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Toxicological evaluation of parathion and azinphosmethyl in freshwater model ecosystems

NN08201.81B :R.J.Dorîland Toxicological evaluationo fparathio nan d azinphosmethyl infreshwate rmode l ecosystems Promotor: dr.J.H . Koeman,hoogleraa r ind e toxicologie. R.J. Dortland

Toxicological evaluation of parathion and azinphosmethyl in freshwater model ecosystems

Proefschrift terverkrijgin gva nd egraa dva n doctori nd elandbouwwetenschappen , op gezagva nd erecto rmagnificus , dr.H.C .va nde rPlas , hoogleraari nd eorganisch e scheikunde, inhe topenbaa rt everdedige n opvrijda g2 0jun i198 0 desnamiddag st evie ruu ri nd eaul a vand eLandbouwhogeschoo l teWageninge n

Centre for Agricultural Publishing and Documentation

Wageningen - 1980 Astract

Dortland,R.J. ,1980 .Toxicologica l evaluationo fparathio nan dazinphosmethy li n freshwatermode l ecosystems.Agric .Res .Rep .(Versl .landbouwk .Onderz. )898 , ISBN9 022 0073 24 ,(viii )+ 112p. ,3 3figs ,4 2tables ,13 1refs ,appendices , Eng.an dDutc hsummaries . Also:Doctora l thesis,Wageningen .

Astud ywa smad eo fth epossibl ehazard so f long-termexposur e offreshwate recosys ­ temst olo w (<1 m gm~3 )concentration s oforganophosphoru s insecticides.Range-fin ­ ding,acut ean dsub-acut e (3weeks )laborator y toxicity trialswer ecarrie d outwit h parathion,azinphosmethyl ,diazinon ,malathio nan dparathion-methyl .Th e rateso f dissipation ofthes ecompound s fromth ewate r phaseo faquari awit han dwithou tplant s andsediment swer estudied .Th emai ntes tspecie swer eDaphni amagna ,Asellu saqua - ticus,Chaoboru scrystallinu s andCloeo ndipterum .D .magn awa s themos t sensitive species,3-wee kno-effec tconcentration s ranged from0. 1 to 1.2m gm"3 .Parathio nwa s themos t toxiccompoun d tested.Parathio nan dazinphosmethy lwer e tested in3 x 1x 1 moutdoo rmode lecosystem s inconcentration s< 1m gm~ 3ove rperiod s ofmor etha n 3months .Wit ha waterproo f partition themode lecosystem swer edivide d intotw osub ­ systems.Withou t insecticide treatment thesesubsystem s developed similarly.Insecti ­ cidetreatmen to fon esubsyste mresulte d indevelopment s dissimilar tothos eo fth e control.Parathio n stronglyreduce dpopulation s ofDaphi asp p (0.5an d 1m g m~3), Simocephalusvetulu s (1m gm~3) ,Chydoru s sphaericus (1m gm"3 )an dChaoboru scrystal ­ linus (1m gm"3) ,N oeffect swer eobserve d at0. 2m gm"3 .Azinphosmethy l at 1m gm~ 3 reducedpopulation so fS .vetulu san dDaphni aspp .N odetrimenta leffect swer eobser ­ ved onCyclopoid aan dOstracoda .

Freedescriptors :hazar dassessment ,toxicity ,EC50 ,no-effec tconcentration ,repro ­ duction,half-life ,diazinon ,malathion ,parathion-methyl ,Daphni amagna ,Asellu s aquaticus,Chaoboru s crystallinus,Cloeo ndipterum ,Cyclopoida ,Ostracoda ,aquati c invertebrates.

This thesiswil lals ob epublishe d asAgricultura l ResearchReport s89 8

© Centrefo rAgricultura l Publishing andDocumentation ,Wageningen ,1980 .

Nopar to f thisboo kma yb ereproduce d orpublishe d inan yform ,b yprint ,photoprint , microfilmo ran yothe rmean swithou twritte npermissio n from thepublishers . Stellingen

1. De in de onderhavige studie gebruikte modelecosystemen zijn goed bruikbaar voor de toetsing van no-effect-concentraties van toxische stoffen in zoetwater.

2. De voorlopige grenswaarde voor de concentratie aan Cholinesterase-remmende stoffen _3 in Nederlandse oppervlaktewateren dient te worden verlaagd van 1 mg.m naar _3 0,1 mg.m paraoxon-equivalenten.

3. Voor organofosforinsekticiden kunnen no-effect-concentraties voor veldsituaties worden afgeleid van 48-uur ECL.-waardeECL.-waarden voor Daphnia magna Straus door deze waarden te vermenigvuldigen met een factor 0,1.

4. De in de Kwartaalverslagen van Rijkswaterstaat als paraoxonequivalenten vermelde concentraties aan cholinesterase-remraende stoffen in het Nederlandse oppervlaktewater zijn, als gevolg van de gebruikte bepalingsmethodiek, van beperkte waarde als indica­ tor voor mogelijke ecologische schade.

5. In tegenstelling tot de bewering van Parker et al., is het gaan drijven van daphnia's geen symptoom van vergiftiging door cholinesterase-remmende stoffen.

B.L. Parker, J.E. Dewey & CA. Back, 1970. Carbamate bioassay using Daphnia magna. J.Econ.Entom. 63(3): 710-714.

6. Bij het industrieel-toxicologische onderzoek van werknemers dienen slechts die expositie- en functietests te worden gehanteerd die relevant zijn met betrekking tot de blootstelling.

7. Vanuit een oogpunt van natuurbehoud is aanwezigheid van milieutoxicologische kennis binnen de teams die met tsetsevliegbestrijding zijn belast van groot belang.

8. De grote invloed van de produkt- en bedrijfsschappen op de formulering van de Be­ sluiten krachtend de Warenwet is, vanuit het standpunt van de consument bezien, vol­ strekt strijdig met de in de Toelichting op deze wet als één der hoofddoelen genoemde 'bevordering van de eerlijkheid in de handel'. 9.D esuggesti e vanCunningha mo mva npatiënte nme t lymfomend e'dietar yhistories ' teonderzoeke n lijktzinvol .

A.S. Cunningham, 1976.Lymphoma s andanimal-protei n consumption.Th eLancet : 1184-1186.

10. Het grootst opgezette onderzoek vanTOO te r ontwikkeling van een nieuwe brood­ soort die 'de smakelijkheid van witbrood moet paren aan de gezondheid van vezelrijk volkorenbrood' is overbodig. R. Kroes, 1980.Voedin ge ngezondheid . Innovatie 38: 1-3.

11.D ebewerin gda td ehiërarchi ei nhe t legerloute rfunctionee l is,word tgelogen ­ straftdoo rd evel eprivilege s died ehogere ni nran ggenieten .

12.Da thede n tendag ekindere n inhu nspe ld ekoms tva nee npolitiewage n aankondigen methe tAmerikaans epioew-pioew-pioe w inplaat sva ntatietata-tatietata ,geef t e denken.

R.J.Dortlan d Toxicological evaluationo fparathio n and azinphosmethyli nfreshwate rmode l ecosystems Wageningen,2 0jun i 1980 Aan mijn ouders Ter herinnering aan Soorti Woord vooraf

Aand etotstandkomin g vandi tproefschrif t hebbenvel epersone n eenbijdrag e geleverd. Prof.dr.J.H . Koemanbedan ki kvoo rhe ti nmi j gesteldevertrouwe ne nvoo rd e intensieve begeleiding bijhe tbewerke nva nhe tmanuscript . De doctoraalstudentenMiek eHiddink-Scholte ,Ludge rva nde rEerden, OlfertCleveringa ,Magd a Bergman,Arn o Peters,Roe lJansen ,Froukj e Balk,Miek eMelis , Aris Schellinkhoute nLiesbet h Smitbedan ki kvoo r hunbijdrage n aandi t proefschrift enhun ,ondank s somsmoeilijk e omstandigheden, inspirerend enthousiasme. Vanhe t instituut voorOnderzoe kva nBestrijdingsmiddele n danki kprof.dr . F.J. Oppenoorth alsmede-opstelle rva n hetprojectvoorstel ,drs .H.R . Smissaertvoo r zijnwaardevolle ,ongezoute n kritieke ndr.ir .H .d eHee rvoo r zijnhul pbi jd egas - chromatografiee nhe t inrichtenva nd e"proefsloten" .Laatstgenoemd ee ndr.ir . M.Leistr a hebbenbovendie nonde rwelhaas t tropische omstandighedenmeegeholpe nme t het ingravenva nproefsloten . Voortswi li kbedanken : -d evakgroe p Erfelijkheidsleer voorhe tte r beschikking stellenva nhe tterrei n voor deproefsloten . -dr .W.F .te n Berge (toenno gwerkzaa m bijAkz o Research)voo rd ehul pbi jhe top ­ zettenva nd ewatervlooienkwee k -mej . dr.J.F.M .Geelen ,drs .J .Cuppe ne nir .A .Klin kvoo rhu nhul pbi jd eidenti ­ ficatieva nd edivers ewate r organismen. -drs .J.J.P . Gardeniersvoo r zijncommentaa ro pdele nva nhe t concept -mej . E.G.M.Harmse nvoo r hetuitvoere nva nd echemisch e waterbepalingen -d ehere nG.J . Hardemane nR . Kleesvoo rd egoed everzorgin g vand ewatervlooien -e n algenkweek -d ehere nH . Groenevelde nH.G . Slijkhuis voorhu nnooi t aflatende hulpvaardigheid, toti nd eavondure ne nweekeinde nto e -d ehere nH . Paardekopere nJ . vand eGoo re nhu nmedewerker sva nd ewerkplaat s voor deonmisbar ee nvakbekwam e technische bijdragenaa nhe t onderzoek -drs .J.C . Beunder,beheerde rva nhe tBiotechnion ,voo r zijnsoepe l inspeleno p mijn annexatiedrift -d ehee rH . Koopmanvoo r zijnhul pbi jhe tinrichte nva nnieuw-geannexeerd e ruimten -d ehee rG .va nGinke lvoo rhe tsne le ngrondi g reinigenva n vele karrevrachten glaswerk -d ehee rC .Rijpm avoo rhe tuitstekend e tekenwerk - ir.K.Ch.A.M . Luybenvoo r zijnhul pbi j hetberekene nva nhalfwaardetijde n -mej . R.Jansse nvoo rhe t typenva n eendee lva nhe t concept enmevr .IV .Korte-Baye r vand eAfdelin gTekstverwerkin g (LH)voo rd e zorgvuldigewi jz ewaaro p zehe tuitein ­ delijkemanuscrip t heeftgetypt . -mevr .Brouns-Murra yvoo r deEngels e correctie -d ehee rR.J.P .Aalpo lvoo r deredactionel e vormgeving vanhe t proefschrift - drs.J . Bergsma,ing .G.P .Hartin g enmevr .F . Petersvoo rhu n koeriersdiensten tussenGruttoweide ,Salverdaplei n enPudoc . Voortsdan k ikall emedewerker s vand evakgroe pToxicologi e voord euiters t plezierige encollegial ewerksfeer . Tenslotte dank ikJoke ,Kaspe r enSterr evoo rd e tijddi e ik - tegen onze beginselen in- va nhe nmoch t roveno mdi tboekj e eindelijk af temaken . Curriculum vitae

RobDortlan dwer do p1 4maar t 1948t eGoud ageboren .N ai n196 6 zijn einddiploma Gymnasium-betat ehebbe nbehaal daa n hetCasimirlyceu mt eAmstelveen ,bego n hiji n datzelfde jaar zijnstudi eaa nd eLandbouwhogeschoo l teWageningen .Septembe r197 3 behaaldehi j zijn ingenieursdiploma (met lof)me tal s hoofdvakd ekenni sva nd e levensmiddelene nal sbijvakke n toxicologie (verzwaard)e npedagogie ke nalgemen e didactiek.He tbijva k toxicologiewer d bewerkto phe tInstituu tvoo r Veterinaire Farmacologiee nToxicologi eva nd eRijk sUniversitei t teUtrecht . Na zijnmilitair e dienst kwamhi j alspromotie-assisten t indiens tva nd e Landbouwhogeschool,bi jd evakgroe pToxicologie . Sinds oktober 1979i shi jwerkzaa m bijd eDirecti eNoordze eva nRijkswaterstaat , alwaar hij zichbezighoud tme t adviseringe nhe tinitiëre nva nonderzoe kme tbetrek ­ kingto td emilieu-toxicologisch easpecte nva n het beheerove rd eNoordzee . Contents

1 Introduction 1

2 Toxicology of organophosphorus insecticides in freshwater ecosystems 4 2.1 Dispersionbehaviou r 4 2.1.1 Volatilization 4 2.1.2 Distribution 4 2.2 Transformation 7 2.2.1 Chemicalhydrolysi s 7 2.2.2 Microbialdegradatio n 9 2.2.3 Photolysis 10 2.2.4 Transformationproduct s 10 2.3 Toxicityt oaquati corganism s 12 2.3.1 Modeo factio n 12 2.3.2 Toxicityt ofis h 13 2.3.3 Toxicityt ocrustacean s 14 2.3.4 Toxicityt oaquati c insects 15 2.3.5 Toxicityo ftransformatio nproduct s 19 2.4 Concluding remarks 19

3 Methods for the measurement of organophosphorus insecticides 21 3.1 Extraction 21 3.2 Gas-liquid chromatography (GLC) 21 3.3 Thin-layer chromatography (TLC) 23

4 Laboratory toxicity tests 25 4.1 Insecticides 25 4.2 Testspecies ;origin ,rearin g andmaintainin g procedures 25 4.2.1 Daphniamagn a Straus 26 4.2.2 Otherorganism s 28 4.3 Assaymethod s 28 4.3.1 General test-conditions 28 4.3.2 Species-specific test-conditions 29

4.3.3 EC50 determination 31 4.4. Results 31 4.4.1 Daphniamagn a Straus 31 4.4.2 Asellus aquaticusL . 35 4.4.3 Cloeondipteru mL . 39 4.4.4 Chaoboruscrystallinu s(d eGeer ) 39 4.4.5 Other species 40 4.5 General discussion 42

5 Half-lives of five organophosphorus insecticides in the waterphase of freshwater aquaria 43 5.1 Aimo fth eexperimen t 43 5.2 Materials andmethod s 43 5.2.1 Calculationo fth ehalf-lif e 44 5.3 Resultsan ddiscussio n 46

6 Model ecosystem (MES} studies 48 6.1 Introduction 48 6.2 Materials andmethod s 50 6.2.1 Designo fth eME S 50 6.2.2 Physico-chemicalmeasurement s 51 6.2.3 Vegetationassessmen t 52 6.2.4 Insecticide applications 53 6.2.5 Populationassessmen to fzooplankto nan dmakrofaun a 54 6.3 Results 56 6.3.1 Developmento fth eME Si nth epre-experimenta lperio d 56 6.3.2 Physico-chemicalmeasurement s 56 6.3.3 Vegetation assessment 62 6.3.4 Insecticide concentrations 66 6.3.5 Populationassessmen to fzooplankto nan dmacrofaun a 67 6.4 Discussion 68 6.4.1 Physico-chemicalmeasurement s 68 6.4.2 Vegetation assessment 70 6.4.3 Insecticide concentrations 70 6.4.4 Populationassessmen to fzooplankto nan dmacrofaun a 71 6.4.5 Similaritiesan ddifference sbetwee ncorrespondin g subsystems 74 6.5 Conclusions 75

7 Comparison of the MES-experiments with the laboratory toxicity trials 77

8 Conclusions and recommendations 79 Surrmary

Samenvatting

Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F

Literature But, thank God, they are not as intelligent as we who kill them; although they are more noble and more able,

ERNESTHEMINGWA Y Theol dma nan d these a 1 Introduction

A numbero forganophosphoru s pesticidesar econtinuousl ypresen ta scontaminant s insurfac ewater si nth e Netherlands.B yga s liquid chromatography, thin-layerchro ­ matographyan dsometime sb ymass-spectroscop y the organophosphorus compounds azin- phosmethyl,chlorfenvinphos ,diazinon ,dimethoate ,malathio nan dparathio na swel la s thecarbamat e insecticidecarbary lhav ebee ndetecte di nth e RiverRhin ean dit sdis ­ tributariesan di nothe r surfacewater s (Grèvee tal. , 1972;Wegma ne tal. ,1978 ; Pons, 1972;Blauw , 1975). Similar findingshav ebee nreporte d from other European countries likeGrea t Britain,Ital yan dWester n Germany (Lowdene tal. , 1969;De l Vecchioe tal. , 1970;Sörensen , 1973). Some figuresfo rth eRive rRhin e aregive ni n Table1 . Thesourc ean dorigi no fthes e insecticides areno talway s known.The yma ypar ­ tially originate from agriculturean dhorticultur ebu tdirec t disposal from factories alsoma y contributet othei r general occurence. (Fritschie tal. , 1978;Coppag ee t al., 1976). The levelsreporte d canb econsidere da srelativel y high,becaus e they areo f the sameorde ra sth elevel s foundt ob etoxi ct osevera laquati c organisms under laboratorycondition s (Table 2). However,i ti sdifficul tt oasses sho whazardou s thesecompound sma yb efo rth eaquati c ecosystems because toxicitydat a obtained under laboratorycondition s oftencanno tb eextrapolate d directlyt ofiel dsituations . The toxicityo fchemical si nwate ri sstrongl y influencedb yvariou s environmental conditions sucha spH ,hardness ,th epresenc eo fsuspende dmatte r andmicro-organisms , which generallyar eno to rcanno tb esimulate d adequatelyi nlaborator ymodels .

Table 1.Averag emas s concentrationo fCholinesteras e inhibitorsi nRhin e water sampled near Lobith.Concentratio n expressed inm gparaoxo n equivalent perm- * water. (AfterRijkswaterstaat , 1973-1978).

Quarter Mass concentration (mgm ) indifferen tyear s

1973 1974 1975 1976 1977 1978

1st 5.73 0.92 4.5 31.0 5.9 1.54 2nd 1.52 0.95 2.9 21.0 5.4 2.01 3rd 2.42 2.74 10.1 2.2 3.8 0.65 4th 3.27 1.64 12.1 3.8 1.9 2.74 Annualave r age 3.24 1.56 7.4 14.5 4.3 1.73 Table2 .Acut e toxicityo fsom eCholinesteras e inhibitors tofreshwate rorganisms . SourceD .pule xi sSander s& Cop e (1966).Otherdat aar ederive d fromreference si n Pimentai (1972).Effec trefer st oimmobilizatio n (D.pulex )o rdeat h (otherspecies) .

