. The traps were kept active for 24 hours. The individuals captured were
stored in thermally-sealed plastic bags and frozen at the site. Several individuals of the
macroinvertebrate
species
Arctosa
cinerea
(Araneae,
Lycosidae),
Geophilus
sp.
(Geophilomorpha, Geophilidae) Parallelomorphus laevigatus (Coleoptera, Carabidae),
Phaleria bimaculata (Coleoptera, Tenebrionidae), Phaleria provincialis (Coleoptera,
Tenebrionidae), Pimelia bipunctata (Coleoptera, Tenebrionidae), Scarites buparius
(Coleoptera, Carabidae), Talitrus saltator (Amphipoda, Talitridae) and Tylos europaeus
(Isopoda, Tylidae) were selected.
All individuals were taxonomically determined, age
classified, washed and oven-dried at 60°C. Finally, they were kept in a dry and dark place
until processing. Four replicates of each species were used for elemental and isotopic
analysis. Replicates consisted of 5-8 whole individuals of the same species. All replicates
were re-dried and milled to a fine homogeneous powder using an agate mortar and pestle.
The powder was stored in a dry environment (relative humidity under 20%). About half of
each one of the replicates was acidified by adding 1 N HCl drop-by-drop until cessation of
bubbling (Nieuwenhuize et al., 1994). Samples were then left in excess of acid for 3 hours.
The other half was kept untreated. The acidified sub-samples were re-dried at 60ºC for 24h,
and milled again to a fine homogenous powder. Samples were not rinsed with distilled water
6
after acidification to avoid the alteration of the isotope values by the lixiviation of some
organic carbon and nitrogen compounds (e.g., Cloern et al., 2002).
Finally, an aliquot of 0.7 mg DW of each sample was weighted and placed in a tin capsule
for solid samples. The encapsulated samples were kept under constant laboratory temperature
(20º C) and humidity conditions until analysis. Elemental and isotopic composition was
determined for the gasses evolved from a single combustion using a Finnigan Delta S isotope
ratio mass spectrometer (Conflo II interface) at the Scientific-Technical Services of the
University of Barcelona. Isotopic values are reported in the VPDB notation relative to the
standards Vienna Pee Dee Belemnite and air for carbon and nitrogen, respectively (sample =
1000 [(Rsample/Rstandard) – 1], R = 13C/12C, or R = 15N/14N). Analytical precision based on the
standard deviation of internal standards (atropine, IAEA CH3, CH6, CH7, and USGS40 –
analytical grade L-glutamic acid, for carbon, and
atropine, IAEA N1, NO3, N2, and
USGS40, for nitrogen) ranged from 0.11 ‰ and 0.06 ‰ (mean = 0.09 ‰) for carbon, and
from 0.06 ‰ and 0.28 ‰ (mean = 0.16 ‰) for nitrogen.
All data were checked for normality (Kolmogorov-Smirnov test) and for variance
homogeneity (Levene’s test). One-way ANOVAs were used to test the null hypothesis of no
difference in the elemental composition and isotopic values between acidified and untreated
samples. A proxy was used to estimate the content of CaCO3 and to assess its role in the
effect of acid washing on the stable isotope ratios (Jacob et al., 2005):
Carbonate proxy = [(C : N)untreated / (C : N)acidified] - 1
[eq. 2]
where [C : N]untreated and [C : N]acidified are the C:N ratios of raw and acidified samples
respectively. This proxy is linearly related to sample CaCO3 content given that tissue C:N is
7
independent of sample CaCO3 (Jacob et al., 2005). All numerical procedures were performed
using the statistics software package STATISTICA 7.1 (STATSOFT, Inc, OK, USA).
