Original article

Acid washing effect on elemental and isotopic compositionof whole beach arthropods: Implications for food webstudies using stable isotopes

Oscar Serranoa, Laura Serranoa, Miguel Angel Mateoa,*, Isabella Colombinib,Lorenzo Chelazzib, Elena Gagnarlib, Mario Fallacib

aCentro de Estudios Avanzados de Blanes, Consejo Superior de Investigaciones Cientıficas. Acceso a la Cala St. Francesc,

14. 17300 Blanes, Girona, SpainbIstituto per lo Studio degli Ecosistemi del CNR Sezione di Firenze, Via Madonna del Piano, Sesto Fiorentino, Florence, Italy

a r t i c l e i n f o

Article history:

Received 1 February 2008

Accepted 17 April 2008

Published online 3 June 2008

Keywords:

Acidification

Carbon

Nitrogen

Carbonates

Isotopic ratios

Macroinvertebrates

a b s t r a c t

Inorganic carbon removal through acidification is a common practice prior to isotopic anal-

ysis of macroinvertebrate samples. We have experimentally tested the effect of acidifica-

tion 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% on average for carbon

and nitrogen, respectively, suggesting that acid washing affects body carbon compounds

other than carbonates. With a few exceptions, d13C and d15N showed no changes following

1 N HCl attack. Based on those exceptions, our results show that only those samples with

a high CaCO3 content result in impoverished 13C as a consequence of acidification. Those

suspected to be carbonate-free are not significantly affected. Concerning d15N values,

only high carbonate species were affected when treated with HCl. As a standard protocol,

it is recommended to acidify only carbonate-rich samples prior to d13C analyses. When

possible, muscle tissue samples should be used instead of the entire organism.

ª 2008 Elsevier Masson SAS. All rights reserved.

1. 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; Fry, 2006). 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 contrib-

ute to the global animal isotopic signature. It is common that

samples are acidified before analysis in order to avoid the

alteration of the d13C values by the high 13C content of this

non-dietary carbon fraction (e.g., Nieuwenhuize et al., 1994;

Yokohama et al., 2005). The chemical reaction that follows

the acidification treatment is:

CaCO3 þ 2HCl/CO[2 þ CaCl2 þH2O (1)

However, the need for and the convenience of using HCl as

part of the sample preparation for duel isotopic analysis is still

a matter of controversy that has only recently begun to be

directly addressed (e.g., Soreide et al., 2006; Mateo et al. (in

press)). A few observations have reported that acidification

* Corresponding author. Fax: þ34 972 337806.E-mail address: mateo@ceab.csic.es (M.A. Mateo).

ava i lab le at www.sc ienced i rec t . com

journa l homepage : www. e lsev ier . com/ loca te /ac toec

1146-609X/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.actao.2008.04.002

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 6

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 690

may also alter d15N 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 di-

vide the sample into two aliquots, one acidified for the d13C

analysis and another one not acidified for the d15N 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 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 grind-

ing (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 a few studies have

presented data on the elemental composition (C and N) and

discussed the effects of acidification on the elemental compo-

sition (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 d13C and d15N values of some repre-

sentative beach arthropods. The effect of acid washing on

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 rela-

tion between the effect of acidification on invertebrate d13C

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 ex-

ception 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 carbonate-rich algae. We hypothesize

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 specif-

ically, we hypothesize that the most common acidification

procedure (i.e., 1 N HCl acid washing until bubbling cessation)

results in the removal of dietary (i.e., not from carbonates) car-

bon and nitrogen. We finally provide strong evidence support-

ing that the carbonate content of the invertebrate sample can

be one of the most useful criteria for deciding on the need for

acidifying the samples prior to isotopic analysis. 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 proxy. In the light of the re-

sults, simple criteria for sample pre-treatment are proposed

for standardization.

