assessment criteria for using the sea-urchin embryo test with sediment elutriates as a tool to...

Environmental Toxicology and Chemistry, Vol. 29, No. 5, pp. 1192–1198, 2010# 2010 SETAC

Printed in the USADOI: 10.1002/etc.136

ASSESSMENT CRITERIA FOR USING THE SEA-URCHIN EMBRYO TEST WITH SEDIMENT
ELUTRIATES AS A TOOL TO CLASSIFY THE ECOTOXICOLOGICAL
STATUS OF MARINE WATER BODIES

IRIA DURANyz and RICARDO BEIRAS*zyLaboratorio de Ecoloxıa Marina, Departamento de Quımica Analıtica e Alimentaria, Facultade de Ciencias do Mar, Universidade de Vigo,

Estrada Colexio Universitario s/n. E-36310, Vigo, Galicia, Spain

zToralla Marina Science Station, Universidade de Vigo, Illa de Toralla, E-36331, Coruxo, Galicia, Spain

(Submitted 23 September 2009; Returned for Revision 4 December 2009; Accepted 14 December 2009)

* T(rbeira

Pub(www.

Abstract—A large, multiyear data set was generated by pooling the sediment elutriate (SET) results collected during previous studiesconducted in the Galician Rias (northwest Iberian Peninsula) that met the acceptability criteria in the controls, to ensure optimum qualityof data (n¼ 162). Two subsets of equal to reference and lower than reference sites were identified by comparing the Percentage NetResponse (PNR) value from each sampling site with nontoxic, cruise-specific, reference sites by using the t test with the unequal varianceassumption. Ecotoxicological Assessment Criteria (EAC0, EAC1, EAC2, and EAC3) were then derived from those two subsets to classifythe SET results into five categories of ecotoxicological status: high, good, moderate, poor, and bad, in line with the European legislation.The 50th and 5th percentiles of the PNR distribution of the equal to reference sites subset were EAC0¼ 0.879 and EAC1¼ 0.694. AnEAC2¼ 0.508 was obtained from the 50th percentile of the lower than reference sites subset. Because the PNR values of the entiredatabase showed a distribution that can be adjusted to two normal populations, the EAC3¼ 0.240 PNR was calculated as the intersectionbetween the first and second normal distributions identified. Power analysis proved that the limit between acceptable and unacceptablestatus (EAC1) corresponded to a detectable PNR difference to control with a confidence level >99% and a power of 95%. Environ.Toxicol. Chem. 2010;29:1192–1198. # 2010 SETAC

Keywords—Sediment toxicity Water quality Ecotoxicological status Paracentrotus lividus

INTRODUCTION

The European Water Framework Directive (WFD) [1]defined a series of biological, hydromorphological, and chem-ical indicators of water quality, established five differentcategories of ecological status according to the degree ofanthropogenic alteration (high, good, moderate, poor, or bad),and stated the ambitious goal to achieve, for all European waterbodies, including marine coastal waters, a good ecologicalstatus by 2015. Therefore, for any given biological responseto anthropogenic impact, site-specific assessment criteria, alsoknown in ecotoxicology as toxicity scores, must be developed toclassify the continuous response into discrete categories. Withregard to marine ecosystems, the ICES has recently made animportant effort to develop these criteria for the more stand-ardized biological assessment tools, such as several molecularbiomarkers (ethoxyresorufin-O-deethylase, vitellogenin, neu-tral red) and toxicity tests (sea-urchin embryo, bivalve embryo,amphipods) ([2]; http://www.ices.dk/products/icesadvice.asp;[3], http://www.ices.dk/products/icesadvice.asp).

In coastal ecosystems, the changes in salinity and the hydro-dynamics of estuaries and other transitional areas cause a flux ofparticulate matter toward the bottom sediments that act as sinksof pollutants originated from continental sources. In fact, 90 to95% of the inputs of trace elements into the sea accumulate incoastal environments [4]. In contrast to water, sediments pro-vide higher and more stable (time-integrated) concentrations of

o whom correspondence may be [email protected]).lished online 1 February 2010 in Wiley InterScienceinterscience.wiley.com).

