Embryo–larvae and juvenile toxicity of Pb and Cd in NorthernChilean scallop Argopecten purpuratus

Patricia Romero-Murillo & Winfred Espejo &

Ricardo Barra & Rodrigo Orrego

Received: 22 November 2016 /Accepted: 28 November 2017# Springer International Publishing AG, part of Springer Nature 2017

Abstract This study aimed to investigate the effectsof Cd and Pb on earlier stage of the commerciallyimportant scallop (Argopecten purpuratus) in thecontamination context of Northern Chile where thisspecie is farmed, through acute exposure bioassaysin embryo–larvae measuring cumulative abnormality(EC50) and juvenile cumulative mortality (LC50) asendpoints, based on environmentally detected con-centrations and available toxicological data fromsimilar species. Embryo–larvae exposure indicates48 h EC50 of 1.55 mg/L Cd, and 0.044 mg/L Pb.

On the contrary, 96 h LC50 in juvenile scallops was0.48 mg/L Cd and 1.47 mg/L Pb. Our results dem-onstrated differential toxicity between embryo andjuvenile scallops that might relate to different pri-mary defense mechanisms or effect in morphologi-cal development of individuals in each ontogeneticstage. Compared to similar bivalve metal toxicitytests, this study demonstrated that A. purpuratusembryos are more sensitive to Pb than most otherbivalve species. Our results indicate that maximumpermitted levels of Pb in marine waters and estuaries(according to Chilean regulation) could pose a riskfor scallops’ first stage of life (embryo–larvae) de-velopment, and needs to be reviewed. Furthermore,Chilean environmental regulations do not have qual-ity standards for marine sediments (currently underdiscussion), where high levels of metals have beencontinuously reported.

Keywords Embryo–larvae . Juvenile . Scallop .

Toxicity . Pb . Cd

Introduction

Metals are natural components of the biosphere, andeven though some metals are essential for life,others such as cadmium (Cd) and lead (Pb) general-ly are deleterious to most living organisms andwould be toxic in low concentrations (Gnassia-Barelli and Romeo 1993; Lane and Morel 2000;Wu et al. 2016). Anthropogenic activities such as

Environ Monit Assess (2018) 190:16 https://doi.org/10.1007/s10661-017-6373-9

P. Romero-MurilloApplied Science PhD Program, Faculty of Marine Science andNatural Resources, University of Antofagasta, Av. Universidad deAntofagasta, Antofagasta 02800, Chilee-mail: patricia.romero@uantof.cl

P. Romero-Murillo : R. Orrego (*)Aquatic Toxicology Laboratory (AQUATOX), Natural ScienceInstitute Alexander von Humboldt, Faculty of Marine Science andNatural Resources, University of Antofagasta, Av. Universidad deAntofagasta, Antofagasta 02800, Chilee-mail: rodrigo.orrego@uantof.cl

W. Espejo :R. BarraAquatic Systems Department, Faculty of Environmental Sciencesand EULA-Chile Centre, University of Concepcion, BarrioUniversitario S/N Concepcion, Antofagasta, Chile

W. Espejoe-mail: winfredespejo@gmail.com

R. Barrae-mail: ricbarra@udec.cl

industrial mining and urban growth increase theavailability of metals in coastal areas by waste dis-posal, mineral losses during transport, or dispersionby wind into the sea (Pan and Wang 2012b). In thesecoastal ecosystems, metals can influence the healthof organisms in two ways, directly by affecting itsmetabolism and physiology and indirectly throughhabitat modifications, food availability, or behavior-al changes, which according to their magnitude andscale may cause changes in other levels of biologicalorganization (Adams 2005).

Northern Chile coastal environments are character-ized by high environmental concentrations of metals inmarine sediments mainly Cd, Cu, Zn, and Pb (47.76 Cd,509.39 Cu, 399.06 Zn, and 237.65 Pb mg/kg) (MMA2013), partially due to natural processes (e.g., diagenesisor coastal upwelling), that can increase the concentra-tions of metals such as Cd (Valdés et al. 2006), and otherlocal anthropogenic processes (mining residues, indus-trial and domestic waste disposal, and fuel combustion)resulting in high Pb levels (Valdés et al. 2014; Valdéset al. 2015).

Several cities of Northern Chile have beenestablished around mining activities, increasing the riskfor metal exposure in coastal areas. Although there is agovernment which required chemical monitoring pro-grams (MMA 2013) and multiple studies mainly relatedto the distribution of metals in sediment, water, andintertidal organisms (Calderón and Valdés 2012;Valdés et al. 2015; Valdés et al. 2010), the establishmentof cause–effect relationships on key marine organismsas tool for potential risk evaluation has not been thefocus of research. According to Valdés et al. (2015), tovalidate historical environmental monitor programs, da-ta is necessary to move towards the effect identificationand evaluation in autochthonous marine aquatic organ-isms in order to establish a more realistic risk assessmentin local programs for future sediment quality guidelinesto develop.