2. Materials and methods

Sample collection was carried out in May 2005 on the beach

of Burano (N 42�2305100; E 11�2204000, Grosseto, Italy) using

pitfall cross traps that intercepted macroinvertebrates. Six

pitfall cross traps were deployed along the beach at 3-m in-

tervals from the shoreline to the base of the dune. The traps

were kept active for 24 h. 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. (Geophilo-

morpha, Geophilidae), Parallelomorphus laevigatus (Coleoptera,

Carabidae), Phaleria bimaculata (Coleoptera, Tenebrionidae),

Phaleria provincialis (Coleoptera, Tenebrionidae), Pimelia

bipunctata (Coleoptera, Tenebrionidae), Scarites buparius (Cole-

optera, Carabidae), Talitrus saltator (Amphipoda, Talitridae)

and Tylos europaeus (Isopoda, Tylidae) were selected. All

individuals were taxonomically determined, age classified,

cleaned with distilled water and oven-dried at 60 �C for

48 h. Finally, they were kept in a dry, dark place until pro-

cessing. Four replicates of each species were used for ele-

mental and isotopic analysis. Replicates consisted of 5–8

whole individuals of the same species. All replicates were

re-dried (placed in the oven overnight at 60 �C) 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 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 h. This procedure was selected be-

cause it is considered to be the most widely used standard.

The other half was kept untreated. The acidified sub-samples

were re-dried at 60 �C for 24 h, and milled again to a fine

homogenous powder. Samples were not rinsed with distilled

water 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 dry weight (�0.05 mg) of each

sample was weighed and placed in a tin capsule for solid

samples. The encapsulated samples were kept under constant

laboratory temperature (20 �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 dVPDB notation

relative to the standards Vienna Pee Dee Belemnite and

atmospheric nitrogen for carbon and nitrogen, respectively

(dsample ¼ 1000 [(Rsample/Rstandard) � 1], R ¼ 13C/12C, or

R ¼ 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 car-

bon, and atropine, IAEA N1, NO3, N2, and USGS40, for nitro-

gen) ranged from 0.11 to 0.06& (mean ¼ 0.09&) for carbon,

and from 0.06 to 0.28& (mean ¼ 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 overall

difference in the elemental composition and isotopic values

between acidified and untreated samples. Post-hoc compari-

sons were then used to test specific differences between pairs

(single species, raw vs. acidified samples). A proxy was used to

estimate the content of CaCO3 and to assess its role on the ef-

fect of acid washing on the stable isotope ratios (Jacob et al.,

2005):

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 6 91

Carbonate proxy ¼hðC : NÞuntreated=ðC : NÞacidified

i� 1 (2)

where (C:N)untreated and (C:N)acidified are the C:N ratios (w:w) of

raw and acidified samples respectively. This proxy is linearly

related to sample CaCO3 content given that tissue C:N is inde-

pendent of sample CaCO3 (Jacob et al., 2005). All numerical

procedures were performed using the statistics software

package Statistica 7.1 (Statsoft, Inc, OK, USA).

3. Results and discussion

3.1. Elemental composition

In many arthropods carbon is contained both in inorganic and

organic forms. Because trophic ecology based on stable

isotopes is mainly interested in the carbon fixed in organic

tissues, the bias introduced by 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 un-

derstood that the carbon lost during acid washing corre-

sponds 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

(w40%, 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 ob-

served a decrease in carbon content in all the samples ana-

lyzed (Table 1). Moreover, despite the fact that carbonates

do not contain nitrogen, we observed a loss of nitrogen asso-

ciated with acid washing, suggesting that organic (i.e., dietary)

nitrogen is also 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, re-

spectively, Table 1). Acidification resulted in a loss of dietary 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% on average as a consequence of acidification,

the maximum decrease being 10.7% (adults of T. saltator) and

a minimum of 4.4% (adults of T. europaeus). Nitrogen content

decreased 1.2% on average after acidification, ranging from

0 to 1.9% (for Geophilus sp., and both Arctosa cinerea and Phaleria

spp. larvae, respectively). The elemental impoverishment af-

ter acid washing was proportionally higher in C than in N

resulting in a lower C:N ratio in acidified samples (Table 1).

Table 1 – Comparison of C and N elemental composition percentage (mean ± SD; dry weight) of no acidified (no acid) andacidified (acid) beach macroinvertebrates

Species %C %C %N %N C:N C:N

Acid No acid Acid No acid Acid No acid

Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD

Arctosa cinerea 37.0 4 1.17 44.0 4 1.11 10.8 4 0.35 12.7 4 0.28 3.4 4 0.1 3.5 4 0.0

Geophilus sp. 46.2 4 0.96 52.6 3 0.32 9.8 4 0.31 9.8 4 1.23 4.7 4 0.2 5.0 4 0.2

Parallelomorphus laevigatus 42.5 5 1.58 51.2 4 1.07 9.6 4 0.38 11.3 3 0.05 4.2 4 0.1 4.1 3 0.1

Phaleria bimaculata 43.8 5 0.78 52.0 4 1.48 9.2 4 0.20 10.6 3 0.05 4.4 4 0.0 4.4 3 0.0

