Effect of land use on pollution status and risk of fish endocrine disruption in small farmland ponds

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1 Hydroiologi (214) 723:13 12 DOI 1.17/s THE ROLE OF PONDS Effect of lnd use on pollution sttus nd risk of fish endocrine disruption in smll frmlnd ponds Syghlirw N. M. Mndiki Virginie Gillrdin Koen Mrtens Dirk Ercken Els De Roeck Tom De Bie Steven A. S. Declerck Luc De Meester Ctherine Brsseur Edwige Vn der Heiden Mrie-Louise Schippo Ptrick Kestemont Received: 15 Novemer 212 / Accepted: 26 July 213 / Pulished online: 23 August 213 Ó Springer Science+Business Medi Dordrecht 213 Astrct To study whether the intensity of griculturl ctivities ffects pesticides lods in pond environment, lrge numer of Belgin frmlnd ponds were surveyed in spring 24. Temporl distriution of pollutnts ws lso investigted over restricted survey ponds smpled three times round yer 27. Sedentry pond Prussin crp juveniles were lso cptured to determine their rin romtse ctivity (AA) nd plsm vitellogenin (VTG) levels. Hevy metl distriution ws lso exmined in vrious pond mtrices. Amongst the pesticides nlysed, only hericides were detected. Contmintion of pond wter y trzine ws frequently oserved during spring 24, while isoproturon nd glyphoste were detected round yer 27. Levels of hericides were inversely relted to the distnce of ponds to crop field, nd vlues peked in April or Octoer. Asence of Guest editors: R. Céréghino, D. Boix, H.-M. Cuchie, K. Mrtens & B. Oertli / Understnding the role of ponds in chnging world S. N. M. Mndiki (&) V. Gillrdin P. Kestemont University of Nmur, URBE-NARILIS, 61 Rue de Bruxelles, 5 Nmur, Belgium e-mil: Roert.Mndiki@fundp.c.e K. Mrtens D. Ercken E. De Roeck Royl Belgin Institute of Nturl Sciences, Vutierstrt 29, 1 Brussels, Belgium T. De Bie S. A. S. Declerck L. De Meester 32, 3 Leuven, Belgium endocrine disruptors in pond wter ws confirmed y lck of modultion in VTG nd AA in mle fish. Hevy metls were present in ll the pond mtrices, ut overll contmintion levels were low. The results demonstrted tht Belgin ponds were minly contminted y hericides nd tht pond sedentry fish were not ffected y endocrine disruptors. They lso demonstrted mrked effect of lnd-use intensity on hericide pollution which cn e mitigted y n djustment of the uffer zones. Keywords Pollution Ponds Prussin crp Endocrine disruption Lnd-use intensity Hericides Hevy metls Introduction Pools nd smll ponds hve in ddition to their esthetic nd griculturl vlues high conservtion vlue (Scheffer et l. 26). Their iotopes re C. Brsseur Lortory of Mss Spectrometry, University of Liège, CART, Allée de l Chimie, 6, Bât B6, Srt Tilmn, 4 Liege, Belgium E. Vn der Heiden M.-L. Schippo Lortory of Food Anlysis, University of Liège, CART, Bld de Colonster, 2, Bât B43, Srt Tilmn, 4 Liege, Belgium

2 14 Hydroiologi (214) 723:13 12 chrcterized y heterogeneous communities of qutic orgnisms, nd they often contin rre or unique species (Willims et l., 23; Wood et l., 23; Nicolet et l., 24). Severl studies hve reported the contriution of smll ponds to regionl diversity to e higher thn tht of other freshwter ecosystems such s lkes nd rivers (Dvies et l., 28; Willims et l., 28). In predominntly griculturl res, smll wter odies offer specil refuge to mny qutic iots nd thus contriute to mitigte the decline in overll iodiversity. Becuse of the limited wter exchnge compred to lrge wter odies, vrious pollutnts cn ccumulte in pond environments, which my impct the qutic orgnisms inhiting the ponds. The effects of lnduse intensity on pesticide ccumultion in the pond environment, nd its effects on pond iot, re, however, poorly documented. Despite the n of trzine in the EU, this hericide is still used in other countries nd, ecuse of its rther resistnt nture, it is still often detected in sediments nd surfce wters (Wu et l., 29; Sun et l., 21). Hericides, such s diuron, isoproturon nd glyphoste hericides, re nowdys widely used worldwide, nd their ccumultion hs een reported in pond wter (Greulich et l., 22; Howe et l., 24; Bonnet et l., 27; Rohr et l., 28). Besides their direct inhiitory ctions on trophic resources, such s qutic plnts nd insects, hericide molecules hve vrious deleterious effects on qutic orgnisms. While dt on the potentil hormonl effects of trzine re still controversil in fish (Spnò et l., 24; Ndzilek et l., 28; Tillitt et l., 21), numerous studies demonstrted tht this pesticide, t environmentlly relevnt concentrtions, hs mrked endocrine disrupting effects tht ffect the centrl nervous nd immune system of frogs nd slmnders (Sprling et l., 21; Forson & Storfer, 26; Klos et l., 29; Lsserre et l., 29; Hyes et l., 21). High concentrtions of glyphoste hericides nd the ssocited surfctnts hve lso vrious detrimentl effects on mphiin tdpoles, such s the disturnce in the metmorphosis process, til dmges nd gond normlities (Howe et l., 24; Brni et l., 27; Brusch & Smith, 27; Dinehrt et l., 29). It hs lso een shown tht comintions of chemicls, including trzine, diuron, isoproturon nd their rekdown products, t environmentlly relevnt concentrtions re more toxic to fish nd mphiins thn the prent compounds in isoltion (Spnò et l., 24; Ftim et l., 27; Bonnet et l., 27). Some pesticides tht re lredy known to e resistnt to degrdtion might e ccumulted in smll pond environments, s there is no dilution or flushing effect. Insecticides or industril compounds such s ldrin or nonylphenol cn induce sex reversl in fish nd mphiins y ltering vrious neuroendocrine pthwys including those controlling reproductive functions. They my induce n increse in the expression nd ctivity of P45 romtse, leding to n increse in oestrogen production tht susequently induces VTG ccumultion in the liver nd loodstrem (Folmr et l., 21; Joling & Tyler, 26; Hyes et l., 26; Wingfield & Muki, 29; Ndzilek et l., 211). Production of VTG induced y n increse in P45 AA in mles hs een used s relile iomrker to ssess the occurrence of endocrine disruption (Folmr et l., 21; Desforges et l., 21; Ndzilek et l., 211). Presence of PAHs my lso e suspected in the different pond mtrices since they re uiquitous in the environment due to mostly industril ctivities ut lso due to other nthropogenic sources. PAHs re known to e strongly persistent with good dispersl properties (Guo et l., 211). Aprt from their known crcinogenic, mutgenic nd endocrine disrupting properties (Brsseur et l., 27; Guo et l., 211; Rhind et l., 211), PAHs hve een shown to lter oth the specific nd non-specific immune system s well s the helth sttus of different fish nd other nimls living in qutic environment (Lmpi et l., 26; Reynud & Deschux, 26; Gillrdin et l., 29; Milinkovitch et l., 211; Dnion et l., 211). Amongst the persistent pollutnts, ccumultion of hevy metls is frequently ssessed for monitoring the helth sttus of wter odies (Ms et l., 21; Rimondi et l., 212). Aprt from geochemicl processes, increse in hevy metls in pond environments my e cused y vrious sources, such s industril nd trffic emissions s well s run-off from hittions, especilly in urnized res. Although it hs een demonstrted tht metl pollution lods re mrkedly higher in urnized industril lndscpes thn in griculturl res (Imperto et l., 23), griculturl opertions such s sewge sludge or fertilizer use my increse metl contmintion in soils, especilly of cdmium (Cd), copper (Cu) nd zinc (Zn) (Nicholson et l., 23; Ms et l., 21). Intensifiction of griculturl ctivities nd other