1192

pollutants, making the monitoring based on sediment more costeffective and reliable than monitoring based on water samples.Therefore, sediment toxicity bioassays are techniques currentlyincluded in the marine environmental monitoring programsworldwide [5–14].

With the goal of developing ecotoxicological assessmentcriteria (EAC) on scientific grounds, the present study hasresorted to the ecotoxicological data set generated in the last10 years of chemical and toxicological studies in the GalicianRias (northwest Iberian Peninsula), consisting of sedimentchemistry and sea-urchin embryo-larval bioassays with sedi-ment elutriates. A posteriori statistical methods were used toseparate two subsets of equal to reference (ER) and lower thanreference (LR) sites, from whose distributions the assessmentcriteria were obtained, and the classification was validated onthe basis of the available chemical data.

MATERIAL AND METHODS

Sediment sampling

Samples were collected at a total of 65 sites as part ofinvestigative monitoring surveys that took place from 1997 to2008 along the Galician coast. Intertidal sediments were col-lected by hand at low tide, and subtidal sediments were sampledfrom the research vessel Navaz (Instituto Espanol de Oceano-grafia) using a modified Bouma box corer (0.0175 m2 surfacearea and 10–20-cm sediment depth). Chemical contaminantswere measured in the surface sediments by standard procedures[6,8] and results were expressed in terms of range and geometricmean in both subsets of samples (ER and LR). Mean effectrange median (ERM) quotients (mERMq) were calculated as

Assessment criteria for the sea-urchin embryo bioassay Environ. Toxicol. Chem. 29, 2010 1193

the average of the ratios between the concentration of eachpollutant in the sample divided by the ERM levels described byLong et al. [15]. Because the list of pollutants measured is notexactly the same for all the surveys included in the data set,three partial mERMq values were calculated—one for metals,one for polycyclic aromatic hydrocarbons and another forpolychlorinated biphenyls (PCBs)—and the global mERMqwas calculated as the average of the partial mERMq values.

Toxicity test

Elutriates for the sea-urchin embryo test were obtained byrotatory mixing 100 g of sediment and 500 ml of control 0.22-mm-filtered sea water (FSW) in airtight polypropylene flaskswith no head space for 30 min [6]. The liquid phase wastransferred into new polypropylene flasks after decantationovernight in darkness at 208C, and gently aerated for 10 minto eliminate potential H2S toxicity. Physicochemical para-meters were checked prior to the bioassay to ensure they laywithin optimum values for sea-urchin development (7.0 < pH<8.5, dissolved oxygen > 2 mg/L, H2S < 0.1 mg/L, and NH3 <40mg/L [16]). Serial dilutions of the elutriates in FSW(a minimum of three per sample) were tested.

The embryo larval bioassays with Paracentrotus lividuswere carried out according to standard methods [16–18]. Game-tes were obtained by dissecting mature individuals. Prior tofertilization, gamete viability was assessed by checking underthe microscope egg roundness and sperm motility, placing thegametes in a drop of FSW. Eggs from a single female werecollected in a 100-ml measuring cylinder with FSW, a few ml ofundiluted sperm collected with a glass Pasteur pipette from asingle male was added, and gentle stirring was provided with aplastic plunger. Four aliquots of 20ml were taken, and totalnumber of eggs and numbers of fertilized eggs, identified bythe fertilization membrane, were counted in a Sedgwick raftercounting cell. Calculations were made to deliver with anautomatic pipette 40 fertilized eggs/ml in the 4-ml test vials.A series of control vials with fertilized eggs were fixed at timezero, and the remaining vials were incubated for 48 h in the darkat 20� 0.58C, fixed with two drops of 40% formaldehyde andobserved under an inverted microscope.

