Environmental and biological monitoring of persistent organic pollutants in waterbirds by non-invasive versus invasive sampling

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Toxicology Letters

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nvironmental and biological monitoring of persistent organicollutants in waterbirds by non-invasive versus invasive sampling

asih Kocagöza, Ortac Onmusb, Ilgen Onata, Beste C agdas a,ehmet Sıkıb, Hilmi Orhana,∗

Department of Toxicology, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, TurkeyDepartment of Biology, Faculty of Sciences, Ege University, 35100 Bornova-Izmir, Turkey

i g h l i g h t s

POP concentrations represent a decreasing trend over time.Venous blood represents promising biomonitor for internal PCB concentrations.Enzyme activities correlate with the liver concentrations of several OCPs.Egg DDE levels are below the threshold for the risk of hatch and reproductive success.

r t i c l e i n f o

rticle history:eceived 14 October 2013eceived in revised form 4 December 2013ccepted 27 January 2014vailable online xxx

eywords:ersistent organic pollutantsnvironmental monitoringield study

a b s t r a c t

Three main groups of persistent organic pollutants (POPs); namely organochlorine pesticides (OCPs),polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) were quantified in waterand sediment samples, as well as in various invasive and non-invasive samples from waterbirds in theBüyük Menderes River (BMR). Liver and muscle tissues, blood, and preen gland oil samples of yellow-legged gull (Larus michahellis) and Euroasian coot (Fulica atra) were collected both from the origin (Is ıklıLake) and the estuary (Söke) of the river, blood and preen gland oil samples of grey heron (Ardea cinerea)and pelican (Pelecanus crispus) were collected from the estuary only. In addition, non-hatched eggs fromseveral above species and Mediterranean gull (Larus melanocephalus), in either station were collected. Inall samples, POP contamination was measured and the potential usefulness of those invasive and non-

aterbirdnvasive samplingon-invasive samplingüyük Menderes Riveregean Sea

invasive sampling for biomonitoring was evaluated. Activities of antioxidant enzymes were measuredas potential indicators of POP exposure and of changes in the cellular defence. Venous blood provedto be a promising biomonitor for the concentrations in liver and muscle, especially for PCBs. Activitiesof antioxidant enzymes were correlated with the liver concentrations of several OCP congeners. Themeasured egg DDE concentrations were below the established threshold concentrations for the risk ofhatch and reproductive success.

. Introduction

Environmental and biological monitoring represent the first andhe most important step of ecological, as well as human risk assess-

ents for the environmental pollutants that pose a health risko humans and ecological species. Waterbirds have been proveduitable for monitoring environmental pollution because they areong-lived and highly mobile, thus they integrate pollutants over a

Please cite this article in press as: Kocagöz, R., et al., Environmentawaterbirds by non-invasive versus invasive sampling. Toxicol. Lett. (2

road area (Furness, 1993), and they are at the top of the food chainFossi et al., 1999). There have been studies to establish POP levels inirds by both invasive and non-invasive ways (Albanis et al., 2003;

∗ Corresponding author. Tel.: +90 232 373 91 73; fax: +90 232 388 52 58.E-mail addresses: [email protected], [email protected] (H. Orhan).

378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2014.01.044

© 2014 Elsevier Ireland Ltd. All rights reserved.

Chen et al., 2009; Custer et al., 2010a,b; Goutner et al., 2001, 2011;Rajaei et al., 2011; Colabuono et al., 2012).

In addition to represent a source of human exposure via edibleseafood (fish, shellfish, waterbirds etc.), environmental pollutionthreats also the existence of those species, especially the ones underthe risk of extinction. In this sense, ecological species can be uti-lized in several ways; estimating the dimensions of the pollutionin a certain area, establishing the levels for human exposure, andanalysing the adverse effects to the species itself due to internaldose of the pollutants. The ideal approach for such purposes wouldbe measuring the concentration of the pollutant in the respec-

l and biological monitoring of persistent organic pollutants in014), http://dx.doi.org/10.1016/j.toxlet.2014.01.044

tive tissue of the species; in the edible parts for estimating humanexposure, in the best representing tissue for environmental concen-trations, and in the target tissue for determining adverse effects ofthe pollutant. In most cases, the life of ecological species should

dx.doi.org/10.1016/j.toxlet.2014.01.044


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e terminated for those purposes. However, this is undesirableoth from ethical and ecological perspectives. As to the ecologicalerspective, several species are facing with the risk of extinction.lthough various aquatic species are already consumed as foody humans, using them for the purpose of monitoring the envi-onmental pollution brings an additional risk for their existence.hese facts prompted researchers in the area of life sciences toeek alternatives to those destructive or invasive approaches. Oneromising non-invasive approach is utilizing preen oil from birdpecies (Johnston, 1976; Van den Brink, 1997; Yamashita et al.,007). Preen oil is secreted from uropygial gland located at the basef the tail feathers. This gland produces lipids which may protecthe feathers from wear, aid in waterproofing and protect againstermatophytes (Soini et al., 2007). It is especially relevant for theollutants to be accumulated which are lipophilic such as OCPs,CBs, and PBDEs. Yamashita and co-workers collected preen glandil samples from 30 seabirds at North Pacific Ocean and determinedCB concentrations (Yamashita et al., 2007). They established aeak but significant correlation between the total PCB levels in

he preen gland oil and adipose tissue, which was improved whenetabolic losses were taken into account. They proposed that the

il values can be used to estimate the abdominal adipose tissueurden (internal dose) of seabirds within one order of magnitudeYamashita et al., 2007). On the other hand, correlations betweenon-invasive samples and various tissues should be evaluated forhe purposes above; whether the non-invasive approach is rele-ant to reflect the chemical concentration in the tissue of interest.rom this point of view, correlation to muscle tissue for estimat-ng human exposure, and correlations to liver tissue for evaluatingoxicity to the ecological species should be analysed. This is whate considered in the present study. The other alternative sam-lings are focusing on blood (Finkelstein et al., 2006), droppingsSun et al., 2006), feathers (Jaspers et al., 2006), and non-hatchedggs (Goutner et al., 2001; Albanis et al., 2003; Antoniadou et al.,007; Wang et al., 2011). As Yamashita et al. (2007) commented,lood sampling requires trained technical skill and dropping samp-