3 Hydroiologi (214) 723: humn ctivities my increse the run-off relese of hevy metls in pond environments, ut the effect of lnd-use intensity on hevy metl pollution is not yet quntified. Accumultion of hevy metls in pond mtrices my induce detrimentl effects on qutic orgnisms, for instnce y ltering their physiologicl nd defence ility (Desouky, 26; Reynders et l., 28; Ng et l., 211; Rimondi et l., 212). The ojective of our study ws to evlute pesticide nd hevy metl pollution sttus s well s risk of fish endocrine disruption in Belgin smll frmlnd ponds in reltion to lnd-use level. To chieve this ojective, (1) we investigted the effect of lnd-use level on hericide lods in pond wter s well s on hevy metl concentrtions in vrious pond mtrices during two lrge surveys covering the entire Belgin territory; (2) we verified whether there is sesonl vrition in pesticide pollution in pond wter, focussing on vrious prolemtic pollutnts; nd (3) we ssessed numer of indictors of reproductive endocrine disruption in Prussin crp (Crssius urtus gielio) juveniles resident in ponds locted in intensive or extensive griculturl res. Mterils nd methods Sesonl chnges in pesticide lods in pond wter Survey designs nd nlysed pesticides Two surveys were conducted in order to investigte whether chnges in pesticide pollution in pond wter vry with lnd-use intensity. In the first survey conducted during My June 24, 126 ponds cross lmost the entire territory of Belgium (Fig. 1) were smpled for wter. For the selection of the investigted ponds, the dominnt lnd-use type in the immedite neighourhood of the ponds ws the min criterion (see lso Declerck et l., 26). We tried s much s possile to uncouple lnd use nd geogrphicl position. To do so, we first selected forty-two geogrphiclly spred clusters such tht ech cluster contined severl ponds tht were locted within circulr re of pproximtely 2 km 2 (Declerck et l., 26). Within ech cluster, we then selected three ponds tht correspond to grdient in griculturl lnd-use intensity, rnging from intensive griculturl ctivities (mostly croplnd; intensive ponds ), over intermedite griculturl lnd use (dominted y pstures; extensive ponds ) to more pristine ( nturl, mostly locted in protected nture res). Crop presence ws noticed round the mjority of ponds: croplnd ws within rdius of 1 or 2 m for intensive or extensive ponds, while for nturl ponds, no crop ctivities ws present or croplnd ws oserved within rdius [1 m. Other detils concerning the investigted sites hve een previously descried y Declerck et l. (26). Pesticide nlyses focussed mostly on hericides (trzine, simzine, propzine, teruthylzine, trizine metolites, diuron nd isoproturon) ut lso on insecticides, nmely endosulfn nd lindne. A second survey ws crried out in 27. This survey ws more limited in numer of ponds ut involved three smples to cover temporl dynmics, involved roder rnge of pesticides, nd lso included industril pollutnts. Fifteen smll ponds were selected covering the three lnd-use ctegories (Fig. 1); these ponds were suset of the ponds smpled in 24. Wter ws smpled three times (April, July nd Octoer 27) with n intervl time long enough to cover the dissiption time of vrious pesticide molecules in wter conditions (Mouvet et l., 1997; Sørensen et l., 23; Tissier et l., 25). The pollutnts tht were quntified were s follows: (1) hericides: trzine, simzine, propzine, teruthylzine, trizine metolites, diuron isoproturon, glyphoste nd minomethyl-phosphonic cid (AMPA); (2) insecticides: ldrine, chlorfenvinphos, -endosulfn nd dicofol; (3) industril compounds: octylphenol, nonylphenol, ldrine nd four polycyclic romtic hydrocrons (PAHs: enzo()fluornthene, enzo(k)fluornthene, enzo()pyrene) nd enzo(ghi)perylene). A summry of the numer of investigted ponds nd types of nlysed pesticides is presented in Tle 1. Wter smpling nd pesticide nlyses For nlyses of the trgeted pesticides, pool of 2 l wter ws tken t eight rndomly selected smpling sttions in ech pond (4 in the middle of the pond nd 4 close to the edge). Just fter smpling, wter ws filtered over 2-lm mesh screen in order to remove lrge orgnic prticles, nd then, one litre of the wter smple ws kept t 4 C. In the lortory, the wter smples were stored t -2 C. Before solid phse extrction (SPE) of pesticides, the wter smples were

4 16 Hydroiologi (214) 723:13 12 Fig. 1 Mp of Belgium showing the loctions of the 42 investigted clusters (circulr symols). Within cluster, three smll ponds were selected long mximl grdient of surrounding griculturl lnd-use intensity, rnging from reltively nturl re ( ponds in nturl res ), pstures with low cttle density ( extensive lnd-use ponds ) to res with intensive griculture, especilly croplnd ( intensive lnd-use ponds ). Loctions of the 15 smll ponds selected in the Northern (Flnders) nd Southern (Wlloni) regions of Belgium in res of intensive or extensive lnd use or in nturl reserves. The ponds were selected mongst the 126 ponds smpled during spring 24 using the sme grdient of lnd-use intensity filtered over 1-lm Whtmn screen to eliminte remining lrge orgnic prticles nd then successively over 2.- nd.6-lm Millipore screens. C 18 crtridges (6-ml Osis column, Wters) were clenconditioned y methnol nd MEQ wter efore smples were loded t rte of 6 ml/min nd eluted y cetonitrile. After SPE, the eluted smples were stored t -2 C until HPLC (only for trizines), or GC MS nlyses were pplied s previously descried in Ndzilek et l. (21). Before injection, 1 ml of the eluted smple ws filtered (.2 lm), nd this filter ws rinsed with 1 ml cetonitrile efore solvent evportion under N 2. The dry residue ws suspended in 9 ll of hexne nd 1 ll of solution (in hexne) of [ 2 H 12 ] chrysene (2 pg injected), used s internl stndrd. The detection limit ws 1 pp for ll pesticides. Glyphoste nd its metolite AMPA were quntified in wter y LC MS method. After SPE clen-up s for other pesticides, smples were derivtized with 9-fluorenylmethyl chloroformte following the method descried y Koeer et l. (21) nd Iáñez et l. (26). Then, eluted concentrted smples were nlysed with n Allince 2,69 liquid chromtogrphy system nd Micromss