Bioassays are reliable tools for contaminant effectanalysis through a biological context; they can be usedduring a first step of environmental quality assessmentas bioavailability indicators and cause–effect relation-ship establishment tools. Besides, most of the toxicityassay methods are focused on the continuous exposureof toxicants at different stages of ontogeny, which wouldexplain the observed differences in their sensitivity andtime of effect creating a better ecotoxicological dataset(Durán and Beiras 2013).

Bivalves are frequently used in toxicity testing andmarine monitoring programs due to their abundance,wide distribution, and high tolerance to environmentalfluctuations, and for being readily available from in-labcultures, from hatcheries, or from field natural popula-tions (Cranford 2006; His et al. 1999; Rainbow 2006).This is the case of A. purpuratus (Pectinidae), a scallopthat inhabits Peruvian and Chilean coasts (Wolff andMendo 2000), existing as natural banks (MiguelAvendaño and Cantillánez 2005), and being intensivelycultured for commercial purposes (Merino et al. 2009;Molina et al. 2012). In Northern Chile, this scalloprepresents over 10% of total national bivalve resourcedisembarkation behindMytilus chilensis which is main-ly cultured in southern Chile (Cox 2014). However,there is a lack of information regarding metal sensitivity,especially comparing different ontogenetic stages suchas embryo–larvae and juveniles.

Metal embryotoxicity tests have been previously de-veloped to evaluate environmental metal contaminationsuch as Cd, Zn, and Cu in sediment and water usingCrassostrea gigas (Geffard et al. 2002; Mai et al. 2012).Besides, induced embryo–larvae abnormalities relatedto DNA damage were recently demonstrated for Cd andCu (Mai et al. 2012). Furthermore, embryotoxicity testsusing bivalves such as Mytilus galloprovincialis andRuditapes decussatus were used to trace metal environ-mental risk assessment in the Galician Rias; as baseinformation to establish protective quality standards ofmarine resources, these research results indicated thatCd does not represent any risk, contrary to the high riskof Cu exposure (Beiras and Albentosa 2004).Embriotoxicity tests are being developed in scallopssuch as Chlamys asperrima that were used to assessmetal toxicity and other contaminants determining lowCd toxicity (0.29 mg/L) compared with Cu (0.0045 mg/L) and Zn (0.045 mg/L) (Krassoi et al. 1997).

These adverse effects of Cd and Pb and their mixtureshave also been demonstrated in Argopecten ventricosusjuveniles, causing a twofold mortality by acute exposureCd with respect to Pb (Sobrino-Figueroa et al. 2007).Moreover, acute toxicity evaluated in C. rhizophoraealso established high Cd toxicity (Chung 1980). A sim-ilar toxicity pattern was also detected in other environ-mental risk assessment using Perna viridis in India Cu >Cd > Pb > Zn (Rajkumar 2012).

This study aimed to investigate the effects of Cd andPb (metals that historically have reached high levels inSan Jorge Bay, Northern Chile), through acute water

16 Page 2 of 13 Environ Monit Assess (2018) 190:16

exposure bioassays using A. purpuratus embryo–larvaeand juveniles to several doses of these metals based onpreviously detected environmental concentrations(almost undetectable in water and high levels detectedin sediments as example of the worst scenario) togetherwith toxicological data for similar species, to estimatethe potential risk associated with metal exposure inNorthern Chile. The endpoints of this study focus onboth impaired embryo–larvae cumulative abnormality(EC50) and juvenile cumulative mortality (LC50), estab-lishing sensitivity thresholds between ontogeny stages.

Material and methods

All plastic and glassware used in this study were washedin nitric acid and hydrochloric acid (10% during 24 heach) followed by five rinses with reverse osmosis(RO)-purified freshwater (Viga Flow RO System Cod.SDC-25-1020, Taiwan) before use.

Collection and maintenance of scallops

The scallop A. purpuratus is a functional hermaphroditespecies with partial self-fertilization (Avendaño et al.2001; Concha et al. 2011); therefore, to avoid self-fer-tilization, a high dilution and massive sperm is recom-mended (Helm et al. 2006). Broodstock scallop adultswere obtained from a local hatchery farm (Santa MariaSpA, Antofagasta, Chile) and transported 30 min undercontinuous aeration at 15–16 °C to the Aquatic Toxicol-ogy Laboratory (AQUATOX), University of Antofagas-ta (Chile). Upon arrival, a total of 70 individuals weresubsequently selected, cleaned, and transferred to a10,000-L tank. Following that, they were induced tospawn by thermal stimulation (with temperature incre-ment of 10–12 °C). After spawning, embryo develop-ment was continuously monitored for fertilization suc-cess (establishing 80% as minimum selection criteria,prior to the bioassay).

Juvenile scallops of A. purpuratus (21 mm in shelllength) were collected from the same farm and accli-matized at 19 ± 1 °C for 2 weeks in continuouslyfiltered natural seawater in aerated tanks until theexposure experiment. During acclimatization time,organisms were daily fed with a microalgae mixture(Isochrysis galbana, Phaeodactylum tricornutum,Nannochloropsis sp., and Tetraselmis suecica, rate1:1:1:1, 90,000 cells day−1) supplied by the

Microalgae Production Laboratory (LEA, Universityof Antofagasta). Natural seawater for all acclimatiza-tion and experimental processes described waspreviously filtered (5, 5, 1 μm and active carbon)and sterilized by UV light (filtered seawater, FSW-UV) and with 34.8 ± 0.5 PSU and 8.05 ± 0.007 pH(Thermo Scientific Orion Star A329 PortableMultiparameter).