3 Speciesteste d Substance EC50(mg m~ ) ExposurEx] e time (h)

Crustaceans: Daphniapule x DDVP 0.,06 6 48 Dipterex 0.,1 8 48 Parathion 0. 60 48 Diazinon 0.9 0 48 Malathion 1.. 8 48 Phosphamid on 8., 8 48 Carbaryl 6 48

Gammaruslacustri s Azinphosme thyl 0. 3 48 Malathion 1.8 48 Parathion 6 48 Diazinon 500 48 Carbaryl 22 48

Insects: Pteronarcyscalifornic a Malathion 6 48 Azinphosme thyl 8 48 Parathion 11 48 Diazinon 60 48 Carbaryl 15 48

Fishes: Percaflavescen s Azinphosme thyl 13 96 Malathion 263 96 Carbaryl 745 96

Salmogairderi i Azinphosmethyl 14 96 Malathion 170 96 Parathion 275 96 Carbaryl 438 96

The aim of the investigations wast oasses sth ehazard so flong-ter m exposureo f aquaticecosystem st oorganophosphoru s insecticides,wit h special emphasiso nth e mostsusceptibl e groupso faquati c organisms like crustaceansan dinsects .A prope r hazard assessment cannotb ebase do nlaborator y toxicitydat aalone .Therefor ea n attemptwa smade t odevelo pa tes tmode l incorporating someo fth emajo renvironmen ­ talvariable st onarro wdow n theris kmargi ni nth eextrapolatio no flaborator ydat a tofiel dsituatio nan dt oenabl ea more realisti c hazard assessment. Research strategy Fiveorganophosphoru s insecticideswere chosenbecaus e theywer e presenti nsurfac ewate r anduse di nagricultur ean dhorticultur ei nth eNetherlands . These fivecompound swer eparathion ,azinphosmethyl ,diazinon ,malathio nan d para- thion-methyl.Thei r formulasan dphysico-chemica l propertiesar eillustrate di n Tables1 5an d16 .O fthes e insecticidesparathio nan dazinphosmethy lwere studie di n most detail.Th eresearc hprogramm econsiste do fthre eparts : -Toxicit y testing inth e laboratory: determination ofacut e andchroni c toxicity of every compound to fourspecie s ofaquati c invertebrates.Thes e specieswer e Daphnia magnaStrau s (Cladocera,Crustacea) ,Asellu s aquaticus L. (Isopoda,Crustacea) , Chaoborus crystallinus (DeGeer ) (Diptera,insecta )an dCloeo n dipterumL . (Ephemerop- tera, Insecta). -A laboratorymode l ecosystem study tomeasur e therelativ e rate ofdissipatio n of thesecompound s from thewate rphase . -Outdoo rmode lecosyste m studies toinvestigat e theeffect s of long-term low-level (< 1m gm )exposur e on theaquati c invertebrate population ina situationwhic h closely resemblesnatura l circumstances. These threepart s aredeal twit h inth eChapter s 4,S an d 6 respectively,wherea s the analytical-chemical methods for themeasurement s of the insecticides used in these three studies aredescribe d inChapte r 3. 2 Toxicology of organophosphorus insecticides in freshwater ecosystems

2.1 DISPERSION BEHAVIOUR

2.1.1 Volatilization

Datao nth evolatilizatio no forganophosphoru s compoundshav eno tbee n foundi n the literatureconsidere di nth epresen t study.A sthes e compoundshav ea hig hmole ­ cularmas san dlo wvapou rpressures ,a lo w rateo fvolatilizatio nwoul db eexpected . DeHee r (1979)calculate d thevolatilizatio n half-livesfo razinphosmethy lan d dimethoatet ob e41 3an d0.17x1 0 days respectively. These calculationswer e basedo nassumption so fpur e solutionsan da rapi ddiffu ­ siono fth esubstanc et oth ewate r surface.I ttherefor e seems realistict oconside r thesedat aa sminimu mhalf-lives .I fthes etw o compoundsar erepresentativ efo r thegrou po forganophosphoru s insecticidesa sa whole ,volatilizatio n fromwate r bodiesca nb econsidere da sbein go fmino r importance.

2.1.2 Distribution

An importantprerequisit efo ra prope rhazar d assessmenti st oconside rth edis ­ tributiono fth e compound overvariou s compartmentso fa necosyste mi norde rt olear n first,whic h organismsar eliabl et obecom e exposedan dsecondly ,ho wlon g theyar e exposedan dt owha t concentrations.I ngenera lth e distributiono fchemica l substan­ cesi nth eenvironmen t dependso nwate r solubility,sorptio ncharacteristics ,volati ­ litiesetc .Withi n thisgrou po forganophosphoru s compounds therei sa larg evariatio n inthes epropertie swhic h canb eattribute d mainlyt oth evariet yo fsubstitute d groupsi nth eX-position .I nTabl e3 som eexample sar egive no fgroup s substituted inthi spositio nan dth eaqueou s solubilitieso fth ecorrespondin g compounds.Al ­ though somecompound s havehig hwate r solubilities,th emajorit ybelong st oth egrou p thatar eno t readilysolubl ei nwater .Th eorganophosphoru s insecticidesuse di nthi s studyals obelon gt othi sgroup .Th ephysico-chemica l parameter,whic h togetherwit h theaqueou s solubilityi so fmai n importancefo rth eultimat edistributio no fa chemical overth e aquatic ecosystem,i sth en-octanol/wate rpartitio n coefficient. This coefficientwa s firstuse dt opredic t theuptak eo fa chemica lb yorganism s (Kenaga,1972 )an dlate rfo rth e assessmento fth e potential forbiomagnificatio n (Lu& Metcalf ,1975 ;Metcalf , 1973;Neele ye tal. , 1974)an dlipophili c storage (Daviese tal. , 1975).Th epartitionin g coefficientmigh t alsob euse dt opredic tth e Table3 .Structura l formulaso fth eX-grou po fselecte d organophosphorus insecticides and theaqueou ssolubilitie so f thesecompounds .

Generalformul ao fa norganophosphoru s insecticide: 0 (S) R, II

H' ^X Compound Structuralformul ao fth eX-grou p Solubilityi n waterm em

Parathion 24 -NO,

Disulfoton -S-CH,—CH~—S—C~Hr 25

Azinphosmethyl -S-CH 2^N ' 33 I

•°VK^(CH3)2 Diazinon 40

CH,

Phorate -S-CH2-S-C2H5 50

-0C=CHC1

,C1 Chlorfenvinphos 145

I CI -S-CH-COOC.H 2 5 Malathion I 145 CH-COOC2H5

-S-CH.-N 2 I

Methidathion 240 I OCH.

•oyN Thionazin 1140

Dimethoate -S-CH.-CO-NH-CH, 2500 distributionove r thedifferen tcompartment so fa necosyste m (Neeley, 1979). Inre ­ lationt oth edistributio no forganophosphoru s compounds inaquati cecosystem sth e results ofman y fieldstudie ssho wa rapi ddissipatio no fthes ecompound s fromth e waterphas e toth ebotto m layer.Mull ae t al., (1966)reporte d adecreas e inpara - thionconcentratio n inpond s from400-51 0m gm to 10m gm after 8day s and to 3m gm aftera fortnight .Concentration s inmu dwer eu p to6 x 10~ mg kg" , lasting 22days .Simila rdat awer eobtaine d forparathio napplie d toa cranberr ybo g (Millere t al., 1967).Thes eauthor s suggested that thehig hconcentration s inwate r found immediatelyafte rapplicatio nwer e causedb ystratificatio no f thepesticid e and thatth erapi ddecreas ewa s aresul to fmixin g anddilution .Observation s after 32 applications of[ P] labelledmalathio nt opadd yric e field (deptho f theirrigate d water 10cm )suppor t thishypothesis .Afte r spraying astron g radioactivitywa s first detected inth esurfac e layer,graduall ydiffusin g intodeepe r layers ofwate r and followedb y adsorptiont omud .Mor e than9 Spe rcen to fth eradioactivit y disappeared from the irrigatedwater swithi n 2day s (Sato& Kubo , 1964).Th e sameauthor sre ­ porteda complet e disappearancefro mirrigatio nwater s (depth 5cm )withi n 7day s aftera ninitia lconcentratio no f 1.7 gm parathion.Simila r resultswer e reported forchlorfenvinpho sspraye d ona 1.5m deeppon da ta dos e rateo f 74k g active ingredient/ha.Her e the concentration inwate rdecrease d from6. 1 gm to less than 1g m " within 19hour s andt o0.1 2 gm " after about 34days .Residue s inmu d increased from0.2 5m g kg" after 5hour st o0.3 2m g kg after 115hours ,decreasin g to0.1 5m g kg" onDa y 34 (Beynone t al., 1971).Agai n the initial rapid decrease of theconcentratio n inwate rwa sprobabl y the resulto fmixin g anddilution .However , a substantialamoun to f insecticidewa sprobabl ydeposite d through sedimentationo f suspendedparticles ,especiall ya s thewate rappeare d turbidbefor e sprayingbu twa s clear 13hour s later.Th eauthor ssuggeste d eithera rapi dprecipitatio n of the sus­ pendedparticle sdu e toadsorptio no fth epesticid e toth eparticle s and subsequent sedimentationo ra destructio no fplanktoni canimals .Rapi d adsorptiont oplant s and bottommateria lwa sals oreporte d forDursba n (Smithe t al., 1966)an dadsorptio nt o plants andalga ewa smentione d forparathio n (King,1969 ;Mulla e t al., 1966)an d fenitrothion (Moodye tal. , 1978). So farn obiologica lmagnificatio no f thesecompound s infis hha sbee nreported . Theirchemica lproperties ,suc ha ssusceptibilit y todegradatio nb yphysical ,chemica l andbiologica lmean s topola rproduct sreadil yaccoun t for this.Th epersistenc e in fishi s low.Mace k (1970)presente da tablei nwhic h thehalf-lif e timeso f fiveorga - nophosphates infis har esummarized . Inthi stabl emalathio nha sa half-lif e of less thanon eday ,whil e thepersistenc eo fDursban ,azinphosmethyl ,parathio nan d diazinon is lesstha non eweek . Ingenera l itca nb e said that, apart fromparathio nan dsom eothe rcompounds , littleinformatio ni savailabl eo n thebehaviou ran d toxicological impacto forgano ­ phosphorus insecticides inaquati cenvironments . 2.2 TRANSFORMATION

Chemicalhydrolysis ,microbia ldegradatio nan dphotolysi sca nb eregarde da sth e mostimportan troute so ftransformatio nfo rth eorganophosphoru sinsecticides .

2.2.1 Chemical hydrolysis

Hydrolyticcleavag ei sth etransformatio npathwa ywhic hha sbee nstudie dmos t intensivelyan dunde rver ydivers econditions ,especiall ywit hregard st otempera ­ ture,p Han dtyp eo fwater .Th etemperatur erange sfro m 10t o7 0 C,an dth emediu m variesfra ndistille dwate ran dbuffe rsolutio nt ora wrive rwater .Som einvestigator s madethei robservation si nacidi csolutions ,whil eother suse dalkalin econdition sfo r theirexperiments .Dat ao nth ecompound suse d inthi sstud yar ecompile d inTabl e4 . Therapi ddisappearanc eo fparathio ni nrive rwate rca nb eattribute dt oa bio ­ logicalreaction .Th eauthor sstate dtha tsinc esuspende dmatte rwa spresen tmicro ­ bialbreakdow nma yhav econtribute d toth edisappearanc eo fth emothe rcompound .Fo r malathionan dparathionmethy lth ehalf-live sar eeve nshorter .D eHee r (1979)foun d

Table4 .Half-live s (ti)o forganophosphoru s insecticides inaqueou s solutions ofneutra lan dalkalin epH . References:(1 )Eichelber g &Lichtenber g (1971); (2)Faus t &Goma a (1972); (3)D eHee r (1978); (4)Heue re tal .(1974) ; (5)Quenti n (1969); (6)Wolf ee tal . (1977); (7)Y u& Sandbor n (1974).

Insecticide Typeo fwate r Temp.(°C ) pH Half-life References inday s parathion riverwate r , 7.3-8.0 72 1 buffersolutio n 20 7.4 108 2 tapwate r 20 7.5 56.5 5 buffersolutio n 20 9.0 22.8 2 standardref.w . 26.7 7.9 15-.1 6 7 azinphosmethyl surfacewate r 10 7.4 624 3 surfacewate r 10-30 7.5-11.0 0.63--7.. 3 3 (outdoortanks ) buffersolutio n 6 8.6 36.4 4 buffersolutio n 25 8.6 27.9, 4 buffersolutio n 20 8.7 6.1 3 buffersolutio n 20 8.8 12.8 3 diazinon buffersolutio n 20 7.4 185 2 buffersolutio n 20 9.0 136 2 malathion riverwate r . 7.3-8.0 7 1 (252le i Et after one week) buffersolutio n 27 8.0 6 5 parathionmethvl riverwate r . 7.3-8.0 ;* 1 (25%le i Et after one week)

1 storage int h edar k 2 storage under sunlightan d fluorescent ligt hconditions . theconversio no fazinphosmethy l inoutdoo r tanks tob emuc h faster than inth e labo­ ratory inth edar k and ascribed this to theeffec t of light.However ,th e differences intemperatur e and pH,an d theireffec t onhydrolyti c conversion ratesma y have been more important.Th ep H inthes eoutdoo r tanksrange d from 7.5 to 11.0ove r atempe ­ rature rangeo f 10-30°C ,whil e inth e laboratory thisrang ewa s only6.4-8. 1 ata fixed temperature of 10°C . The slowconversio n rateo fdiazino n inwate r ofneutra l ormoderatel y alkaline pHmigh t explain theconcentratio n ofdiazinon ,foun d indrainag e ditches in the GreatLak eRegio n ofCanada ,bein g higher than those ofethio n andparathion ,a s found byHarri s& Mile s (1975). Malathion isth ecompoun dwhic h atpH> 7 ismos t rapidlyhydrolysed ,bu t it isextremel y stablea t pH<7. (Spiller, 1961;Weis s & Gakstätter, 1964). Although there aren o studies on thehydrolysi s ofparathionmethy lunde r labora­ toryconditions ,i tca nb epredicte d that thiscompoun dwil lhydrolys emor e rapidly than itsethy l analogue because of thegreate r electrophilic character of themethy l group (Ruzicka et al., 1967). Thehighe r rateo fconversio n ofparathion-methy l found inrive rwate r comparedwit h thato fparathion ,i si n linewit h thisprediction . From thedifferen t studieso nhydrolysi s somegenera l conclusions canb edrawn : - Organophosphorus insecticides are fairly stable inneutra l and acidic conditions (Faust& Gomaa, 1972;Grahl ,1973 ;Weis s &Gakstätter , 1974).A nexceptio n is diazinon which isals o hydrolysedunde r acidiccondition s (Gomaae t al., 1969). Themor e alka­ line theaqueou s solution,th emor e rapid isth ehydrolysis .Malathio n for instance shows instantaneous hydrolysis atpl i1 2 (Spiller, 1961). - Temperature increase greatlyaccelerate s thehydrolyti c cleavage (Ruzicka et al., 1967). From theactivatio n energies involved,Faus t &Goma a (1972)conclude d thata t lower temperatures hydrolysisma y slowdow n tosuc h a lowrat e that thecompoun dbe ­ comes fairlypersistent .Thi scoul d alsopartl y explain thesometime s high concentra­ tions ofCholinesteras e inhibitors found in the Rhinedurin gwintertime . -Th ephophorothionate s and thiolothionatesar emor e stable than the corresponding oxygen analogues.Parathio n is anexceptio n since this compound hydrolysesmor e rapid­ lytha nparaoxo nunde r acidic orslightl y alkaline conditions (Faust &Gomaa , 1972). -Thos e organophosphorus insecticides inwhic h thealky l (R)grou p (Table 3) isa methyl group,hydrolys emor e rapidly than their ethyl analogues due toth e greater electrophilic character of themethy l group (Ruzicka et al., 1967). - Forcertai n organophosphorus compounds theproces s ofhydrolysi s seems tob een ­ hanced byadsorption .Pionk e &Cheste r (1973)gav e examples ofdiazino n and malathion which appeared todegrad emor e rapidly inassociatio nwit h colloids or suspended solids.O n theothe r hand theyals omentione d aprolonge d persistence ofDursba n due toadsorptio no norgani csolids . 2.2.2 Microbial degradation

Many studieshav ebee nmad eo nth emicrobia l degradationo forganophosphorou s compounds.Mos to fthes erefe rt osoi l systems.A goo d reviewo nthi sbreakdow ni n soilwa sprepare db yT u& Mile s (1976).The y listed bacteria,actinomycete san dfung i capableo fdegradin gon eo rmor e insecticides. Lessi sknow nabou tmicrobia l degradationi nwater ,bu ta si nsoil ,war m tempe­ raturesan dth epresenc eo forgani cmatte rwil l generallypromot e bacterial activity and,consequently , themetabolis mo fpesticides . For instance,Yasun oe tal . (1975)explaine dth erapi d inactivationo fparathio n useda sa larvicid ei npollute dwate r (incontras tt opur ewater )b ybacteria ldegra ­ dation.A nimportan tphenomeno ni nth emicrobia l breakdowno fpesticide si sth e adap­ tationo fth emicroflor at othes ecompounds .Sethunatha n (1973) found thati nfloode d rice fieldsrepeate d applicationo fdiazino nmarkedl y increasedth e degrading capacity comparedwit ha singl eapplication . Arthrobacter sp.,Corynebacteriu m sp.an da facultativ e anaerobic Flavobacterium were isolatedan dfoun d capableo fmetabolizin g diazinon. A similar adaptationwa s found afterparathio n treatment.Bacteri a capableo f hydrolysing parathionan ddegradin g its hydrolysisproduc tp-nitropheno lwer e isolated fromparathion-amende d flooded soils.The ywer e identifieda sPseudomona s sp. and Bacillus sp. (Siddaramappae tal. , 1973). This findingi si ncontras tt osevera l studiesi nwhic h nitrogroup reductioni sregarde da sth emajo rpathwa yo fparathio n degradationb ymicro-organism si nbot h soil,wate ran dpur eculture s (e.g.Graet ze t al., 1970;Zuckerman , 1970).A synergisti c actionwa s reportedb yGunne r& Zuckerma n (1968)fo rArthrobacte r sp.an dStreptomyce ssp .i nth e degradationo fdiazinon .I t isconceivabl e that similar adaptation processesa sth eone s referredt oabov eca n developi nwate rbodie s thatar erepeatedl yo rcontinuousl y contaminatedwit horgano - phosphorus insecticides,suc ha sth eRhin ean dit s distributariesi nth e Netherlands. The scientists engagedi nthi s typeo fresearc hd ono tagre eo nwhethe ro rno t aerobico ranaerobi c conditions favourmicrobia l degradation.Sethunata n (1973)sug ­ gested thatalternativ eoxidizin gan dreducin g conditionsma yprovid emor e suitable conditions fora complet e destructiono fpesticide s thanoxidatio no rreductio nalone , especially forcompound spossessin ga rin gmoiety ,sinc e ring cleavage reactionsre ­ quire oxygen.Thi svie wi ssupporte db yth efindin go fGraet ze tal . (1979) thatunde r anaerobic conditionsparathio nwa s convertedt oaminoparathion ,bu ttha tn ofurthe r degradation tookplace . A similaralternatio no faerobiosi san danaerobiosi s canb efoun di norganicall y polluted waterswit ha larg ealga lbiomas san dperhap so nth esurfac eo fhighe rwate r plantsi nalternatin g periodso fphotosynthesi san drespiration .Anaerobi c conditions canals ob efoun di nth e bottom layerso fwate rbodies .I nth e waterphas eth e aero­ bic conditionswil l generally prevail.Therefor ei nmos t aquatic ecosystems conditions arepresen t that enablemicrobia l degradationo forganophosphorou sinsecticides . 2.2.2 Photolysis

Photolysiso forganophosphorou sinsecticide sb yultr aviole t lightha s been studiedb ysevera l investigators (Cook, 1954,1955 ;Coo k& Oltes , 1959;Mitche le t al., 1968;Lian g& Lichtenstein , 1972;Heue re tal. , 1974). From these studiesth econclusio nca nb edraw n thatb ythi smechanis morgano - phosphorus compounds canb econverte d into transformationproducts .Mos t studieswer e made underartificia l conditionsan donl yth estudie sb yLian g& Lichtenstei nan d Heuere tal .refe rt oth ephotolyti c conversioni naqueou s solutiono fa norganophos - phorus insecticide (azinphosmethyl).Lian g& Lichtenstei n (1972)foun da rapi d conver­ siono fazinphosmethy li naqueou s solutions into sevenbreakdow n products.Bu tthi s finding doesno tguarante ea simila r rapidityunde rnatura l conditions,sinc e sunlight passing down througha wate rbod y decreasesi nintensit yan dmarke d changesi nit s spectral distribution occur.Therefor e therear einsufficien t datat oconclud e that photolysiso forganophosphoru scompound si so fimportanc ei naquati cecosystems .