5 Hydroiologi (214) 723: Tle 1 Summry of pesticide surveys covering lmost the entire Belgium territory (spring 24) or the min pesticide pulveriztions periods (April, July nd Octoer) over 27 Once smpling over Spring 24 Three-month smpling over 27 Numer of ponds Pesticide types: Hericides trzine, simzine, propzine, teruthylzine, trizine metolites, diuron, isoproturon trzine, simzine, propzine, teruthylzine, trizine metolites, diuron, isoproturon, glyphoste, minomethyl-phosphonic cid (AMPA) Insecticides endosulfn, lindne ldrine, chlorfenvinphos, endosulfn, dicofol Industril compounds octylphenol, nonylphenol, 4 polycyclic romtic hydrocrons (PAHs: enzo()fluornthene, enzo(k)fluornthene, enzo()pyrene) nd enzo(ghi)perylene) Quttro Ultim Pltinum triple qudrupole mss spectrometer (Wters/Micromss, Mnchester, U.K.). The quntifiction limit of oth glyphoste nd AMPA ws 1 pp. Reproductive disruption in fish The ojective of this prt of our reserch ws to evlute the risk of reproductive disruption in sedentry pond fish. Prussin crp (Crssius urtus gielio, goldfish suspecies) ws selected s resident fish sentinel ecuse it ws one of the most undnt fish species present in the ponds (results not shown). Six intensive or extensive ponds of the 15 ponds lredy surveyed for pesticide lods in 24 nd 27 were selected. Ten juveniles (5 mles nd 5 femles) (35 65 g) from ech pond were cptured in 28 nd 29, weighed nd lood smpled for plsm to determine plsm VTG levels. Then, fish were dissected for gond nd rin smples to estimte the gondosomtic index (GSI, 1 * gond weight/ ody weight) nd the rin romtse ctivity. Blood smples were tken from the cudl vessel using heprinized syringes. The smples were centrifuged for 2 min t 4,5 rpm, nd the plsm ws collected nd stored t -8 C until ssyed for VTG. Plsm concentrtions of VTG were quntified y ELISA, using crp commercil VTG ELISA kit (SPID BIO, Frnce) with detection limit of 1 ng/ml nd n intr- nd inter-coefficient of vrition of 4.85 nd 5.85%, respectively. Brin romtse ctivity ws quntified y rdioimmunossy enzymtic method descried y Noksson et l. (21) nd Gonzles& Piferrer (23) (see lso Mndiki et l., 25). Briefly, the rin homogente smple ws mixed with generting solution (NAA) contining 1 mm NADPH (Sigm N-55, Steinheim, Germny), 25nM ndrost- 4-ene-3,17-dione-[1-H 3 ] (NEN, Boston, USA) nd 275 nm ndrost-4-ene-3,17-dione (Sigm, Steinheim, Germny). More specificlly, 1 ll of tissue homogente, representing 1 mg of rin tissue, ws mixed with 15 ll of 3 nm NAA nd 5 lluffersolution nd incuted for 6 min in wter th t 3 C with gentle shking. A control tissue homogente mixed with sustrte without NADPH to prevent romtse ctivity ws co-incuted simultneously. The enzymtic rection ws stopped y the ddition of trichlorocetic cid (TCA, 1%), nd then, smples were centrifuged (8, rpm, 1 min). The concentrtions of proteins in the pellets were determined y the Lowry method. The superntnt ws extrcted with chloroform Tle 2 Summry of the numer of ponds nd pond mtrices tested for hevy metls (Cd, Sn, Zn, Cu, P nd Hg) during summer 23 nd spring 24 Summer 23 Spring 24 Wter Suspended solids 99 Sediment 68 Snils No smpling

6 18 Hydroiologi (214) 723:13 12 for the queous phse, which ws mixed with dextrncoted (5%) chrcol (.5%) nd quntified using liquid scintilltion counter. The intr- nd inter-coefficients of vrition of the ssys were 5.55 nd 6.85%, respectively. Hevy metl distriution During summer 23 or spring 24, we smpled different pond mtrices (wter, suspended solids, sediment nd snils) to nlyse the following hevy metls: cdmium-cd, tin-sn, zinc-zn, copper-cu, led- P nd mercury-hg (the ltter only in wter smples of 24). The numer of ponds nd pond mtrices investigted is summrized in Tle 2. For ech pond, wter ws smpled t 8 loctions s in pesticides design using tue smple integrting the entire wter column. The wter tken t the different loctions ws pooled nd filtered over 2 mm to remove lrger orgnic nd other mterils. Then, 2 litres of wter of ech pond ws rought to the lortory in cooling ox (4 C). Upon rrivl in the lortory, the wter smple ws filtered through 125-lm Teflon mesh screen to remove lrge orgnic prticles nd then over 1.2-lm filter. These filters were used for nlysing hevy metl concentrtions in suspended solids, wheres 1 ml of the filtrte ws used to quntify hevy metl concentrtions in the wter. Filters were clened with HNO 3 1% prior to use. For Hg nlyses in wter smples, cid-clened (1% HNO3) glss ottles were used nd filtrtion ws performed s soon s possile with pproprite filtrtion equipment pretreted for trce metl nlysis to void contmintion. Sediment smples were tken from the upper sediment lyer ( 3 cm) t eight smpling sttions in ech pond (4 in the middle of the pond nd 4 close to the edge). They were plced in plstic ottles nd dried t 6 C during 48 h. Before nlyses, they were clcinted t Fig. 2 : Atrzine occurrence () nd concentrtions () in wter smpled during spring 24 in ponds locted in reltively nturl res, res with extensive nd res with intensive griculturl lnd use. Atrzine contmintions were higher (P \.5) in intensive lnduse ponds thn in extensive lnd-use ponds or ponds in nturl ponds. n = numer of ponds in which trzine ws detected 1 75 % 5 25 Presence Asence C B A c Intensive Extensive Nturl 1, (n = 23) Atrzine (ug/l),5 (n = 13) (n = 6), Intensive Extensive Nturl