Phaleria provincialis 42.7 3 0.41 49.3 4 0.67 9.7 4 0.23 10.9 4 0.11 4.8 3 0.1 4.9 4 0.2

Phaleria spp. (larvae) 38.6 4 1.95 44.6 4 1.54 9.1 4 0.54 11.0 4 0.35 4.5 4 0.1 4.5 4 0.1

Pimelia bipunctata 42.6 4 0.17 48.3 4 0.72 9.4 4 0.03 10.6 4 0.10 4.5 4 0.0 4.6 4 0.1

Scarites buparius 45.0 4 1.33 52.8 4 0.94 9.4 4 0.32 10.3 3 0.60 4.8 4 0.3 5.0 3 0.4

Talitrus saltator (adult) 25.2 4 1.52 35.9 4 1.77 5.7 4 0.26 7.3 3 0.31 4.6 4 0.4 4.9 3 0.3

Talitrus saltator (juvenile) 24.6 4 2.07 35.2 4 0.15 5.6 4 0.34 7.4 4 0.10 4.4 4 0.1 4.8 4 0.1

Tylos europaeus (adult) 25.7 4 1.24 30.1 4 1.65 5.6 4 1.10 5.5 3 0.15 4.9 4 0.5 5.3 3 0.1

Tylos europaeus (juvenile) 19.5 4 0.82 29.5 4 0.67 4.1 4 0.12 5.3 4 0.17 4.7 4 0.1 5.6 4 0.1

Average 36.1 49 9.18 43.8 47 8.06 8.2 48 2.20 9.4 43 2.36 4.5 48 0.42 4.7 48 0.56

Species %C %N C:N

MS f P MS f P MS f P

Arctosa cinerea 97.796 75.54 *** 7.080 72.54 *** 0.003 9.72 NS

Geophilus sp. 69.045 116.37 *** 0.000 0.00 NS 0.123 3.35 NS

Parallelomorphus laevigatus 166.764 86.65 *** 5.213 60.18 *** 0.006 4.19 NS

Phaleria bimaculata 148.171 114.70 *** 2.965 45.63 ** 0.024 0.78 NS

Phaleria provincialis 73.544 218.10 *** 3.114 96.34 *** 0.000 0.00 NS

Phaleria spp. (larvae) 70.699 22.98 ** 6.922 33.20 ** 0.064 6.40 NS

Pimelia bipunctata 65.974 238.58 *** 2.866 548.70 *** 0.002 0.85 NS

Scarites buparius 121.008 91.39 *** 1.484 7.20 * 0.113 1.00 NS

Talitrus saltator (adult) 229.466 84.20 *** 4.216 53.20 *** 0.179 1.24 NS

Talitrus saltator (juvenile) 223.167 103.77 *** 6.353 103.54 *** 0.283 33.29 **

Tylos europaeus (adult) 38.752 18.19 ** 0.008 0.11 NS 0.284 2.37 NS

Tylos europaeus (juvenile) 199.443 354.13 *** 2.629 128.67 *** 1.513 78.78 ***

Average 1204.966 15.3 *** 39.310 7.61 ** 0.008 4.14 NS

Top: elemental carbon and nitrogen composition values and C:N ratios (w:w); bottom: results of post-hoc tests. P, significance level: *P � 0.05,

**P � 0.01, ***P � 0.001; NS, non-significant difference (P � 0.05).

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 692

The mean 0.2 decrease in C:N ratio, however, was not signifi-

cant. 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 these samples lost

proportionally more carbon than nitrogen as a result of acidi-

fication in most cases. A positive relationship exists when

comparing the sample C and N content of non-acidified and

acidified samples (r2 ¼ 0.75 and 0.84, respectively; Fig. 1).

This relationship may be a consequence of differences in the

species’ carbonate content. The bulk total carbon and

nitrogen content of species containing higher amounts of

carbonates may be proportionally carbon and nitrogen impov-

erished due to the low-carbon and negligible nitrogen content

of carbonates relative to that of soft tissues. Two sub-groups

were identified corresponding to the species belonging to

Malacostraca (low carbon and nitrogen content) and to the

other groups (high carbon and nitrogen content). After acidifi-

cation, 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 propor-

tion 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 other groups as a consequence of acid

washing (1.15 and 1.34%, respectively). Our results suggest

that 1 N HCl attack systematically removes C and N from all

samples as a function of sample carbonate content (see the

following sections).