2.2.4 Transformation products

Many transformationproduct so forganophosphoru s insecticides havebee nidenti ­ fied,especiall yb ystudie swit hmode lecosystem so fth eMetcalf-typ e (see for in­ stance referencesi nMetcalf , 1977).Th etransformatio nproduct so fth ecompound s usedi nth epresen t studyar eliste di nTabl e5 .Amon g themajo r tranformationpro - Table 5.Metabolite so ffou r organophosphorus insecticides used inthi s study. For parathion-methyln oliteratur e datawer e found,bu tit sbreakdow ni sprobabl y analogous totha to fparathion . Sources: parathion:Sethunatha ne tal . (1977);Y u& Sandbor n (1974); azinphosmethyl: Liang4 Lichtenstei n (1972); diazinon:Sethunatha n& Patha k (1972), Gomaae tal . (1969);malathion :Wolf ee tal . (1977). Parathion (a)Diethylphosphorothioi c acid (C-H.OKP-OH

(b)Diethylphosphoroi c acid (C2H 0)2P-OH

Aminoparathion (C^O^P-O-^ Q_)

Paraoxon (C^OljP-O-ZQV N02

p-Nitrophenol H0-(O)-N02

p-Aminophenol HO— / () \ — -NH2

10 Table5 .Continued .

Azinphosmethyl (c)Dimethylphosphorodithioi c acid SH (CH30) *f (d)Dimethylphosphorothioni c acid (CH30) SH $ Dimethyl benzazimidesulfide R-CH2- S-CH2-R

Dimethyl benzazimidedisulfide R-CH2-•S-S -CH2-•R

N-methyl benzazimide CH3-R benzazimide H-R azinphosmethyloxo n (CH 0)-P-C H- R S

Diazinon (a)+ (b)

CH(CH3)2 2-Isopropyl-4-methyl-6- hydroxypirimidine (IMPH)

Malathion (c)+ (d) Malathionß mon o acid (CH,0)-P-S-CH-C00C,H 3 | I 2 5 c S CH2-C00H Diethylfumarate CH-C00C.H C2H5OOC

Malathiona mon o acid (CH 0)2-P-S-CH-COOH S CH2-C00C2H Malaoxon (CH..0)„-P-S-CH-C00.H, i l | 25 CH2-C00C H

Diethylthiosuccinate HS-CH-C00C.H I 25 c CH -C00CH

ductso fthes e insecticides are,o fcourse ,th ediethy lan ddimethy l phosphorodi- thioic,phosphorothioi c andphosphori c acids. Oxygen analogues havebee n found only forparathio nan dazinphosmethyl . Ina model ecosystem studyY u& Sandbor n (1974)detecte d 0.30,0.4 7an d1. 4m gm" 3o f parathion,paraoxo nan dp-nitropheno l respectivelyi nth ewate r phase aftera perio d of3 3days .Th ehig h concentrationo fparaoxo ni ssurprisin ga sn oothe r authors have reported theoccurrenc eo fthi s oxygen analoguei naquati c systems.Lian g& Lichtenstein (1972) found substantial amountso fth eoxyge n analogueo fazinphos ­ methyl under laboratory conditions.Th eoxyge n analogues generally hydrolyse more

11 rapidly thancompound s containing sulphur (Ruzickae tal. , 1967). Formalathio n sixmor emetabolite swer ementione db yBende r (1969)tha nar e listedi nTabl e5 .Onl y thosear eliste dwhich ,accordin gt oWolf ee tal . (1977) and Parise tal . (1975)ca nb econsidere da simportant .

2.3 TOXICITYT OAQUATI CORGANISM S

S.S.I Modeof action

Itseem swel l established thatth einsecticida l actiono forganophosphoru s insec­ ticidesi sprimaril y basedo nth einhibitio no fcholinesterases ,presen ti nth e ner­ vous system.Th eoxyge nanalogue so fth ethio-compound sreac twit hth eenzym ean d blockth e hydrolysiso fth enorma l substrate,acetylcholin e (Corbett, 1974).Th ehig h reactivityo fthes ecompound sfo rth e enzymesi scouple dwit ha lo wdephosphorylatio n rate.Thi sma yresul ti na naccumulatio no facetylcholine ,causin g excessive stimu­ lation,followe db ya tota lbloc ko fimpuls e transmission.Th esymptom so fpoisonin g canb eexplaine do nth ebasi so fthi smod eo faction .Poisone d organisms initially showhyperactivity , followedb yconvulsiv ean duncoordinate dmovements ,endin gi n paralysisan ddeath . Althoughth eorganophosphoru s insecticides aremainl y useda sinsecticides ,the y are alsoeffectiv e againstmites ,ticks ,parasiti cnematode si nanimal san dplant s andmollusc s (Corbett,1974) .The yar eals o toxict overtebrates ,du et oth e samemechanis ma si ninsects .I nmammal s acetylcholine isno tinvolve d onlyi nth e transfero fimpulse sacros ssynapti c junctionsi nth ecentra lan dautonomi cnervous - system,bu tals oi nneuro-muscl e transmission.Th emajo r causeo fdeat hi nwarm ­ blooded vertebrateswhe n fatallypoisone dwit h organophosphorus compoundsi sasphyxia , resulting from respiratory failure (Murphy, 1975). Acetylcholinesterase isno tth eonl yenzym e inhibitedb ythes e insecticides. Various otheresterase sar einhibite db yorganophosphoru s compounds,suc ha sothe r cholinesterases,aliphati c (ali-)esterasesan daromati c esterases.On egrou po fthes e enzymesi sth eso-calle dpseudo-cholinesterase s which occuri nbloo dan dothe r tissues ofma nan dothe rmammals .Th edegre eo finhibitio no fthi senzym ei sconsidere dt ob e an indicationo fa certai ndegre eo fexposure . Infis hmeasuremen to facetylcholinesteras e inhibitioni nth ebrai nca nb euse d asa bio-assa y techniquet omeasur e levelso fCholinesteras e inhibitorsi nwate r (Weiss& Gakstätter , 1974). Datao nth einhibitio no festerase si naquati c inverte­ bratesar eno treadil yavailable .Fro mth epoisonin g symptoms described fordaphnid s (Section6.4.1 )an dAsellu s aquaticus (Section6.4.2 )th econclusio nca nb edraw n thatth emod eo factio ni ncrustacean s issimila rt otha ti ninsects .Othe r esterases are inhibiteda swell .Th e enzyme aliesterasei nDaphni amagn afo rinstanc ei sstrong ­ lyinhibite db yi nviv oexposur et oorganophosphate s inconcentration s less than 1m gm " (Dortland, 1978).A sth ebiologica lmeanin go fothe r esterases thanacetyl -

12 Cholinesterasei sno tknown ,i ti sno tpossibl et ojudg eth etoxicologica lconsequen ­ ceso fthei rinhibition .

Z.Z.Z Toxicity to fish

Comparativestudie so nth etoxicit yo fdifferen tpesticide so nestuarin e (Eisler, 1970)an d freshwater fish (Macek& McAllister ,1970 )sho wtha tth eorganophosphoru s insecticidesar egenerall y lesstoxi ct ofis htha nth eorganochlorines .I nth elatte r studya nassessmen twa smad eo fth etoxicit yo fthre eorganochlorine ,fou rorgano ­ phosphorusan dtw ocarbamat einsecticide so ntwelv especie so ffreshwate rfish , belongingt ofiv edifferen tfamilies .Th e96-hou rTL ™ valuesfo rth eorganochlorine s rangedfro m2 t o13 1m gm" J,whil efo rth eothe rtw ogroup so finsecticide sthes e valuesvarie dfro move r10 0t omor etha n1 000 0m gm .Th eonl yexceptio nwa sazin - phosmethylo fwhic hvalue ssmalle rtha n1 0m gm werefoun d (Table6) .Thi stabl e showstha tther ei sa marke dspecie sdifferenc eamon gth efis hi nsusceptibilit yt o

-3 Table6 .Acut e toxicity (inm gm )o fparathion ,azinphosmethyl ,diazinon ,malathio n andparathion-methy lt oeigh tspecie so ffreshwate r fish.Mai nsourc eMace k& McAllister (1970).Othe rsource sar ereference s in(1 )Pimente l (1972)an di n (2)U.S .Environmenta l ProtectionAgenc y (1973). _, Unlessstate d otherwise thedat arefe r to96-hou rLC,. n (mgm ).

Species TemperatureParathio nAzinphos - Diazinon MalathionParathion - methyl -methyl

Pimephalus promelas 18 °C 1410 (2) 235' • 8650 8900 (fathead minnow) Lepomis macrochiris 18 °C 65 (2) 22 52 (1) 103 5720 (bluegill) (24 h 24 °C) Micropterus salmoides 18 °C 190 (2) 5 285 5220 (largemouth bass) Salmo trutta 13 °C • 4 • 200 4740 (brown trout) Salmo gairdnerii 13 °C • 14 380 (1) 170 2750 (rainbow trout) (24 h 13 °C) Cyprinus carpio 18 °C )2 695 6590 7130 (carp) Ictaluris punctatus 18 °C • 3290 • 8970 5710 (channel catfish) Perca flavescens 18 °C • 13 • 263 3060 (yellow perch)

1.Adelma ne tal .(1976 )foun d0.5 1 mgm asn oeffec -3t leveli n afecundit ytest . 2.Pimente l (1972)give sa no-effec t level (96h )o f50 0m gm ;temperatur eno t mentioned.

13 organophosphorus insecticides.Compare dwit h other freshwater organisms likecrusta ­ ceansan daquati c insects fishca nb econsidere da srelativel y tolerantt othi s class of insecticides.A sub-letha l effecto fa norganophosphoru s insecticidei nfis hwa s reported formalathion .Eato n (1970)describe dth eappearanc eo fspina l deformities inth ebluegil l (Lepomismacrochirus ) after severalmonth s exposuret o7. 4m gm ". This crippling effectdi dno t occur after elevenmonth s exposuret o3. 6m gm . The toxicological implicationso flo wdegree so finhibitio no fbrain-acetylcholinesteras e in fish,a sreporte db yWeis s (1959)ar eno tclear .

2.3.3 Toxicity to crustaceans

With2 500 0specie sth ecrustacean s forma larg ean dvarie d groupo finvertebrat e organisms.Amon gth elarge r speciesar ecrabs ,lobster san dshrimp sbu tfa rmor e abundantar eth esmalle r species.A classificatio no fcrustacean s which occuri n freshwateri nth eNetherland si sgive ni nAppendi xA .Man yo fthes e represent impor­ tant food organismsfo rfish ,bot hmarin ean dfreshwate ran dtherefor e forma n essential linki nth efoo d chaino faquati c life (Crosby& Tucker , 1966). Thisproba ­ blyi son eo fth ereason swh yrepresentative so fthi sclas shav e frequentlybee nuse d todetermin eth etoxicit yo fvariou s substancesfo raquati c life.Th efamil y Daph- niidaewit h thewell-know n generaDaphni aan dSimocephalu sha sbee n studiedmos t frequently.Especiall y Daphnia spp.ar epredominan t zooplankterswhic har ever y sus­ ceptiblet otoxi c substances likepesticides . (Sanders &Cope , 1966). Generallyth e suborder Cladocera,o fwhic h theDaphniida ear ea family ,ar emos t sensitivet oorga ­ nophosphorus insecticides.Othe rcrustacean s likeCopepod a (Muirhead-Thompson, 1971) and Ostracoda (Muirhead-Thompson, 1971;Khudair i& Ruber , 1974)ar eles s sensitive toorganophosphoru s insecticides.Th etoxicit y forAmphipod aclosel y resembles that for theCladocera .A comparativ e studyb ySander s& Cop e (1966)wit hDaphni a pulex andSimocephalu s serrulatus showed thatdaphnid s aremor e sensitivet othes e insecti­ cides than theyar et oth eorganochlorin e insecticidesan dherbicide s (Table7) . Exceptions areDDT ,TD Ean dmethoxychlo rwhos e toxicityt odaphnid si scomparabl e with thato fth eorganophosphoru s compounds. A similar trend canb eobserve d fromth etoxicit y data forAmerica n amphipods (Table 8).Th e gammaridsar emor e sensitivet oorganophosphate s thant oorganochlo - rines,wit hth eexceptio no fDDT ,TDE ,methoxychlo ran daldrin . Sucha clea rdiffer ­ encewa sno t foundb yLüdeman n& Neuman n (1962)fo rth eEuropea n speciesCarinogam - marus roeselii.However ,thi sdifferenc emigh t have beenobscure db yth eshor t time ofexposur e (24hours )an dth erelativel y largeconcentratio n intervals thatwer e usedi nthi s study.Therefor en oaccurat e assessmento fth eLC5 0coul db emade .Fo r G. pseudolimnaeus subacute toxicity datawer e established ina 3 0da ytestin g period. Noeffec t concentrations amountedt o0.20 ,0.1 0an d0.00 8m gm ~ fordiazinon ,azin - phosmethylan dmalathio n respectively, indicating that evenver ylo wlevel sma yb e hazardous (U.S.Environmenta l ProtectionAgency , 1973).Th edat agive nb yLüdeman n&

14 Table7 .Acut etoxicit yo fselecte dorganophosphoru s andorganochlorin e insecticides fortw ospecie so fCladocer a (95%confidenc e limits inparentheses) .Figure s represent 48-hourEC. ,immobilizatio nvalue sa t 15.6 C (mgm ). Source:Sander s& Cop e (1966).

Insecticide Organism

Daphniapule x Simocephalus serrulatus

Organophosphates: Baytex 0.80 (0.56-1.1) 0.92 (0.66-1.3) Diazinon 0.90 (0.67-1.2) 1.8 (1.4-2.2) Dibrom 0.35 (0.22-0.78) 1.1 (1.0-1.3) Dichlorvos 0.066(0.049-0.088 ) 0.26 (0.16-0.42) Dipterex 0.18 (0.13-0.25) 0.70 (0.56-0.87) Guthion 3.2 (1.8-5.8) 4.2 (2.9-6.1) Malathion 1.8 (1.4-2.4) 3.5 (2.6-4.8) Parathion 0.60 (0.39-0.79) 0.37 (0.23-0.57) Phosdrin 0.16 (0.11-0.22) 0.43 (0.33-0.57) Phosphamidon 8.8 (6.2-12.0) 12.0(8.0-18.0 )

Organochlorines: Aramite 160(110-220 ) 180(120-270 ) Aldrin 28(20-39 ) 23(17-30 ) Chlordane 29(23-36 ) 20(12-32 ) Chlorobenzilate 870(690-1100 ) 550 (460-650) DDT 0.36 (0.28-0.47) 2.5 (1.9-3.3) DDD (TDE) 3.2 (2.3-4.4) 4.5 (3.1-6.6) Dieldrin 250(220-290 ) 240(200-280 ) Endrin 20(13-32 ) 26 (18-36) Heptachlor 42(21-63 ) 47(32-68 ) Lindane 460(390-550 ) 520 (450-600) Metoxychlor 0.78 (0.57-1.1) 5 (3.8-6.6) Toxaphene 15(11-20 ) 19(12-29 )

Guthion• azinphosethy l

Neumann (1962)show ,tha tfo rbot hth eorganochlorin ean dorganophosphoru sinsecti ­ cidesth eisopo dAsellu saquaticu s isgenerall ya ssusceptibl ea sC .roeselii ,an d alsotha torganochlorine sar emor etoxi ct othi sisopo d thanorganophosphates .Onl y thelas tconclusio ni si nlin ewit hth etoxicit ydat afo rAmerica nspecie s (Table8) . Onth ewhol ether ear efe wdat areferrin gt oexposur eperiod slonge rtha n9 6 hoursan d Ifoun dn odat aa tal lo neffect so fthes einsecticide so nreproduction .

2.3.4 Toxicity to aquatic insects

Ofth eaquati c insects,som ear etrul yaquati ci nth esens etha tthe yliv ei n thewate rthroughou tthei rlife :The yonl yleav eth ewate rt ofl yfro mon ewate rbod y toanothe r (e.g.Heteroptera ,Coleoptera) .Other sonl ypas son eo rmor estage so f theirlif ei nth ewater .Eithe rthe yliv ei nth ewate ra slarva e(Ephemeroptera , Odonata),a slarva ean dpupa e (Diptera)o ra slarva ean dimagine s (certainColeopte -

15 Table8 .Acut e toxicityo fselecte dorganophosphorou san dorganochlorin e insecticides toNorth-America nspecie so f Isopodaan dAmphipoda . Figuresrepresen t the96-hou rLC5 0value s (mgm~3) . Source:Reference s inU.S .Environmenta lProtectio nAgenc y (1973).

Insecticide Organism

Isopoda Amhipoda

Asellusbrevicaudu s Gammarus lacustris G.fasciatu s

Organophosphates: Azinphosmethyl 21.0 0.15 0.10 Diazinon . 200 . Dichlorvos . 0.50 0.40 Dursban . 0.11 0.32 Fenthion 1800 8.4 110 Malathion 3000 1.0 0.76 Naled 230 110 14 Parathion 600 3.5 2.1

Organochlorines: Aldrin 8 9800 4300 DDT 4 1.0 0.8 Dieldrin 5 460 600 Chlordane , 26 40 Endrin 1.5 3.0 0.9 Heptachlor . 29 40 Lindane 10 48 10 Methoxychlor 3.2 0.8 1.9 Toxaphene . 26 6

ra).Wit hregar dt othi sgrou po finsect smos ttoxicit ystudie swit hinsecticide s havebee nmad eo nDiptera .Severa lo fthes einsecticide swer edevelope despeciall y forapplicatio ni naquati cenvironment s inorde rt ocontro lbitin gan dnon-bitin g mosquitoesan dflie sb ydestructio no fthei rlarva ei nth ewater . Inthes etoxicit ystudie sth eDipter awer etarge torganisms ,an dtherefor emos t datarefe rt o2 4hou rLC9 0value san dn otoxicit ydat afo rDipter aar eliste di nth e WaterQualit yCriteri a 1972o fth eU SEnvironmenta lProtectio nAgenc y (1973),wher e onlynon-targe torganism sar ementioned .Whe nwate rbodie sar eunintentionall yconta ­ minatedwit h insecticides,however ,thes elarva ed ohav et ob econsidere da snon - -targeta swell ,sinc ethe yfor ma nintegrate dpar to fth eaquati cecosystem .I ti s thereforecorrec ttha tLüdeman n& Neuman n (1962)include dDipter alarva ei nthei r studieso ntoxicit yo finsecticide s tonon-targe taquati corganisms .The yfoun dth e

24hou rLCr n forChironomu splumosu st ob e 12,29 ,10 5an d 11m gm formalathion , parathion,diazino nan dchlorthio nrespectively .Thes evalue swer eo fth esam eorde r asthos efo rth eorganochlorin e insecticidesthe ytested .Chaoboru splumicorni swa s foundt ob eles ssensitiv et obot hclasse so finsecticides . Mullae tal .(1969 )foun d that2 4hou rLCS 0value sfo rchironomi dlarva erange d

16 Table9 .Toxicit yo fselecte dorganophosphoru s andorganochlorin einsecticide st o severalspecie so fstonefl ynaiads .Figure srepresen t96-hou r LCCQ values (mgm ). Source: (1)Sander san dCop e (1968); (2)Jense nan dGaufi n (1964).