7 Hydroiologi (214) 723: C nd then cidified (HClO 4 HF HNO3) t 11 C overnight efore eing evported nd stored in 5% HNO3 solution. In ddition to the iotic pond mtrices, ioccumultion of hevy metls ws tested in snils of the genus Lymne tht were collected in the ponds. Snils were present in 14% (18/99) nd 27% (34/126) of the smll ponds smpled during summer 23 or spring 24, respectively. A totl of 48 ponds were investigted with 3 45 snils for ech pond depending on popultion size of the snils. In the lortory, snils were lyophilized nd digested y cidified solution (H 2 O 2 HNO 3 ), nd stored in nitric cid (5%HNO 3 ). Then, concentrtions of hevy metls were mesured in the digested solutions using highresolution qudruple-inductively coupled plsm-mss spectrophotometer (HR-ICP-MS). Multi-element stndrd solutions t different concentrtions (,.2, 1, 5, 2, 1 nd 2 pp) were used for clirtion. Totl vrition coefficients of four replicte mesurements vried etween 1.66 nd 5.45%. Hevy metl levels in wter were compred with the Belgin nd interntionl reference qulity for surfce wters: 1, 5, 5, 2 3 lg l -1 nd 77 ng l -1 for Cd, P, Cu, Zn nd Hg, respectively (Deleuck 27; Reynders et l., 28; USEPA, 29; Rimondi et l., 212), nd those in the sediment were compred with the prole effect concentrtions reported y McDonld et l. (2): 4.98, 149, 128 nd 459 lg l -1 for Cd, Cu, P nd Zn, respectively. Sttisticl nlyses Vlues re men ± SD, nd dt expressed in percentges were log-trnsformed efore sttisticl Fig. 3 Sesonl chnges (, n = numer of ponds) nd vrition in isoproturon levels (, n = numer of smples) in wter smpled three times in ponds locted in intensive nd extensive griculturl res or in nturl reserves during 27. Isoproturon contmintion ws more frequent in Octoer nd April, nd vlues were higher (P \.5) in ponds locted in griculturl res thn ponds in nturl res Isoproturon (ng/l) (n = 12) (n = 9) April July Octoer (n = 15) 3 Isoproturon (ug/l) 2 1 (n = 15) (n = 12) (n = 9) Intensive Extensive Nturl

8 11 Hydroiologi (214) 723:13 12 Fig. 4 Sesonl chnges (, n = numer of ponds) nd vrition in glyphoste nd AMPA levels (, n = numer of smples) in wter smpled three times in ponds locted in intensive nd extensive griculturl res or in nturl reserves during yer 27. Glyphoste nd AMPA contmintion ws more frequent in Octoer nd April, nd vlues were higher (P \.5) in intensive lnd-use ponds thn in extensive lnd-use ponds or in ponds in nturl res Concentrtions (ug/l) 2 1 Glyphoste AMPA April July Octoer (n = 5) (n = 6) (n = 9) 3 Glyphoste AMPA Concentrtions (ug/l) 2 1 c Intensive Extensive Nturl (n = 14) (n = 3) (n = 3) d e nlyses. Homogeneity of dt ws tested y Brtlett s test, nd then, ANOVA-II ws used to test the min effect of the three types of ponds nd seson. Significnce of differences etween groups ws estimted using the Scheffe s multiple rnge tests with significnce level t P \.5. Clcultions were performed using Sttistic softwre (STATSOFT, Tuls, OK, USA). Regression nlysis ws used to test the reltionships etween trzine (the more frequent hericide) levels in wter nd distnce to crop fields s well s etween sediment concentrtions nd snil concentrtions for ech metl. Results Pesticide levels in pond wter During the wter smpling of 24, trzine ws found in 42 ponds, t concentrtions vrying etween.11 nd lg l -1. Atrzine ws detected in 33% of the 126 investigted ponds, with their numer incresing with lnd-use intensity: 6% of the ponds in nture reserves, 13% of the extensive lnd-use ponds nd 23% of the intensive lnd-use ponds (Fig. 2). In those ponds where trzine ws detected, concentrtions were significntly (P \.5) higher in intensive lnd-

9 Hydroiologi (214) 723: use ponds thn in extensive lnd-use ponds or ponds in nturl res (Fig. 2). Overll, trzine levels in the ponds were inversely relted (R =-73, P = ) to the distnce from the nerest crop field. Presence of other hericides molecules ws generlly low ut showed lso trend of more contmintion in intensive ponds thn in extensive ponds. Indeed, while no trizine metolites were detected, simzine ws detected in 5 intensive lnd-use nd 3 extensive lnd-use ponds (6.3% of the investigted ponds), t low concentrtions ( lg l -1 ). Diuron ws detected in 8.7% of the ponds, gin predominntly locted in griculturl res (6 intensive lnd-use ponds, 3 extensive lnd-use ponds, nd 2 ponds in nture reserves), t low concentrtions vrying etween lg l -1. No other trgeted pesticides (isoproturon, endosulfn nd lindne) were detected during the 24 smpling cmpign. During the 27 smpling cmpign, no trizines or other pesticides were detected except for isoproturon nd glyphoste. Concentrtions of these two hericides were reltively low ut showed importnt sesonl vritions. Isoproturon levels were significntly (P \.5) higher in Octoer nd April thn in July (Fig. 3). All intensive lnd-use ponds nd ll extensive lnd-use ponds were contminted y isoproturon, nd ll ut three extensive lnd-use ponds hd isoproturon irrespective of smpling seson. All five ponds in nturl res were lso contminted y isoproturon in Octoer, ut three smples of April nd July did not revel contmintion. Similr to our oservtions for trzine in 24, isoproturon concentrtions were higher (P \.5) in intensive lnd-use ponds ( lg l -1 ) compred to extensive lnd-use ponds ( lg l -1 ) or ponds in nturl res (.1.61 lg l -1 ) (Fig. 3). Fig. 5 Profiles of plsm vitellogenin levels () nd rin romtse ctivity () in sedentry Prussin crp juveniles smpled in ponds locted in intensive nd extensive griculturl res in 28 nd 29. N = 3 juveniles. No modultion ws oserved in plsm VTG or rin romtse ctivity in mles, whtever the type of pond the fish were smpled from VTG (ug/ml) Extensive Intensive Femles Mles Femles Mles AA (fmol/mg prot/min Extensive Intensive Femles Mles Femles Mles