RESULTS AND DISCUSSION
Elemental composition
In many arthropods carbon is contained both in inorganic and organic forms. Because trophic
ecology based in stable isotopes is mainly interested in the carbon fixed in the organic
tissues, the bias introduced by the inorganic carbon is usually eliminated by acid washing
prior to analysis (e.g. Rau et al., 1983; Jackson et al., 1986; Hobson and Welch, 1992). It is
understood that the carbon lost during acid washing corresponds to the inorganic carbon that
forms part of their exoskeletons. Carbonates contain a low proportion of carbon (12%) if
compared to other animal tissues like muscle (40%, e.g., Gorokhova and Hansson, 2000) or
chitin (40.3% C, e.g., Amiji, 1998). Following the rationale above, the %C should increase
after acidification. On the contrary, we have observed a decrease in carbon content in all the
samples analyzed (Table 1). Moreover, despite carbonates do not contain nitrogen, we
observe a loss of nitrogen associated to acid washing, evidencing that also organic (i.e.,
dietary) nitrogen is being removed. One-way ANOVA showed highly significant differences
in both %C and %N between acidified and non-acidified samples (P < 0.001 and P < 0.01,
respectively, Table 1). Acidification resulted in a systematically loss of dietary C and N in
almost all species. The exceptions are represented by the lack of changes in nitrogen content
in Geophilus sp. and in adults of T. europaeus. The proportion of carbon in the samples
decreased 7.7% in average as a consequence of acidification, the maximum decrease being of
10.7% (adults of T. saltator) and the minimum of 4.4% (adults of T. europaeus). Nitrogen
content decreased 1.2% in average after acidification, ranging from 0% to 1.9% (for
Geophilus sp., and both Arctosa cinerea and Phaleria spp. larvae, respectively). The
8
elemental impoverishment after acid washing was proportionally higher in C than in N
resulting in a lower C:N ratio in acidified samples (Table 1). The mean 0.2 decrease in C:N
ratio, however, was not significant. An exception was the case of juveniles of T. saltator and
T. europaeus, with significant decreases in C:N ratios of 0.4 and 0.9, respectively. This result
suggests that the carbon and nitrogen is lost proportionally during acidification in most of the
cases. A positive relationship exists when comparing the sample C and N content of nonacidified and acidified samples (r2 = 0.75 and 0.84, respectively; Figure 1). This relationship
may be a consequence of differences in the species carbonate content. Species containing
high amounts of carbonates may be carbon and nitrogen impoverished due to low-carbon and
negligible nitrogen content of carbonates. Two sub-groups were discriminated corresponding
to the species belonging to Malacostraca (low carbon and nitrogen content) and to the rest of
groups (high carbon and nitrogen content). After acidification, the %C and %N decreased
proportionally as a function of the initial content for all species. T. saltator and T. europaeus
species were those which significantly lose a higher proportion of carbon during
acidification, in comparison with the other arthropods (P < 0.01; 8.9% and 7.0%,
respectively). Malacostraceans were not significantly nitrogen depleted in comparison with
the rest of groups (1.15% and 1.34%, respectively) as a consequence of acid washing. Our
results suggest that HCl attack removes systematically C and N from all samples as a
function of sample carbonate content (see further).
13C and 15N values
Acidification did not show an overall significant effect on macroinvertebrate isotopic ratios
(Table 2). More specifically, 13C values showed an average decrease of 0.3o/oo after
acidification, whereas 15N was unaffected. Taking into account the large effect observed in
the elemental composition, it is somehow surprising the lack of effect of acidification on the
9
isotopic signatures. One plausible explanation for such an unexpected result is simply that the
samples analyzed had no or very little carbonate content and leaching of dietary carbon and
nitrogen-bearing compounds occurs without or with negligible isotopic fractionation. An
alternative hypothesis is the selective removal of dietary
compensate for the loss of
13
13
C-depleted compounds which
C-rich carbonate carbon (with a 13C around 0o/oo, e.g., Kump
and Arthur 1999). The treatment with HCl has been found to remove part of the acid-soluble
proteins from the acetyl groups of the chitin and tissues (chitin 13C = -23.6 o/oo as reported in
Schimmelmann and DeNiro, 1985) through hydrolytic depolymerization and deacetylation
(DeNiro and Epstein, 1981; Percot et al., 2003). Both hypotheses are also consistent with the
general decrease of the elemental carbon and nitrogen composition.