Insecticide Organism

Pteronarcys Acroneuria A.lycoria s Claassenia californi :a pacifica sabulosa (1) (2) (2) (0 (O

Organophosphates: Azinphosmethyl 1.5 22 8.5 . Diazinon 25 . . . Dichlorvos 0.10 • . . Dursban 10 . . 0.57 Fenthion 4.5 26.5 5.1 . Malathion 10 . . 10 2.8 Naled 8.0 . . • . Parathion 36 30 1.5

Organochlorines: Aldrin 1.3 180 200 . Chlordane 15 . . . DDT 7.0 . . 3.5 Dieldrin 0.5 39 24 0.58 Endrin 0.25 2.4 0.32 0.76 Heptachlor 1.1 . . 2.8 Lindane 4.5 . . . Metoxychlor 1.4 . . 1.3 Toxaphene 2.3 • • •

from0.4 2 (Dursban,i nChironomu ssp. )t o5 4m gm ~ (fenthion,i nGoldichironomu s holoprassinus).

Forcomparison :th e2 4hou rLC 5Q valueso fstonefl ynaiad s (Pteronarcyscalifor - nica)fo r1 9organophosphoru s insecticidesrange d from1 4t o250 0m gm withth e majorityi nth e14-5 6m gm~ 3range .(Sander s& Cope ,1968) .Stoneflie sar eth emos t intensivelystudie dfreshwate r insects ininsecticid estudies .Th eresul to ftw ocom ­ parativestudie sar esummarize d inTabl e9 .Th evalue spresente db yJense n& Gaufi n (1966)ar esubstantiall yhighe rtha nth ecorrespondin gvalue so fSander s& Cop e (1968).Th elatte rauthor sattribut ethi st odifference s intestin gmethods ,tempera ­ ture,siz ean dag eo fth enaiads ,althoug hmos to fthes edifference scanno tb esee n fromth earticl eb yJense nan dGaufin .Difference si nlif ehistor yo fth enaiad sa s wella sth eus eo femulsifier si nth eJense n& Gaufi ntest sals oma yhav eplaye da role.Acut ean dsubacut etoxicit ydat afo rsevera lothe rspecie so faquati cinsect s aresummarize di nTabl e10 .Th edat aindicat etha ti nothe raquati cinsec tfamilies , speciesma yb epresen twhic har ever ysensitiv et oorganophosphoru sinsecticides , especiallywher eexposur ei slon gterm . Asfo rcrustaceans ,virtuall yn oinformatio nexist so nlong-ter meffect san d reproduction (e.g.pupation ,emerging) .

17 — es m — CM

-* CM GO 0) CS O O) u O ON o •H l/V-' M •UCJ 3 CS m O tJ "O tu /-N x: CT\o to >> « i ö ca o « ö rn > ^-N *H *aO tl

H U ß^

3 W 3 M - <ü •-« O OO nj j= mc > w 1-9 J* c O

O 3 fi oo

"4-1 fi o to o ai u>» oC 4uJ uO •H ü) Q) Ü

•o .,4 e 3 4J 4-> > o 3 3 O s c CO O at u N-> o 1-4 CJ a» n < «c •J «o •U i-i — •«H •H a• ^4 •4-1 O& ai en 3 • P4 V4 14 m cj 0) 0) co I c E E •H o ffla ai 0) 4J o 14 X. .e 0) rö er o er» o a m < < w u& m 2. 3.S Toxicity of transformation products

Ingenera l conversionan ddegradatio no forganophosphoru s insecticides result inles sharmfu lproducts .Diptere xhowever ,i sconverte db ymil d alkalit oth emuc h more toxicDDV Pan dconversio no fphosphorothionate san dphosphorothiolothionate st o their oxygenanalogue sgenerall y increases their toxicity.O fth e transformation pro­ ductso fth ecompound suse di nth epresen t study (seeTabl e 5)onl ya fe wca ni n themselvesb eregarde da shazardous .Amon g thesear eth eoxyge n analogues.p-Nitro - phenoli sa methaemoglobi ninduce r (Smith,1974 )an dma y causea toxicologica l stress either directlyo rb yth eformatio no fnitrite . Bender (1969)i nstudie swit h the fatheadminno w foundth e knownbasi c hydrolysis product diethylfumaratet ob emor e toxic than themothe r compound.H eals onote da certain synergistic effect forsom emetabolite sbu tcalculate d thatonl ydiethyl ­ fumarate couldb eproduce di nsufficien t quantitiest oproduc eth esynergisti c effect innature .Dat apertainin gt oth etoxicit yo fmetabolite so fazinphosmethy lan d dia- zinoni naquati c environments areno treadil yavailable .Th etoxicit yo fmetabolite s ofazinphosmethy l hasbee n indicatedb yth eWH Oa sa nare afo rth efutur e research (WHO/FAO,1974) .O fth e diazinonmetabolit e IMPHi tca n onlyb esai dtha ti ti s rapidly convertedt ocarbo ndioxid e (Sethunathan& Pathak , 1972).

2.4 CONCLUDING REMARKS

Thedat ai nth eforme r sections clearlysho w thatcrustacean san daquati c insects arever ysusceptibl et oorganophosphoru s insecticides.Fis har egenerall y less sus­ ceptible.Anothe r importantobservatio ni stha t almostal ldat a refert oacut etoxici ­ ty studieso fn omor e thanfou r days.Thi s implies thatpossibl e long-term effects including thoseo nreproductio nar e largelyunknown . From toxicitydat aavailabl ei tcanno tb econclude d thatorganophosphoru s insec­ ticidesar eles stoxi ct oaquati corganism s thanorganochlorines .However ,organo ­ phosphorus insecticidesd ohav eth eadvantag e that theyd ono t showbiomagnificatio n toan yappreciabl e extent.Als o theyappea rt ob emor e easilydegrade db yprocesse s sucha schemica l hydrolysis andmicrobia l breakdown resultingi nshorte rexposur e times.Especiall y thegenerall yhig hp Hvalue si nth e surfacewater si nth e Nether­ landswil l favour chemical hydrolysis.I nthi srespec ti tseem s thatth econtinuou s presenceo forganophosphoru s compoundsi nthes ewater sha st ob eattribute dt oconti ­ nuous immisionrathe r thant oretarde d degradation. Accordingt oth etoxicit ydat ath econcentration si nth esurfac ewater s inth e Netherlandsma yrepresen ta toxicologica l risk forCladocer aan dcertai naquati cin ­ sects.Sinc eadsorptio nt osuspende dparticle san dbotto mmu da swel la sbioconcen ­ trationi nalga eha sbee nreported ,th e questionremain swhethe ro rno tcertai nothe r groupso forganism sar erunnin ga nextr a risk.Suc h groupsmigh tb eth efilterfeeders , filtering suspendedparticle sfo rfood ,organism s that scrape sessilealga e from

19 stones,plant san dothe r surfaces (e.g.certai nCladocera ,Cyclopoida ,Ephemeroptera ) and species that feedo ndecayin gorgani cmatter ,o rburro w inth emu d likeTubifici - daean d certainChironomida elarvae .

20 3 Methods for the measurement of organophosphorus insecticides

3.1 EXTRACTION

Sampleso f1 o r1. 5d m ofwate rwer e extractedwit h aliquotso f150 ,5 0an d 50c m ofdichloromethan esuccessivel yi na 2 d m separatory funnelwit ha Teflo n cork and stopcock,b yvigorou smanua l shakingfo ron eminute .Th ecombine d extracts were driedwit h anhydrous sodium sulphatean dconcentrate d ina Kuderna-Danis h evaporative concentrator ina waterbat ha t6 0C .Th econcentrat e was transferred quantitatively intoa calibrate d 10c m cylinderwit hglas s stopper.Thereafte rth edichloromethan e wasevaporate d undernitroge n flowan d thesedimen twa sredissolve di naceton eu pt o a totalvolum eo f1 c m. Thi ssolutio nwa suse d forbot hth egas-liqui dan dthin-laye r chromatography analysis.

3.2 GAS-LIQUID CHROMATOGRAPHY(GLC )

Measurements werecarrie d out on aTraco r 550ga sChromatograp h witha flam e photometric detector operatingi nbot hth ephosphoru san dsulphu rmode s (526n man d 394n mfilter s respectively).Th ephosphoru s detection wasuse dfo r quantitative ana­ lysis,whil eth esulphu r response onlyserve da sa qualitativ e checko nth einsecti ­ cides.Th ega sChromatograp hwa sconnecte dt oa Yokogaw a 3046 dualpe nrecorde r operateda ta spee do f1 cm/min .Th ecolum n consistedo fa 6 0c mlong ,2. 7m mi.d . coiledglas s tubepacke d with 4«.Silicon e SE-30an d6% SP-240 1o nSupelcopor t 100-120 mesh,obtaine d fromSupelc o Inc. (PleugerNederland ,Amstelveen) . Gas-chromatographic conditionsar eillustrate di nTabl e 11.Th esample swer e injected withmicrolite r syringesa tvolume so f5 an d1 0m m. Linearityo fth ephosphorus-respons e was checked withmalathio n (1-10 ng),para - thion (1-10ng )an ddiazino n (0-5ng) , respectively.Th eresult s indicatea goo d linear relationshipi nthi srang eo fconcentration s (Table12) . Recoverieso fth einsecticide s fromwate rcollecte d from theoutdoo rmode l ecosystemswer e assessedb yanalyse so fspike d sampleso f1 d m ofwate rwit h ali­ quotso f0. 5an d1 p go fth ecompounds ,administere d inacetone .Th erecoverie s were invariably high (Table 13)an dtherefor eth eresult so fth eanalyse swer eno tcor ­ rected.Th edetectio n limitsfo rth evariou s insecticides arepresente di nTabl e14 .

21 Table 11.Gas-chromatographi c conditions for themeasuremen t oforganophosphoru s insecticides. ProgrammeI foran ymixtur eo finsecticide s Programme II forparathio nalon e ProgrammeII Ifo razinphosmethy lalon e ProgrammeI V additional toProgramm eI t oseparat emalathio nan dparathion-methyl . 3 -1 Carrierga sN . (cmmi n ) 40 (I,II ,III ) 55(II )

Detectorgase s

3 _1 H„ (cI m mi • n i) 150 0_ (cmmi n ) 15 air (cmmi n ) 30

Programme I 180(1- 2min ) 180-250 (2-4min ) 250 (4-8min ) Programme II,I V 160(0- 8min ) Programme II 250(0- 8min ) injection block 220 detector 220

Table 12.Pea kheigh trespons efo ramoun to finsecticid e injected (10m m total injectionvolume) ,usin g thephosphoru sdetector .

Parathion Malathion Diazinon

injected response injected response injec ted response (ng) (cm) (ng) (cm) (ng) (cm)

0 0 0 0 0 0 0.20 0.31 0.21 0.40 0.20 0.64 0.40 0.72 0.41 0.56 0.40 1.00 0.80 1.40 1.03 1.10 0.80 2.06 1.60 2.72 2.06 2.00 1.00 2.56 3.20 5.56 5.14 4.54 2.00 4.68 6.40 11.40 10.28 8.82 5.00 12.70 10.00 17.21

correlationcoefficien t correlationcoefficien t correlationcoefficien t 0.9999 0.9996 0.9993

Table 13.Recover ypercentage safte rextractio no f 1d m spikedwate rsample s obtained from theoutdoo rmode lecosystems .

Insecticide Average 7. Standard Min.an d Numbero f recovery deviation max.rec . samples

parathion 99.5 2.6 96-105 9 azinphosmethyl 104.2 ' 9.5 93-120 9 diazinon 105.0 7.1 100-110 2 malathion 103.0 4.3 100-106 2 parathion-meithy l 96.5 5.0 93-100 2

22 Table 14.Detectio n limitsfo rfiv eorganophosphoru s insecticides inaceton e solution.Tota l injectionvolum e 10mnW .Detectio n limita tabou t2 x noise .

Insecticide Detectionlimi t (ng) parathion 0.4 azinphosmethyl 0.5 diazinon 0.2 malathion 0.3 parathion-methyl 0.4 1 T-T 3 Thedetectio n limit inm gm calculated for 1d m ofwate r isfoun db y dividing thesefigure sb y10 .

3.3 THIN-LAYERCHROMATOGRAPH Y (TLC)

Thin-layer chromatographywa s used fora qualitativ e confirmationo fresult s obtained with GLC. Themetho d accordingt oErns t& Schurin g (1970)wa sused ,wit htw o minormodifications - Theinsecticide swer e oxidizedb ysprayin g theplat ewit h1 5c m H bromine solution insteado fusin g brominevapour .Th esolven t systemuse d consisted of 93'.7v/ vinstea do fa 94: 6v/ vchloroform-ethe rmixture .Chromatospot swer e made by applying 20m m ofth eextract si naceton eo nprecoate d TCLplate s (Silicage l 60F 254o fMerck) . Beeswer eobtaine d from the Departmento fEntomolog yo fth e Agricultural University and storeda t-20°C .

23 O o z z o o X e /-> 0 O ^-N ••-) ^ (J - « M OU «•OU « >i'^ • 4-t « e B o ca o eu M *-' C J3 Pk O co o C >> u ceo ffl O o C T3 " 4J ca t->>> q E ca H i-I < co PM £ O ai M w G w 1 'S O S O e v-' C N N o « m s 0 o O O O eu S co c t-l 0 -— C c •H C O O -i C *H T-l •H O JS U o J5 •-I 4J TJ O O J= J= ja 0) •o a. e eu •M u no «H -r-l TJ •^ 4-1 U u "O U CO O •o •H O O «4-1 JS 3 N O 'H Ä CO CO co m r-l •-! Ij CO 4-1 «10 CO CO O U 4J r-4 i-l 1—1 O J3 -H O 3 3 CO 'H CU 3 >N ca CO !0 CO o CU i-H >iH fa H Z fa O pq O Z Z u SE X X o fa S ca z /—s

>. CO 41 1 4J .C O o1 4-1 0 CU 4J .c u CO u 0 4J CU eu û. 4J O 4-1 •H >N o •H -c u o 4-1 X 'H e J5 0 4J CU u O O J3 i 4-> 1 0 U Cl) N X 4J Pu O o. kl CO B e I -~- 4-1 'H 1 u i o CU v^ cu cu f-t i-H CU T3 O o o JC 1 jo a !"-. >* A 1 a f-i n 1 o O. B U r-l •-I .ac. •H r-. i-l co en i O -H C0 >-> co ï», 0 J^= J 5 JZ>, Ü Oi £ £ 4J 4-1 CM 3C O..H 4J jo= o; — *J o. CU -r4 • CO 0 B CU o. co w CU e 3 CO -H S i-I ~1- \-* •H V4 e f-H •H ÙO •a !•. •a n 1 >. •o c i c i ai »-»<>-l 1» 1 c CA 'H o CU O 4J >> c "O cl CU 3 J= J3 « CA JS -H o £ H 4J o a O 01 4J N woo. * O M

10 o «o J= ix e o z C " ra M oo co m pr n X X CN o CN O " O 41

— 03 O 41 CU CJ Pu i-I M JS 3 c CO O 10 H CO X

24 4 Laboratory toxicity tests

4.1 INSECTICIDES

Theinsecticide suse di nthes etest swer eparathion ,azinphosmethyl ,diazinon , malathionan dparathion-methyl .Thei rnames ,formula san dphysico-chemica lcharacter ­ isticsar etabulate di nth eTable s 15an d 16.Al linsecticide swer eo fanalytica l grade (991purity )(Pestanal ,purchase dfro mRiedl-d eHäen ,Aktiengesellschaf t Seelze-Hannover (GDR)).Theywer estore da t-2 0° Ci nth edark .

4.2 TESTSPECIES ;ORIGIN ,REARIN GAN DMAINTAININ G PROCEDURES

OnlyDaphni amagn awa sculture d inth elaboratory .Al lothe rtes tspecie swer e eithercollecte d inth efiel do rfro mth eoutdoo rmode lecosystems .

Table 16.Physico-chemica lpropertie so fth eorganophosphoru sinsecticide suse di n thisstudy . Sources: (1)Merc k Index (1976), (2)Marti nan dWorthin g (1977), (3)Th eChemagr o DivisionResearc h Staff (1974).

Insecticide Molecular Melting po int Vapourpressur e Solubility (2) mass(1 ) °C(1 ) (2)(mPa ) -3 parathion 291.27 6 5.0 at2 0° C 24g m inwate ra t 25°C ;solubl e inman y organicsolvent s azinphosmethyl 317.34 73- 7 4 <51a t2 0° C 14g m'l at 15° C(3 ) 29g m" ^a t2 5° C(3 ) 47g n i at3 5° C(3 ) soluble inmos torgani c solvents,excep t aliphatics -3 diazi 304.36 83- 8 4 186a t2 0 C 40g m atroo mtem ­ perature;solubl ei n many organicsolvent s -3 malathion 330.36 2.9 5.3 at3 0' 145g m atroo mtem ­ perature;solubl ei n many organicsolvent s parathionmethyl 263.23 37- 3 8 1.29 at2 0 55-60g m" 3 at2 5°C ; 35- 3 6 solublei nmos torgani c solvents

25 4.2.1 Daphnia magna Straus

Thedaphnid swer eculture di na nartificia lmedium .A smediu mth eStandar dTes t Medium (STM)accordin gt oFrea r& Boy d (1976)wa sused .Initiall ya modifie dFlücki - germediu m (Flückiger& Flück ,1949 )wa sused ,bu tthi swa sabandone dbecaus epreci - patesoccurre d inth estoc ksolutions .Th eST Mwa sprepare d in1 0d m glassbottle s withglass-distille dwate rb yaddin g 100cm 3 20k gNaHCO ,nf 3, 100cm 3 44.4k g -3 3 -3 3

CaCl2.6H,0m and1 0c m 2.6k gK^SO ,m toa tota lvolum eo f 10d m .Th emediu m wasthe nvigorousl yaerate dovernigh tan dquietl yaerate dfo ra tleas t2 4hour sprio r touse .Characteristic so fbot hth eST Man dmodifie dFlückige rmediu mar egive ni n Table17 . Chlorellapyrenoidos awa sreare d forfoo dusin ga nutrien tsolutio nan dproce ­ dureaccordin gt oth eDutc hStandardizatio n Institute (1976).Th ealga ewer e concentratedb ycentrifugatio nan dth epelle tresuspende di nSTM .Th econcentratio n wasestimate db ygraphi cinterpolatio no fth eextinctio na t54 0mat a 1:25dilutio n ofthi ssuspensio ni nSTM .Th estoc ksuspensio nusuall yha da concentratio no fabou t

Table 17.Compositio no f theStandar dTes tMediu m (STM)an d themodifie d Flückiger Medium.

Salt STM (g m 'h Mod.Flück .Med . (gm-3 )

NaHCO. 100 200

CaCl2.6H20 187. 5 444 26 K2S04 MgS04.7H20 18.5 KCL 6 Na.HPO..2H.0 23 2 4 2 KHPO. 2 4 CaCO 3 2

Table 18.Feedin g schedule forD .magn aStrau swit h themonocellula r algaChlorell a pyrenoidosa.