10 112 Hydroiologi (214) 723:13 12 Concentrtion (ng/l) 4 2 Nturl Extensive Intensive Concentrtion (ng/l) Nturl Extensive Intensive * Cd Sn P Cu Zn Hg Concentrtion (ng/g) Nturl Extensive Intensive Concentrtion (ng/g) Nturl Extensive Intensive Cd Sn P Cu Zn c 6 Nturl Extensive Intensive 4 Nturl Extensive Intensive Concentrtion (ug/g) Concentrtion (ug/g) Cd Sn P Cu Zn d 5 Nturl Extensive Intensive 3 Nturl Extensive Intensive Concentrtion (ug/g) 3 Concentrtion (ug/g) 2 1 Cd Sn P Cu Zn

11 Hydroiologi (214) 723: Fig. 6 Vrition in hevy metl concentrtions in wter (), suspended solids (), sediment (c) or snil homogentes smpled in ponds in intensive nd extensive griculturl res or in ponds in nturl reserves during summer 23 or spring 24. No significnt effect of lnd-use intensity on hevy metl concentrtions ws oserved whtever the pond mtrix Overll, glyphoste concentrtions were low, with overll higher concentrtions (P \.5) in Octoer thn in July nd April (Fig. 4). All five intensive lnduse ponds were contminted y glyphoste hericides whtever the smpling seson, while this ws oserved in only three extensive lnd-use ponds nd ponds in nturl res. In ddition, in those ponds where glyphoste hericides were detected, concentrtions were significntly higher (P \.5, Fig. 4) in intensive lnd-use ponds ( lg l -1 ) thn in other pond types (extensive lnd use: lg l -1, nturl res:.6.19 ponds). AMPA levels showed the sme profiles s for glyphoste hericides (Fig. 4 ). Concentrtions of insecticides (ldrine, chlorfenvinphos, -endosulfn, dicofol) nd industril compounds (octylphenol nd 4-(pr)-nonylphenol nd 4 PAH molecules) were elow the detection limits in ll ponds. Reproductive disruption fetures was expected, plsm VTG levels were mrkedly higher (P \.1) in femle (16 88 lg ml -1 ) thn in mle ( lg ml -1 ). VTG vlues were lower thn the detection limits (.1 lg ml -1 )for5%ofthe tested mles (Fig. 5). Levels of rin romtse ctivity were mrkedly lower (P \.1) in mles ( fmol mg -1 prot -1 min -1 )thninfemles ( fmol mg -1 prot -1 min -1 ), nd vlues did not differ etween fish from intensive nd extensive lnd-use ponds (Fig. 5). Only one intersex individul ws oserved in ech type of ponds (2 out of 12 fish screened = 1.7%). In these intersexul fish, high level of romtse ctivity (19 23 fmol mg -1 prot -1 min -1 ) nd plsm VTG vlues (48 71 lg ml -1 ) comprle to vlues oserved in femles ws oserved. Both for femles ( %) nd mles ( %), GSI vlues vried strongly etween individuls, nd no significnt differences were oserved etween mles from intensive lnd-use (.85 ±.1%) nd extensive lnd-use (.94 ±.3%) ponds. The two intersex fish displyed higher GSI vlues (2.45%) thn norml mles nd femles. Hevy metls For ll nlysed hevy metls, no effect of lnd-use intensity ws oserved in ny of the studied pond mtrices (Fig. 6 d). In the pond wter, totl concentrtions (ng l -1 ) of Cd (rnge..44), Sn (rnge..14), P (rnge ), Zn (rnge ) nd Hg (rnge ) were ner the detection limits (Fig. 6). Concentrtions of Zn were significntly (P \.5) higher in 27 compred to 24, ut concentrtions were generlly low (\.1 lg l -1 ) in most of the ponds. In suspended mterils (Fig. 6), Zn levels were the highest ( lg g -1 ) of ll metls, nd concentrtions were very low for the other metls, vrying etween nd 2 ng l -1 for Cd nd Sn nd.1.9 lg g -1 for P nd Cu. As expected, contents of hevy metls in sediment (Fig. 6c) were significntly higher thn in wter (P \.1) or suspended mterils (P \.1). The lowest vlues in sediment were oserved for Cd or Sn ( or lg g -1 ) nd the highest ones for Zn ( lg g -1 ). Levels for P nd Cu vried etween nd lg g -1, respectively. For ll the investigted hevy metls in sediment, vlues were lower thn the minimum effect concentrtions, except in 8.8% of the griculturl or nturl ponds. Accumultion of hevy metls in snil homogentes (Fig. 6d) reflected tightly the levels found in the sediments nd not those in wter or suspended mterils. Indeed, sediment concentrtions were significntly correlted to snil concentrtions (Tle 3) for ll metls except for Cu nd Sn; specificlly P (R =.99, P =.) nd Zn (R =.65, P =.1) sediment concentrtions were well relted to snil concentrtions. Tle 3 Coefficients of correltions etween vlues for hevy metl (Cd, Zn, Cu, P) concentrtions in snils nd in sediments smpled in ponds locted in intensive nd extensive griculturl res or in nturl reserves during spring 24 R Cd P.99. Cu Zn.65.1 Sediment concentrtions were closely relted to snil vlues, except for only Sn; Hg ws not ssyed in snils or sediment P