Acidification only affected the isotopic composition of two of the cases studied. Concretely,
the juveniles of T. europaeus showed a highly significant decrease both in 13C and 15N
values after acidification of 1.9 and 0.6o/oo, respectively. Adults of T. europaeus and
juveniles of T. saltator presented a slight significant decrease only in the carbon isotopic
ratio (1.1 and 0.3 o/oo, respectively). The isotopic compositions of the rest of species did not
show any significant change following acidification. 13C was higher in malacostraceans than
in the rest of species both before and after acid washing (mean±SD before: -19.7±1.5 and 22.3±1.8, respectively; after: -20.6±1.2 and-22.3±1.8, respectively), whereas 15N showed no
significant differences. Macroinvertebrates carbon content and 13C of non-acidified samples
were negatively correlated (r2 = 0.58, P < 0.001; Figure 2). These results suggest that
acidification only affects the carbon isotopic composition of those species containing
relatively high proportions of carbonate, such is the case of the malacostraceans studied here
(T. europaeus and T. saltator).
The carbonate proxy and acid washing effect
10
The macroinvertebrates considered in our experiments belong to four different Classes
(Insecta, Malacostraca, Arachnida and Chilopoda). Their exoskeletons may differ in
structural and molecular characteristics. Arthropods harden their new cuticle by a process
called sclerotization (proteins-polysaccharides cross-linking), whereas most of the
crustaceans proceed also by calcification (Travis, 1963; Giraud-Guille and Quintana, 1982).
The amphipod T. saltator and the isopod T. europaeus belonging to Malacostraca, which
origins are marine, are highly modified for living in the supralittoral zone, and hence are
physiologically and morphologically pre-adapted for terrestrial environments (e.g., Little,
1983; Powers and Bliss, 1983; Spicer et al., 1987). Three of the four layers forming the
Isopod and Amphipod cuticles are mineralised, essentially by precipitation of calcium
carbonate into the chitin-protein cholesteric matrix (e.g., Luquet and Marin, 2004).
We compared the carbonate proxy (Eq. 2) of our samples with the differences in the isotopic
signatures between untreated and treated samples (Figure 3a, b) to assess the effects of
acidification on the isotopic composition as a function of carbonate content. The differences
in the carbon isotope ratios were positively related to the sample CaCO3 proxy (r2 = 0.54, N
= 43, P < 0.001), being those species with greater carbonate content the most affected by acid
washing. As expected, species with high carbonate proxy values where
13
C-depleted after
acid washing, as a result of the removal of carbonates. The differences in the nitrogen isotope
ratios were not related to the CaCO3 proxy (N = 43, P > 0.05). Bosley and Wainright (1999)
and Carabel et al. (2006) reported the lack of effects of carbonate removal on the 15N values
in a wide spectrum of zooplankton size fractions and crabs, supporting our findings. If we
accept that acid washing implies removal of nitrogen, then the overall lack of effect in 15N
following HCl attack should be ascribed to the absence of isotopic fractionation during the
process. Exceptions were the samples of juveniles of T. europaeus, which presented a
significant decrease both in the elemental and in the isotopic values. A few observations have
11
reported that acidification can alter 15N values (Goering et al., 1990; Bunn et al., 1995;
Pinnegar and Polunin, 1999), supposedly as a consequence of partial loss of cuticle matrix
compounds.
Adults and juveniles of T. europaeus are the species which estimated carbonate proxy was
highest (mean±SD = 0.13±0.06 and 0.21±0.02, respectively). Also, the carbonate proxy of T.
saltator adults and juveniles was found to be high (0.10±0.04 and 0.09±0.04, respectively) in
relation to the other beach macrofauna studied (average, 0.01±0.03, ranging from -0.04 to
0.04). The analysis of presumably CaCO3-free species, such as Phaleria spp. larvae, showed
negative carbonate proxy values (-0.04±0.03).
Carbonates contain a low proportion of carbon and do not contain nitrogen. Coherently,
malacostraceans (which yielded the highest carbonate proxy values) also present the lowest
carbon and nitrogen relative content (mean ± SD = 32.7 % ±3.2 and 6.4 %±1.0, respectively).
Similarly, species with low carbonate proxy values present higher carbon and nitrogen
content (48.9 % ±3.9 and 11.0 % ±0.9, respectively).