ing requires cooling during preservation and transportation whichs a potential problem in remote areas. Collecting feathers is a non-nvasive approach; however, it may not accurately reflect the bodyurden of the pollutant as they are exposed to physical environ-ent and are open to outer effects.Since a large proportion of OC compounds are known to bio-

agnify in the egg yolk (Kleinow et al., 1999), contaminant levelsn waterbird eggs serve as an important tool for monitoring changesn the environmental quality. In addition, collection of eggs is a rel-tively non-invasive technique that has minimal adverse effects onhe bird community (Connell et al., 2003). Hence, collecting non-atched eggs prevents the destructive effect on next generations.o the best of our knowledge, contaminant concentrations in bothreen gland oil and non-hatched eggs have not been reported forhe same waterbird species yet.

Toxic effects of OCPs, PCBs and PBDEs in aquatic life formsnclude endocrine disruption (such as reduction of testosterone lev-ls in the blood) (Tanabe, 2002; Brouwer et al., 1989; Reijnders,990; Boon et al., 1992), thinning the eggshell (causes death ofmbryo before hatch) (Ratcliffe, 1967; Hickey and Anderson, 1968)nd induction of oxidative stress (Bainy et al., 1993; Padmini andijaya Geetha, 2007; Glauert et al., 2008; Gurer-Orhan et al., 2006).herefore, OCPs have played an important role in the decline ofaterbird populations (Fox et al., 1991). Measuring the activity of

ntioxidant defence enzymes such as SOD, CAT, GPx and GST is uti-ized as a marker of POP pollution (Regoli et al., 2002; Ozmen et al.,


008; Richardson et al., 2008), alteration in the activity was pro-osed as early signals of environmental disturbance (Santos et al.,004). Oxidative stress induced by OCPs was reported in rat (Bainyt al., 1993) and in Mugil cephalus (Padmini and Vijaya Geetha,

PRESStters xxx (2014) xxx–xxx

2007). As a consequence of oxidative stress, peroxidation of mem-brane lipids may result in degradation of membrane integrity andsubsequently cell damage. POPs have been shown to induce mem-brane lipid peroxidation in CHO cell culture in vitro (Gurer-Orhanet al., 2006).

In the present study, we aimed at evaluating the potential appli-cability of both preen gland oil and blood, as well as egg samplesof waterbird species in estimating the internal dose of the POPs;liver and muscle concentrations. Beside, any possible correlationbetween POP concentrations of liver and muscle to non-invasivesamples was also evaluated. In order to achieve this, the destruc-tive liver and muscle tissues were obtained from Euroasian coot andyellow-legged gull, which are not under the risk of extinction. Fromgrey heron and pelican, however, only non-destructive preen glandoil and blood samples, as well as non-hatched egg samples werecollected. Egg samples were also collected from Euroasian coot andMediterranean gull. Such a monitoring study for OCPs, PCBs andPBDEs in both blood and preen gland oil of endangered species suchas grey heron and pelican, and in liver, muscle, egg, blood and preengland oil samples of Euroasian coot and yellow-legged gull has beenconducted for the first time. In addition, the present study enabledus to compare any possible difference between the origin and theestuary of BMR, and also between various biosamples of differentspecies in terms of POP pollution, as the latter was reported for con-centrations in egg samples from different species (Dong et al., 2004;Luo et al., 2009). Antioxidant enzyme activities were evaluated aspotential early sign of chemically-induced oxidative stress.

2. Material and methods

2.1. Chemicals

Acetone pestanal and n-hexane pestanal were purchased fromFluka (USA). Dichloromethane (DCM) suprasolv and silica gel 60were obtained from Merck (Germany). Organochlorine pesticidemix 3, PCB mix, PCB103 and PCB198 were purchased from Dr.Ehrenstorfer (USA). PBDE mix was from Wellington (USA). All otherreagents were of analytical grade.

2.2. Sampling

Biotic and abiotic samples were collected from two stations(Is ıklı and Söke) in the BMR. Is ıklı is the origin and there is rela-tively fewer agricultural activity around the lake compared to Sökestation. Söke is the estuary of the river and one of the two mostintensive agricultural plateaus in Turkey. Surface water sampleswere collected and filtered from 55 micron pore width plankton-filter into 2.5 L borosilicate bottles. After closing tightly, they weredelivered to the laboratory and were kept at 4 ◦C until analysis.Surface sediment samples were collected into 1 L of borosilicatejar, closed tightly, and kept at 4 ◦C until analysis. After drying inthe oven at 75 ◦C, sediment samples were sieved through vibratingstainless steel sieves with mesh size of 50 �m. Sampling period forboth abiotic and biotic samples was between May and July 2009.Grey heron and pelican were chosen as they are the species underthe risk of extinction. Euroasian coot and gull species (yellow-legged, and Mediterranean gull) were chosen as they integratepollutants over a broad area of the river estuary, and represent anideal biomonitor. The required official permissions from the Min-istry of Environment for systematic sampling were provided priorthe study. Upon catching the waterbird; blood, liver and muscle


as invasive samples were obtained from yellow-legged gull andEuroasian coot, and only blood samples were obtained from greyheron and pelican due to the risk of extinction. Preen gland oil andnon-hatched eggs as non-invasive samples were obtained from all


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pecies above and additionally from Mediterranean gull, except eggrom yellow-legged gull. Sample types and sampling locations wereummarized below:

Sampletype

Yellow-leggedgull

Euroasiancoot

Grey heron Pelican Mediterraneangull

Is ıklı/Söke Is ıklı/Söke Is ıklı/Söke Is ıklı/Söke Is ıklı/Söke

Liver 3/3 2/2 −/− −/− −/−Muscle 3/3 2/2 −/− −/− −/−Blood 3/3 3/3 −/3 −/3 −/−Preen

gland oil3/3 3/4 −/3 −/3 −/−

Egg −/− 3/− −/3 1/2 3/5

Blood, liver and muscle tissues were rapidly dissected andmmediately frozen in liquid nitrogen until analyses. Preen glandil and egg samples were kept at 4 ◦C until analysis.