Ageo f the daphnids Number of alp;alcell s DenDensits y of thedaphnid s inday s given in1 0» cells/ intth l ecultur evessel s dm3p e r2 ! i de iphnids per (dm 3

0-2 1 1000 3-4 2 1000 5-6 4 1000 7- 8 6 25 8-9 8 25 >10 10 25

26 Figure 1.Sketc ho fa cultur evesse luse d for therearin go fDaphni amagna .Whe n ( )th evesse l isdarkene d and thewatc h glassremoved ,th edaphnid s swimdow n into thebottle ,whic hca nb escrewe dof fafte r theta pi sclosed .Whe nth ewatc h glassi sreplace db ya nylo nsiev e (1mmmesh )onl y theyoun gdaphnid sca nreac h thebottle . 1- all-glas scultur evesse l (10d m) 2- ai r inlet;on ebubbl eever y twosecond s 3- watc hglas s 4 - tap with Teflon screw-cork 5 - glass bottle 6 - screw couplings 7 - perspex top

10 cells/m .Th ealga ewer estore di nth edar ka t 1 C.Alga ethu sprepare dan d storedremaine dviabl efo ra tleas t5 days .Th edaphnid swer efe ddail yaccordin gt o theschem egive ni nTabl e 18.The ywer ereare d in1 0d m vessels (Fig.1)i nS - 7 d m ofmedium .Th edaphnid swer ekep ta ta densit yo f2 5daphnid sdm ". Thevessel swer ecleane dtwic ea wee kan dth eyoun gremoved .Thereafte rth eol d mediumwa sfiltere dove ra siev ewit ha mes hsiz eo f0. 1m man d2 0pe rcen to fthi s oldmediu mwa suse dt omak eu pth ene wmediu m (onepar tol dsupplie dwit h4 part s newSTM) .Daphnid swer ekep tunti lth eag eo ffou rweeks . Forth etoxicit ytest sonl ydaphnid syounge rtha n2 4hour so fag ean dwit h motherso f2 4+ 4 day sol dwer eused .

27 4.S.2 Other organisms

Asellus aquatiaus L, This speciesbecam eavailabl ei nlarg enumber si nNovembe r197 6 duringth eroutin ecleanin go fth eME S. Onl yspecimen s fromth euntreate dME Swer e usedfo rtesting .Specie s identificationbase do nth epatter no fth ehea d shield showed thatal lspecimen sbelonge dt oth especie sA .aquaticus .The ywer ekep ta t roomtemperatur ei nlarg epolythen econtainer so f8 0x 4 0x 1 5c mfille du pt oa heighto f1 0c mwit hSTM . Elodeanuttallii ,Potamogeto ncrispu san dsom e smalldea d fish fromth etrou t hatcheryi nth elaborator ywer esupplie dfo rfood .

Cloeon dipterum L. Thelarva eo fthi smayfl y specieswer ecollecte d froma loca l pond.The ywer ekep ti nplasti c aquaria (60x 3 0x 3 0cm )wit h5 c mST Man dElode a assubstratum .Fo rfoo dChlorell apyrenoidos a fromth estoc k suspensiono fth e Daph- niacultur ewa sprovide da sa fil mo nth e bottom.Temperatur ewa skep ta t1 8+ 2 C. Specimenswer e identifieda tth een do fth e test,accordin gt oMaca n (1970).

Chaoborus arystallinus (de Geer) Thelarva ewer ecollecte d froma smal l localcanal . Theywer ekep ti n2 0d m allglas saquari a filled withST Ma troo m temperature.Th e larvaewer efe do nyoun gdaphnid s fromth ecultur ean dlarva eo fArtemi asalina . All specimenswer e checked individuallyaccordin gt oJohannse n (1973)a tth een do fth e tests.

Other teat species Allothe r specieswer ecollecte di neithe rth euntreate dME S (Culex,Herpobdella ,Tricladida )o rth e pondreferre dt oabov e (Notonecta,Cloeon , Sigara).unles sstate dotherwise ,the ywer ekep ti nST Ma t18+ 2 C,supplie dwit h foodan dsubstratu mwher enecessary .

4.3 ASSAYMETHOD S

4.3.1 General test-conditions

All toxicitytest swer econducte d ina climati c chambera t18+ 2 C.Th etempe ­ ratureo fth emediu mi nth etes tbeaker srange d from 17-19 C.Th eligh t regimewa s 14hour sligh tan d1 0hour sdark .I nmos t testsST Mwa sused ,bu tsom eo fth eearl y testswit hD .magn awer econducte di nmodifie d Fliickigermedium .Th etest swer eru n in1 d m glassbeakers ,fille dwit h0. 8d m STMan dcovere dwit ha watc hglass . The testmedi awer eprepare da sfollows .O feac h insecticide stocksolution swer emad e of80 0g m " ingredienti nacetone .Thes e solutionswer edilute d 1:1000wit hSTM .A n

1 MESstand sfo rMode l ecosystem,se esectio n6.2. 1

28 aliquotwa spipette dou to fthes edilute d solutions intoth etes tbeake r containing 0.4d m STM.The nth evolum ewa smad eu pt o0.7 5d m withSTM .Afte rth edaphnid s were transferredt othi sglas sbeake rth evolum ewa sadjuste dt o0. 8d m withSTM . Ineac hexperimen ttw ocontrol swer e run,on e withoutaceton ean dth eothe rwit hth e highest acetoneconcentratio nuse di nth etes trange ,bu t nevermor e than2 5gm . Theaceton econcentratio ni nal l insecticide concentrationso fth etes t rangewa s adjustedt otha ta tth ehighes tconcentratio no finsecticide . Four typeso ftest swer eperformed : - acute toxicity testsyieldin gth e48-hou r EC™ - 8ub-aoute toxicity testsprovidin gth e3-wee kEC ™ andfo rD .magn aals oth e reproduction rate - comparative toxicity testsi nwhic hal l five insecticideswer e simultaneously testedi nthre econcentration sfo rtes tperiod su pt othre eweek s - range-finding toxicity testsyieldin gth e4 8o r96-hour sEC ™ valuefo rth especie s whichwer edifficul tt omaintai nfo rlonge rperiod so ro fwhic honl y limited numbers wereavailable . Inal l sub-acute tests themediu mwa srenewe d everyMonday ,Wednesda yan dFriday . Inth esub-acut e testswit hdaphnid s themediu mconsiste do fol dmediu m fromth e Daphniacultur ean dne wST Mi na rati oo f1: 3(v/v) ,sinc e testsperforme d with10 0 percen t freshmediu m resultedi na considerabl emortalit yi nth econtrol san dbrea ­ kingo fth esecon dantennae . Inth eacut ean drange-findin g teststh eorganism swer eno t fed.Al lspecie s exceptth edaphnid swer e acclimatizedfo ra tleas t4 8hour sprio rt otestin gi nST M at1 8+ 2 °C ,unles s statedotherwise .Durin g thisperio d theywer eprovide d with substratuman dfoo da dlibitum .

4.3.2 Species-specific teat-conditions

Daphnia magna wasfe ddail yaccordin gt oth esam eschem ea suse di nth ecultur e (Table 19).Th eamoun to ffoo dwa scorrecte d formortalit yonl ywhe n themortalit y ratewa smor e than1 0pe rcent .Offsprin g wasremove dan dcounte dwhe n themediu m was refreshed.

Asellus aquaticus wasacclimatize di nST Mfo ra tleas ton e weeki nth eclimati c chamber.I nth etes ta fe wsprig so fElode awer euse da ssubstratu man dfood .Dea d aselliwer e removedan dthei r lengthsmeasured .Sometimes ,however ,thi swa simpos ­ sibledu et oscavengin gb yth elivin gaselli .

Cloeon dipterwn larvaewer e acclimatizedfo ra tleas ton e weeki nST Ma t1 8+ 2 °C . Chlorellapyrenoidos a wasapplie da sa fil mo nth ebotto mo fth ebeake rglass .A sprigo fElode awa sprovide da ssubstrate .Dea d specimens were removed, identified accordingt oMaca n (1970)an dthei rbod y lengthswer emeasured .Sometimes ,however ,

29 33 33 H H H H f-i m to ta w c c« C c O o •H C •H 4-1 O 4J eu Os N •>u . 4->) . 4J M •H l-t •r-t •r4 C t-t H-HT3 i-l •H •H es X) O eu B 4-1 X> 4J O IJ 4J u U« a O h e a rJ u U o O o o .-H g S S S tw •H

U 'H O c a o< a a a a m c u 3 3 3 3 3 3 3 XI (U U 6ÛH T3 T3 T3 T3 'O 13 -O U] (A •o S 00 kl n c a 0) M 'r4 3 o o a- -t-t « -o o

eo I at «w c00 1c3 CU -H M »4-t

I 'r4 r-t . a ai 4J (0 4-t en e •H d 3 -H x: 4-1 x: u •H X X X X X XX XX -a

X X X X X

r-l >> r-t r-4 t-t i-t >.J= J= *J X>T . J>.= J>=> x>: * 4J 0) 4-1 4-1 4J 4-1 ai S 0) di O CU en esse e S C g C S C S c r4 U r-l M -4 H -r* W 'H tl -H u o ai eu N eu cu •«H eu N CU N CU N CU N CU CM o *-> o. « a e •o a a O. cö O. CU O. « a 0 » C 3 C

f-t XI 4J «1 a 3 3 o tn 4J 00 CU O 3 c «Vd X O r-t o CU 5) 4J 00 r o •r4 3 X • a z VJ O

30 thiswa simpossibl esinc ethe ywer epartiall yeate nb yth especimen swhic hwer estil l alive.

Chaoborus arystallinus wasacclimatize dfo ra tleas t4 day si nST Mi nth eclimati c chamber.Tw ohour sbefor eth etest sstarte dthe ywer esupplie dwit ha nabundanc eo f newlyhatche dArtemi alarvae .I nth eexperiment sonl yspecimen swit ha ful lintestin e wereuse da scoul db esee nfro mth ereddis hcolou rdu et oth econsumptio no fth e Artemialarvae .I nth esub-acut etest sthe ywer efe dtwic edail ypredominantl ywit h daphnidsfro mth eculture . A surveyo fth etest sconducte dwit hth edifferen ttes tspecie s isgive ni n Table 19,togethe rwit hsom etes tconditions .

4.3.3 EC,„ determination

Thepercentag eo feffec tpe rconcentratio nwa splotte do nlogarithmic-probabili ­ typape rfo rdeterminatio no fth eEC ™ valuefo rth edifferen ttim eintervals .Th e methodo fLitchfiel d& Wilcoxo n (1949)fo revaluatin gdose-effec texperiment swa s usedt odetermin eth econfidenc elimit so fth eEC™ . Ifth enumbe ro fpoint sbetwee n 0an d 100?ower eno tsufficien tt oappl ythi smethod ,a graphica l interpolationwa s carriedou tbetwee ntw oadjacen tpoints .I nth etable sth econcentration scorrespond ­ ingwit hthes epoint sar epresente dbetwee nsquar ebrackets . Theresult so fth ecomparativ etest sar epresente di ngraphica lform ,b yplottin g medianeffectiv etim eagains tconcentration .Thi smedia neffectiv etim e (ET™)i sth e timea twhic hhal fth etes tpopulatio n ina certai npesticid econcentratio nexhibit s therelevan teffect .Usuall ythi sET ™ valuei sfoun db yinterpolatio no nlog-probali - typape rbetwee ntw osuccessiv emoment so fobservatio nan dthei rcorrespondin gper ­ centageso feffect .

4.4 RESULTS

4.4.1 Daphnia magna Straus

Symptoms of poisoning Thesymptom so fpoisonin gb yCholinesteras e inhibitorswer e describedb yWasserburge r (1952)an dParke re tal . (1970).Fro mth eman ytest scarrie d outi nthi sstud yth efollowin gconsecutiv estage sca nb edescribed . 1.Hyperactiv e swimming:th edaphnid shan gforwar ddurin grapi dswimming ,instea do f havingthei rnorma lvertica lattitude . 2.Uncoordinate dswimming :the ymak ecircula rmotion si nal lplane san dforwar dan d backwardsummersaults . 3.Paralysis :th eanimal sli eo nth ebottom ,bu toccasionall yhav eshor toutburst s ofuncoordinate d swimmingactivity .Som eo fth edaphnid ssho wtremors . 4.Tremors :th edaphnid sstic kt oth ebotto mwit hheav yan dconstan ttremor so fth e

31 secondantennae ,sometime srotatin gslowl yaroun dthei raxis .I nthi sstag ethe y usuallyremai ni non eplace . 5.Immobilization :th eanimal sar egenerall yimmobil ewit hsom eincidenc eo fantenna l movement. Ingenera lal lindividual sexhibite dal lstages ,bu ta thig hconcentration ssom eo f thestage sma yhav ebee nmissed . Themeasuremen to feffec twa smainl ybase do nparamete rimmobilization ,corres ­ pondingwit hstage s4 an d5 ,althoug hoccasionall ya nindividua lexhibitin gstag e3 mayhav ebee nincluded .Stage s4 an d5 wer einvariabl efoun dt ob emoribun d inbot h theacut ean dsub-acut etests .Whethe ro rno tth efirs ttw ostage sar et ob econsider ­ eda smoribun dprobabl ydepend so nth etim einterva lafte rwhic hthe yar eobserved ; theshorte rth einterval ,th eearlie ra stag ewil lb emoribund . Thefloating ,describe db yParke re tal . (1970)wa sneve robserve dan dprobabl y hast ob eattribute dt osurface-activ ecompounds ,unintentionall yadde di ncombinatio n withth einsecticide suse di nthei rstudies .

Toxicity data Theresult so fth eacut ean dsub-acut e testsar egive ni nTabl e2 0an d Fig.2.Th edat asho wtha tdifference sar eminima lbetwee nduplicat eseries ,bu ttha t differenttes tserie swit hth esam ecompoun dma yresul ti nEC ™ valuestha tdiffe ra

-3 Table20 .Estimate d £CJQ immobilizationvalue sin gm forD .magn aexpose dt o organophosphorusinsecticides .Temperatur e 18+^2 °C.Al l testswer ecarrie dou ti n duplicate.Figure s inparenthese s representP « 0.0 5 confidence limits.Figure s betweensquar ebracket srefe r toconcentratio n intervaluse d forinterpolatio n (see Section4.3.3) .

Insecticide 48 hourEC. . 21da yEC^ .

acute sub-acute

parathion 1.3 (1.1-1.6) 0.99 (0.72-1.4) 0.33 (0.3-0.7) 1.3 (1.2-1.6) 0.91 (0.60-1.2) 0.35 (0.3-0.7)

1.4(1.2-1.6 ) 1.3 (1.2-1.5)

0.7 (0.5-0.9) 0.7 (0.5-0.8)

azinphosmethyl 1.6 (1.5-1.7) 1.6 (1.2-2.1) 0.28 (0.22-0.30) 1.6 (1.5-1.7) 1.5 (1.2-2.0) 0.26 (0.22-0.35)

diazinon 1.5 (1.3-1.6) 0.8 (0.7-1.0) 0.22 (0.2-0.3) 0.7 (0.6-0.9) 0.24 (0.2-0.3) malathion 2.1 (0.14-14) 1.6 (1.2-2.4) 0.34 (0.3-0.6) 2.2 (1.6-3.1) 1.7 (1.2-2.4) 0.38 (0.3-0.6)

parathion-methyl 9.1 (7-14) 8.0 (4.8-6.9) 2.0 (1.2-2.4) 8.5 (6-12) 7.8 (4.8-9.6) 2.0 (1.2-2.4)

32 3 EC50l m gm" ) 10-

-i r- 4 12 16 20 timeldays) Figure2 .ECj gvalue sfo rfiv eorganophosphoru s insecticidesversu s timeo fexposure . Valueswer eobtaine db ycalculatio no rinterpolatio n (Section 4.3). (A)parathion , (X)azinphosmethyl ,(• ) diazinon,(o )malathion ,(o )parathion-methyl .

factortw oo rmore .Therefor eal linsecticide swer eteste di nparalle l tests,t oob ­ taincomparabl eresults .(Fig.3) . Inal ltest sparathion-methy lwa sth eleas ttoxi co fal lfiv ecompounds ,follow ­ edb ymalathion .Th emos ttoxi ccompound swer eparathion ,azinphosmethy lan ddiazinon . Hardlyan ydifferenc ei ntoxicit ywa sfoun damon gt elatte rcompounds ,althoug ha preliminaryconclusio n fromth ecomparativ etes tmigh tb etha tdiazino ni sslightl y moretoxi ctha nth efirs ttw oinsecticides .I twa sals ofoun dtha tdifference sbecam e smallerwit hlonge rexposur eperiods .Al lcompound sexcep tparathion-methy l canb e considered asver ytoxi cfo rDaphni amagna . Datao nreproductio nan dth eapproximat eno-effec tlevel sar epresente di n Table21 .Reproductio nwa sonl yimpaire dwit h0. 4m gm azinphosmethyl.The nth e numbero fyoun gproduce dwa smarkedl yles sthe ni nth econtrols ,whil ea tthi sleve l thereproducin gadult sdi dno tsho wan ysymptom so fpoisoning .

33 r-1 *J •U (0 O C 1-1 R 0) 0) 0) W ,Û >-* £

a c o -u J-l (Ü -U t-H

a) R ki h O n 3 C0 S B Ofl 4-1 'H .-1 tu u Pu 3 J= a •o 4J eu O — m u e•n 4J O O eu a) n kl •n j= eu u 9) a •^o •w NI •>H o u C i-l o

•H 0-,-H J=

3 t-i eu h m ev 4-1 Ü 4-1 u to J= U ci a, o ft CO •o o fi J= c J= tft u •f t — en a Cl) 'M o 3 CO O CD c .f3i eu m eu to 00 u tn eu b eu 4J -c o X p-t k> 4J 3 C o •o eu CN IM „"< ,-. ^ 4-1 ^iftf4»Ni005»0-TOO c 01 ki ooe^sotnu-i^oinr^o — OO SS'

o T) eu eu 1 a o ^ 3 S X -H g 4J c eu V4 3 eu 0 00 eu c M c eu ex •H e 4J eu tfl N u 3 u ,e eu to c u n u ki •H o to eu 3 Q cj 1 4-1 o 00 -a 4J •H 3 to (44 M >y C n o eu• Coo -1a •4*J 4J •H — u to U est o o 3 Ci. •H> rH •o -i o r-4 eu V4 a J:3* tu > o. 3 4J n C 0 •o eu 1 00 c B S ffl TJ *H c to S "J •H o 00 C -o J3 S • £ eu U O. Q O. 4J 3 e o tfl G 0 4J• •H a »4-1 T3 eu tfl N o o — o to •a eu u 3 »-j eu eu 4J < o o o e ti x •M x: o a a u eu •r4 u J= 4J 00 T3 10 4J M o es c o O 3 -M tO y-i *w 73 U eu 0 0\ O vO O"* 00 fï ^ o o*« u «3 N-ï »O S ^O CTi -* O 1—1 koi wa- eou eu •o 0) a>H u 3 c CN o O r- CM • o co CM n eu ew to eu ^* m m u-i -s* sO -3" >~' "-' >QJ K OH 4J en 3 CO eu i _ • B u 3 u-t kl c o — HJ < H C 3 >-J3 o o

34 E^ (days) 12

-•experiment terminated

4 -

0.5

0.25

X.0.7 1.4 2.8 5.6 concentrationImgm -3)

Figure 3.Comparativ e testo nth etoxicit yo ffiv eorganophosphoru s insecticidest o Daphniamagna .N omortalit ywa sobserve d in0. 7m gm~ 'parathion-methy la tth een do f theexperiment .(A )parathion ,(X )azinphosmethyl ,(• ) diazinon,(n )malathion , (o)parathion-methyl .

A lowerbirt hrat ewa sobserve dwit hdiazino nan dmalathion .However ,thi scoin ­ cidedwit htoxi ceffect si nth eadults ,whic hshowe dparalysi san dtremor so nDa y2 0 and21 ,resultin gi nunbor nan dstillbor noffspring .Th ever ylo wreproductio nfigure s atsom eo fth ehighes tconcentration swer edu et o10 0pe rcen tmortalit yo fth eadult s beforeDa y14 . Asca nb esee ni nTabl e22 ,th etoxicit ydat aobtaine d inth epresen tstud y agreewel lwit hdat afoun di nth eliterature ,i nspit eo fdifference s intemperatur e andtes tspecies .