12 114 Hydroiologi (214) 723:13 12 Discussion Pesticide pollution Regulr follow-ups of pesticide pollution lods re often mde for lrge lkes nd rivers, ut informtion is generlly lcking for smll wter odies such s ponds. Here, we tested whether ponds suffer from pesticide contmintion nd whether this is relted to current lnd use nd lnd-use intensity. In our survey of 126 ponds, trzine ws detected in 33% of the investigted ponds, ut the overll concentrtions were low. Given the n on this hericide y the Europen Union (24/248/EG), the fct tht we still detected it in 1/3 of the ponds smpled in 24 my indicte tht trzine molecule does not rek down esily in pond wter conditions. In other surfce wters, the hlf-time for the dissiption of trzine is reported rnging etween 4 nd 11 months depending to wter physico-chemicl conditions (Mouvet et l., 1997; Sørensen et l., 23; Tissier et l., 25). But prt from possile long-term stility of trzine in the pond wter conditions, the current oservtion could lso e n evidence of continued use of trzine since the smpling cmpign ws conducted not fr from the n regultion. The percentges of trzine contmintion incresed mrkedly with the increse in lnd-use intensity, with oth the numer of contminted ponds nd the verge concentrtions of trzine in the contminted ponds eing higher in intensive lnd-use compred to the other ponds nd higher in the extensive lnd-use ponds thn in the ponds in nturl res. In our 27 survey, no trizine hericides, insecticides or industril compounds were detected. In this survey, however, we detected the presence of the hericides isoproturon nd glyphoste (nd its derivtive AMPA) in the mjority (glyphoste) or even in ll ponds. Such results indicte historicl chnges in the use of pesticides in reltion to the EU regultion concerning trzine, which led to the use of other pesticide molecules. For oth the hericides detected, there ws n increse in concentrtion from ponds in nturl res over extensive to intensive lnd-use ponds. Overll, our nlyses did not revel very high concentrtions ut did show tht hericides re detected frequently, with cler impct of lnd use on the proility of occurrence nd oserved concentrtions. Declerck et l. (26) showed tht lnd-use intensity in the immedite neighourhood (1 2 m) of the ponds strongly impct wter qulity vriles such s turidity nd nutrient concentrtions. Here, we show tht croplnd nd griculturl ctivities in generl in the neighourhood of the ponds led to incresed concentrtions of hericides in the wter. Irrespective of this cler impct of lnd use, considerle frction of ponds in nture reserves ws lso contminted. Overll, our results do revel lower concentrtions of hericides thn mesured in rivers nd lkes (Deleuck, 25, 28). Given the importnce of distnce to croplnd nd lnd-use intensity in the immedite neighourhood of the ponds, our results suggest tht the cretion of sizele uffer zone (5 m) where no crops my e cultured round ponds might help to mitigte spry-drift input in ponds. Due to the current widespred use of phenyl ure in griculturl production nd s totl hericide in urn res, isoproturon is mongst the contminnts most frequently encountered in rivers, strems, lkes, mrine wters nd groundwter in Belgium nd other Europen countries (Thurmn et l., 2; Gerecke et l., 21; Sørensen et l., 23; Deleuck, 25, 28, 21). It hs lso een reported tht hevy rinfll susequent to hericide pplictions in spring my led to contmintion of qutic environments y run-off (Polrd et l., 211). Indeed, it is well known tht hericide contmintion negtively ffects the welfre of qutic nimls y ltering the food vilility nd hitt or some interctions etween iots such s dominnce of predtors. All hericides detected in the present study hve een reported to ct directly on vrious physiologicl pthwys of qutic nimls. High levels of trzine re still of concern for groundwter in Belgium (Deleuck, 21) s in the US nd other countries (Gillion et l., 26 in Tillitt et l., 21). Atrzine hs een reported to ct s endocrine disruptor, ut the dt re still controversil in fish (Kzeto et l., 24; Spnò et l., 24; Hinfry et l., 26; Ndzilek et l., 28; Tillitt et l., 21). Studies on mphiins converge to the conclusion tht trzine used t environmentl concentrtions hs mrked endocrine disrupting effects tht ffect the centrl nervous nd immune system of frogs nd slmnders (Sprling et l., 21; Forson & Storfer, 26; Klos et l., 29; Lsserre et l., 29; Hyes et l., 21, ). Despite mssive use of isoproturon for vrious purposes nd its possile relese in the environment, informtion is still scrce out the long-

13 Hydroiologi (214) 723: term effects of exposure to chronic low levels of this hericide in nimls. Greulich et l. (22) reported tht levels of 1 lg l -1 of isoproturon in wter cn ffect the detoxifiction enzymtic system in tdpoles of vrious mphiin species. Our study confirms erlier oservtions tht isoproturon nd glyphoste hericides re nowdys mongst the most frequently encountered pesticides (Deleuck, 25, 28). High concentrtions of glyphoste hericides hve een found in pond environments in oth Europe nd the USA, with deleterious effects on qutic nimls, including the disturnce in the metmorphosis process, til dmges, endocrine disruption nd gond normlities (Howe et l., 24; Brusch & Smith, 27; Dinehrt et l., 29, 21). Since glyphoste hericides re now mssively used everywhere, more studies re needed to clrify the long-term effects of low concentrtions of these pesticides on qutic orgnisms. Simzine nd diuron were detected t very low concentrtions nd in only few ponds (survey 1). None of the tested insecticides (ldrine, chlorfenvinphos, -endosulfn, dicofol) or industril compounds (octylphenol, nonylphenol nd 4 PAHs) were detected in survey 2. This my e due to the fct tht the ponds re reltively isolted in the lndscpe, nd thus less likely to ecome contminted except for ppliction in their immedite neighourhood. Despite the fct tht these insecticides hve een nned in Europen countries, they re still found in rivers nd strems (Deleuck, 27) t low concentrtions due to their persistence in sediments nd regulr soluiliztion process through the wter column. Mny studies on rivers nd strems do show contmintion y phenolic compounds (Micic & Hofmnn, 29) nd PAHs (Deleuck, 27, 28, 21; Wu et l., 211; An et l., 212). Endocrine disruption in resident fish To test whether the presence of some pesticides or other pollutnts ws missed in the pond wter nd to etter understnd their effects on qutic pond nimls, reproductive disruption iomrkers were exmined in resident Prussin crp. This suspecies of goldfish is mongst the more undnt fish species found in frmlnd ponds in Belgium nd hs lredy een used s sensitive sentinel for detecting exposure to vrious endocrine disruptors such s 17- estrdiol nd 4-nonylphenol (Soverchi et l., 25; Popesku et l., 28). In the present study, intersex incidence (1.7%) ws comprle to tht oserved in other regions in reference rivers for some cyprinid fish species ( 41%) such s chu Leuciscus cephlus, roch Rutilus rutilus nd gudgeon Goio goio (Vn Aerle et l., 21; Joling et l., 22; Rndk et l., 29; Hinfry et l., 21). Moreover, VTG concentrtions in mles ( lg ml -1 ) did not indicte disturnce in endocrine production nd were in the sme order of mgnitude s those mesured in some mle cyprinid fish such s roch ( lg ml -1 ) from reference strem sites (Tyler et l., 25). VTG vlues were lower thn vlues reported for mle cyprinid chu from contminted strem sites, which cn rech 49 lg ml -1 (Hinfry et l., 21). In reltion to VTG levels, low rin romtse ctivity confirmed the lck in upregultion of endocrine production. Vlues for romtse ctivity were indeed comprle to those reported for some cyprinid fish from river systems (Noksson et l., 21; Hecker et l., 27; Hinfry et l., 21). Since the two ltter investigted endocrine fetures hve een reported s relile iomrkers of pesticide exposure for vrious qutic orgnisms (Folmr et l., 21; Hyes et l., 26; Wingfield & Muki, 29; Desforges et l., 21; Ndzilek et l., 211), the lck of their up-regultion in fish inhiting the ponds reflects the sence (or presence t very low concentrtions) of oestrogenic pollutnts in the pond wter. The results of the present study indicte tht fish inhiting the frmlnd ponds studied were not ffected y endocrine disruptors. Hevy metls For the tested hevy metls (Cd, Sn, P, Cu, Zn, Hg), levels in wter nd suspended mterils were out 1 times lower thn those usully mesured in the sme mtrices from rivers nd strems in Belgium (Bervoest & Blust, 23; Deleuck, 27). Aprt from Sn, the tested hevy metls re considered prolemtic in surfce wters in Belgium nd in mny other countries, ut in the present study, none were found t concentrtions higher thn the Europen or US criteri of good qulity surfce wter (Deleuck, 27; Reynders et l., 28; USEPA, 29; Rimondi et l., 212). Zn concentrtions incresed mrkedly in 24 compred to 23, ut vlues were still mrkedly