.3. POP analyses

The 17 targeted OCP compounds were grouped in five chemicalub-groups as DDTs (p,p′-DDD, p,p′-DDE, p,p′-DDT, methoxychlor),CHs (�-HCH, �-HCH, �-HCH and �-HCH), CHLs (heptachlor, hep-

achlor endoepoxide), DRINs (aldrin, dieldrin, endrin and endrinldehyde), and SULPHs (�-endosulfan, �-endosulfan and endosul-an sulphate). Indicator PCBs (PCB28, 52, 101, 118, 138, 153 and80) and PBDE congeners (PBDE17, 47, 66 and 100) were mea-ured in both abiotic and biotic samples. Sediment samples wererocessed for POP analyses according to the published method (Pazit al., 2011) after minor modifications. 10 ng of PCB103 and 5 ng ofCB198 were added as internal standards to the 10 g of dried andieved sediment samples. 40 mL hexane:DCM (50:50) was addedo the samples and the mixture was stirred for 16 h with a mag-etic stirrer. They were further extracted in an ultrasonic bath for

h. Samples were filtered and concentrated to 2 mL under gentleitrogen stream; 2 g of metallic copper is added for the removal ofulphur. Water samples were analysed according to the publishedethods (Kidd et al., 1998; Kucuksezgin et al., 2001; Crimmins et al.,

002). The details of the analyses were given in the accompany-ng paper (Karaca et al., 2014). Heparinized blood samples wereaken from the heart and/or the abdominal area of the body ofon-endangered, and from the wing vein of endangered waterbirdpecies and frozen until analyses. After adding the internal stan-ards, POP contents of the blood samples were extracted 2 timesith 5 mL of hexane:acetone (9:1, v:v), stirred vigorously by vortex

or 1 min. and combined organic phases were concentrated to 5 mL.reen gland oil samples were kept at 4 ◦C until analysis, and samerocedure was also applied to preen gland oil samples. Before anal-sis, surface of eggs was washed with distilled water and hexane.he content was homogenized in a blender and 7 g was used for POPnalysis similar to liver or muscle. Although acidification causesartial degradation of some OCP congeners such as dieldrin, endrinnd methoxylchlor (Manirakiza et al., 2002), all waterbird biologi-al samples were treated with acidic silica due to the high content ofat. They were applied to specially designed and manufactured glassolumns (1.5 × 23 cm; i.d. × length) which was packed with 6 g sil-ca gel (activated by heating to 200 ◦C for 4 h and deactivated by 20%

/w H2SO4) and 1 g anhydrous Na2SO4 and columns were condi-ioned with 5 mL n-hexane. After adding the concentrated eluents,CPs, PCBs and PBDEs were eluted with 50 mL hexane, 25 mL hex-ne: DCM (1:1; v/v) and 25 mL DCM. Eluents were combined andurther concentrated to 200 �L under a gentle nitrogen stream.


CPs, PCBs and PBDEs were quantified by gas chromatography-lectron capture detection (GC-ECD). Details of the instrument andnalysis were described in an accompanying paper in this issue ofhe journal (Karaca et al., 2014). Identification of the compounds

PRESStters xxx (2014) xxx–xxx 3

was carried out by comparing sample peak retention times withthose obtained for standards by GC-ECD. Quantification was per-formed using the ratio of the sample peak area to the standardpeak area. Standard curves for each analyte were constructed inthis way between 5 and 20 pg/�L, and the resultant equation wasused to calculate the sample concentrations. Quality control wasdetermined by calculating precision, accuracy, limit of detection(LOD) and limit of quantification (LOQ) and monitoring the repro-ducibility by Shewhart charts as described in the accompanyingpaper (Karaca et al., 2014). All values were satisfacting for enablinga high quality of analytical measurement. Two g of homogenateswere used for fat analysis according to the method of Bligh andDyer (1959). Preen gland oil samples were collected in tubes whichtheir tares were determined previously.

2.4. Measurements of enzyme activities

Antioxidant enzyme activities were measured according to themethods described in the accompanying paper (Karaca et al., 2014).Protein levels were determined by Lowry method which was mod-ified by Miller (1959).

2.5. Statistical analysis

Data were expressed as the mean value ± standard error (S.E.).The student t-test was used to analyze the significance of differ-ences between the groups. Associations between variables wereinvestigated using Pearson product moment coefficient of corre-lation (r). A value of p < 0.05 was considered significant both fordifferences and correlations.

3. Results

3.1. POP concentrations in water and sediment

All targeted pollutants (OCPs, PCBs, and PBDEs) were found tobe measurable in river water and in sediment, which were givenin Table 1. Concentrations were represented as the sum of the con-geners in five chemical groups for OCPs as described in Section 2.3.Two locations were used as sampling sites at Söke; estuary and5 km prior the estuary (plateau) in order to comprehensively ana-lyse the broad area of waterbirds. Total PCBs and PBDEs were belowthe LOD in water samples of plateau, and the latter was also belowLOD at Is ıklı, while all POPs were measurable in all sediment sam-ples (Table 1). Among three groups of POPs, OCPs were the highestat all stations both in water and in sediment samples. CHLs weremeasurable only in sediment of estuary. DDTs were the major pol-lutant in general (the highest in the water of Is ıklı Lake). Due tolow solubility of POPs in water, they quickly become associatedwith particulate matter and accumulate in sediments. Assumingthat water concentrations of ng/L are comparable to sediment con-centrations in ng/kg (ng/g in Table 1), concentrations of pollutantsare relatively higher in sediment compared to water, as expected.However, only total PCB levels at estuary were found to be statis-tically significantly higher compared to Söke plateau, due to highvariation in the measurements.

3.2. POP concentrations in biological samples

Concentrations of the POPs in liver, muscle, preen gland oil,blood and egg samples of waterbirds were given in Tables 2–4,respectively. Similar to river water and sediment samples, OCPs


were relatively higher compared to PCBs and PBDEs in liver sam-ples, which is believed to reflect recent exposure. When comparingthe pollutant groups to each other, although there were not sta-tistical significances between mean levels due to high variation,


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Table 1Concentrations (ng/g dry weight for sediment and ng/L for water) of persistent organic pollutants in sediment and water from sampling sites.