The SET data analysis

For each vial, the size (maximum dimension) of the first35 individuals observed (either normal larvae or earlier devel-opmental stages) was recorded, and size increase, compared tothe initial diameter of the eggs, was calculated. Results wereexpressed as percentage of the FSW control.

For each environmental sample two toxicity parameterswere obtained: the percentage net response (PNR), which isthe control-corrected size increase in the undiluted elutriate, andthe toxic units (TU), which takes into account all the dilutionstested. Toxic units were calculated as TU¼ 100/ED50, whereED50 is the theoretical dilution of the elutriate causing aninhibition of 50% in size increase. This ED50 was obtained bylinear regression of size increase versus dilution in logarithmicscale.

The PNR value takes into account the information from theundiluted elutriate only, reducing the number of test vials threeor more times (rapid and cost-effective screening procedure).In addition, another advantage of the PNR value is that it allowsstraight-forward statistical comparison to reference response.However, the TU values show two advantages. They summarizemore toxicological information (three or more dilutions persampling site), and their comprehension is more intuitive

because, unlike PNR, TU are set to 0 in undisturbed sitesand the value increases as toxicity increases.

On the other hand, the calculation of TU shows somelimitations. Aiming at feasibility in routine monitoring, testsmust be designed on the basis of a certain a priori knowledge ofthe expected toxicity. Particularly toxic points may still retaintheir toxicity with the highest dilution routinely assayed. In thatcase, only an approximate estimate of the TU value can beobtained. Second, because TU are calculated from the linearregression of the biological response versus the dilution of theelutriate (in log scale), it requires a minimum of toxicity, that is,a slope of that regression significantly lower than zero.

Ecotoxicological assessment criteria

A large, multiyear data set was generated by pooling the SETresults collected during the previous investigative monitoringsurveys conducted in the Galician Rias. To ensure the quality ofthe data set, bioassays not meeting the acceptability criteria of a218-mm size increase in the control [16] were excluded. ThePNR data of the remaining experiments (n¼ 183) were used forthe calculation of the EAC.

With this aim, reference stations were first identified.Although each survey included at least one sampling siteconsidered a priori as nonpolluted, reference sites for eachsurvey were empirically defined as those promoting the highestlarval growth in the undiluted elutriate, that is, those exhibitingthe highest PNR value. For each survey, the PNR value of all thesampling sites were compared to the respective reference site byusing the t test with the unequal variances assumption [19,20],and those not significantly different (p� 0.05), were pooledinto a subset of data termed equal to reference sites. The50th and 5th percentiles of the PNR distribution in this equalto reference subset were calculated, and these values wereconsidered as the EAC0 and EAC1, that is, the limits betweenhigh and good and good and moderate ecotoxicological status,respectively. The EAC0 value is also useful to set a minimumvalue of 0 TU to undisturbed treatments.

Next, stations with a response significantly different to thecorresponding reference station for each survey and exceedingthe EAC1 were pooled into a second subset of data termed lowerthan reference. The 50th percentile of this distribution wascalculated and this value was considered as the EAC2, that is,the threshold dividing water bodies with moderate and poorecotoxicological status, sensu the WFD.

The EAC3 value, separating poor from bad ecotoxicologicalstatus, was derived taking into account the distribution of PNRin the entire data set. The PNR data showed a bimodal dis-tribution and were thus adjusted to two overlapping normallydistributed populations, setting the EAC3 as the intersectionbetween both distributions. The data were first adjusted withFISAT II program (FAO-ICLARM Stock Assessment Tools,Food and Agriculture Organization of the United Nations) totwo distributions using the Bhattacharya method [21]. Thecalculated preliminary values were then improved by usingthe R ([22], http://www.R-project.org) statistical package andMixdist program [22] to obtain the final normal distributions.