3.2. d13C and d15N values

Acidification did not show an overall significant effect on mac-

roinvertebrate isotopic ratios (Table 2). More specifically, d13C

N (% dry weight)

2 4 6 8 10 12 14

C (%

d

ry w

eig

ht)

10

20

30

40

50

60

Other arthropods - acidified Cl. Malacostraca - acidified Other arthropods - no acidified Cl. Malacostraca - no acidified

Fig. 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 shown. Non-acidified samples:

y [ 13.764 D 3.145 3 x, r2 [ 0.753, P < 0.001, N [ 47.

Acidified samples: y [ 4.802 D 3.868 3 x, r2 [ 0.843,

P < 0.001, N [ 48.

values showed a non-significant average decrease of 0.3&

after acidification, whereas d15N was unaffected. Taking into

account the large effect observed in the elemental composi-

tion, the lack of effect of acidification on the isotopic signa-

tures is somewhat surprising. 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 alterna-

tive hypothesis is the selective removal of dietary 13C-depleted

compounds which compensate for the loss of 13C-rich carbon-

ate carbon (with a d13C around 0&, 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 d13C ¼ �23.6& as reported in

Schimmelmann and DeNiro, 1985) through hydrolytic depoly-

merization 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 d13C and d15N

values after acidification of 1.9 and 0.6&, respectively. Adults

of T. europaeus and juveniles of T. saltator presented a slight sig-

nificant decrease only in the carbon isotopic ratio (1.1 and

0.3&, respectively). The isotopic compositions of the other

species did not show any significant change following acidifi-

cation. d13C was higher in malacostraceans than in the other

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 d15N showed no signif-

icant differences. Macroinvertebrates carbon content and d13C

of non-acidified samples were negatively correlated (r2 ¼ 0.58,

P < 0.001; Fig. 2), suggesting that acidification only affects the

carbon isotopic composition of those species containing

relatively high proportions of carbonate, as is the case with

the malacostraceans studied here (T. europaeus and T. saltator;

see Section 3.3).

3.3. The carbonate proxy and acid washing effect

The macroinvertebrates considered in our experiments be-

long to four different Classes (Insecta, Malacostraca, Arach-

nida 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 crusta-

ceans proceed also by calcification (Travis, 1963; Giraud-Guille

and Quintana, 1982). The amphipod T. saltator and the isopod

T. europaeus belonging to Malacostraca, the origins of which

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 mineral-

ized, essentially by precipitation of calcium carbonate into the

chitin–protein cholesteric matrix (e.g., Luquet and Marin,

2004).

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 6 93

Table 2 – Effect of acid washing on the C and N isotopic ratios of beach macroinvertebrates