Metal preparation

Metal stock solutions (1000 mg/L) were preparedfrom analytical grade Titrisol® standard cadmiumchloride (CdCl2) and lead nitrate Pb(NO3)2 (MerckMillipore, Darmstadt, Germany) in RO-purifiedfreshwater. Exposure concentrations were based ona similar species toxicity test (Beiras and Albentosa2004; Fathallah et al. 2013; Mai et al. 2012; Sobrino-Figueroa et al. 2007) and previously reported fromsites with significantly high concentrations in marinesediments and water of San Jorge Bay in NorthernChile (Castro and Valdés 2012; MMA 2013; Valdéset al. 2010; Valdés et al. 2011). Subsequently, testsolutions were prepared before toxicity test usingplastic containers by diluting the stock solution inFSW-UV. A pilot study was conducted to definemetal treatments, based on a previously reported Pbsolubility (Angel et al. 2015).

Metal analysis

Water samples of metal treatment and experimentalcontrols (50 mL) were analyzed before toxicity testsfor embryos and juveniles in the Chemistry Labora-tory of the Environmental Science Centre (EULA-Chile, University of Concepcion Chile) (ISO 17025Certified) for Cd and Pb by inductively coupledplasma optical emission spectrometry (ICP-OES, inICP Perkin Elmer Optima 8000) following standardanalytical procedure (American Public Health et al.2011). Standards for Cd # 10008-1 and Pb # 100028-1 were used (supplied by High-Purity Standards,USA), and 0.0005 mg/L for Cd and 0.001 mg/L forPb detection limits were measured. The quality ofthe analytical method was checked by analyzing thematerial certified Inorganic Ventures (J2-CD02063;K2-PB03074).

Environ Monit Assess (2018) 190:16 Page 3 of 13 16

Toxicity tests

Embriotoxicity assay

After spawning, eggs were periodically observed undera microscope (Sedgewick Rafter cell, Microscope LeicaCME 10×) until 80% of success fertilization that wasverified as expulsion of the first and second polar cor-puscles (De la Roche et al. 2002). Subsequently, eggswere sieved (39 and 24μm) and washed several times toavoid organic matter contamination (Nadella et al. 2013)and potential damage caused by other organisms. Em-bryo density (50 cells mL−1) was established in triplicate(Sedgewick Rafter cell counting chamber) and random-ly transferred to 6-well sterile cell culture plates(Corning Incorporated, New York, USA).

The exposure started by mixing 4.5 mL of the differ-ent treatment solutions (Table 1) with 0.5 mL of seawa-ter (concentration included embryo volume) in six rep-licates (n = 10 ± 1 embryos for replicate). The cultureplates with embryos were incubated to 19 ± 1 °C for48 h in the dark. An experimental control was made byadding 4.5 mL of FSW-UV. After 48 h, larval develop-ment was verified under an inverted microscope (Olym-pus CKX41, ×20) to determine embryo abnormality

percent per treatment according to the criteria describedby His et al. (1999) as embryos that have not reached theD-larvae stage (not D-shell larvae with a convex hinge,incomplete shell, or protruding mantle), and to deter-mine EC50 (experimental concentration that inhibits50% of normal D-larva development of the fertilizedeggs). There were no significant differences in pHamong treatments.

Juvenile exposure

A total of 400 juvenile scallops were placed in 5-Lplastic tanks in five replicates for Cd and triplicate forPb (n = 10 ind. per replicate) continuously aerated (4.5 ±0.5 mg/L), with a 12-h light/dark photoperiod cycle in acontrol temperature room (18 ± 1 °C), 8.05 ± 0.007 pH,and salinity of 34.5 ± 0.1 PSU. The exposure to differenttreatments (Table 1) was conducted under static condi-tions without significant differences in pH or salinity.

Mortality and routine observations were recorded for0, 12, 24, 36, 48, 60, 72, 84, and 96 h post exposure. A96-h LC50 (lethal concentration required to kill 50% ofthe experimental population) was calculated. At the endof the test, the remaining scallops were euthanized byanesthetic overdose (MS-222). All animal handling

Table 1 Nominal and mean measured seawater concentrations of Cd and Pb

Nominal Cdconcentration(mg/L)

Measured Cdconcentration(mg/L)

pH Cd Salinity(PSU)

Nominal Pbconcentration(mg/L)

Measured Pbconcentration(mg/L)

pH Pb Salinity(PSU)