The relationship between carbonate content and the acid washing effect on the carbon isotope
signatures suggests that only the carobn isotope rario of those beach arthropods with a
carbonate proxy equal or higher than 0.09, is significantly altered after acidification. Jacob et
al. (2005) observed the same trends studying a wide range of species; those samples with
high carbonate proxies were
13
C depleted after being acidified. Our results suggest that acid
attacks carbonates when present, but also alters chitin structure, proteins and other tissues.
Other studies report that non-acidified whole macroinvertebrates with high proportions of
hard parts can be enriched in 13C by 2.5 o/oo compared to acidified duplicates (Carabel et al.,
2006). Bunn et al. (1995) also reported that when the proportion of exoskeletons to body
mass is small, the effect of acid washing on the 13C values may not be detectable. Soreide et
al. (2006) studied the controversy around the effect of the different methodologies used prior
12
to isotopic analysis and published C and N elemental composition data of the marine species
Gammarus wilkitzkii and Thysanoessa inermis. We calculated an average carbonate proxy
including all data from Soreide et al. 2006 when its calculation was possible (mean ± SD =
0.77±0.3, N = 12). The carbonate proxy calculated for that data showed much higher CaCO3
content in comparison with our data. Aquatic crustaceans are expected to be CaCO3 enriched
because the lower impact of gravity allows them to carry stronger and heavier protective
structures (Little, 1983). Unfortunately, data on elemental composition of macroinvertebrates
is very scarce in the literature making a detailed carbonate proxy modelling impracticable.
CONCLUSIONS
One first conclusion from all above is that, in the light of the results presented, the processes
taking place as a consequence of acidification are more complex than initially thought. This
is a consequence of the high chemical inter- and intra-specific heterogeneity of the different
macroinvertebrates (Welinder, 1974). In the case of semi-terrestrial crustaceans, the changes
observed in the 13C are large enough to critically confound food web analysis. As shown,
the problem associated to acid washing (1 N HCl) is that some dietary carbon- and nitrogenbearing compounds are being removed together with non-dietary ones resulting in a
substantial change in the C and N content of the samples. The removal of carbonates would
require a technique that ensured no effect on non-carbonate carbon fractions. Percot et al.
(2003) proposed and demonstrated that a concentration of HCl of 0.25M during 15 min was
enough for complete demineralization minimizing the hydrolysis of chitin polymers of the
exoskeletons, being the most appropriate protocol for the acid washing treatment. In order to
avoid isotopic variability due to acid washing samples we recommend using non-acidified
soft tissues whenever possible for isotopic analyses. The results presented strongly support
that acidification of beach macroinvertebrates could be omitted in all cases except when the
13
study is interested in the 13C values of invertebrates with a carbonate proxy > 0.09 (as for
the case of semi-terrestrial crustaceans in this study). In this last case, untreated aliquots
should be analysed separately for 15N.
ACKNOWLEDGEMENTS
This study has been supported partially by funds of a bilateral programme between the CNR
(Italy) and the CSIC (Spain), and partially by the EU-INCO project WADI (CT2005-15226).
14
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Table 1. Effect of acid washing on the C and N elemental composition of beach
macroinvertebrates. Top: elemental carbon and nitrogen composition values and C:N ratios;
bottom: results of individual univariate tests of variance. P, significance level, *P ≤ 0.05,
**P ≤ 0.01, ***P ≤ 0.001; NS, non-significant difference (P ≥ 0.05).
21
%C
acid
mean
N
Arctosa cinerea
Geophilus sp.