Location Samples IS IKLI SÖKE (Plateau) SÖKE (Estuary)

Water Sediment Water Sediment Water Sediment

Total PCBs 0.5 ± 0.1 0.3 ± 0.02 <LOD 0.3 ± 0.01 2.3 ± 0.04 0.8 ± 0.03a

Total PBDEs <LOD 0.2 ± 0.04 <LOD 1.3 ± 0.3 0.3 ± 0.04 1.2 ± 0.3Total HCHs 1.9 ± 0.3 0.01 ± 0 <LOD 0.2 ± 0.03 12.9 ± 3.2 0.1 ± 0.02Total DDTs 5.4 ± 1.3 1.9 ± 0.3 <LOD 2.5 ± 0.5 2.8 ± 0.0 1.4 ± 0.3Total CHLs <LOD <LOD <LOD <LOD <LOD 0.1 ± 0.03Total DRINs 0.7 ± 0.2 0.1 ± 0.01 6.2 ± 1.5 0.2 ± 0.02 2.0 ± 0.2 0.4 ± 0.02b

Total SULPs 2.7 ± 0.5 0.1 ± 0.01 6.8 ± 1.0 0.3 ± 0.03 0.9 ± 0.3 0.3 ± 0.04Total OCPs 10.7 ± 0.3 2.1 ± 0.1 12.9 ± 0.4 3.2 ± 0.1 18.6 ± 0.7 2.2 ± 0.1

LOD, limit of detection.a Significantly different (p < 0.05) compared to Söke (Plateau) station.b Significantly different (p < 0.05) compared to Is ıklı station.

Table 2Concentrations (mean ± SE, ng/g lipid weight) of persistent organic pollutants in liver and muscle tissues of waterbirds.

Location Bird species IS IKLI SÖKE

Yellow-legged gull Euroasian coot Yellow-legged gull Euroasian coot

Liver (n = 3) (n = 3) (n = 3) (n = 3)Lipid (%) 4.9 ± 0.8 1.5 ± 0.6 3.8 ± 0.4 1.6 ± 0.8Total PCBs 261 ± 126 184 ± 30 670 ± 276 367 ± 235Total PBDEs 60 ± 14 233 ± 99 141 ± 44 489 ± 315Total HCHs 19 ± 4 167 ± 56 38 ± 6 155 ± 75

Total DDTs 273 ± 56 729 ± 248 334 ± 76 386 ± 184.2Total CHLs 88 ± 31 370 ± 109 106 ± 9 712 ± 502Total DRINs 68 ± 28 313 ± 89 57 ± 30 69 ± 23Total SULPs 73 ± 32 85 ± 21 387 ± 181 97 ± 63Total OCPs 521 ± 102 1164 ± 381a 922 ± 286 1418 ± 829

Muscle (n = 3) (n = 3) (n = 3) (n = 3)Lipid (%) 5.9 ± 0.5 4.6 ± 0.1 6.1 ± 0.4 3.7 ± 0.2Total PCBs 463 ± 387 21 ± 3 269 ± 123 36 ± 11Total PBDEs 47 ± 25 29 ± 14 31 ± 12 19 ± 7Total HCHs 5 ± 1.3 3 ± 0.1 7 ± 1 8 ± 2Total DDTs 267 ± 218 40 ± 11 165 ± 85 40 ± 8Total CHLs 15 ± 4 26 ± 4 18 ± 4 24 ± 3Total DRINs 65 ± 42 21 ± 6 262 ± 144 23 ± 3Total SULPs 122 ± 105 5 ± 1 100 ± 49 7 ± 1Total OCPs 476 ± 370 94 ± 21 551 ± 204 102 ± 14

a Significantly different (p < 0.05) compared to the yellow-legged gull.

Table 3Concentrations (mean ± SE, ng/g lipid weight for preen gland oil, ng/mL for blood) of persistent organic pollutants in preen gland oil and blood.


Yellow-legged gull Euroasian coot Yellow-legged gull Euroasian coot Pelican Grey heron

Preen gland oil (n = 3) (n = 3) (n = 3) (n = 4) (n = 3) (n = 3)Total PCBs 3735 ± 1744 3997 ± 901 1904 ± 635 2106 ± 554 21,545 ± 8740b 6766.6 ± 1598.1a,b

Total PBDEs 127 ± 60 23 ± 89 52 ± 16 127 ± 92 3771 ± 3430 153 ± 32a

Total HCHs 86 ± 7 217 ± 82 70 ± 21 94 ± 34 1643 ± 886b 1070 ± 152a,b

Total DDTs 152 ± 94 91 ± 21 1270 ± 688 117 ± 40 5037 ± 1739 3215 ± 2069Total CHLs 5 ± 5 28 ± 19 3 ± 1 15 ± 11 455 ± 231 174 ± 33a,b

Total DRINs 37 ± 17 41.3 ± 19 30 ± 8 51 ± 36 886 ± 397 757 ± 91a,b

Total SULPs 20 ± 7 38 ± 19 23 ± 4 31 ± 19 4438 ± 3142 1288 ± 280a,b

Total OCPs 300 ± 66 416 ± 153 1397 ± 669 308 ± 137 12,459 ± 4990b 6505 ± 2511b

Venous blood (n = 3) (n = 3) (n = 3) (n = 3) (n = 4) (n = 3)Total PCBs 8 ± 4 3.0 ± 0.6 7.6 ± 2 18.6 ± 7 22 ± 15 10 ± 4Total PBDEs 1.6 ± 0.4 0.9 ± 0.2 1.0 ± 0.2 7.2 ± 6 5.4 ± 4 5 ± 3Total HCHs 1.4 ± 0.3 0.8 ± 0.2 0.9 ± 0.3 2.5 ± 1 61 ± 58 1.4 ± 0.5Total DDTs 3.1 ± 0.5 3.3 ± 0.3 3.0 ± 1.0 2 ± 1 9 ± 4 11 ± 5Total CHLs 0.3 ± 0.1 0.1 ± 0.1 0.3 ± 0.1 2 ± 2 nd ndTotal DRINs 1.4 ± 0.2 0.7 ± 0.3 1.7 ± 0.5 3.5 ± 3 2.2 ± 1.2 4.6 ± 1.7Total SULPs 1.6 ± 1 0.3 ± 0 1.2 ± 0.1 1 ± 0.5 4 ± 3 1.8 ± 0.3Total OCPs 7 ± 1.5 5.2 ± 0.3 7.2 ± 0.9 11 ± 5 91 ± 72 22 ± 8

n


d: not detected.a Significantly different (p < 0.05) compared to the yellow-legged gull from Söke stationb Significantly different (p < 0.05) compared to the EuroAsian coot from Söke station.