14 116 Hydroiologi (214) 723:13 12 lower (\.1 lg l -1 ) thn those reported for some lkes in Wlloni (.93 lg l -1, Deleuck, 27) nd contminted sites in rivers in Flnders (19 2,369 lg l -1, Reynders et l., 28). This increse in Zn levels my e relted to higher rinfll during the smpling cmpign in 24 compred to tht in 23, resulting in higher run-off of hevy metls in the pond wter or in n incresed in situ geochemicl trnsfer of some metls from the sediment lyer to the wter column. As expected, concentrtions of hevy metls were higher in sediment thn in wter nd in suspended solids, ut vlues were still lower thn those found in other, lrger wter odies (Deleuck, 27; Reynders et l., 28). In some ponds, levels for Cu nd P were higher thn those typiclly reported for rivers in Flnders (Bervoest & Blust, 23), ut for ll the tested hevy metls, vlues were lower thn miniml effect concentrtions s determined for qutic orgnisms (McDonld et l., 2). No significnt effect of lnd-use intensity on hevy metl concentrtions ws oserved. It hs een shown tht the concentrtions of some hevy metl, such s Cu, Cd nd Zn, my e incresed in griculturl res due to sewge sludge nd fertilizer use (Nicholson et l., 23; Mss et l., 21), ut overll especilly industril ctivities re historiclly ssocited with hevy metl pollution (Losk et l., 24; Reynders et l., 28). Isolted ponds such s those tested in the current study my e reltively spred from pollution y hevy metls nd industril such s PAHs. Exceptions would e ponds locted in the immedite vicinity of sources or ponds tht re regulrly flooded y lrge wter. We estlished significnt reltion etween hevy metl concentrtions in sediment nd ccumultion in the snils, indicting tht snils re relile sentinel species for hevy metl pollution in pond environment. Concentrtions of hevy metls in snils were for some metls, such s Cd nd Cu, comprle to those reported in other snil species (such in Melnopsis premors nd Theodoxus jordni) or higher thn vlues reported for other species (such in Pern pern, Dosini sp. or Donx rugosus), even though the wter odies in these other studies were chrcterized y higher concentrtions of hevy metls (Swilek et l., 1994; Sidoumou et l., 26). In contrst, while Sn ws present in ll pond mtrices, only trce levels were oserved in the snils, indicting low uptke or high elimintion rte (Grosell & Brix, 29; Moloukhi & Sleem, 211) of this metl y the snils. It hs een reported tht snil species differ strongly in hevy metl ccumultion nd re more sensitive to metl pollution thn other qutic nimls such s fish (Voets et l., 26; Ng et l., 211; Moloukhi & Sleem, 211). In generl, metl exposure is followed y rpid ctivtion of detoxifiction pthwy defence, such s the increse in specific grnules in the digestive glnd cells for metl sequestrtion nd the induction of metllothionein production y lysosoml grnules in the endoplsmic reticulum for metl inding nd excretion (Desouky, 26, Voet et l., 26; Ng et l., 211). Despite the efficiency of the detoxifiction mechnism, long-term exposure of qutic nimls to metl pollution is detrimentl to their fitness ecuse the energy requested for detoxifiction defence is re-llocted from norml physiologicl functions such s growth nd reproduction (De Coen & Jnssen, 23; Ng et l., 211). Some toxicologicl studies hve lso suggested link etween metl exposure nd reproductive impirment in fish popultions (Boyle et l., 28); high levels of some hevy metls such s Cd could dely oocyte mturtion nd ovultion y disturing the vitellogenesis process rther thn the pituitry pthwy control in Prussin crp (Szczerik et l., 26). A recent study demonstrted tht Cu or Cd itself hs no direct effect on VTG expression ut my increse the oestrogenic-vtg induction level compring to single oestrogenic exposure in goldfish (Chng et l., 211), ut no norml increse in the levels of plsm vitellogenin ws oserved oth in mles nd femles in the current study. In the current study, concentrtions Cu or Cd were low in ll the tested pond mtrices, nd no ltertion in rin romtse ctivity or circulting vitellogenin level ws oserved in Prussin crp inhiting representtive pond environment of the Belgin intensive or extensive lnd-use ponds. Conclusions The overll results of our survey study demonstrted tht smll Belgin ponds were minly contminted y some hericides nd spordic high dischrges of hevy metls in sediment nd snils, nd tht pond sedentry fish were not ffected y endocrine disruptors. Our results lso demonstrted mrked effect of griculturl lnd-use intensity on hericide pollution,

15 Hydroiologi (214) 723: which would e mitigted y n djustment of the lnd use in the immedite neighourhood of the pond or the use of sufficiently lrge uffer zones. Acknowledgements We thnk A. Evrrd for help with the fieldwork. This study ws finncilly supported y the Belgin federl Science Policy (Belspo) in the frmework of the PODO II-progrm, project MANSCAPE (EV/1/29E), nd the SDDprogrm, project PONDSCAPE (SD/BD/2). References An, L., B. Zheng, L. Wng, Y. Zhng, H. Chen, X. Zho, L. Zhng & K. Lei, 212. Biomrker responses nd genotoxicity in the mud snil (Bullct exrt) s indictors of costl contmintion. Mrine Pollution Bulletin 64: Brni, S., E. Boncompgni, A. Grosso, V. Bertone, I. Freits, M. Fsol & C. Fenoglio, 27. Evlution of Rn snk escult lood cell response to chemicl stressors in the environment during the lrvl nd dult phses. Aqutic Toxicology 81: Bervoest, L. & R. Blust, 23. 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Smith, S. T. McMurry, T. A. Anderson, P. N. Smith & D. A. Hukos, 29. Toxicity of glufosinte nd severl glyphoste-sed hericides to juvenile mphiins from the Southern high plins, USA. Science of Totl Environment 47: Dinehrt, S. K., L. M. Smith, S. T. McMurry, P. N. Smith, T. A. Anderson & D. A. Hukos, 21. Acute nd chronic toxicity of Roundup Wethermx nd Ignite 28 SL to lrvl Spe multiplict nd S. omifrons from the Southern High Plins, USA. Environmentl Pollution 158: Ftim, M., S. N. M. Mndiki, J. Douxfils, J. Silvestre, P. Coppe & P. Kestemont, 27. Comined effects of hericides on iomrkers reflecting immune-endocrine interctions in goldfish. Immune nd ntioxidnt effects. Aqutic Toxicology 81: Folmr, L. C., N. D. Denslow, K. Kroll, E. F. Orlndo, J. Enlom, J. Mrcino, C. Metclfe & L. Guillette, 21. Altered serum sex steroids nd vitellogenin induction in wlleye (Stizostedion vitreum) collected ner metropolitn sewge tretment plnt. Archives of Environmentl contmintion nd Toxicology 4: Forson, D. D. & A. Storfer, 26. Atrzine increses rnvirus susceptiility in the tiger slmnder, Amystom tigrinum. Ecologicl Applictions 16: Gerecke, A. C., C. Tixier, T. Brtels, R. P. Schrzench & S. R. Muller, 21. Determintion of phenylure hericides in nturl wters t concentrtions elow 1 ng L -1 using solid-phse extrction, derivtiztion, nd solid-phse microextrction gs chromtogrphy-mss spectrometry. Journl of Chromtogrphy A 93: Gillrdin, V., F. Silvestre, M. Dieu, E. Delive, M. Res, J. P. Thomé & P. Kestemont, 29. Protein expression