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Phaleria spp. (larvae)
Pimelia bipunctata
Scarites buparius
Talitrus saltator (adult)
Talitrus saltator (juvenile)
Tylos europaeus (adult)
Tylos europaeus (juvenile)
37,0
46,2
42,5
43,8
42,7
38,6
42,6
45,0
25,2
24,6
25,7
19,5
AVERAGE
36,1
Species
SD
%C
no acid
mean
N
4
4
5
5
3
4
4
4
4
4
4
4
1,17
0,96
1,58
0,78
0,41
1,95
0,17
1,33
1,52
2,07
1,24
0,82
44,0
52,6
51,2
52,0
49,3
44,6
48,3
52,8
35,9
35,2
30,1
29,5
49
9,18
43,8
%C
Species
SD
%N
acid
mean
N
SD
4
3
4
4
4
4
4
4
4
4
4
4
1,11
0,32
1,07
1,48
0,67
1,54
0,72
0,94
1,77
0,15
1,65
0,67
10,8
9,8
9,6
9,2
9,7
9,1
9,4
9,4
5,7
5,6
5,6
4,1
4
4
4
4
4
4
4
4
4
4
4
4
0,35
0,31
0,38
0,20
0,23
0,54
0,03
0,32
0,26
0,34
1,10
0,12
12,7
9,8
11,3
10,6
10,9
11,0
10,6
10,3
7,3
7,4
5,5
5,3
4
4
3
3
4
4
4
3
3
4
3
4
47
8,06
8,2
48
2,20
9,4
%N
%N
no acid
mean N
SD
C:N
acid
mean
SD
C:N
no acid
mean
N
N
SD
0,28
1,23
0,05
0,32
0,11
0,35
0,10
0,60
0,31
0,10
0,15
0,17
3,4
4,7
4,2
4,4
4,8
4,5
4,5
4,8
4,6
4,4
4,9
4,7
4
4
4
4
4
4
4
4
4
4
4
4
0,1
0,2
0,1
0,0
0,1
0,1
0,0
0,3
0,4
0,1
0,5
0,1
3,5
5,0
4,1
4,4
4,9
4,5
4,6
5,0
4,9
4,8
5,3
5,6
4
4
4
4
4
4
4
4
4
4
4
4
0,0
0,2
0,1
0,0
0,2
0,1
0,1
0,4
0,3
0,1
0,1
0,1
43 2,36
4,5
48 0,42
4,7
48 0,56
C:N
MS
f
P
MS
f
P
MS
f
P
Arctosa cinerea
Geophilus sp.
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Phaleria spp. (larvae)
Pimelia bipunctata
Scarites buparius
Talitrus saltator (adult)
Talitrus saltator (juvenile)
Tylos europaeus (adult)
Tylos europaeus (juvenile)
97,796
69,045
166,764
148,171
73,544
70,699
65,974
121,008
229,466
223,167
38,752
199,443
75,54
116,37
86,65
114,70
218,10
22,98
238,58
91,39
84,20
103,77
18,19
354,13
***
***
***
***
***
**
***
***
***
***
**
***
7,080
0,000
5,213
2,965
3,114
6,922
2,866
1,484
4,216
6,353
0,008
2,629
72,54
0.00
60,18
45,63
96,34
33,20
548,70
7,20
53,20
103,54
0,11
128,67
***
NS
***
**
***
**
***
*
***
***
NS
***
0,003
0,123
0,006
0,024
0,000
0,064
0,002
0,113
0,179
0,283
0,284
1,513
9,72
3,35
4,19
0,78
0,00
6,40
0,85
1,00
1,24
33,29
2,37
78,78
NS
NS
NS
NS
NS
NS
NS
NS
NS
**
NS
***
AVERAGE
1204,966
15,3
***
39,310
7,61
**
0,008
4,14
NS
Table 2. Effect of acid washing on the C and N isotopic ratios of beach macroinvertebrates.
Top: 13C and 15N values; bottom: results of individual univariate tests of variance. P,
significance level, *P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001; NS, non-significant difference (P ≥
0.05).
22
13C
acid
mean
N
Arctosa cinerea
Geophilus sp.
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Phaleria spp. (larvae)
Pimelia bipunctata
Scarites buparius
Talitrus saltator (adult)
Talitrus saltator (juvenile)
Tylos europaeus (adult)
Tylos europaeus (juvenile)
-20,9
-23,2
-20,8
-21,5
-21,1
-21,0
-24,9
-25,2
-21,9
-21,2
-19,0
-20,5
AVERAGE
-21,8
Species
SD
13C
no acid
mean
N
4
4
4
4
4
4
4
4
4
4
4
4
0,10
0,61
0,15
0,33
0,25
0,41
0,14
0,97
0,34
0,15
0,62
0,22
-20,8
-23,3
-21,0
-21,6
-21,1
-20,7
-24,7
-25,7
-21,5
-20,9
-17,9
-18,6
48
1,81
-21,5
13C
Species
df
Arctosa cinerea
Geophilus sp.