Control 0.0005 8.05 ± 0.007 34.95 ± 0.07 Control 0.007 8.05 ± 0.007 34.9 ± 0.07

0.1 0.095 8.05 ± 0.02 34.9 ± 0.14 0.03 0.025 8.05 ± 0.007 35.05 ± 0.07

0.4 0.05b 8.05 ± 0.007 35.15 ± 0.4 0.08 0.05b 8.05 ± 0.007 35.09 ± 0.01

1.2 0.41c 8.06 ± 0.007 35.2 ± 0.14 0.1 0.1b 8.06 ± 0.007 35.2 ± 0.14

1.5 0.983 8.06 ± 0.006 35.09 ± 0.01 0.5 0.14 8.06 ± 0.007 35.09 ± 0.01

2 0.806b 8.06 ± 0.01 35.25 ± 0.07 0.7 0.57b 8.05 ± 0.01 35.2 ± 0.03

4.5 2b 8.07 ± 0.007 35.1 ± 0.28 1.15 0.73 8.05 ± 0.007 35.2 ± 0.14

9 4b 8.08 ± 0.007 35.18 ± 0.17 1.5 1 8.07 ± 0.007 35 ± 0.14

1.6 1.2c 8.07 ± 0.007 35.15 ± 0.07

1.8 1.4c 8.05 ± 0.007 35.05

12 8.018 8.07 ± 0.007 35.4 ± 0.2 2a 1.59 8.05 ± 0.007 35.08 ± 0.03

25 16.71 8.08 ± 0.007 35.2 ± 0.25 2.1 1.6c 8.07 ± 0.007 35.2 ± 0.14

50 48.78 8.07 ± 0.007 35.3 ± 0.07 2.2 1.8c 8.05 ± 0.007 35.4 ± 0.2

aMaximum Pb solubility in seawater, according to Angel et al. (2015)b These were concentrations only used in embryosc These were concentrations only used in juveniles. Concentrations without symbol used in embryos and juveniles. Besides, it was registerpH (mean ± SD) of embryo and juvenile bioassays

16 Page 4 of 13 Environ Monit Assess (2018) 190:16

procedures were in accordance with the University ofAntofagasta Bioethics Committee and under NationalCommission of Science and Technology (CONICYT)Ethic and Biosafety Protocols.

Statistical analysis

Statistical analysis was carried out with STATISTICA7.0 software (StatSoft, Tulsa, OK, USA). Data normal-ity was checked by using Shapiro–Wilk W test (at α =0.01), and for homogeneity of variances using Brownand Forsythe’s test (at α = 0.01). One-way analyses ofvariance (ANOVA) were used to analyze overall differ-ences among treatments and controls. Significant

differences (p < 0.05) were then confirmed by Tukey’sHSD post hoc test. Nonparametric analysis (Kruskal–Wallis test) was used to analyze differences amongtreatments and control in time for juveniles. PreliminaryEC50 and LC50 data was analyzed by standard curveanalysis using SigmaPlot 11.0 (Systat Software Inc.).

Results

Sub-lethal effects (embriotoxicity assay)

Differences between normal and abnormal scallop larvaeaccording to His et al. (1999) are shown in Fig. 1. The

Pb Concentration (mg/L)0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

0

20

40

60

80

100

Cd Concentration (mg/L)0 2 4 6 8 10 12 14 16 18

)%(

ytilamro nbA

eavr aLde pah S-

D

0

20

40

60

80

100 *

*

* * * * ******

- 16.71 mg/L

- 8.018 mg/L

- 4.0 mg/L

- 2.0 mg/L

- 0.98 mg/L

- 0.8 mg/L

- 0.09 mg/L

- 0.05 mg/L

- 0 mg/L

- 1.59 mg/L

- 1.0 mg/L

- 0.73 mg/L

- 0.57 mg/L

- 0.14 mg/L

- 0.1 mg/L

- 0.05 mg/L

- 0.025 mg/L

- 0 mg/L

a b

c d

Fig. 1 Embriotoxicity assay in A. purpuratus. aMicrography of anormal scallop D-larvae stage (×20). bAbnormal scallop BD-larvawith abnormality^: black arrow = protruding mantle, gray arrow =

shell without D-shape (His et al. 1999) (×20). Percentage ofabnormality larvae after 48-h exposure of embryos to differentconcentrations (mg/L) of individual dissolved Cd (c) and Pb (d)

Environ Monit Assess (2018) 190:16 Page 5 of 13 16

normal larval development stage has a straight dorsalhinge giving the larva the characteristic symmetricalshape of a capital letter D (D-larvae; Fig. 1a). At thisstage, the larvae begin to feed and the abnormality regis-tered as asymmetrical shape, concave D-line, carvedshells, and protruding mantle. Abnormalities above2 mg/L Cd and 0.05 mg/L Pb showed significant differ-ences from control, reaching 100% abnormality mainlyobserved in 8.018 and 16.71 mg/L Cd, and 0.728 and1.59 mg/L Pb, respectively (Fig. 1c, d). The seawatercontrol larvae showed that 10% of abnormality developedin D-larvae of A. purpuratus. Cumulative abnormalitywas evaluated in each concentration for EC50 calculationsin fertilized eggs 48 h post exposure (Fig. 2a, b) (48 hEC50 values for Cd, 1.55 mg/L, and Pb, 0.044 mg/L).