Power analysis

In addition, the minimum significant difference (MSD)approach was used to conduct a power analysis of the test[14,20,23]. With this aim, the PNR values from the whole dataset were used. MSD values were calculated following the

PNR

0.0 0.2 0.4 0.6 0.8 1.0

TU

0

5

10

15

20

25

30

35

PNR

0.0 0.2 0.4 0.6 0.8 1.0

TU

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

a

b

Fig. 1. Regression between toxic units (TU) and percentage net response(PNR) with the lower than reference subset. (a) Including all the dataavailable in the subset. (b) Removing of the dataset extremely toxic points.

1194 Environ. Toxicol. Chem. 29, 2010 I. Duran and R. Beiras

expression [23].

MSD ¼ tcritical

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffis2

1

n1

þ s22

n2

� �s(1)

where tcritical is the t value from the standard statistical table for aconfidence levela and the appropriate degrees of freedom, s2

1 ands2

2 are the variances for the FSW control and the elutriate, and n1

and n2 are the number of replicates for the control and theelutriate.

Statistical analyses were performed with SPSS1 statisticalpackage for Windows, version 17.0 (SPSS Inc.). The frequencydata were adjusted to normal distributions using FISAT II 1.2.2and R version 2.8.1.

RESULTS

The SET results: PNR and TU

As explained above, TU are calculated from the linearregression of the biological response versus the dilution ofthe elutriate (in log scale); it requires a minimum of toxicity,that is, a slope of that regression significantly lower than zero.This requirement is not met in the nontoxic samples, limitingthe applicability of the TU concept to the LR data subset.Therefore, the TU value is arbitrarily set to zero in the sampleswith a PNR > EAC0. The correlation between PNR and TU forthe remaining sites shows a R2¼ 0.50 ( p< 0.01; n¼ 141).When samples from an extremely toxic sampling point (withTU above 30) were removed, the goodness of fit improved toR2¼ 0.80 (Fig. 1), and the following equation was obtained:

TU ¼ �3:247PNR þ 2:511ðp< 0:01; n¼ 137Þ (2)

This equation can be used to estimate a TU value for the caseof data sets where only the PNR value is available because serialdilutions had not been tested.

Ecotoxicological assessment criteria

EAC0 and EAC1. To obtain the threshold between good andmoderate ecotoxicological status, a t test with the unequalvariances assumption was performed with the PNR values,and 56 of the 176 cases were not statistical different to refer-ence. Those sites not significantly different from the referencewere added to the equal to referenc data set, and the5th percentile of the resulting distribution was established asEAC1, with a value of 0.694 PNR.

As the WFD also makes a difference between high(undisturbed) and good ecological status, in addition, anEAC0 was derived as the limit between both categories asthe 50th percentile of the equal to reference data set. TheEAC0 so calculated was 0.879 PNR.

EAC2 and EAC3. The second data subset included thosesites statistically different from the reference samples andexceeding the EAC1, termed lower than reference. TheEAC2 was established as the 50th percentile of this subset.Thus, the limit between moderate and poor ecotoxicologicalstatus resulted in 0.508 PNR.

In addition, the WFD separates between poor and badecological status. Aiming at identifying those priority sites withthe lowest ecotoxicological status, the entire data set was fitted(Fig. 2) to a distribution composed by two normal distributions:the first, constituted by the bad sites, with a mean PNR of 0.024(s¼ 0.109), and the second with a mean PNR of 0.734(s¼ 0.184). There was no significant difference between the

observed PNR values and the sum of the overlapped normaldistributions ( p¼ 0.76). The EAC3 was then calculated as theintersection between the first and second normal distributions,and a value of 0.240 PNR was obtained.

Power analysis

For a given confidence level, a, the power of the test (1 � b)can be related to the MSD, also termed detectable difference,which is the smallest difference between control and treatmentthat can be considered statistically significant. The MSD is acharacteristic of the test that depends on the natural variabilityof the response and the test design, and it can be assessed fromits previous performances. Figure 3 depicts this relation settinga to the standard values of 0.05 and 0.01, respectively. When a

and b values are changed, the MSD varies as described inTable 1. A reduction in the detectable difference causes anincrease in the value of a and/or b, thatis, an increase in theprobability of type I and/or II error.