Species d13C d13C d15N d15N

Acid No acid Acid No acid

Mean N SD Mean N SD Mean N SD Mean N SD

Arctosa cinerea �20.9 4 0.10 �20.8 4 0.10 10.0 4 0.13 10.1 4 0.06

Geophilus sp. �23.2 4 0.61 �23.3 4 0.5 7.4 4 0.76 7.5 4 0.75

Parallelomorphus laevigatus �20.8 4 0.15 �21.0 3 0 11.6 5 0.17 11.7 4 0.08

Phaleria bimaculata �21.5 4 0.33 �21.6 3 0.2 8.9 4 0.17 8.9 4 0.28

Phaleria provincialis �21.1 4 0.25 �21.1 4 0.2 9.2 4 0.12 9.1 4 0.26

Phaleria spp. (larvae) �21.0 4 0.41 �20.7 4 0.4 9.3 4 0.83 9.4 4 0.58

Pimelia bipunctata �24.9 4 0.14 �24.7 4 0.1 4.7 4 0.14 4.6 4 0.12

Scarites buparius �25.2 4 0.97 �25.7 3 1 3.5 5 0.21 3.4 4 0.37

Talitrus saltator (adult) �21.9 4 0.34 �21.5 4 0.3 6.5 5 0.16 6.5 5 0.12

Talitrus saltator (juvenile) �21.2 4 0.15 �20.9 5 0.2 7.1 4 0.18 7.3 5 0.13

Tylos europaeus (adult) �19.0 4 0.62 �17.9 3 0.2 8.9 5 0.43 8.9 4 0.10

Tylos europaeus (juvenile) �20.5 4 0.22 �18.6 4 0.2 7.7 4 0.08 8.3 4 0.08

Average �21.8 48 1.81 �21.5 45 2.12 7.9 52 2.23 8.0 50 2.24

Species d13C d15N

df MS f P MS f P

Arctosa cinerea 1 0.009 0.87 NS 0.003 0.26 NS

Geophilus sp. 1 0.018 0.06 NS 0.003 0.00 NS

Parallelomorphus laevigatus 1 0.053 3.66 NS 0.043 2.33 NS

Phaleria bimaculata 1 0.014 0.16 NS 0.010 0.21 NS

Phaleria provincialis 1 0.003 0.06 NS 0.003 0.06 NS

Phaleria spp. (larvae) 1 0.191 1.20 NS 0.033 0.06 NS

Pimelia bipunctata 1 0.056 4.50 NS 0.012 0.71 NS

Scarites buparius 1 0.431 0.44 NS 0.002 0.03 NS

Talitrus saltator (adult) 1 0.281 3.53 NS 0.009 0.40 NS

Talitrus saltator (juvenile) 1 0.193 6.28 * 0.103 4.51 NS

Tylos europaeus (adult) 1 1.981 8.48 * 0.008 0.07 NS

Tylos europaeus (juvenile) 1 7.220 196.9 *** 0.605 121 ***

Average 1 1.952 0.5 NS 0.215 0.04 NS

Top: d13C and d15N values; bottom: results of individual post-hoc tests. P, significance level: *P � 0.05, **P � 0.01, ***P � 0.001; NS, non-significant

difference (P � 0.05).

C (% dry weight)

25 30 35 40 45 50 55

13C

(%

o)

-28

-26

-24

-22

-20

-18

-16

Arctosa cinerea

Geophilus sp.Phaleria spp. (larvae)Parallelomorphus laevigatus

Phaleria bimaculata

Phaleria provincialis

Pimelia bipunctata

Scarites buparius

Talitrus saltator adultTalitrus saltator juvenileTylos europaeus adultTylos europaeus juvenile

Fig. 2 – Relationship between non-acidified d13C values and

carbon elemental compositions: y [ L12.717 L 0.207 3 x,

r2 [ 0.582, P < 0.001, N [ 48.

We compared the carbonate proxy (Eq. (2)) of our samples

with the differences in the isotopic signatures between un-

treated and treated samples to assess the effects of acidifica-

tion on the isotopic composition as a function of carbonate

content. The differences in the carbon isotope ratios were pos-

itively related to the sample CaCO3 proxy (r2 ¼ 0.54, N ¼ 43,

P < 0.001; Fig. 3a), those species with greater carbonate con-

tent being the most affected by acid washing. As expected,

species with high carbonate proxy values were 13C-depleted

after acid washing, presumably as a result of the removal of

carbonates. The differences in the nitrogen isotope ratios

were not related to the CaCO3 proxy (N ¼ 43, P > 0.05; Fig 3b).

Bosley and Wainright (1999) and Carabel et al. (2006) reported

the lack of effects of carbonate removal on the d15N values in

a wide spectrum of zooplankton size fractions and crabs, sup-

porting our findings. If we accept that acid washing implies re-

moval of nitrogen, then the overall lack of effect in d15N

following HCl attack should be ascribed to the absence of iso-

topic fractionation during the process. Exceptions were the

samples of juveniles of T. europaeus, which presented a signif-

icant decrease both in the elemental and in the isotopic

values. A few observations have reported that acidification

can alter d15N values (Goering et al., 1990; Bunn et al., 1995;

Pinnegar and Polunin, 1999), supposedly as a consequence of

partial loss of cuticle matrix compounds.

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 694

Adults and juveniles of T. europaeus are the species for

which estimated carbonate proxy was highest (mean �SD ¼ 0.13 � 0.06 and 0.21 � 0.02, respectively). Also, the car-

bonate 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. Malacostraceans (which yielded the highest

carbonate proxy values) also present the lowest carbon and

nitrogen relative content (mean � SD ¼ 32.7% � 3.2 and

6.4 � 1.0%, respectively). Consistently, species with low car-

bonate proxy values show higher carbon and nitrogen content

Carbonate proxy

-0,1 0,0 0,1 0,2-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

13C

cru

de -

13C

acid

-0,10 -0,05 0,00 0,05 0,10 0,15 0,20 0,25-0,4

-0,2

0,0

0,2

0,4

0,6

0,8

1,0

15N

cru

de -

15N

acid

Carbonate proxy

a

b

Arctosa cinerea

Geophilus sp.Phaleria spp. (larvae)Parallelomorphus laevigatus

Phaleria bimaculata

Phaleria provincialis

Pimelia bipunctata

Scarites buparius

Talitrus saltator adultTalitrus saltator juvenileTylos europaeus adultTylos europaeus juvenile

Fig. 3 – (a) Relationship between the carbonate proxy and

the difference in d13C between acid washed and untreated

samples ( y [ 0.025 D 0.085 3 x, r2 [ 0.54, P < 0.001,

N [ 43). (b) Relationship between the carbonate proxy and

the difference in d15N between acid washed and untreated

samples.