Acute effects (juvenile exposure)

Cumulative mortality of juvenile scallops exposed todifferent Cd and Pb treatments is shown in Fig. 3a, b,with an LC50 0.48 mg/L for Cd. Besides, after three testsof Pb exposure (two failed and two successful), LC50

was 1.42 mg/L after 96 h.Temporal trends for both metal treatments show sig-

nificant differences from control in cumulative mortality(Fig. 4a); all treatments with the exception of 0.09 mg/Lshowed significant differences from control after 60 h ofexposure to Cd. In contrast, juvenile scallops were ex-posed to Pb where the observed 1.2–1.8 mg/L showedsignificant differences with respect to controls after72 h. There was a significant increase in cumulative

Cd Concentration (mg/L)0.001 0.01 0.1 1 10 100

)%(

yt ilamronb A

eavra LdepahS-

D -20

0

20

40

60

80

100

120

48-h EC50= 1.55 mg/L Cd

Pb Concentration (mg/L)0.001 0.01 0.1 1 10

-20

0

20

40

60

80

100

120

48-h EC50= 0.044 mg/L Pb

a b

Fig. 2 a Cd and b Pb EC50 values at 48 h for scallops exposed to different concentrations. Embryo cumulative abnormality (%) ± standarderror. Forty-eight-hour EC50 values for Cd, 1.55 mg/L (95% CI 1.547, 1.552), and Pb, 0.044 mg/L (95% CI 0.043, 0.044)

Pb Concentration (mg/L)0.01 0.1 1 10

)% (

ytil atroM

ev ita lumu

C

0

25

50

75

100

125

Cd Concentration (mg/L)0.01 0.1 1 10 100

)%(

ytilatroM

evitalumu

C

0

25

50

75

100

125

96-h LC50=0.48 mg/L 96-h LC50=1.42 mg/L

a b

Fig. 3 Cd and Pb LC50 values for juvenile scallops exposed to different concentrations. a Cd 96-h cumulative mortality. b Pb 96-hcumulative mortality ± SE. Ninety-six-hour LC50 values for Cd, 0.48 mg/L (95% CI 0.138, 0.832), and Pb, 1.42 mg/L (95% CI 1.28, 1.56)

16 Page 6 of 13 Environ Monit Assess (2018) 190:16

mortality in juvenile scallops exposed to Cd (0.9–48.7 mg/L) and Pb (1.59–1.8 mg/L) after 96 h of expo-sure, compared to the filtered seawater control group(Fig. 4b).

Other behavioral effects observed in scallops ex-posed to 48.78 and 16.71 mg/L Cd, and 1.8, 1.6,1.59 mg/L Pb were as follows: immediately lockedvalves at the beginning of exposure, followed by valvestotally open prior to the complete lack of response tostimulus.

Discussion

The present study demonstrated that A. purpuratusshow both sub-lethal and acute-lethal effects to Cd andPb exposure. Bioassays were based on toxicologicalinformation available for other bivalve species and es-pecially to the persistent contamination issue of thesemetals historically studied in Northern Chile (Valdéset al. 2014; Valdés et al. 2015). Furthermore, our interestwas to evaluate A. purpuratus differential sensitivity

between two key ontogenetic stages (embryo–larvaeand juvenile) as a potential real-time biosensor of metalcontamination issues at local environment level.

Pb and Cd embryotoxicity assay

Bivalve embryos have been considered for decades asthe most sensitive stage to metal exposure (His et al.1999; Martin et al. 1981). Moreover, experiments onthis stage have proven to be of short duration andinvesting few resources with reliable results in hours(Cragg 2006; His et al. 1999). Our acute exposure testresults indicate a two order of magnitude higher toxicityof Pb compared with Cd in A. purpuratus embryo–larvae (Table 2), with EC50 of 0.044 mg/L Pb and1.55 mg/L Cd causing detectable abnormalities (im-paired D-larvae development).

The sensitive differences to metals between speciescould be related to changes for climate, habitat, temper-ature, salinity, or exposure history of adult making itdifficult to compare (da Cruz et al. 2007; Weng andWang 2014). However, our EC50 results are consistent

PbCd

*

*

*

Concentration (mg/L)

Control 0.09 0.4 0.9 8.01 16.7 48.7

)%(

yt ilatroM

evitalumu

C 0

20

40

60

80

100

Concentration (mg/L)

0.02 0.14 0.72 1 1.2 1.4 1.59 1.6 1.8

****

Time (h)

0 24 48 72 96

)%(

ytilatroM

evitalumu

C

0

20

40

60

80

100

120

48.78 mg/L

16.71 mg/L

8.01 mg/L

0.98 mg/L

0.4 mg/L

0.09 mg/L

0 mg/L

PbCd*

Time (h)