4.4.2 Asellus aquatiaus L.

Thesymptom so fpoisonin gfoun dwer ehyperactivity ,followe db ywha tcoul db e describeda suncoordinate d 'dancing',wit hrapi dgil lmovements .A ta late rstag eth e animalsla yo nthei rbacks ,waverin gthei rlegs .Th egil lan dleg-movement sgraduall y

35 Table22 .Comparativ e toxicitieso forganophosphoru sinsecticide s forD .magn a andD .pulex .Figure srepresen tE C immobilizationvalue s inm gm (1)Value sdetermine d inthi sstud y (2)Value s reportedb ySander s& Cop e (1966). (3)Value sreporte db yreference si nU.S .Environmenta l ProtectionAgenc y (1973).

D.magn a D.pule x D.magn a D.magn a 48-hourEC, . 48-hour EC 21-day 21-day EC 50 50 18+ 2 ° C 50 15.6° C 18+ 2 §> temp.? (1) (2) (0 (3) parathion 1.3 0.60 0.34 . azinphosmethyl 1.6 . 0.27 . diazinon 1.5 0.90 0.23 0.26 malathion 2.2 1.8 0.36 0.6 parathion-methyl 8.8 • 2.0 •

-3 Table23 .Estimate d LC valuesan dno-effec t levels inm gm forAsellu saquaticu s exposed toparathio nan aazinphosmethy la t 18+ _2 C.Onl ypar to fth etes ta tth e highconcentratio nrat eo fth esub-acut e testwa scarrie d outi nduplicate .Al lothe r testswer esingle .Figure s inparenthese srepresen tP » 0.0 5 confidencelimits . Figuresbetwee nsquar ebracket srefe r toconcentratio ninterva luse d forinterpola ­ tion (seeSectio n6.3) .

Parathion Average Azinphosmethyl Average length length (mm) (mm)

48-hourL C acutetes t 20(13-30 ) 5.2 4.8 (4.0-5.6) 5.0 12(18-18 ) 3.1 48-hourL C sub-acutetes t 23(17-32 ) 4.9 6.7 (5.5-8.1) 4.4 22[ 20-40 ] 5.0 7.5 (6.2-9.2) 4.4 21-day LC50 4.8 (3.1-7. 3) 5.0 2.4 (1.7-3.5) 4.6

21-dayn oeffec t level 1.0- 2. 5 5.0 0.5 - 1.0 4.6

becameslowe runti lth einterval sbetwee nth egil lmovement sincrease dan dbecam e irregular,followe db ydeat ho fth eanimals .Mos tstage so fpoisonin gwer efoun dt o becompletel yo rpartiall yreversibl eafte rth eanimal swer etransferre d toST Mwith ­ outinsecticid eafte ra nexposur eperio do f4 8hour st oparathion .N orecover ywa s foundwit h (nine)animal sshowin ginfrequen tgil lmovements .Th eresult so fth eacut e andsub-acut etest sar esummarize d inTabl e23 .Thes edat aindicat etha tazinphosme ­ thyli smor etoxi ctha nparathio ni nal ltests .Th eno-effec tlevel sfo rmortalit y were 1.0m gm ~ and0. 5m gm forparathio nan dazinphosmethyl ,respectively .Th e levelwher en osymptom sa tal lwer eobserve dwa s 1.0m gm " forparathio nan d 0.25m gm ~ forazinphosmethyl .Fro mth eresult so fth ecomparativ e testi tca nb e concludedtha tazinphosmethy l isth emos ttoxi co fth efiv ecompound steste dan d parathion-methylth eleas ttoxi c (Fig.4).Toxicit yincrease si nth efollowin gorder : parathion-methyl,diazinon ,malathion ,parathion ,azinphosmethyl .A compariso nwit h 48-hour LCPQ valuesfo rth eAmerica nspecie sA .brevicaudu s (azinphosmethyl21.0 ;

36 ET50(days) 20 -experiment terminated o—__l*)

8 -

2 -

*8.4 0.5 -

0.25 ^V -i— 75 15 30 concentration(m grrr 3)

figure4 .Comparativ e testo n thetoxicit yo ffiv eorganophosphoru s insecticides to Asellusaquaticus .Figure sindicat eth eaverag e length (mm)o fth eorganism si n eachtes tseries .(* ) A.sudde nincreas ei nmortalit y occurred inth econcentration s '.5an d 15m gm~ 3o fparathion-methyl ,probabl ydu e toerroneou s concentrationsafte r thetes tmediu mwa srenewe da tDa y 15.(A )parathion ,(X )azinphosmethyl , (•) diazinon,(• ) malathion,(o )parathion-methyl .

malathion600 ;malathio n300 0m gm ~) indicate stha tA .aquaticu si sprobabl yfa r woresusceptibl et oth etoxi cactio no forganophosphoru sinsecticides . Fromth edat ao fLüdeman n& Neuman n (1962)i tca nb ededuce dtha tth e24-hou r

LCg0value sfo rA .aquaticu s inthes etest swoul db ebetwee n1 0an d5 0m gm ~ for -3 -3 parathion,5 0an d 100m gm fordiazino nan d 1an dS Om gm formalathion .Thes e resultssee mi nlin ewit hthos eobtaine di nth epresen tstudy .Fo rinstanc eth e 24-hourmortalit y inth eacut etest swit hparathio namounte d to2 0an d6 0pe rcen t at4 0m gm~ Jwit ha naverag elengt ho f2. 9an d5. 4mm ,respectively .

37 Table24 .Estimate dL C valuesan dno-effec t levelsi nm gm forCloeo ndipterum , exposed toparathio nan dazinphosmethy l at 18 +_2 C.Acut e testscarrie dou ti n duplicate.Figure s inparenthese s representP » 0.0 5 confidence limits.Figure s betweensquar ebracket s refer toconcentration-interva l used forinterpolatio n (see Section6.3).

Parathion Average Azinphosmethyl Average length length (mm) (mm)

48-hourI X .(acut etest ) 2.5 (1.7-3.5) 6.1 13.3 (10.8-16.4) 6.2 2.6 (2.0-3.4) 6.4 12.0(9.9-14.1 ) 6.0 48-hourL C (sub-acute test) 1.7[ 1.25-2.5 ] 6.0 6.0 11.6[8.0-16.0 ] 5.8

21-day LC50 0.38 (0.28-0.52) 6.6.00 3.4[2.0-4.0 ] 5.8

21-dayno-effec tleve l 0.15 - 0.1 6.0 2.0- 4. 0 5.8

E1"50l<3ays) 2o J*—experiment terminated

10

0.5

0.25

V 2.5 75 225 concentration(m gm" 3)

Figure 5.Comparativ e testo n thetoxicit yo ffiv eorganophosphoru s insecticidest o Cloeondipterum .Th efollowin g percentagesmortalit ywer efoun do nDa y 21:diazino n 2.5m gm~3 : 11.1%;malathio n 7.5an d2. 5m gm~3 ; 33.3%an d0 % respectively.Number s indicate theaverag elengt h (mm)o f theorganism sfo reac h testseries .(A )parathion , (X)azinphosmethyl ,(• ) diazinon,(a )malathion ,(o )parathion-methyl .

38 4.4.3 Cloeon dipterum L.

The symptomso fpoisonin gwer e roughlyth esam ea sthos e described forAsellu s exceptfo rth egil lmovement swhic hwer eno tobserve di nsufficien t detail.N oat ­ temptwa smad et oestimat e themoribun d stageo fpoisoning . The resultso fth eacut ean dsub-acut e testsar esummarize di nTabl e 24.Para - thioni sfa rmor e toxic thanazinphosmethyl ,no tonl y after acute exposure,bu tals o after sub-acute exposurean dthe nth edifferenc ei seve nmor epronounced .Th eno - effect level forparathio ni scompetitiv ewit hth ecorrespondin g valuefo rD .magna . Thecomparativ e tests showa nincreasin g toxicityi nth e ordermalathion ,diazinon , azinphosmethyl,parathionmethy lan dparathio n (Fig.5).

4.4.4 Chaoborus crystallinus (de Geer)

The firstsympto mo fpoisonin g washyperactivity ,wit hth elarva ean dpupa e rushing throughth eglas sbeake rwit h rapid spasmodican djerk ymovements .Th efollow ­ ingstag ewa s that theyhun gagains t thewatersurface ,stil lcapabl eo fdivin g from timet otime ,bu tapparentl y unablet osta ydeepe ri nth ewater . Finally both larvae and pupae lost theirbalanc ean dstarte d floatingo nthei rside .Som e individuals evertedth e alimentary canal through themouth ,a phenomeno nwhic hha s alsobee n reported forestonefl ynaiad s (Jensen& Gaufin , 1966).Thi s floatingi spermanen tan d irreversiblebecaus e allth eanimal sdi ei nthi sposition .Result so fth eacut ean d sub-acute testsar esummarize di nTabl e 25.Al llarva ewer e about1 0m mlon gan d showed littlevariatio ni nlength . Sinceapproximatel y 10-15 percen to fth e larvaeemerge d duringth esub-acut e tests,mortalitie swer e calculatedi nrelatio nt oth e numbero flarva ean dpupa e remaininga ta give npoin to ftime .Th edat a show thatparathio ni sfa rmor e toxic thanazinphosmethy li nbot hth e acutean dsub-acut e tests.Fo rparathio na no-effec t levelcoul dno tb eestablishe d since0.2 5m gm wasth elowes tconcentratio ni nth e series.Th enex t higherconcentratio n (0.5m gm )showe d 100pe rcen tmortalit ya t day1 4an d16 .Fo razinphosmethy la no-effec t levelo f2. 0m gm wasfound .Mortalit y

Table25 .Estimate dE C floatingvalue san dno-effec t levelsi nm gm forChaoboru s crystallinus exposed toparathio nan dazinphosmethy l at 18+^2 C.Figure si nparen ­ thesesP - 0.0 5 confidence limits.Figure sbetwee nsquar ebracket s refer toconcen ­ trationinterva luse d forinterpolatio n (seeSectio n6.3).

Parathion Azinphosmethyl

48-hourE C (acutetest ) 1.0 (0.75-1.4) 67 (50-89) 48-hourEC* " (sub-acute test) 1.4 (0.99-2.1) 33(22-52 ) 21-dayEC, „ 0.25 10.6[ 8.0-16.0 ] 50 0.20(0.0-0.25] 10.2(8.0-16.01

21-dayno-effec t level <0.25 2.0- 4. 0

39 ET50 (days) 20 y— experiment terminated

8-

1-

0.5

0.25

0125 —r- V 6 36 concentra tio n(m gm" 3)

Figure6 .Comparativ e testo nth etoxicit yo f fiveorganophosphoru s insecticidest o Chaoboruscrystallinus .Th efollowin gmortalit y percentageswer e foundo nDa y21 : azinphosmethyl Im gm~^ :31.6% ;diazino n 1m gm"-* :15.8% ;malathio n6 an d 1m gm"^ : 26.4%an d 20%,respectively . (A)parathion ,(X )azinphosmethyl ,(• ) diazinon,(o ) malathion,(o )parathion-methyl .

at4. 0m gm was4. 7an d 14.7pe rcen to nda y 14an d21 ,respectively .Th ecompara ­ tivetes tshow stha tparathio nan dit smethyl-analogu ear eequall ytoxic .Th eorde ro f increasingtoxicit y ismalathion ,azinphosmethyl ,diazinon ,parathio nan dparathion - methyl (Fig.6).

4.4.5 Other species

Theresult so fth eacut etest swit hvariou sothe raquati cinvertebrate sar e presented inTabl e26 .

Culex 8p. Thedifferenc ei nsusceptibilit ybetwee nlarva ean dpupa eamount s toabou t threeorder so fmagnitude .I norde rt otes tthei rreactio nth epupa ewer egentl y

40 Table26 .Acut e toxicitieso fparathio nan dazinphosmethy l tovariou sspecie so f aquatic invertebrates.Figure si nparenthese s representP • 0.0 5 confidencelimits .

Organism Criterium Exposure EC50 of effect time (hours) parathion azinphosmethyl

Culex sp. immobilization 48 1.1 (0.7-1.7) • larvae Culex sp. immobilization 48 >1000 . pupae Notonecta sp. mortality 48 5.1 (3.9-6.6) • adults + larvae Sigara sp. floating 48 2.6 (2.0-3.4) • adults + larvae 2.3 (1.8-2.8) Herpobdella mortality 96 >16 0 >160 octoculata Dugesia mortality 96 >16 0 >16 0 lugubris

touchedwit ha Pasteu rpipette .A t 1m gm ~ theyshowe da slowe rreactio ntha ni nth e control,bu tnon eo fth eindividual sdie dwithi nth etes tperiod . llotoneota sp. Althoughi ti svirtuall yimpossibl et oidentif yspecie so fHeteropter a inth elarva lstages ,i ti smos tlikel ytha tal lspecime nuse di nth etes tbelon gt o thespecie sN .glauc asinc eal ladult sfoun da tth esamplin g sitebelonge dt othi s species.Th e individualssuffere d fromextr astres sdu et oa pani cescap ereactio n uponever ymovemen ti nth etes troom .

Sigara sp. Forth esam ereaso na sgive nfo rNotonect asp .al llarva eprobabl ybelong ­ edt oth especie sS .lateralis .Th eindividual stha tstarte d floatingcoul dno tdiv e anymor ean dwer econsidere dmoribund . Thetest swit hth ethre eaforementione d specieswer ecarrie dou ti nfiltere d waterfro mNE SIV .

Herpobdella octoeulata With 160m gm parathiontwent ypercen tmortalit ywa sfoun d inthi sleec hspecies .Th eothe rindividual sa tthi sconcentratio nan dsom ea t 80m gm exhibiteduncoordinate d swimmingwhe ntouche dgentl ywit ha Pasteu rpipett e inorde rt otes tthei rreaction .Afte rexposur et oazinphosmethy lmortalit yamounte d totwent ype rcen ta t 160m gm .Uncoordinate d swimmingwa sexhibite db y individuals exposedt oth econcentration s40 ,8 0an d 160m gm ~ ,respectively .

Dugesia lugubris Bothi nth econtro lan di nth etes tconcentration s theindividual s usuallywer emotionless .The yadhere dt oth ewal lo fth eglas sbeake ran dmove donl y incidently.Thei rreactio nt ogentl estimulatio nwit ha pipett ewa sinvariabl eslo w

41 Table27 .Orde ro fdecreasin g toxicityo ffiv eorganophosphoru s insecticides tofou r specieso faquati c invertebrates,base do nth ecomparativ e toxicitytests .

Parathion Azinphos- Diazinon Malathion Parathion- methyl methyl

Daphniamagn a 1-3 1-3 1-3 4 5 Asellusaquaticu s 2 14 3 5 Cloeondipteru m 1. 3 4 5 2. Chaoboruscrystallinu s 1-2 4 3 5 1-2 1 exequ o

inal lconcentrations ,a swel la si nth econtrols .U pt oth emaximu mconcentratio no f 160m gm usedi nth etes tn oadvers eeffect scoul db eobserved .

4.5 GENERALDISCUSSIO N

Ofth efou rspecie stha twer eextensivel yteste dwit hrespec t tothei rsensiti ­ vityt oa numbe ro forganophosphoru scompound sD .magn awa sth emos tan dA .aquaticu s theleas tsensitive .Ther edoe sno tsee mt ob ea marke ddifferenc e insusceptibilit y betweenCloeo ndipteru mlarva ean dth elarva eo fChaoboru scrystallinu s inth eacut e test.Th esusceptibilit yo fth elas ttw ospecie swit hregar d tosub-acut etoxicit y andno-effec tleve lfo rparathio nclosel yresemble stha to fDaphni amagna . Itca nb econclude dfro mth ecomparativ etest stha tparathio n isth emos ttoxi c ofth efiv ecompound steste d (Table 27).Parathion-methy lappear st ob eb yfa rth e leasttoxi cfo rth ecrustaceans ,whil emalathio ni sleas ttoxi cfo rth einsec tlar ­ vae.Th etoxicit yo fth eothe rcompound sdoe sno tvar ymarkedl yfro mspecie st o

species.O fth especie sonl yteste di nth erange-findin g tests,th eparathio n EC^0 valuesfo rth einsects ,viz .Cule xsp. ,Notonect asp .an dSigar asp .ar eo fth esam e ordera sfo rCloeo ndipteru man dChaoboru scrystallinus .I tcanno tb eexclude dtha t adultsmigh tescap epoisonin gb yflyin gawa yfro ma contaminate dwate rbody .However , therei sn o informationavailabl ewhic hsupport sthi shypothesis .Fo rth eadul t leechesan dflatworm steste dther eseem st ob elittl edange ro fthei rbecomin gpoison ­ edb yorganophosphoru s insecticidesi nth elowe rrang eo fconcentration sapplie di n thepresen tstudy . Theconclusio nca nb edraw ntha tchroni cexposur et oconcentration s inth eorde r oftenth so fm gm oforganophosphoru s insecticidesma yendange r thesurviva lan d thereproductiv epotentia lo fa tleas tsom especie so fCladocer aan daquati cinsects .

42 5 Half-lives of five organophosphorus insecticides in the waterphase of freshwater aquaria

5.1 AIMO FTH EEXPERIMEN T

Theai mo fth eexperimen twa s threefold. Firstt oge ta nimpressio no fth edis ­ sipationrate s thatcoul doccu ri nth eoutdoo rMES ,an ds oarrang ea schem efo r maintaininga certai n concentration.Secon dt ocompar eth erat eo fdissipatio nfo r the five insecticides used,an dthir dt oestimat eth einfluenc eo fwate rplant san d bottom sedimento ntha t rate.

5.2 MATERIALSAN DMETHOD S

Of three8 5d m all-glass aquaria,tw o(number s 1an d2) wereprovide d witha 1 -8 c mbotto m layero fsan dan dver y finemud .Thi smateria lwa sobtaine d fromth e samepon da sreferre dt oi nSectio n 4.2.2.Th ewate ro fthi spon d was analysedfo r Cholinesterase inhibitors.However ,th eoutcom eo fthes emeasurement swa snegative . All threeaquari awer e then filledwit hwate r fromth euntreate d outdoor WS and placedi na climati c rooma ta temperatur eo f2 0+ 1 °C .I nAquari a 1an d222 0gra m (wetweight )o fElode a densawer eplanted .Th ethir d aquarium (No.3)wa sno t provided witheithe rbotto mmateria lo rwate rplant ss otha tth einfluenc eo fsedimen tan d plantso nth edissipatio n rate couldb eevaluated .Th eaquari awer e coveredwit ha Plexiglassto pt oreduc e evaporation but still permitting gasexchang ean d photo­ chemical reaction.Tw ofluorescen t daylight lampso f4 0wat teac hwer eplace d3 0c m aboveth eplasti c covero fth eaquari at ogiv e1 4hou rdiurna l illumination.Afte r a fortnighto facclimatization , thewate rwa sanalyse dfo rCholinesteras e inhibitors byTLC .Th eresult so fthes eanalyse swer enegative .Th eaquari awer e subsequently treatedwit hparathion ,azinphosmethyl ,diazinon ,malathio nan dparathion-methyl , all ata concentratio no f1 m gm ,calculate dfo rth ewatervolum e aboveth ebotto m layer. The compoundswer e injected froma stoc k solutiono f10 0m gdm " inaceton ean dsub ­ sequently stirredwit ha smal l batterymixer .On ed m aliquotswer ewithdraw n from theaquari a after 1,20 ,50 ,90 ,152 ,216 ,355 ,S2 5an d100 5hour s after dosing. Zooplanktoni nthes e sampleswa s sievedof fwit ha 0, 1m msieve ,kille di n5 pe rcen t formaldehydean dcounted . Thewate r sampleswer eextracte dan danalyse db ygas-liqui d chromatography accordingt oth eprocedure s giveni nChapte r 3.Durin gth eexperimen tp Han dth e concentrationo fdissolve doxyge nwer emeasure da tintervals .