16 118 Hydroiologi (214) 723:13 12 profiling in the Africn clwed frog Xenopus levis tdpoles exposed to the polychlorinted iphenyl mixture roclor Moleculr Cell Proteomics 29(8): Gonzles, A. & F. Piferrer, 23. Chrcteriztion of romtse ctivity in the se ss (Dicentrrchus lrx L.) rin. Distriution nd reltion to ge, sex, nd the nnul reproductive cycle. Generl nd Comprtive Endocrinology 132: Greulich, K., E. Hoque & S. Pflugmcher, 22. Uptke, metolism nd effects on detoxiction enzymes of isoproturon in spwn nd tdpoles of mphiins. Ecotoxicology nd Environmentl Sfety 52: Grosell, M. & K. V. Brix, 29. High net clcium uptke explins the hypersensitivity of the pulmonte snil, Lymne stgnlis, to chronic led exposure. Aqutic Toxicology 91: Guo, J., F. Wu, X. Luo, L. Zhng, H. Lio, R. Zhng, W. Li, X. Zho, S. Chen & B. Mi, 211. Anthropogenic input of polycyclic romtic hydrocrons into five lkes in Western Chin. Environmentl Pollution 158: Hyes, T. B., P. Cse, S. Chui, D. Chung, C. Heffele, K. Hston, M. 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AïtAïss, P. Noury, E. Gomez, P. Gerudie, C. Minier, F. Brion & J. M. Porcher, 21. Endocrine disruption in popultions of chu (Leuciscus cephlus) in contminted French strems. Science of the Totl Environment 48: Howe, C. M., M. Berril, B. D. Puli, C. C. Heling, K. Werry & N. Veldhoen, 24. Toxicity of glyphoste-sed pesticides to North Americn frog species. Environmentl Toxicology Chemistry 23: Iáñez, M., O. J. Pozo, J. V. Sncho, F. J. López & F. Hernández, 26. Re-evlution of glyphoste determintion in wter y liquid chromtogrphy coupled to electrospry tndem mss spectrometry. Journl of Chromtogrphy 1134: Imperto, M., P. Admo, D. Nimo, M. Arienzo, D. Stnzione & P. Violnte, 23. Sptil distriution of hevy metls in urn soils of Nples (Itly). Environmentl Pollution 124: Joling, S. & C. R. Tyler, 26. Introduction: the ecologicl relevnce of chemiclly induced endocrine disruption in wildlife. Environ Helth Perspectives 114: 7 8. Joling, S., N. Beresford, M. Noln, T. Rodgers-Gry, G. C. Brighty & J. P. Sumpter, 22. Altered sexul mturtion nd gmete production in wild roch (Rutilus rutilus) living in rivers tht receive treted sewge effluents. Biology Reproduction 66: Kzeto, Y., A. R. Plce & J. Trnt, 24. Effects of endocrine disrupting chemicls on the expression of CYP19 genes in zerfish (Dnio rerio) juveniles. Aqutic Toxicology 69: Klos, W., I. Lutz, T. Springer, H. Krueger, J. Wolf, L. Holden & A. Hosten, 29. Does trzine influence lrvl development nd sexul differentition in Xenopus levis? Toxicology Science 17: Koeer, R., C. Fiescher, F. Lnz, K. S. Boos, B. Sellergren & D. Brcelo, 21. Evlution of multidimensionl solidphse extrction pltform for highly selective on-line clen-up nd high-throughput LC-MS nlysis of trizines in river wter smples using moleculrly in printing polymers. Anlyticl Chemistry 73: Lmpi, M. A., J. Gursk, K. I. McDonld, F. Xie, X. D. Hung, D. G. Dixon & B. M. Greenerg, 26. Photoinduced toxicity of polycyclic romtic hydrocrons to Dphni mgn: ultrviolet-medited effects nd toxicity of polycyclic romtic hydrocron photoproducts. Environmentl Toxicology Chemistry 25: Lsserre, J. P., F. Fck, D. Revets, S. Plnchon, J. Renut, L. Hoffmnn, C. Arno, A. C. Gutle, C. P. Muller & T. Bohn, 29. Effects of the endocrine disruptors trzine nd PCB 153 on the protein expression of MCF-7 humn cells. Journl of Proteome Reserch 8: Losk, K., D. Wiechul & I. Korus, 24. Metl contmintion of frming soils ffected y industry. Environment Interntionl 3: Ms, S., R. Scheifler, M. Benslm, N. Crini, E. Lucot, Z. Brhmi, S. Benycou & P. Giudoux, 21. Sptil distriution of hevy metl concentrtions in urn, suurn nd griculturl soils in Mediterrnen city of Algeri. Environmentl Pollution 156: McDonld, D. D., V. G. Ingersoll & T. A. Berger, 2. Development nd evlution of consensus-sed sediment qulity guidelines for freshwter ecosystems. Archive Environmentl Contmintion Toxicology 39: Mndiki,S.N.M.,I.Bik,J.M.Bopopi,F.Leprieur&P.Kestemont, 25. Effects of sex steroids nd their inhiitors on endocrine prmeters nd gender growth differences in Eursin perch (Perc fluvitilis) juveniles. Steroids 7: Micic, V. & T. Hofmnn, 29. Occurrence nd ehviour of selected hydrophoic lkylphenolic compounds in the Dnue River. Environmentl Pollution 157: Milinkovitch, T., A. Ndiye, W. Snchez & S. Le Floch, 211. Thoms-Guyon H. Liver ntioxidnt nd plsm immune responses in juvenile golden grey mullet (Liz urt) exposed to dispersed crude oil. Aqut ic Toxicology 11: Moloukhi, H. & S. Sleem, 211. Bioccumultion, fte nd toxicity of two hevy metls common in industril wstes in two qutics molluscs. Journl of Americn Science 7:

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