0 24 48 72 96

0

20

40

60

80

100

120

1.8 mg/L

1.6 mg/L

1.59 mg/L

1.4 mg/L

1.2 mg/L

1 mg/L

0.72 mg/L

0.14 mg/L

0.02 mg/L

0 mg/L

*

* *

*

*

*

*

****

b

a

Fig. 4 a Cumulative mortality ± SD of juvenile scallops exposedto measured concentrations of Cd and Pb from 12 to 96 h. Signif-icant difference from control denoted by asterisks (*Kruskal–Wallis test, p < 0.05). b Cumulative mortality ± SD of juvenile

scallops exposed to concentrations measured on Cd and Pb (96 h).Significant differences from control are denoted by asterisks(*one-way ANOVA test, p ≤ 0.05 confirmed by Tukey post hoctest, p ≤ 0.05)

Environ Monit Assess (2018) 190:16 Page 7 of 13 16

with values observed in another bivalve species withsimilar embryotoxicity pattern Pb > Cd found inmusselsM. edulis (Martin et al. 1981), M. galloprovincialis(Beiras and Albentosa 2004), oyster C. virginica(Calabrese and Nelson 1973), clams Meretrix meretrix(Wang et al. 2009), and R. decussatus (Fathallah et al.2013) among other several species (His et al. 1999)(Table 2), showing low tolerance to Pb. Our study hasdemonstrated that embryo scallops are highly sensitiveto Pb by one to two orders of magnitude from Cd morethan other bivalves. On the contrary, our results differfrom the pattern found in oyster C. gigas (Cd > Pb) withhigh sensitivity to Cd (Martin et al. 1981). These re-searches suggest that in the early-life stage of bivalvethere is an incidence of metal, especially Pb.

In early-life stages, the morphological developmentcould be affected by metals that may influence individ-ual survival. During initial shell formation (reaching

trocophora stage), embryos–larvae need to increaseCa+2 concentration uptake (Cragg 2006), where Pbcould compete affecting shell formation and growth,increasing its availability and transport through calciumchannels displacing iron, zinc, and calcium (Martinez-Finley et al. 2012). The tolerance to metal observed inour embryo exposure experiments might relate withthese defense mechanisms to metal toxicity.

According to several researches, proteins’ induc-tion defense mechanisms such as metallothionein(MT), obtained via maternal transmission, allow theembryo to deal with stress caused by metal exposure,the increase in survival of progeny, and tolerance tometal (Mao et al. 2012; Meistertzheim et al. 2009;Weng and Wang 2014).

In Mytilus galloprovincialis, low Cd toxicity withlargest proportion of total soluble Cd (50%) associatedwith MT in comparison with Zn (12%) and Hg (6%)

Table 2 EC50 values of different bivalve species

Response Specie Exposure(days)

EC50–LC50 Cd(mg/L)

Exposure Cd(mg/L)

EC50–LC50 Pb(mg/L)

Exposure Pb(mg/L)

Reference

EC50 embryosabnorm.

Mytilus trossulus 2 0.502 0.005–0.5 Nadella et al. (2009)

M. trossulus 2 0.045 0.003–1 Nadella et al. (2013)

M. edulis 2 1.2 0.476 Martin et al. (1981)

Meretrix meretrix 1 1.01 0.001–10.16 0.297 0.0019–7.15 Wang et al. (2009)

M. galloprovincialis 2 1.925 0.221 Beiras andAlbentosa (2004)

Crassostrea gigas 1 0.212 0.01–1 Mai et al. (2012)

C. gigas 2 0.611 0.758 Martin et al. (1981)

C. iredalei 2 0.45 0.05–2 Ramachandran et al.(1997)

C. rhizophorae 1 0.282 24–75 da Cruz et al. (2007)

C. virginica 2 3.8 1–6 2.45 0.5–6 Calabrese et al.(1973)

Ruditapesdecussatus

2 0.424 Beiras andAlbentosa (2004)

R. decussatus 1 0.57 0.12–2.03 0.256 0.062–1.03 Fathallah et al.(2013)

Chlamys asperrima 2 0.29 0.050–1 Krassoi et al. (1997)

Argopectenpurpuratus

2 1.55 0.09–16.71 0.044 0.025–1.59 This study

LC50 juvenilesmort.

A. ventricosus 4 0.396 0.156–2.5 0.83 0.28–5 Sobrino-Figueroaet al. (2007)

Perna viridis 4 2.53 0.01–100 3.16 0.01–100 Rajkumar (2012)

A. purpuratus 4 0.48 0.09–48.78 1.42 0.025–1.8 This study

Argopecten purpuratus,Mytilus trossulus,Meretrix meretrix,M. edulis,M. galloprovincialis,Crassostrea gigas,C. iredalei,C. rhizophorae,C. virginica, Ruditapes decussatus, and Chlamys asperrima; LC50 of A. ventricosus and Perna viridis

16 Page 8 of 13 Environ Monit Assess (2018) 190:16

(Pavicic et al. 1994) was observed. Besides, it has beendemonstrated that Crassostrea sikamea offspring resis-tance to Cu and Zn was correlated with the concentra-tion of metal experienced by their parents that inducedhigh MT level in embryos (Weng and Wang 2014). Thisdefense mechanism depends on MT efficiency againstmetal damage. Furthermore, it has been previouslyestablished that MTs have the highest affinity for Cdproducing a high metal tolerance (Pavicic et al. 1994;Roesijadi et al. 1996). Therefore, probably A. purpuratusadults frequently would be exposed to Cd with offspringmore tolerant.