The value dividing high and good categories (EAC0) was setto 0.879 PNR, a 0.121 difference from the control, and thatdividing good and moderate categories (EAC1) to 0.694 PNR, a0.306 difference from the control. From Figure 3 it can becalculated that a 0.121 difference corresponds to a b of 0.15 and0.30 for a¼ 0.05 and 0.01, respectively. This means that theSET, using an EAC0 of a difference of 0.121, can detect slightlyaltered sites (good category) with a 95% confidence and a power

Fig. 2. Distribution of PNR data in our dataset. Blue line represents thehistogramobtained from the complete dataset, red lines represent the adjustednormal distributions, and green line is the sum of the two normal distributionsidentified. Red triangles are the mean of each distribution. Distribution 1:mean¼ 0.024; s¼ 0.109; Distribution 2: mean¼ 0.734; s¼ 0.18.

Table 1. Minimum significant difference (MSD) of the sea-urchin testobtained for different probabilities of type I and type II error

a b MSD

0.05 0.05 0.2180.05 0.1 0.1460.01 0.05 0.2740.01 0.1 0.218


of 85%. Similarly, a 0.306 difference from the control corre-sponds to b values of 0.02 and 0.05 for a¼ 0.05 and a¼ 0.01,respectively. This means that the SET, using an EAC1 of adifference of 0.306, can detect altered sites (moderate category)with a 99% confidence and a power of 95%.

Ecotoxicological status of the Galician coast

Figure 4 shows the different ecotoxicological status and theEACs that establish the limits between them. The EACs herepresented were useful to assess the general ecotoxicologicalstatus of the Galician Rias, where 57% of the sites sampled werehigh or good, and only 20% poor or bad (Fig. 5), and alsoidentified hot spots, particularly in the inner part of Ria of

0

10

20

30

40

50

60

70

80

90

100

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

MSD

Pow

er (1

-β)

α = 0.01

α = 0.05

Fig. 3. Power of the sea-urchin embryo test at different a and b values(MSD¼minimum significant difference).

Pontevedra (Spain), where protective measures are urgentlyneeded (Fig. 6).

DISCUSSION

Although the WFD defines five different categories ofecological status, the proposed first approach when interpretingthe SET results, is the use of two thresholds, EAC1 and EAC2,which allows a classification of the response into three cate-gories: good, moderate, and poor, conveniently correspondingin the traffic light symbols of green, yellow, and red. Once thesecategories were confirmed in routine assessment, and if furtherdetail is necessary, the green and red types can easily besubdivided into two classes by using the EAC0 and EAC3 togenerate the high (undisturbed) and good and the poor and badcategories.

The most vital threshold is EAC1, that is, the divisionbetween acceptable (good) and non acceptable (moderate)ecotoxicological status (corresponding to the green and yellowtraffic lights). The EAC1 is also useful in screening surveyswhere only undiluted elutriates are tested. Samples showingPNR below EAC1 require further testing of serial dilutions tocalculate TU values. To establish this criterion, a series of sitesneed to be identified suitable to represent unaltered, or slightlyaltered and thus acceptable, ecotoxicological status. This wasachieved by testing the differences between the local reference(the one exhibiting the highest biological response in eachsurvey) and every other site in that survey by using the t testwith unequal variance assumption [19]. This data subset ofequal to reference sites showed sediment chemistry consistentwith well-known international environmental quality criteria[24–26].

Table 2 shows the concentrations (range and geometricmean) of contaminants in the equal to reference and the lowerthan reference subsets. Concentrations (geometric mean) ofpollutants in sites from the LR group were from 0.8 to 2.1times higher than in the reference subset.