43 5.2.1 Calculation of the half-life

Thehalf-lif etim eo fth einsecticid eca nb econsidere da sa measur e forth e rateo fdissipatio ni nth ewate rphase .Althoug hsevera lfactor scontribut e todissi ­ pation,accordin gt oGrah l (1973),th eoveral lproces sca nb echaracterize db yon e rateconstant ,resultin gi na firs torde requation : dc/dt= k c (1) inwhic h c =mas sconcentratio no fth einsecticid e t= tim e k •rat ecoefficien to fdissipatio n Integrationo fEq n1 give s

k= -I n(c/c 0)/t (2) inwhic h c„= concentratio na ttim ezero .

Figure 7-9.Rat eo fdeclin eo f theconcentratio no f fiveorganophosphoru s insecticides inth evate rphas eo faquariu m systemswit h (Fig.8,9)an dwithou t (Fig.7)botto m material andwate rplants .Curve dwer efitte d bylinea r leastsquare sanalysis . (A)parathion ,(+ )azinphosmethyl ,(X )diazinon ,(o )malathion ,(o )parathion-methyl , (•) initialconcentratio n (calculated).

massconcentratio n( m gm" 3) 1.0

0.1 -

Fig. 7: Aquarium 1; with bottom material and water plants.

0.01 200 400 600 800 1000 time (hours)

44 mass concentration(m g m"3) 1.0

0.1

0.01

1.0 -,

0.1

-[A]

Fig. 9: Aquarium 3; without bottom material and water plants.

0.01 800 1000 time(hours)

45 Thusth eslop eo fa I nc versu stim eplo tprovide sk .Th ehalf-lif eo fth einsecti ­ cidesca nthe nb efoun dwit h t,= 0.693/ k 2

5.3 RESULTS

Theconcentratio no fth efiv einsecticide s isplotte do na lo gscal eagains t timei nth eFigure s7-9 .I nthes egraph sth econcentration smeasure d att = 1 ar eno t plotted,becaus eth eresult sindicat etha ta ttha tmomen tth einsecticide swer eno t yetfull ymixe dwit hth ewate rphase .Th ecalculate dconcentration swer euse dfo rth e concentrationa tt = o .Th ederive dhalf-live sar epresente di nTabl e28 .Th eresult s showa fai ragreemen tbetwee nth eAquari a 1an d2 .However ,half-live si nthes etw o aquariadiffe rconsiderabl yfro mthos ei nAquariu m3 ,wit hth eexceptio no fth ehalf - lifefo rmalathio nwhic hi salmos tth esam ei nal lthre eaquaria .Thes edifference s canonl yb eattribute dt oth epresenc eo fbotto mmateria lan dwate rplant si nAquari a 1an d2 ,bot hbein gabsen ti nAquariu m3 .Moreove rth ehighe rp Hi nAquariu m 3migh t havefavoure dhydrolysi si nthi saquarium , (seeTabl e 29).Th epathway stha tma yhav e

Table28 .Tim ei nhour srequire d forhalvin g theinitia lconcentratio no ffiv e insecticides inth ewate rphas eo faquariu msystem swit han dwithou tbotto mmateria l andwate rplants . Aquarium 1:water ,botto mmaterial ,wate rplant s Aquarium2 :ide m Aquarium 3:water . Figuresi nparenthese sgiv eth ehalf-lif etime sa sa percentag eo f thecorrespondin g half-livesi nAquariu m3 .

Insecticide Initial concentration Half-life (hours ) (mgm"-* )

1 2 3 I 2 3

parathion 0.98 0.95 0.92 112(34 ) 122(37 ) 333 azinphosmethyl 0.98 0.95 0.92 138(28 ) 151 (31) 488 diazinon 0.98 0.95 0.92 227(63 ) 231 (64) 361 malathion 0.98 0.95 0.92 34(103 ) 41 (124) 33 parathion-methyl 0.98 0.95 0.92 78(34 ) 99(43 ) 231

Table 29,Averag ep Han dconcentratio no fdissolve d oxygeni nth eaquari adurin g theexperimenta lperiod .

Aquarium pH Dissolved oxygen (gm )

average standard numbero f average standard numbero f deviation measurements deviation measurements

1 7.4 0.23 21 8.0 1.63 25 2 7.7 0.52 22 7.6 2.29 26 3 8.2 0.21 21 9.2 0.52 22

46 contributed tothi srapi d dissipation in thepresenc e ofmu d andbiota ,ar emicrobia l degradationan dadsorptio n toparticulat ematte r and biota.Durin g theexperimen t the biomasso fElode a increased considerably,resultin g ina greate r adsorptionsurface . InTabl e 28th e relativehalf-live s forth evariou s insecticides aregive n in parenthesesa spercentage so fth ehalf-lif e inAquariu m 3.Thes e figures give anin ­ dication towha texten t thesepathway s (apart fromhydrolysis )ma yhav e contributed toth edissipatio n from thewate rphase ,viz .th e smaller thepercentage ,th e higher thecontribution .Ther e isa meaningfu l contribution forazinphosmethyl ,parathio n andparathion-methy l and,t oa lesserextent ,fo rdiazinon . Inbot h the aquariawit h plantsan d sediments theorde ro fth ehalf-live s for the five insecticides isth e same:diazino n >azinphosmethy l >parathio n >parathion-methy l >malathion . On thebasi so fth eresult so f thisexperimen ta calculation could bemad e of the frequencyb ywhic h the twopesticide sparathio n and azinphosmethyl had tob eap ­ plied toth eME S (seeSectio n 6.2.4). The results ofth e zooplanktoncounting s dono tpermi tmor e thansom eprelimina ­ ryconclusion s (seeAppendi xB) .

47 6 Model ecosystems (MES) studies

6.1 INTRODUCTION

Over thepas t decademode l ecosystems (MES)hav e comet opla ya nincreasingl y important rolei nbiologica lan dtoxicologica l investigationso fbot haquati can d terrestrial ecosystems.I ngenera la ME Sca nb eregarde da sa nattemp tt ocreat ea natural ecosystemo na mode l scale.Thes emodel sca n vary from verysimpl e systemst o systemswhic hclosel y resemblenatura l ecosystems with regardt ostructura lan dfunc ­ tional relationships.Aquati cME Susuall y consisto faquari ai nwhic h someo fth e variouscomponent so fnatura l ecosystems like bottommaterial ,water ,plant san d animalsar ebrough t togethert osimulat ea natura l situation.Bot hth ebehaviou ran d fateo fa chemica lo rit stoxi ceffect so nbiot aca nb estudie di nsuc ha system .Th e MESuse da sinstrument si nbiologica lan dtoxicologica l studies aremainl y laboratory MES, also referredt oi nth eliteratur ea smicrocosm so rmicro-ecosystems .Thes ear e incontras tt oso-calle d outdoorMES ,whic h aregenerall y larger andma y even consist ofpart so fnatura l ecosystems isolated from themai n environmentb yartificia len ­ closures.Review so nthes emicrocosm swer e givenb yTau b& Pearso n (1974),Metcal f (1977),Ringelber g& Kerstin g (1978).Th efirs ttw oreview s dealwit hth eenvironmen ­ talbehaviou ro fradionuclide san dtrac emetal san dth edegradatio no finsecticides , respectively.Th elas t study reviews themicrocosm s inrelatio nt oaquati c biology ingeneral ,withou t special referencet otoxicologica l studieso fchemicals . Among themicrocosm s useda stool si ntoxicologica l studiesth esystem s developed byMetcal fan dcoworker sar ethos emos t frequentlyuse d (Metcalf, 1977).Kit h these systems information canb eobtaine d aboutth efollowin g parameters:degradatio n path­ ways,transport ,distributio nove rth ecompartment so fth e systeman dth epotentia l forbioconcentratio nan dbiomagnification .I ngeneral ,emphasi si so nth ebehaviou r of thecompoun d rather thano nth e toxic effects,mainl ybecaus emos t laboratoryNE S areto osimpl efo ran ydetaile d investigationo feffect so npopulation so rcommunit y structure.I femphasi si so nth eeffect so nbiota ,anothe r typeo fNE S shouldb eused , especially wherei ti sth eai mt ostud y long-term ecological changes,suc ha sshift s incommunit y sizean dstructur ea sa resul to fth eintroductio no fa chemical .Then , severalcondition s havet ob efulfilled : -Severa l trophican dothe rrelationship s representativeo fth enatura l ecosystem shouldb eincorporate d because themor emeaningfu l ecological effectso ftoxi c com­ poundsar elikel yt oresul t fromchange si ncompetitio nfo rnutrients , food-selection and otherprocesse swhic h determine community structure (Menzel& Case , 1977).

48 -Th e system should beabl e tosustai n itselfove r a longperio d of time.Thi sre ­ quires sufficientmineralizatio n and recycling ofnutrient s anda continuous survival ofth e species,(Ringelber g &Kersting , 1978).Th e system should be operatable long enought ostud yan yalteration s in,fo rinstance ,life-cycl ebiolog yan d the seasonal changes inpopulatio n since thesewil l be slowan d often initially invisible,especi ­ ally inrespons e toexposur e tochroni c low levels ofspecifi c toxicchemicals . -Th e system should be sufficiently large.Thi s condition isno t only toenabl e the species toescap e overgrazing and overpredation,bu tals o tocreat e a certainrepre ­ sentative diversity of species.Finall y the system should also be largeenoug h to permit a regularchemica l andbiologica l sampling,withou t disturbing the system. -Th e system shouldb eoperatabl e ina wa y todistinguis h betweennatura l changes, sucha sseasona l changes due toclimate ,an d those inducedb y thechemica l introduced. This last condition requires that anadequat e control situation is available whichpreferabl y shouldb e identical toth e system treated. Such acontro l system could eitherb ea n 'internal'o r 'external'blank .A n internal blank implies that the changesobserve d inth eperio do f treatment arecompare d with thepre-treatmen tsitu ­ ation.Her e sufficient base-line information on the system should be available to servea sa reference for thedevelopmen t of thesyste m thatwoul d have takenplac e without experimental intervention. For anexterna l blank thedevelopmen t of the treated system iscompare dwit h thato fa systemwhic h closely resembles,an dprefer ­ ably is identical to,th etreate d system.Th emor e complex the system,th emor ediffi ­ cult it ist oobtai na blank .Therefor e controls and experimentally manipulated sys­ tems arereplicate d so thatth eexperimen t canb e statistically evaluated. Itdoe sno t seempossibl e tomee t the fourrequirement s on a laboratory scale. Morepromisin g seems anapproac h inwhic h outdoorME S are involved. Sucha n outdoor MES formarin e environmentswa sdevelope d inth e framework ofa research programme calledControlle d Ecosystem Pollution Experiment (ŒPEX) andwa s describedb y Reeve et al. (1976)an dMenze l &Cas e (1977). Inthes e experiments largebag so f flexible transparent plastic areuse d toenclos e a largewate r column (upt o 2000m ) inorde r tocaptur e arepresentativ e parto f the aquatic ecosystem.Th e impounded ecosystems caneithe rb e treatedwit ha chemica l or leftuntreated , thus serving as replicate manipulated systemso rcontrols ,respectively . Thebasi c ideao fCEPE X was to study theeffect so fchemical s on a systemo fa nalmos t natural degree ofcomplexit ywit h adequate controls.Thi s ideaals oapplie s to theME Suse d inth epresen t study,whic h canb econsidere d asa nexampl e ofa noutdoo r systemwhic h closelyresemble s a fresh­ water ditchcommunit y inth eNetherlands .Thes eNE S consisted ofpolythen e or Perspex containers of 3x1x1 meter,whic hwer edu g into the soil ina trial field of the Agricultural University.Afte r the introduction ofbotto mmaterial ,wate r andbiota , the systemswer eallowe d todevelo pundisturbedl y for 18month sbefor e the trials started.Durin g twoconsecutiv e summers thecontainer s weredivide d crosswise into halveswit h awaterproo fpartition .Th eai mwa s toobtai n twoalmos t identical sub­ systemso fwhic hon ewa suse d for treatmentwit ha norganophosphorou s insecticide

49 whileth eothe rserve da scontrol .I twa santicipate d thatonl ymino rdifference s woulddevelo pi nthes esubsystem sdurin gsumme ri fther ewa sn oexperimenta linter ­ vention.I nthi swa yth esystem swer econsidere dt omee tth efou rbasi cprerequisite s discussedabove .

6.2 MATERIALSAN DMETHOD S

6.2.1 Design of the MES

Fourplasti ccontainer swer edu gint oth esoi lo na plo tnea rth elaborator y (Fig.10).Thre econtainer swer emad eo fwhit epolythen ean dwer e3x1x 1meter .Th e fourthcontaine rwa smad eo ftransparen tPerspe xan dwa s2.70x0.87x0.9 0meter .Th e containerswer esurrounde db ya woode nconstructio nt oprotec t theside sagains t waterpressur efro mth einsid ean dth epressur eo fth esoi lfro mth eoutside .Th e rimsprotrude dapproximatel y 30c mabov eth esoi lsurfac et oavoi dcontaminatio nfro m thesurroundin gsoil .Stainless-stee lwire-netting s (meshsiz e 11mm )wer eplace do n topo fth econtainer st opreven tcontaminatio no fth ewate rb y leaves,litte ran d animals.Th econtainer swer eprovide dwit ha botto mlaye ro fapproximatel y0. 1m thick.Th ebotto mmateria lwa scollecte dfro ma comparativel yclea rwatercours ewith ­ outsignifican turba ninfluences ,calle d the 'TutenburgseWetering' ,situate dnea r Giesbeek (Provinceo fGelderland) .Th ewatercours eha dbee nenlarge d threeyear s beforesamplin gdurin glan dconsolidatio nwork .Th euppe r 10c mo fth ebotto mmateria l fromth ewatercours ewa scollecte dwit ha basket-typ ecutte rhavin ga fine-meshe d wire-netting insideth ebasket .Thi smateria lwa spartiall ydrie di nai ran dthe n sterilizedfo ron eda yb ysteamin gunde ra sheet .Subsequentl y thecontainer swer e filledwit hta pwater ,originatin g fromdee pgroundwater .Th epolythen econtainer s werefille di nOctobe r 1975,th ePerspe xon ei nAugus t 1976.Th evolum eo fwate rad ­ dedwa smeasure dwit ha water-meter ,th eris ei nwate rleve lwit ha stainless-stee l rule,fixe dt oth eeast-sid eo feac hcontainer .Fro mth ecalibratio ncurve ,th evo ­ lumei neac ho fth econtainer scoul db einferre da tan ytim efro mth ewate rlevel . Aboutthre eweek safte rth econtainer swer efille dwit hwater ,smal lplant so f waterthym e (Elodeanuttallii) ,hornwor t (Ceratophyllumdemersum) ,wate r starwort (Callitrichesp. )an dpondwee d (Potamogetoncrispus ,P .berchtoldii )wer eintroduced . Foreac hcontaine rth esam ewet-weigh to fplant swa sused ,t opromot ea simila rde ­ velopment.Th eplant swer ecollecte dfro ma smal lwatercours ealon gth erive rLing e nearHeteren .The ywer ekep ti nbucket san drinse dwit h tapwate r forthre esuccessiv e dayst oremov epossibl econtaminants .Th efourt hcontaine rwa ssupplie dwit hplant s obtained fromth eothe rthre eMES .Snails ,leeches ,flatworm san doligochaet eworm s wereintroduce d throughth eplan tmaterial .Furthermor ethe ywer einoculate dwit h about2 d m well-mixedfres hbotto msludge ,containin gmicro-organisms ,smal lcrus ­ taceansetc .Al l fourcontainer swer edivide dint otw osubsystem swit ha crosswis e waterproofpartitio n (Fig.10)a tth estar to fth eexperimenta lperiod swhic husuall y

50 2 -& //v u

•

A A A A 1— B B B B Üb • ^ m I II m 17 II

SECTION D-D SECTIONC- C figure 10.Sketc ho f theoutdoo rmode l ecosystems (MES)use d in thepresen tstudy , (a)arrangemen to f theMES ,(b )to pview ,(c )sid eview , (d)fron tview . 1• waterproo f partitioning-sheet 2» beech-hedg e 3* cylinder s ofstainless-stee lwire-nettin g (mesh 1cm ) 4• samplin gpoint s 5» fish-lin e 6• over-flo w 7• stainless-stee l sheet-holder 8• rubbe rflap s 9 "botto mmateria l 10= wate rsurfac e

lastedfro nMa yt oOctober .Thereafte r thesheet swer eremove d inorde rt orestor e pre-experimentalconditions .I neac hsubsyste mfou rcylinder so fstainless-stee l wire-nettingwer eplace duprigh ti nth ewate ran dpartiall ysun kint oth ebotto m layer.Thes ecylinder swhic hwer e90-a nlon gan dha da diamete ro f1 5c mwer eneces ­ saryfo ra prope rsamplin go fbiot a (seeSectio n6.2.5) .

6.2.2 Physico-chemical measurements

Physico-chemicalwate rparameter swer erecorde d forth efollowin gpurposes : -t ocompar eth ephysico-chemica lcharacteristic so fth eMES-wate rwit hthos eo f

51 Table 30.Physico-chemica lwate rparameter smeasure d inth eoutdoo rME Si n197 7 and1978 .

Parameter Unit Frequency pH daily -3 dissolvedoxyge n gm daily min.an dmax .temperatur e °C daily

-3 Totalphosphat e (asP0. ) gm everytw o weeks -3 Orthophosphate (asPO. ) gm everytw o weeks -3 N0~ gm everytw o weeks N0~ gm 3 oncea mont h -3 NHt gm oncea mont h I* -3 Hardness (asCaO ) gm everytw o weeks -3 HCO" molm everytw o weeks -3 Ca gm oncea mont h 2+ -3 Mg' gm oncea mont h -3 K+ gm oncea mont h -3 Na+ gm oncea mont h -3 Cl' gm everytw o months Electric conductivity mSm twicea yea r

surfacewater si nth eNetherlands , -t ose ewhethe ro rno tth eseparat edevelopmen to fth esubsystem swoul dresul ti n differentphysico-chemica lcharacteristic so fthei rwaters , -t osignaliz econdition swhic hmigh tb eunfavourabl e foran yo fth eorganism smoni ­ toredan dthu sinterfer ewit hth eexperiments . A surveyo fth ephysico-chemica lmeasurement s isgive ni nTabl e30 .Concentratio n ofdissolve doxygen ,p Han dtemperatur ewer emeasure ddail ybetwee n8h0 0an d8h30 . Occasionallymeasurement swer emad ea t4-hou rinterval sfro m6h0 0unti l18h00 . Dissolvedoxyge nwa smeasure db ya WT WOx y5 6portabl eoxygen-mete rwit hmembrane - electrode (purchased fromWissenschaftlich-Technisch eWerkstätt eGmbH ,Keilheim , WestGermany) .Fo rth ep Hmeasurement sa portabl epH-mete rWT Wp H5 6wa sused .Bot h pieceso fapparatu sha da naccurac yo f0. 1unit .

6.2.3 Vegetation assessment

A surveyo nth evegetatio ni nth eME Swa smad ethre etime sa year ;a tth een do f May,half-wa yAugus tan da tth een do fOctobe rfo reac hsubsyste mseparately .O feac h plantspecie sth ecoverin gpercentag e inbot hth efloatin gan dsubmers e layerwa s

52 estimated.T ofacilitat ethi sestimation ,nylo nfish-lin ewa sstretche dcrosswis e andlengthwis eove rth eME St odivid eth esurfac e intoeigh tequa lparts ,whic hwer e separatelyexamine d (Fig.10).Th epercentag ewa srelate dt oth etota lwatersurfac e ofa subsyste mminu stha twithi nth ecylinders .