Contrasting research focused in Cd embryotoxicitywith our results would indicate that A. purpuratus ismore sensitive to Cd exposure than Mytilusgalloprovincialis (Beiras and Albentosa 2004) andC. virginica (Calabrese and Nelson 1973), andA. purpuratus has the highest tolerance when comparedto Meretrix meretrix (Wang et al. 2009), M. trossulus(Nadella et al. 2009), M. edulis (Martin et al. 1981),C. iredalei (Ramachandran et al. 1997), R. decussatus(Beiras and Albentosa 2004), C. asperrima (Krassoiet al. 1997), C. rhizophorae (da Cruz et al. 2007), andC. gigas (Mai et al. 2012) (Table 2).

Acute toxicity in juveniles

Surprisingly, juvenile scallops showed an opposite pat-tern in metal toxicity compared to embryos, Cd > Pb(Table 2). According to Amiard et al. (2006) and Wal-lace and Luoma (2003), MT defense mechanismsshould have controlled and detoxified the Cd accumu-lation as it was observed in embryos. However, ouracute toxicity assay would have caused an excess ofCd accumulation and faster scallop mortality. In thisrespect, it has been suggested that concentrations rang-ing between 2 and 4 mg/L Cd could inhibit MT induc-tion in mussel Perna canaliculus juvenile–adult toxicityexperiment (Chandurvelan et al. 2013). A similar resulthas also been described during Bin vitro^ toxicity testusing hemocytes of C. virginica exposed to Cd concen-trations over 1.5 mg/L Cd (Butler and Roesijadi 2000).According to this, our results indicate that the observedhigh mortality in juveniles exposed to 8.01 to 48.78 mg/L of Cd during the first 12 h and for 0.98mg/L after 36 hmay be caused by an inhibited defense mechanismmediated by MT.

Additionally, it has been observed that metalbiokinetics in Crassostrea hongkongensis change

strongly after high stress by Cd depressing its clearancerate and dissolved uptake rate (Pan and Wang 2012a).Besides, MT binds to Cd making its depuration slowerthan other metals (Liu and Wang 2011; Pan and Wang2012a). Moreover, a previous study using A. ventricosusjuveniles exposed by 72 h to sub-lethal concentrationsof Cd (0.04, 0.09, 0.2 mg/L) and Pb (0.09, 0.176,0.41 mg/L) showed that normal filtration rate and rateof oxygen consumption were drastically decreased inCd-exposed scallops (EC50 0.01 mg/L and EC50

0.07 mg/L, respectively) when compared to Pb (EC50

0.12 mg/L and EC50 0.38 mg/L) (Sobrino-Figueroa andCáceres-Martínez 2014), demonstrating differentialphysiologic effects by this metal exposure, similar toour findings (effect evident during first hours in Cdexposure).

This lower Pb toxicity observed might be related todecreased Pb uptake in hemolymph (Mosher et al. 2010)with Pb translocating by hemocyte lysosomes (Metianet al. 2009), and its subsequent detoxification and accu-mulation in mantle (Jing et al. 2007). Furthermore, it hasbeen identified in Pecten maximus and Chlamys varia alow assimilation of Pb from seawater with subsequentfast depuration which determines a low Pb bioaccumu-lation (Metian et al. 2009).

Compared with other bivalve species (Table 2), ourtoxicity pattern Cd > Pb was similar to assay previouslydeveloped in A. ventricosus (Sobrino-Figueroa et al.2007) and P. viridis (Rajkumar 2012). However, theindividual experimental size used in A. ventricosus ex-periments (3 mm) (Sobrino-Figueroa et al. 2007) com-pared to 21 mm used in our assay may account for thedifferential Pb sensitivity observed, because after meta-morphosis in juveniles’ early life-stage somatic growth,shell structural development and metabolic rate remainhighly intensive making them more vulnerable(Beninger and Le Pennec 2006; Cragg 2006). Therefore,for environmental monitoring and risk assessment pur-poses, we suggest using juvenile scallops over 20 mm(avoiding significant metamorphosis and early juvenilestage metabolic interferences).

It is important to highlight that although ourmeasured Pb experimental concentrations were or-ders of magnitude less than Cd, we observed a highPb toxicity especially in embryos. According toAngel et al. (2015), it is impossible to identify Pbtoxicity at concentrations over 2 mg/L in seawater(due to its solubility). However, this situation israrely reported and discussed especially in marine

Environ Monit Assess (2018) 190:16 Page 9 of 13 16

aquatic organism toxicity test research papers.Therefore, previously cited EC50 of M. meretrixand LC50 of A. ventricosus and P. viridis would beunderestimated and should be revised. On the otherhand, although it is highly difficult to work with Pbexposure, further research is needed to evaluate Pbeffect in these endemic organisms, especially be-cause of the continuous stress caused by this metalin San Jorge Bay.

In spite of previously described patterns with em-bryos, more sensitive to metals than juveniles (Hiset al. 1999), our results indicate that this general trendapplies only to some cases, that metal toxicity chang-es writhing organism development and would cer-tainly depend in intrinsic biological aspects in thespecie (A. purpuratus).