.



R. Kocagöz et al. / Toxicology Letters xxx (2014) xxx–xxx 5

Table 4Concentrations (mean ± SE, ng/g lipid weight) of persistent organic pollutants in eggs.


Euroasian coot (n = 3) Pelican (n = 1) Mediterranean gull (n = 3) Grey heron (n = 3) Pelican (n = 2) Mediterranean gull (n = 5)

Lipid (%) 18 ± 2 6 10 ± 0.5 8 ± 1 8 ± 1 16 ± 1Total PCBs 25 ± 21 1556 670 ± 145a 1096 ± 379 718 ± 82 524 ± 147Total PBDEs 7 ± 3 129 33 ± 7a 46 ± 5b 70 ± 13 14.0 ± 7Total HCHs 13 ± 6 89 7 ± 0.3 63 ± 8b 31 ± 8 11 ± 2Total DDTs 68 ± 12 13,878 1076 ± 347a 17,625 ± 4342b 6785 ± 94 2798 ± 1657Total CHLs 4 ± 2 149 8 ± 0.3 64 ± 30 50 ± 1 17 ± 3c

Total DRINs 22 ± 9 345 35 ± 1 319 ± 93 246 ± 26 156 ± 44Total SULPs 9 ± 3 1491 37 ± 17 204 ± 69 509 ± 10 101 ± 19c

Total OCPs 115 ± 28 15,952 1164 ± 349a 18,276 ± 4512b 7622 ± 85 3084 ± 1688

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a Significantly different (p < 0.05) compared to Euroasian coot from Is ıklı station.b Significantly different (p < 0.05) compared to Mediterranean gull from Söke statc Significantly different (p < 0.05) compared to the Is ıklı station at the same bird s

otal PBDE concentrations were higher in Euroasian coot in bothtations compared to total PCB concentrations (Table 2). The orderf the magnitude of the pollutants was generally similar to livern muscle samples; OCPs > PCBs > PBDEs. Total PBDE levels in mus-le of Euroasian coot at Is ıklı was slightly higher than OCPs andCBs, while this was not the case at Söke. At Is ıklı, PCB levels iniver of yellow-legged gull and in blood of Euroasian coot wereignificantly lower compared to the same bird species and tissuesrom Söke (p; 0.046 and 0.010, respectively). Unlike to the sedimentamples, aldrin was not detected in any liver and muscle samples,nly its degradation product, dieldrin was detected. Although sed-ment results suggested that there may be recent entry of aldrin tohe ecosystem, this may not be a continuing input and also aldrinegradation to dieldrin may be faster in living tissues compared toediment. Unlike to liver and muscle tissues, as well as to abioticamples, levels of PCBs were the highest in preen gland oil samplesf all waterbirds, followed by OCPs and PBDEs (Table 3). Betweenhe species, pelican represented the highest levels for all pollutants,ollowed by grey heron. PCB and OCP levels were comparable inlood samples of the species, although pelican showed the high-st blood values as in preen gland oil. PBDE levels were the lowestn preen gland and blood samples among different species. Whenomparing levels in preen gland oil (ng/g) to the blood (ng/mL),he former contained much higher levels of all pollutants (Table 3),

ost likely because all pollutants are highly lipid-soluble. The eggnd tissue (liver and muscle) samples could not be obtained fromhe same species (Tables 2 and 4) due to risk of extinction for peli-an and grey heron, and due to absence of relevant egg samples forellow-legged gull and Euroasian coot. Nevertheless, correlationnalyses between POP concentrations in eggs and in tissues weretill performed, whether egg concentrations reflect tissue concen-rations within the same species. In addition, egg concentrationsere compared to the corresponding concentrations of the same

nd different species in other studies. All egg samples from var-ous species in the current study had well measurable levels ofOPs (Table 4). Pelican eggs contained the highest values in gen-ral, with the exceptions of total PCBs and total OCPs in the eggs ofrey heron at Söke. Although there was only one pelican egg froms ıklı, all pollutant levels were roughly doubled compared to theelican eggs from Söke (Table 4), while it was vice versa in the eggsf Mediterranean gull for OCP levels (Table 4). Concentrations ofhe POPs in Mediterranean gull were significantly higher than theoncentrations of Euroasian coot at Is ıklı (p < 0.05).

.3. Correlations between POP concentrations in biological


amples

Among three main groups of pollutants, PCBs were the most cor-elated ones positively and significantly (six correlations) between

s.