For all chemicals, the concentrations measured in the refer-ence sites are below the ERM limit [24], copper and lead are theonly metals with content above the PEL value (probable effectlevel as described in McDonald et al.) [25]. In contrast, forarsenic (As), copper (Cu), chromium (Cr), mercury (Hg), lead(Pb), and PCBs, the nontoxic reference sites frequently exceedthe upper level of the Convention for the Protection of the

Fig. 4. Ecotoxicological assessment criteria (EAC) for the sea-urchinembryo test (SET) that establish the five categories of ecological statusmentioned in the European Water Framework Directive (WFD). Toxic units(TU) were calculated according to Equation 2. Percentage net of response(PNR) calculated as described in the text.

Fig. 5. Percentage net response (PNR) results of the elutriates and percentage of samples of the complete dataset from the Galician coast falling into each of theWFD categories of ecological status, according to the EAC values shown in Figure 4. Each bar is a sampled station.


Marine Environment of the North-East Atlantic Ecotoxicolog-ical Assessment Criteria (OSPAR EAC), indicating that theselater criteria bear little predictive value, even when using ahighly sensitive toxicity test such as SET.

Caution must be taken when using sediment quality criteriaoutside the regional context used for their calculation. It waspreviously found that ERM values, but not other more con-servative criteria, showed predictive value when studying thetoxicity of sediments form the Galician Rias [6]. The mERMqvalues were thus calculated separately for the reference andlower to reference data subsets. Concerning the equal to refer-ence data set, the partial mERMq ranged from 0.1 to 0.7,indicating again that these criteria are in general useful withinregional context of the present study. According to the mERMqclassification in Long et al. [15], for the reference data set, thesamples have less than 30% probability of toxicity. However,the mERMq values for metals were always higher than those fororganic pollutants. This might be due to different mineralogicaltraits of the sediments from the Galician Rias. This seems to bethe case for the As and Pb ERL value.

Fig. 6. Ecological status of the Galician Rias (NW Iberian Peninsula) according toGalician Coast.

In conclusion, the reference sites have been empiricallyidentified as those with the lowest toxicity and built up anequal to reference data sub-set by comparing each site with thetoxicity of the corresponding reference site. For some of thesesites, analytical chemistry information is available. In all casesand for all the contaminants measured, the sediment concen-trations in the clean sites were below ERM values. When thecontents of metals, hydrocarbons, and PCBs were available,mERMqs were always below 0.2.

Power analysis allowed the reliability of the criterion toseparate acceptable (good) from non acceptable (moderate)sites to be tested. The MSD approach quantifies the probabilityof type I and type II error when using the EAC1 establishedlimit. For example, when using sea-urchin larval morphology asendpoint, Carr and Biedenbach [14] reported a MSD of 0.21 fora¼ 0.01 and b¼ 0.05. In the present study, the MSD obtainedfor the more exigent values alpha and beta tested (a¼ 0.01,b¼ 0.05) was 0.27, slightly higher than the EAC1 (0.306 unitsof reduction in PNR). Therefore, when EAC1 is used to includea sample in the good ecological status, there is just a 1%

the EAC values shown in Figure 4. Dots represent sampled points along the

Table 2. Range, mean, and number of samples in both equal to reference (ER) and lower than reference (LR) subsets and OSPAR ecotoxicological assessmentcriteria

Equal to reference Lower than referencea Enrichment factor (LR/ER)