(48.9 � 3.9% and 11.0 � 0.9%, respectively). Due to the limited

number of species studied in our experiments, other causes

for the C/N ratio differences between groups should not be

discarded.

The relationship between carbonate content and the acid

washing effect on the carbon isotope signatures suggests

that only the carbon isotope ratios of those beach arthropods

with a carbonate proxy equal to or higher than 0.09, are

significantly altered after acidification. Jacob et al. (2005) ob-

served the same trends studying a wide range of species;

those samples with high carbonate proxies were 13C-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& compared to acid-

ified 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 d13C values

may not be detectable. Soreide et al. (2006) studied the con-

troversy around the effect of the different methodologies

used prior to isotopic analysis and published C and N ele-

mental composition data of the marine species Gammarus

wilkitzkii and Thysanoessa inermis. We calculated an average

carbonate proxy including all suitable data from Soreide

et al. (2006) (mean � SD ¼ 0.77 � 0.3, N ¼ 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 protec-

tive structures (Little, 1983). Unfortunately, data on the ele-

mental composition of macroinvertebrates is very scarce in

the literature, making detailed carbonate proxy modeling

impracticable.

The carbonate proxy proposed by Jacob et al. (2005) presup-

poses that there is no effect of acidification on dietary carbon

and nitrogen. If there are no carbonates in the sample, the

quotient between the C/N ratios of raw and acidified aliquots

is 1, Eq. (2) becoming 0. It has been shown that the elemental

composition of either carbonate-free or carbonate-containing

samples is also apparently altered by acid washing. This

means that even under the assumption of a carbonate-free

sample, Eq. (2) would provide a value. A clear example of

this is represented by the carbonate proxy negative value of

Phaleria larvae in Fig. 3a,b. These samples are supposed to be

carbonate-free. A negative value strongly suggests a loss of di-

etary nitrogen due to acidification. In consequence, C:Nacidified

becomes higher than C:Nuntreated and hence Eq. (2) yields

a negative value. So in the light of our results, the carbonate

proxy becomes, in our opinion, less meaningful. In fact, it

should be considered a ‘‘carbonate þ acid-soluble com-

pounds’’ proxy, rather than a carbonate proxy alone. The

outcome of the application of the carbonate proxy to our

results (linearity with the suspected carbonate content of

the samples) indicates that, as already discussed, the isotopic

shifts are larger when the carbonate content of the sample is

high. The use of soft acidification procedures (see Section 4)

that limit the removal of dietary carbon and nitrogen would

result in a much more meaningful applicability of the carbon-

ate proxy.

a c t a o e c o l o g i c a 3 4 ( 2 0 0 8 ) 8 9 – 9 6 95

4. Conclusions

One initial conclusion is that, in the light of the results pre-

sented, the processes taking place as a consequence of acidifi-

cation 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 d13C are large enough to critically confound

food web analysis. As shown, the problem associated with

acid washing (1 N HCl) is that some dietary carbon- and nitro-

gen-bearing 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 from

semi-terrestrial crustaceans 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 0.25 M for 15 min was enough for complete deminer-

alization minimizing the hydrolysis of chitin polymers of the

exoskeletons, this being the most appropriate protocol for

the acid washing treatment. In order to avoid isotopic variabil-

ity 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 study is interested in the d13C values of inverte-

brates with a carbonate proxy >0.09 (as for the case of semi-

terrestrial crustaceans in this study). In this last case, a soft

decalcification should be applied and untreated aliquots

should be analyzed separately for d15N.

Other important methodological procedures (lipid extrac-

tion, distilled water washing, gut content, etc.) critically af-

fecting isotopic signatures of marine macroinvertebrates are

reviewed in Mateo et al. (in press).

Acknowledgements

The authors are grateful to the two reviewers of this paper for

their helpful contributions. 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).

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