different destructive and non-destructive biological samples ofwaterbirds (Table 5). More correlations were observed with PBDElevels, however, four out of eight were negative and weaker com-pared to PCBs. Four positive, but again weaker correlations wereobserved with OCPs (Table 5). The most and strongest correlationswere observed between liver and muscle tissues of yellow-leggedgull at Is ıklı for OCPs, PCBs and PBDEs. At Söke, POP concentrationsin these tissues of gull were also correlated significantly exceptOCPs (Table 5). In the same tissues of Euroasian coot from Is ıklı andSöke, only OCPs were significantly correlated. Although there wereno correlations between the total OCP concentrations of differenttissues, significant correlations for OCP sub-groups were observed.SULPs were positively correlated between blood and liver, muscle,and preen gland oil, also between preen gland oil and liver andmuscle in yellow-legged gull from Is ıklı (Table 5). However, onlythe significant correlation for SULPs was observed between bloodand preen gland oil in the same species from Söke. Instead, DRINswere correlated between blood and muscle and preen gland oil, alsobetween preen gland oil and muscle in this station. CHLs betweenpreen gland oil and muscle were correlated in yellow-legged gullfrom both sampling sites, and only between blood and muscle in thesame species from Is ıklı. HCHs were also correlated between bloodand muscle in yellow-legged gull from Is ıklı. In Euroasian coot, totalHCHs were correlated between blood and muscle, CHLs betweenpreen gland oil and egg, and between liver and muscle, and SULPsbetween preen gland oil and muscle at Is ıklı. In the same speciesfrom Söke, DDTs were correlated between blood and liver, CHLswere correlated between preen gland oil and liver and muscle, andbetween muscle and liver (Table 5). In the two species under therisk of extinction, grey heron and pelican, DDTs were significantlycorrelated between blood and egg. SULPs were correlated betweenblood and preen gland oil both in pelican and grey heron from Söke.

3.4. Antioxidant enzyme activities and correlations to POPconcentrations

Activities of liver GST were found to be significantly higher inboth yellow-legged gull and Euroasian coot from Söke comparedto Is ıklı (Fig. 1). Activity of GPx was also significantly higher inthe latter species. Activities of total SOD were not different inboth species between sampling sites. However, when activities ofthe SOD isozymes were measured individually, the mitochondrialform Mn-SOD, was found to be significantly higher in Euroasiancoot from Söke, while it was not different in yellow-legged gull.The potential of antioxidant enzyme activities to reflect pollutant


concentrations were investigated in liver of Euroasian coot andyellow-legged gull by analysing correlations. Activities of Mn-SOD,CAT, GPx and GST were significantly correlated with HCHs (r: 0.87,0.64, 0.73 and 0.53, respectively) and with SULPs (r: 0.70, 0.62, 0.56



6 R. Kocagöz et al. / Toxicology Letters xxx (2014) xxx–xxx

Table 5Cross-correlations between OCPs, PCBs and PBDE concentrations of various biological samples.

HCHs DDTs CHLs DRINs SULPs �OCPs �PCBs �PBDEs

Yellow-legged gull-Is ıklıBlood–liver 0.91** 0.82**

Blood–muscle 0.81** 0.94** 0.94** 0.85**

Blood–preen gland oil 0.94** 0.91**

Preen gland oil–liver 0.96**

Preen gland oil–muscle 0.90**

Liver–muscle 0.92** 0.97** 0.81** 0.99** 0.75**

Yellow-legged gull-SökeBlood–liver −0.59*

Blood–muscle 0.89**

Blood–preen gland oil 0.77** 0.70*

Preen gland oil–muscle 0.91* 0.94*

Liver–muscle 0.59* 0.75** 0.78**

Euroasian coot-IsıklıBlood–liver −0.61*

Blood–muscle 0.84** 0.73**

Blood–preen gland oil 0.76*

Blood–egg 0.72**

Preen gland oil–liver −0.64*

Preen gland oil–muscle 0.79*

Preen gland oil–egg 0.96**

Liver–muscle 0.89* 0.58**

Euroasian coot-SökeBlood–liver 0.94**

Preen gland oil–liver 0.82** 0.75**

Preen gland oil–muscle 0.93**

Liver–muscle 0.77** 0.56**

Pelican-SökeBlood–preen gland oil 0.60*

Blood–egg 0.60*

Grey heron-SökeBlood–Egg 0.67* 0.68**

Blood–preen gland oil 0.78*

Preen gland oil–egg −0.72*

r

actDlalct

4

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, Correlation coefficient.* p < 0.05.

** p < 0.01.

nd 0.83, respectively) in Euroasian coot. Activity of Cu,Zn-SOD wasorrelated with PCBs (r: 0.70) in the same species.Activity of theotal SOD (Cu,Zn-SOD + Mn-SOD) was correlated with liver CHLs,RINs and OCPs (r: 0.65, 0.54 and 0.58, respectively) in yellow-

egged gull. CHLs were also correlated with Cu,Zn-SOD enzymectivity (r: 0.74) in the same species. HCHs in gull liver were corre-ated with GPx activity (r: 0.63), HCHs, SULPs, PCBs and PBDEs wereorrelated with GST activity (r: 0.77, 0.63, 051 and 0.62, respec-ively).

. Discussion

The main objective of the present study was to evaluate thepplicability of non-invasive preen gland oil and venous bloodrelatively invasive) samples for estimating concentrations of theollutants in critical tissues of various waterbirds. We also aimedt analysing the pollution in biotic and abiotic media of BMR in aomparative manner to the literature. In order to provide data forhis purpose, POP concentrations in muscle and liver tissues were

easured and correlated to the concentrations in non-destructiveamples of the same waterbirds. In addition, the levels of POPs inlood and egg samples of grey heron and pelican, as well as in tis-ues of Euroasian coot and yellow-legged gull, were determinedor the first time at the estuary of BMR, which runs into Eastern

editerranean (Aegean) Sea, and at its origin, Is ıklı Lake.


.1. Discussion of POP concentrations in water and sediment

In a study of PCB levels in fresh water samples from Lakeerkini (northern of Greece) were in the same order of magnitude

with the present study, although seemed somewhat higher(6.6 ng/L vs. 0.5 and 2.3 ng/L) (Antoniadou et al., 2007). In 2008and 2009, Harman et al. (2013) analyzed POPs in the water andsediment samples of Bosna River; concentrations in water rangedbetween 0.12–0.24 ng/L (