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Phaleria spp. (larvae)
Pimelia bipunctata
Scarites buparius
Talitrus saltator (adult)
Talitrus saltator (juvenile)
Tylos europaeus (adult)
Tylos europaeus (juvenile)
1
1
1
1
1
1
1
1
1
1
1
1
AVERAGE
1
SD
15N
acid
mean
SD
15N
no acid
mean
N
N
SD
4
4
3
3
4
4
4
3
4
5
3
4
0,10
0,46
0,02
0,23
0,19
0,39
0,07
1,01
0,25
0,19
0,17
0,17
10,0
7,4
11,6
8,9
9,2
9,3
4,7
3,5
6,5
7,1
8,9
7,7
4
4
5
4
4
4
4
5
5
4
5
4
0,13
0,76
0,17
0,17
0,12
0,83
0,14
0,21
0,16
0,18
0,43
0,08
10,1
7,5
11,7
8,9
9,1
9,4
4,6
3,4
6,5
7,3
8,9
8,3
4
4
4
4
4
4
4
4
5
5
4
4
0,06
0,75
0,08
0,28
0,26
0,58
0,12
0,37
0,12
0,13
0,10
0,08
45
2,12
7,9
52
2,23
8,0
50
2,24
15N
MS
f
P
MS
f
P
0,009 0,87
0,018 0,06
0,053 3,66
0,014 0,16
0,003 0,06
0,191 1,20
0,056 4,50
0,431 0,44
0,281 3,53
0,193 6,28
1,981 8,48
7,220 196,91
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
*
***
0,003
0,003
0,043
0,010
0,003
0,033
0,012
0,002
0,009
0,103
0,008
0,605
0,26
0,00
2,33
0,21
0,06
0,06
0,71
0,03
0,40
4,51
0,07
121
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
***
1,952
NS
0,215 0,04 NS
0,5
CAPTIONS FOR FIGURES
Figure 1. Relationship between C and N elemental composition in macroinvertebrate
samples considering two groups: Malacostraca (T. saltator and T. europaeus); and ‘other
arthropods’ (the rest of species studied). Significant linear relation existed in both cases
(before and after acidification), not showed. Non-acidified samples: y = 13.764 + 3.145 * x,
23
r2 = 0.753, P < 0.001, N = 48. Acidified samples: y = 4.802 + 3.868 * x, r2 = 0.843, P <
0.001, N = 48.
Figure 2. Relationship between non-acidified 13C values and carbon elemental
compositions: y = -12.717 – 0.207 * x, r2 = 0.582, P < 0.001, N = 48.
Figure 3. (a) Relationship between the carbonate proxy and the difference in 13C between
acid washed and untreated samples (y = 0.025 + 0.085  x, r2 = 0.54, P < 0.001, N = 43). (b)
Relationship between the carbonate proxy and the difference in 15N between acid washed
and untreated samples.
24
60
50
%C
40
30
20
Other arthropods ac
Cl. Malacostraca ac
Other arthropods no ac
Cl. Malacostraca no ac
10
2
4
6
8
%N
Figure 1
10
12
14
25
-16
-18
-22
13
 C (%o)
-20
-24
-26
-28
25
30
35
40
45
50
55
%C
Arctosa cinerea
Geophilus sp.
Phaleria spp. (larvae)
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Pimelia bipunctata
Scarites buparius
Figure 2
Talitrus saltator adult
Talitrus saltator juvenile
Tylos europaeus adult
Tylos europaeus juvenile
26
a)
2,5
2,0
 C crude -  C acid
1,5
13
1,0
0,5
13
0,0
-0,5
-1,0
-1,5
-0,1
0,0
0,1
0,2
Carbonate proxy
b)
1,0
0,6
0,4
0,2
15
15
 N crude -  N acid
0,8
0,0
-0,2
-0,4
-0,10
-0,05
0,00
0,05
0,10
0,15
0,20
0,25
Carbonate proxy
Arctosa cinerea
Geophilus sp.
Phaleria spp. (larvae)
Parallelomorphus laevigatus
Phaleria bimaculata
Phaleria provincialis
Pimelia bipunctata
Scarites buparius
Figure 3
Talitrus saltator adult
Talitrus saltator juvenile
Tylos europaeus adult
Tylos europaeus juvenile