According to our results, Cd- and Pb-dissolved con-centrations in seawater which have been continuouslyreported in Antofagasta coastal areas in Northern Chile(MMA 2013; Valdés et al. 2015) should not cause sub-lethal or lethal effects in embryos and juvenile scallops,respectively (Table 3). The Cd maximum concentrationlevels established by the Chilean Primary Water QualityStandard Regulation (Human health protection standard,Decreto-144 2009) would not harm early-life and juve-nile stages of this specie. However, maximum levelestablished in Chilean Regulation for Pb (0.11 mg/L)could implicate a risk to A. purpuratus embryos. More-over, when comparing our EC50 and LC50 results withsome international environmental regulations (Australia,

USA and Canada Table 3), it is possible to observe thatthere are three to four orders of magnitude below whichwould certainly safeguard (unlike the Chilean regulation)the first stages of life of scallops.

Valdés et al. (2014) determined that Pb concentrationregistered in San Jorge Bay would have occasionaladverse effects in benthic organisms. Besides, it hasbeen previously demonstrated that Cd and Pb bioavail-ability in sediments is principally associated with thesoluble fraction (Chapman et al. 1998; Simpson et al.2017). Therefore, our study focuses on dissolved metalexposure effects in scallops based on detected sedimentconcentration (most critical scenario reported in SanJorge Bay).

Then, considering the Cd and Pb EC50 and LC50

result of this study, the sediment concentrations his-torically registered for these metals in Northern Chi-le (MMA 2013; Valdés et al. 2014) (Table 3) couldpose a risk to scallop embryo’s survival and wouldexert mortality in juvenile scallops. Under naturalconditions, scallops are settled on the seabed andalso most of the farming activities are developed incoastal shallow areas exposed to environmental in-teractions such as upwelling and tidal waves. Thisinformation as a first step should preserve benthicaquatic organism life and economically importantspecies (A. purpuratus) cultivated in shallow coastalareas continually affected by dynamic natural pro-cess or with influence of anthropogenic activitiesincreasing metal bioavailability in Northern Chile.

Table 3 Summary of Cd and Pb toxicity (EC50–LC50, mg/L) estimated by dose–response curve analysis in this study, metal concentrationsfor site in marine water (mg/L) and sediment (mg/kg), Northern Chile

Metal This study Northern Chile Chilec USAd Australiae Canadaf

EmbryoEC50

Juv-96 hLC50

Max. level watera Max. levelsedimentb

Max.level

Criterioncontinuous

Levelprotection99%

Protection aquaticlife-long time

Cd 1.55 0.48 0.00003 ± 0.00001 34 ± 6 0.033 0.0088 0.0007 0.00012

Pb 0.044 1.47 0.00009 ± 0.00001 185 ± 20 0.11 0.0081 0.0022 No data

a Valdés et al. (2015)bMinisterio del Medio Ambiente de Chile (MMA 2013)c Primary quality standards for the protection of marine waters and estuarine suitable for recreation with direct contact (Decreto-144 2009)d US Environmental Protection Agency National-recommended water quality criteria (2009)e Australian and New Zealand Guidelines for Fresh andMarine Water Quality (Australian and New Zealand Environment and ConservationCouncil 2000)f Canadian Environmental Quality Guidelines (Canadian Council of Ministers of the Environment 2014). All values are in milligrams perliter with the exception of marine sediment

16 Page 10 of 13 Environ Monit Assess (2018) 190:16

Conclusions

This study demonstrated high sensibility of embryoscallops of Argopecten purpuratus for Pb more thanCd, the case contrary for juvenile scallops. Factorsas defense mechanisms or biological process foreach ontogenetic stage and metal behavior influencethis toxicity. This study shows that especially scal-lop embryos would be more suitable as a monitoringtool for Pb marine pollution. Scallop embryos wouldbe more sensitive to Pb than any other bivalvespecies. Further research is required in order toestimate Cd and Pb toxicity under real environmentand to elucidate the mechanisms of action of toxicmetals at different stages of ontogeny in thisbioindicator specie. Regarding Cd, primary qualitystandards for the protection of marine waters andestuarine established for Chilean coastal areas wouldpreserve scallop embryos and juveniles the same asthat of Pb quality standards, where the embryoscould be at risk. This information would be relevantfor metal environmental monitoring program deci-sions. Both scallop embryo and juvenile bioassaysare useful tools that can be used to evaluate toxicityof field samples of seawater containing Cd and Pb.

Acknowledgements This study is part of PR-M PhD thesis(Applied Science PhD Program, University of Antofagasta) andwas financially supported by FONDECYT-Chile (Project1140164), and PhD Scholarship for thesis research. R. Barrathanks MUSELS NUCLEUS funded by MINECON NC120086and FONDAP CRHIAM 15130015. The funding sources had noinvolvement in the collection, analysis, and interpretation of data,in the writing of the report, or in the decision to submit the paperfor publication.

Compliance with ethical standards

Conflict of interest The authors declare that they have no con-flict of interest.

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