∑PCBs), 0.002–0.04 ng/L (

∑PBDEs) and

0.03–0.2 ng/L (∑

OCPs). Our findings in water were <LOD-2.3(∑

PCBs), <LOD-0.3 (∑

PBDEs) and 11–19 ng/L (∑

OCPs). Thereported concentrations in Bosna River are lower compared tothe present concentrations, especially OCPs (Harman et al., 2013).However, Harman et al. analysed only 8 compounds in OCP group,while 17 compounds were analysed and reported in the presentstudy. Including the other compounds in Harman et al.’s studywould have increased the concentrations significantly. In sedimentsamples of the same river, PCB levels were ranged from 0.8 to16 ng/g dry weight and PBDE levels were ranged between 0.4 and40 ng/g dry weight (Harman et al., 2013). In the present study, PCBand PBDE levels in sediment samples ranged between <LOD-2.3and <LOD-1.3 ng/g dry weight, respectively. Unlike to water, sedi-ment concentrations in Bosna River are 2.5–20 fold higher for PCBsand 2–30 fold higher for PBDEs compared to the present study.OCP derivatives in sediment samples were within the same range,however, the number of analysed OCP compounds were again notequal to the present study, as discussed above for water samples. Asstated in our accompanying paper in this issue (Karaca et al., 2014),the ratio of parent OCPs to their metabolites indicates the time ofexposure or entry to the environment, and the ratio of DDE/DDT <1


indicates a recent entry of DDT. Upon analysing the ratio in water-birds, DDE/DDT was below only in one yellow-legged gull from theestuary. The other two values of the gulls from the same stationwere 1.2 and 13, these ratios were lower compared to the gulls from



R. Kocagöz et al. / Toxicology Letters xxx (2014) xxx–xxx 7

F e. a, ss

Itptatt1bw�twal0osb

4

tf

ig. 1. Liver antioxidant enzyme activities of waterbird species from Is ıklı and Söktation.

s ıklı. Although close to 1 in two of the gulls, the ratios suggestedhat there is no recent entry of DDT to the estuary, which is also sup-orted by the similar analyses of the sediment samples. Dieldrin ishe degradable product of aldrin (Vander Pol et al., 2004), a value ofldrin/dieldrin >1 also indicates new input of aldrin to the ecosys-em. In the analysis of sediment samples, it has been observed thathe ratios of aldrin/dieldrin at Is ıklı and Söke-plateau stations were.3 and 1.8, respectively. This shows that use of aldrin may haveeen continuing during the period of sampling, although its useas already banned. The ratio of the technical mixture of �- and-endosulfan is 7:3, and these two isomers have different degrada-

ion times in soil (Barlas et al., 2006). The ratios of �-/�-endosulfanere 0.8, 0.9 and 0.1 in sediment samples from Is ıklı, Söke-plateau

nd Söke-estuary, respectively. The concentrations of the metabo-ite endosulfan sulphate in the corresponding stations were 0.02,.03 and 0.09 ng/g dry weight, respectively. Relatively low amountf the metabolite at all stations suggested recent input of endo-ulfan to the ecosystem. This is because the use of endosulfan wasanned in 2009, during the collection period of the samples.

.2. Discussion of POP concentrations in biological samples


In a study from Greece, researchers analyzed p-p′-DDE levels inhe eggs of pelican (Pelecanus crispus). Their results were rangedrom 14,183 to 21,653 ng/g lipid weight (Crivelli et al., 1989). In the

ignificantly different (p < 0.05) compared to the corresponding species from Is ıklı

present study,∑

DDT levels varied between 6785 and 13,878 ng/glipid weight, relatively lower. The difference would be increased ifDDT and DDD would be added in the former study (Crivelli et al.,1989). This difference in concentrations can be explained by thepossible cease of DDT exposure due to prohibiting the use of OCPsin both countries starting from 1980s. There is 20 years betweenthe former and the present study, which may have caused lowerconcentrations in the present study. Antoniadou et al. (2007) ana-lyzed PCBs in the eggs of grey heron from Lake Kerkini in Greece.They reported the concentration as 937 ng

∑PCB/g lipid weight.

This is very comparable to the present concentration of 1096 ng/glipid weight. In two consecutive studies again from North East-ern Mediterranean–Greece, authors analysed PCB and OCP levelsin the eggs of yellow-legged, and audouin’s gulls (Goutner et al.,2001; Albanis et al., 2003). The pollutant concentrations in eggswere reported as ng/g dry weight, while as ng/g lipid in the cur-rent study. Taken the mean value of water content of bird egg as75% (w/w) (Jones, 1979; University of Illinois Extension, 2013), andthe mean value of lipid as 8% (w/w) (current study), total PCB lev-els vary between 309 and 1893 ng/g lipid in the study of Goutneret al. (2001), and between 358 and 788 ng/g lipid in the study of


Albanis et al. (2003), while the present values vary between 25 and1096 ng/g lipid. In those studies, PCB153 was not included in theanalyses, while it was included in the current study. The other dif-ferences between those and the present studies are, which were


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ll performed in the Aegean Sea, the species (yellow-legged andudouin’s gulls in those studies versus Euroasian coot, pelican, andediterranean gull in the current study), and most importantly,

ime of the collection period of the eggs (1997–1998 in those stud-es versus 2009 in the current study). Since preventive measuresuch as prohibiting OCPs have been taking in both countries beforehose years, a gradual decrease in environmental concentrations

ay be expected. Such decreases were also observed in the OCPevels (CHLs levels at Is ıklı and HCHs levels at both Is ıklı and Söke)f the current study.

.3. Correlations between POP concentrations in biologicalamples

The observed most and significant correlations between POPoncentrations in liver and muscle tissues were not expecting sinceiver concentrations were considered to reflect recent, and mus-le concentrations to reflect chronic exposure. These correlationsuggested a constant input of the pollutants into this ecosystem.he major aim of the current study was to evaluate whether bloodnd preen gland oil levels can represent liver and/or muscle lev-ls of POPs. Consistent correlations were observed between bloodnd muscle or liver tissues for PCBs in both yellow-legged gullnd Euroasian coot, but not for total OCPs and PBDEs (Table 5).ontrary to our initial assumption, significant correlations wereot found between the concentrations in preen gland oil and in

iver or muscle. Yamashita et al. (2007) reported weak but signif-cant correlations between PCB concentrations in preen gland oilnd abdominal adipose tissue in 30 seabirds belong to 13 speciesrom North Pacific Ocean. Correcting for the metabolic loss of PCBsn the basis of congener profiles, since PCB metabolism is mucheaker in the gland compared to well perfused inner organs, sig-ificantly improved the correlation (Yamashita et al., 2007). Theeason of the absence of such correlation in the current study mighte the difference in bird species between two studies, and more