Range Mean n Range Mean n Mean ERM OSPARb

As (mg/g) 5.5–24.9 13.3 23 6.3–76.9 15.6 45 1.2 70 10Cd (mg/g) 0.1–0.6 0.3 7 0.01–93.5 0.4 218 1.2 9.6 1Cr (mg/g) 18.3–138 54.2 23 17.4–534 70.3 46 1.3 370 100Cu (mg/g) 10–191.4 33.6 23 3.3–7574.7 65.5 46 1.9 270 50Hg (mg/g) 0.2–0.6 0.4 7 0.04–2.7 0.3 18 0.8 0.71 0,5Ni (mg/g) 2.2–33.3 12.1 23 25–82.4 15.6 46 1.3 51.6 50Pb (mg/g) 37.2–143.1 69 23 18.5–1423 84.7 46 1.2 218 50Zn (mg/g) 23.7–287 111.7 22 36.6–17609 203.7 46 1.8 410 500BaAc (mg/kg) 2–131 40.7 6 3.1–1484 63 18 1.5 1,600 1,000BaP (mg/kg) 1.4–154 42.1 6 203–1637 64.4 18 1.5 1,600 1,000BkF (mg/kg) 1.2–70.1 23.8 6 2–734 35.5 18 1.5Chr (mg/kg) 2.6–173.6 49.6 6 4.1–1635 75.3 18 1.5 2,800 1,000Phe (mg/kg) 1.4–146.7 34.7 6 4.12–1298 72.6 18 2.1 1,500 1,000Pyr (mg/kg) 2.4–225.5 66.5 6 7.5–2431 117.4 18 1.8 2,600 500PCB14 (mg /kg) 2.7–221.6 22.4 7 0.9–227.7 17 18 0.8 180 10mERMQ 0.13–0.7 0.03–18.2% <65mm 0.8–93.5 17.5 22 1.6–94 21.1 33% OC 0.1–6.2 1.21 27 0.2–8.5 1.7 43

a Upper range exceeding twice or more the ERM marked in bold.b Upper level of the range set forth by OSPAR (Convention for the Protection of the Marine Environment of the North-East Atlantic).c BaA¼Benzo[a]anthracene; BaP¼Benzo[a]pyrene; BkF¼Benzo[k]fluoranthene; Chr¼Chrysene; Phe¼ Phenanthrene; Pyr¼Pyrene; PCB14¼ sum of 14

PCBs; mERMQ¼mean ERM quotients; OC¼ organic carbon; As¼ arsenic; Cd¼ cadmium; Cr¼ chromium; Cu¼ copper; Hg¼mercury; Ni¼ nickel;Pb¼ lead; Zn¼ zinc.


probability of type I error and less than 5% probability of type IIerror. Adoption of EAC1 as criterion to classify sediment intoacceptable and nonacceptable categories of ecotoxicity, on thebasis of their SET results, enables a reduction in the probabilityof both kinds of error below the less strict standards of a¼ 0.05and b¼ 0.10, frequent in marine ecotoxicology. As stated byThursby et al. [20], a false negative error may create a falsesense of security, allowing continued environmental degrada-tion to occur.

A different approach to the problem of assessment criteria isthe one taken by Losso et al. [27]. In their work they developedtoxicity scores according to two different scales: an effectsemiscore based on the samples that are statistically differentfrom the control (using the MSD approach), and a toxicitysemiscore, based on arbitrary ranges of toxic units. In theirstudy, the limit between toxic (low toxicity) and nontoxicsamples was established at 89.1% of the control, a level ofresponse similar to the EAC0 of the present study. In addition,the limits for medium, high, and very high toxicity are arbi-trarily set to >1, >2, and >4 TU [27]. This is analogous tolimits for moderate, poor, and bad status in the present study, setat 0.27, 0.86, and 1.73 TU (Fig. 4), which are more exigent as aresult of the higher level of impact found in the Venice Lagooncompared to the more oceanic Galician Rias.

Acknowledgement—This study was partially funded by Research ProjectsREN2003-00958 and CTM2006-13880-C03-01/MAR. We thank the crew ofthe research vessel Navaz (Instituto Espanol de Oceanografıa). I. Duran wassupported with an Formacion de Personal Universitario-fellowship from theSpanish Ministry of Education and Science. We acknowledge the technicalsupport of M. Espineira, A. Abalo, and R. Rendo during the course of thisstudy. We thank B. Castro for his help with FISAT and R software. We thankfour anonymous reviewers for comments that contributed to greatly improvethe manuscript.

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