ikely seeking correlation for pollutants between preen gland oilnd liver/muscle tissues instead of abdominal adipose tissue. Theignificant correlations between blood and liver or muscle tissueor PCBs were supported also by a significant correlation betweenlood and egg samples from Euroasian coot (Table 5). Similar cor-elations between egg and blood were observed also in grey heron.owever, the egg samples were collected randomly between non-atched ones, and it is not known whether they belong to the

ndividuals that blood samples were drawn. An interesting obser-ation was that PBDE concentrations between liver and blood ofellow-legged gull, between liver and blood, also liver and preenland oil of Euroasian coot, and between preen gland oil and eggf grey heron were negatively correlated (Table 5). This suggestedhat blood and preen gland oil could not be a surrogate for tissueBDE concentrations. In order to further investigate, more data areequired on distribution kinetics of the studied POPs from tissuesnd blood towards preen gland. To the best of our knowledge, suchata are not available in literature yet. As the eggs could not bebtained from the same birds whose tissues were excised from, aimilar conclusion for eggs can not be inferred, as it was concludedn the study of Albanis et al. (2003) for organochlorines.

.4. Antioxidant enzyme activities and correlations to POPoncentrations

Activities of liver tissue antioxidant enzymes were generallyigher at Söke station compared to Is ıklı (Fig. 1), activity of GST in


ellow-legged gull and activities of both GST and GPx in Euroasianoot were significantly higher at Söke station. Although the analyt-cal variations prevented statistical significance, CAT activity tendso be higher in both species at Söke. Activities of the two isozymes of


SOD were also measured in addition to the total activity, and a sig-nificantly higher Mn-SOD activity of Euroasian coot was observedagain at Söke compared to Is ıklı. Mn-SOD is the mitochondrial formof the SOD, while Cu,Zn-SOD is the cytosolic form. A similar pat-tern of the Mn-SOD activity was also observed in common carp(Cyprinus carpio) caught from a different sampling station on BMR(Sarayköy), which is in between the origin and the estuary of theriver (accompanying paper; Karaca et al., 2014). In that relativelymore industrialized area, Mn-SOD activity, but not Cu,Zn-SOD, wasfound to be significantly increased. Mitochondria are responsiblefor breakdown of inhaled oxygen to water via electron transportchain -ETC- (cellular respiration). During this biochemical pro-cess, tolerable amount of hazardous superoxide anion radical (O•−)escapes from the chain, which is subsequently detoxified mainlyby Mn-SOD. An increase in the activity of Mn-SOD suggests anincrease in the amount of O•− formed. It is known that OCPs, PCBsand PBDEs cause intracellular oxidative stress. Therefore, as dis-cussed in the accompanying paper, OCPs and/or other POPs maybe responsible for the increased Mn-SOD activity via disturbingthe ETC and causing excess production of O•−. Significant correla-tions between activities of all antioxidant enzymes (SOD isozymes,GST, GPx and CAT) and concentrations of various POPs in liver ofwaterbirds provide additional support for oxidative toxicity of thepollutants.

4.5. Discussion of toxicological thresholds

In the study of Henny et al. (1984), the eggshell thickness of 220eggs from night heron was negatively correlated with residues ofDDE. Based on a pooled data, they concluded that the percentageof nests successful, clutch size, and to some extent the number ofyoung per successful nest decline as DDE residues increase above8 ppm. Comparing this threshold to the present DDE concentrationis required an assumption; pollutant concentrations were reportedper g lipid in the present study instead of wet weight in that study.With an assumption that the eggs of night heron would contain 8%of lipid as in the current study (Table 4), 8 ppm would be 100 ppm(100.000 ng/g lipid). The mean levels in the current study for theeggs from Mediterranean gull, Euroasian coot and pelican fromIs ıklı were 0.98, 0.06 and 12.3 ppm, respectively. The concentra-tions in the eggs of Mediterranean gull, pelican and grey heronfrom estuary were 2.6, 6.6, and 17.6 ppm, respectively. None of theDDE concentrations were above the threshold. In another study,egg p-p′-DDE levels were associated with reproductive success inthe brown pelican (Pelicanus occidentalis) and threshold level was3000 ng/g w.w. (3 ppm) (Blus, 1996, 2003). Converting this thresh-old to a value of g lipid, again none of the current values are abovethe threshold.

5. Conclusion

In aggregate, prohibiting the use of OCPs since 1980s causeda decrease in environmental and biological concentrations, simi-lar decreases were also observed for PCBs. Not preen gland oil, butvenous blood taken from the wing is a promising surrogate for esti-mating total PCB concentrations in liver and muscle of waterbirds.Venous blood is also capable to accurately reflect the concentra-tions of SULPs, CHLs and HCHs in liver or muscle. Surprisingly,preen gland oil, the relatively non-invasive sample compared toblood, was correlated only with SULPs in blood. The activities ofantioxidant enzymes (Mn-SOD, CAT, GPx and GST) were correlated


with SULPs and HCHs, which suggested a potential use of theseenzymes as biomonitor for these OCP groups. The significantlyincreased activity of mitochondrial Mn-SOD in liver of waterbirds,and the strongest correlations of this isozyme to various pollutants


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n the present study confirmed the similar observation in commonarp sampled from the same river (accompanying paper). Thesebservations strongly suggest that one of the cellular targets of thenalysed POPs may be mitochondria. In terms of hatch and repro-uctive success, none of the measured egg concentrations of DDEere above the established thresholds. This is in accordance with

he observation of Albanis et al. (2003) that environmental andiological POP concentrations, especially PCBs, in Eastern Mediter-anean are well below the corresponding levels measured in otherarts of Mediterranean.

onflict of interest

None of the authors has a conflict of interest related to this study.

ransparency document

The Transparency document associated with this article can beound in the online version.

cknowledgements

This study was supported by a grant from TÜBITAK, Scientificnd Technical Research Council of Turkey (108Y049). The authorshank Nuriye Serra Istanbullu and Ferhat S en for technical assis-ance. Dr. Fatih Perc in was acknowledged for his contribution tohe field specimen collection.

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