6.2.4 Insecticide applications

A schemefo rth einsecticid etreatmen to fth eME Si nth etw osumme rseason sdu ­ ringwhic htrial swer ecarrie dou ti sgive ni nTabl e 31.Fo rth eapplicatio no fth e insecticidest oa subsyste mo fth eME Sth erequire ddos ewa scalculate d forth etota l watervolum eo fth ecompartmen tconcerne da ttha tmoment .Wit ha stainless-stee l bucketa volum eo fabou t 15d m waswithdraw nfro mth esubsystem .Thi swate rsampl e wasspike dwit hinsecticid efro ma 80 0g m stocksolutio ni nacetone ,stirre dan d pouredbac kint oth esubsyste mi nfiv edashes ,abou tequall ydistribute dove rth e watersurface .Th ebucke twa ssubsequentl yrinse dtwic ewit h2 d m waterfro mth e subsysteman dth erinsin gwa spoure dback .I twa santicipate dtha tthi sprocedur e wouldthoroughl ymi xth einsecticid eove rth esystem .Som etemporar yshor tdisturban ­ ceo fth evegetatio ncoul dno tb eavoided .Onl ywher educkwee dcovere dth ewate r surfacedi dth edisturbanc elas tsom etim e (ordero fdays )becaus epar to fthi swee d becameentangle di nth esubmers evegetation .Th esan eprocedur ebu twithou tinsecti ­ cidewa sperforme dwit hth ecorrespondin gcontro lsystem .

Table 31.Schem e forth einsecticid e treatmento fth emode lecosystems .

Year MES Insecticide Intended concentration (mgm )

1977 IA I A parathion 1 IB

IIA IIB

IIIA azinphosmethyl IIIB

IVA IVB

1978 IA I A azinphosmethyl I IB 1

IIA IIB parathion 0.2

IIIA IIIB parathion 1

IVA IVB parathion 0.5

S3 insecticide concentration

intended cone,leve l

time Figure 11.Concentratio npatter no fparathio nan d azinphosmethyl,respectively ,i nth e waterphas eo f themode lecosystem s (MES),c -intende dconcentration ,c »concen ­ trationmeasure db yGLC .c .- calculate d concentration.

Tomaintai na constan tinsecticid econcentratio nth efollowin gprocedur ewa s used (Fig.11).A tt theinsecticid ewa sadde d toth esyste mt omak eu pth erequire d 3 concentrationc .A tt. ,usuall y4 8hour slater ,a naliquo to f1 d m wassampled , extractedan danalyse db yGLC ,yieldin gc.. .Th erat ecoefficien to fdissipatio nwa s calculatedfro mEquatio n2 (Sectio n5.2.1 )wit hc=c .an dt=t.- t .Subsequentl yc ?> thecalculate dconcentratio na tth etim eo fredosing ,wa sderive d fromth esam e equationb yusin gt=t ?-t..,usuall ya perio do f2 4hours .Thu s2 4hour safte rsamplin g theinitia lconcentratio ncoul db erestore db yaddin g (c-c 2)v,i nwhic hv i sth e watervolum eo fth esyste ma tt, .Thi sprocedur eresulte di na schem econsistin go f twoanalyse spe rwee k (Monday,Thursday )an dtw ore-application so finsecticid e (Tuesday,Friday )pe rtreate dsubsystem .I ncas eo fabnormall yhig hc.-value sth e normalre-applicatio nprocedur ewa sused .However ,i fc .wa sunusuall y low,les sthe n thecalculate ddosag ewa sadde di norde rt oavoi doverdosin gi ncas eth eactua lcon ­ centrationpresen ta tth etim eo fanalysi swoul dhav ebee nunderestimated . Ineac h analysisa wate rsampl eo fa nuntreate dsubsyste mto owa sanalyse d forinsecticide . Theuntreate dsubsystem swer eanalyse di nrotation ,whic himplie sa frequenc yo fa t leastonc ea fortnight .Th esamplin gtechniqu ewil lb edescribe d inSectio n6.2.5 .

6.2.5 Population assessment of zooplankton and macrofauna

Forsamplin go fbot hzooplankto nan dwate r forinsecticid eanalysi sa 1- mlong , 44c mi.d . transparentPerspe xtub ewa suse d (Fig.12).Throug hthi stub ea nylo ncor d wasfixe dt oa rubbe rbal lwit ha diamete ro f5 cm ,loosel yse ti na rin goutsid eth e tube.B ypullin gth enylo ncor dth erubbe rbal lcoul db edraw nou to fth erin gt o lockth etube .A n iron-wireconstructio nwa sfitte dt oth elowe rsid eo fth etub et o preventbotto mmu d frombein g sampledtoo .Withou tthi sconstructio n itwa svirtuall y impossiblet ocoun ta sampl equantitativel ydu et oth efin edetritus .Fo rth epurpos e ofwate rsamplin g forinsecticid eanalysis ,th eope ntub ewa ssun kslowl yan dverti ­ callyint oon eo fth ecylinders .Whe nth ewir econstructio nreache d thebottom ,th e tubewa slocke dan dth esample dwate rcolum nwa spoure dint oa 2 d m glassbeaker .

54 -2.

-3. waterleve l T-TII- I vegetation — M\{SX

... V-f bottommud— % /, /////^f-////

a. Figure 12.Samplin gprocedur ewit hPerspe x tube.Th esamplin g tubei splace d inside thecylinder so fstainless-stee lwire-nettin g (a),whic hi sthe npulle d toth esid e whilea t thesam etim e thetub ei spushe ddownward s toth ebottom .Whe n thebotto m isreache d thenylo nrop ei spulle d toloc k thetub e (b).Figur ec represent sa mor e detailed sketcho f thebotto m endo f thesamplin gtube . 1= samplin gtub e 2= nylo nrop e 3= cylinde ro fstainless-stee lwire-nettin g 4» rubbe rbal l 5» iro nwir econstructio n topreven t thebotto men d of thetub efro mcomin gi n contactwit h themudlaye r 6 =holde r forth erubbe rbal l 7= clam pfo rwire-constructio n andball-holde r

Fromthi ssampl ea 1 d m subsamplewa suse dfo rth eGL Canalysis . Forzooplankto nsampling ,th ecylinde rwa sslante d intoth evegetation ,th e Perspextub ewa srapidl ysun kint oth ecylinde ran dclose dwhe nth ewir econstruc ­ tionreache dth ebotto m (Fig.12).Kit hth ewate rflo wth ezooplankto ndrifte dfro m thevegetatio n intoth ecylinder ,wher ei twa ssubsequentl ysampled .Th ecylinder s werefoun dt ob enecessar ybecaus eo fth edens evegetatio nwhic hdevelope ddurin g thesummer .Withou tthe mth etub epushe dmos to fth evegetatio ndownward san dresto ­ rationo fth evegetatio nstructur ethe ntoo kapproximatel yon eweek .Repeate dsam ­ pling thuswoul dhav eresulte di na permanentl ycompresse d layero fwate rplant si n thelowe rhal fo fth econtainer . Anextr aadvantag eo fthes ecylinder swa sth ediversificatio no fth e^E Sb yth e creationo fope nspots ,sinc evegetatio ndi dno tgro winsid eth ecylinders .Popula ­ tionso fDaphni apule xan dD . longispinawer e foundalmos texclusivel y insidethes e cylinders.A zooplanktonsampl eo fa subsyste mwa smad eu po fsi xsubsamples .Th e arrangemento fthes epoint si sgive ni nFig.10 .Th e totalvolum eo fwate rsample d amounted toapproximatel y7 d m .Thi svolum eo fwate rwa scollecte d ina stainless - steelbucket ,sieve d througha Hydro-Bio splankton-ne twit ha nunscrewabl e tipcu p withgauz ebotto m (mesh5 5um) .Durin gsievin gth elowe rpar to fth ene twa ssuspend ­ edi nth ewate rt ominimiz ewithdrawa lo fwate rfro mth e^E San ddisturbanc eo fth e

55 system.Th eplankto nwa skille dwit h5 pe rcen t formaldehydean dseparate d intotw o fractionswit ha doubl e sieveo f1m man d0.0 1m mmesh ,respectively .Th eplankto n wascounte di nth etota l fractions.Th ecrud efractio nusuall ycontaine d somemacro - faunaan da fe wlarge r specimenso fDaphnia ,Simocephalu san dCyclopoid aan dwa s preservedi n9 0pe rcen tethanol .Thi ssampl ewa scounte di na Petr idish .Th efine r samplewa spreserve di n 1%formaldehyd ean dcounte do na slide ,covere dwit ha cove r slip.Thi sslid e (7.5x 2. 5cm )wa sdivide d into longitudinal stripso f2m mwhic h were successivelyexamine dunde r themicroscope . Inadditio nt oth ecollectio no fmacrofaun ai nth eweekl y zooplanktonsamples , amomentar y surveyo fth emacrofaun apopulatio nwa smad ea tth etim e theME Swer e cleaneddurin gNovembe r 1976an d1977 .A ttha t timehal fo fth evegetatio no fth e MESwa sremove dt opreven t thevegetatio nbecomin g chokedth eyea r following,a swel l ast osimulat eth ecleanin g regimenormall ycarrie dou ti nditche si nth e Netherlands. The aquaticplant scollecte di nthi swa ywer e then searched formacrofaun awhic hwer e subsequently identifiedan dcounted .

6.3 RESULTS

6.5.1 Development of the MES in the pre-experimental period

In thecours eo f197 6th efirs tthre eME Sdevelope d intobasin swit ha varie d abundanceo fflor aan dfauna .The yshowe d similarityt oa ditc h systemo fa rela ­ tivelyundisturbe dnature ,a soccasionall yobserve dunde r fieldconditions . Thewate rwa sinvariabl y transparant,permittin ga clea rvie wo fth ebotto man d sub­ merged vegetation.Despit eth eattemp tt ocreat e similarsystems ,th eME S differedi n bothabundanc ean dcompositio no fth eflora .I nal l threebasin sElode anuttalli iwa s thedominan t species:i n^E SI ,I Ian dII Ii tcomprise d 70,5 0an d9 5pe rcen t res­ pectively,o fth ehighe rwate rplants .Ceratophyllu mdemersum ,bein gon eo fth e moredominan t specieso fplant si nlate ryears ,wa svirtuall y absenti nth e pre-experi­ mentalperiod .Mos tdivers ei nplan t specieswa sNE S II.Th evegetatio nwa s densei n allNE Sexcep t forNE SIII . Inth esumme ro f197 6differen t samplingan dcountin g techniquesfo rzooplankto n andmacrofaun awer e triedout .Th eresult s showed that therewer en omeaningfu ldif ­ ferencesi ncompositio no fzooplankto nan dmacrofauna .Neithe rdi dth especie scom ­ positiono fth ezooplankto no fth esyste malte rver ymuc hi nth efollowin gyears , with theexceptio no fCeriodaphni aquadrangul a thatwa sabundan ti n^E SI an dI I in 1976bu twa sno tfoun d anymorei n197 7an d1978 .

6.3.2 Physiao-ahemiaal measurements

Water temperature Themaximu man dminimu mwate r temperaturesfo rth eseason s197 7 and 1978ar epresente di nFig.1 3an dTabl e 32.Th efigur e givesth etemperatur epat -

56 Table32 .Averag ep Han d theminimu man dmaximu m temperatureo fth ewate r inth e MESdurir g theexperimenta l seasoni n 1977an d1978 .

MES Average Avera Average ge o pH min. temp.( C ) max.temp . (C )

1977 1978 1977 1978 1977 1978

I A 8.9 9.3 13.2 13.0 16.6 15.6 I B 9.1 9.3

II A 7.6 8.0 12.9 13.2 15.9 II B 8.3 8.3 15.3

III A 8.1 8.9 13.3 12.9 16.7 15.4 III B 8.9 9.0

IV A 9.4 8.4 13.0 12.9 15.0 14.7 IV B 9.5 8.4

water temperatur e (°C) 25n

MAY ' JUNE ' JULY ' AUG. ' SEPT.' OCT. Figure 13.Minimu man dmaximu m temperatureo fwate ri nME SI i n 1977(a)an d 1978(b), respectively.

ternfo rME SI a sbein grepresentativ e forth eothe rME Sa swell ,whic h isno tsur ­ prisingsinc eth eatmospheri c temperaturei sth emajo rdeterminan to fthi spattern . Amongth edifferen tME Sther eare ,however ,difference s inth edail yamplitude ,a s canb ederive d fromTabl e32 ;th eamplitud eo fME S I,I Ian d IIIbein ghighe rtha n thato f^E SIV .Thi sdifferenc ei smainl ycause db yth eshadin go fthi slas tME Sb y abeech-hedg e (seeFig.10) .A shadin geffec tb yduckwee dcove ri sreflecte d inth e lowermaximu mtemperature so fME SI Ii nbot h 1977an d 1978.

57 i2n 11 10

8 7 6 5

12 11 10 9 8 7- 6 5

12 11 10 9 8 7 6 5 MAY JUNE' JULY 'AUG .' SEPT .'

Figure 14.Value sfo rp Hi nth ewate ro fME SI/I V (Figs,a/d ,respectively )i n1977 . treated subsystem (exceptME S II,IV ) control.

Measured pH values Theseasona ldevelopment so fthi sparamete r foral lME Sar epre ­ sentedi nFig.1 4a- dan dFig.1 5a- d for 1977an d 1978,respectively .Th eaverag ep H valuesfo rbot hseason sar egive ni nTabl e32 .Th epattern sdiffe rpe rseaso nan dpe r system.Amon gth ecorrespondin gsubsystem sth edifference si np Har egenerall ysmall .

58 MAY JUNE ' JULY AUG. ' SEPT. ' OCT.

Figure 15.Value sfo rp Hi nth ewate ro fME SI/I V (Figs,a/d ,respectively )i n1978 . ~— treated subsystem control.

Onsevera loccasion sp Hwa smeasure da t4-hou rinterval sdurin g theday .Som eo f thesedat aar etabulate di nTabl e3 3t oillustrat etha teve no nsunn yday sth ediur ­ nalfluctuatio ngenerall ydi dno texcee d 2pH-units ,an dals otha toccasionall yp H valuesexceede da valu eo f10 .

59 DISSOLVED OXYGEN (m g m3). 15-J

15-

/\ r\

10- -_-S •v V- ''"/ \ r-

5- '—I r

15-

10

5-Lr MAY JUNE JULY AUG. SEPT.

Figure 16.Dissolve d oxygeni nME SI/I V (Figs,a/d ,respectively )i n 1977. treated subsystem (exceptME SII ,IV ) control.

60 DISSOLVED OXYGEN (nngm^)

15-

15- \ b _ N.\ 10- ^/ I / V / \\ A . V't\ / \ /\ 5 - WV v =vA x \ V

0 - T" 1 T— •• i 1 15

MAY ' JUNE ' JULY ' AUG. ' SEPT. ' OCT.

Figure 17.Dissolve d oxygen inME S I/IV (Figs, a/d, respectively) in 1978. treated subsystem control.

61 Table33 .Developmen to f thep Hi nth edifferen tsubsystem so f theME So non e cloudyan d twosunn ydays .

Day Time MESI MES II MESII I MESI V Weathercondition s

A B A B A B A B

20Jul y 6.00 8.4 9.0 6.6 7.5 6.7 8.8 9.0 9.5 cloudy 1977 10.00 8.8 9.4 7.0 7.8 6.9 8.9 9.0 9.4 max. temp.2 4 C 14.00 9.0 9.4 7.4 8.6 7.4 9.0 9.0 9.5 hourssunshine :4 18.00 9.0 9.3 7.7 9.2 7.9 9.2 8.9 9.4

4Jul y 6.00 9.6 9.9 8.9 9.4 9.4 9.4 9.7 10.0 sunny 1977 10.00 10.0 10.4 9.4 9.7 9.6 9.8 9.8 10.1 max. temp.2 7 C 14.00 10.2 10.5 9.6 9.9 9.7 10.3 9.9 10.2 hourssunshine :1 4 18.00 10.1 10.5 9.7 9.9 10.2 10.4 10.0 10.1

31Ma y 6.00 8.0 8.1 8.9 9.1 8.9 8.6 8.0 8.2 sunny 1978 10.00 9.3 9.5 9.2 9.4 9.1 9.0 8.3 8.4 max. temp.27. 5 C 14.00 9.6 9.8 9.5 9.6 9.2 9.4 8.5 8.8 hours sunshine:1 4 18.00 9.6 9.7 9.6 9.7 9.5 9.6 8.5 9.0

Dissolved oxygen Theseasona lfluctuation s inth econcentration so fdissolve doxyge n arepresente d inFig.1 6a- dan dFig.1 7a- d for197 7an d 1978,respectively .Concentra ­ tionswer egenerall yhig han dofte nth ewate r inth eME Swa ssupersaturate dwit h oxygen.

Other parameters Theothe rphysico-chemica lvariable smeasure dar esummarize di n Table34 .I ngenera lbot hth edifference sbetwee ncorrespondin gsubsystem san damon g separateME Swer esmall .ME SII Idiffere d fromth eother si nit slo wconcentration s oforthophosphat e in1977 .Fiv etime sou to f1 4n oorthophosphat ecoul db emeasure d inSubsyste mA an d6 time sou to f 14i nSubsyste mB .Th epotassiu mconten to fth e wateri nal lth eME Swa susuall y lesstha n1 g m " andofte nunde rth edetectio n limito f0.0 1 gm" 3.

6.3.3 Vegetation assessment

Thevegetationa lcompositio no fth eME Si spresente d inTabl e35 .I tshow stha t thesystem svarie di ncompositio nan dabundance .Th evegetatio nals ovarie dfro m yeart oyear .I n197 7hardl yan yElode awa sobserved ,bu ti twa srathe rabundan ti n 1978.I n197 7an d 1978Ceratophyllu mdemersu mdominate d inal lMES .Sinc eth estar t ofth eMES ,specie slik eRanunculu saquatili san dNasturtiu mmicrophyllu mgraduall y havedisappeared ,whil eCallitrich esp .i n197 8wa sonl yfoun d inNE SII . Generallyth evegetatio nwa sdense .However ,larg eope nspace swer epresen ti n MESII Ii n197 7an di nME S IIan dI Vi n1978 .I nth efirs ttw oME S largequantitie s offilamentou salga ewer epresent .I nSubsyste mA ofME S IIIOedogoniu msp .forme d largealga lmat s incontras tt oonl yon esmal lma ti nB .Simila rmat swer eremove d

62 Table 34.Chemica l characteristics of water samples from the four outdoor MES in 1977 and 1978.

Characteirist iLc s Unit MES I MES II MES III MES IV

'77 '78 '77 '78 '77 '78 '77 '78

Total phosphorus-P g m-3 A 1.8 0.94 1.6 1.1 1.4 0.94 1.8 1.2 B 1.6 1.2 1.5 0.75 1.4 1.1 1.9 1.6 -3 A 0.40 0.52 0.14 0.30 0.08 0.28 0.39 0.54 Orthophosphate-P g m B 0.44 0.60 0.12 0.19 0.04 0.52 0.32 0.86 -3 N0~-N g m A 0.13 0.05 0.10 0.07 0.10 0.06 0.08 0.08 B 0.16 0.06 0.09 0.07 0.09 0.06 0.07 0.07 -3 N0~-N g m A 0.03 <0.01 0.02 <0.01 0.02 <0.01 0.02 <0.01 B 0.02 <0.01 0.01 <0.01 0.01 <0.01 0.02 <0.01 -3 NH*-N g m A 0.27 0.18 0.29 0.33 0.16 0.22 0.20 0.21 4 B 0.27 0.17 0.29 0.21 0.20 0.21 0.14 0.27

-3 A 56 72 54 80 46 67 57 Hardness (as CaO) g m 74 B 78 56 68 50 67 45 64 65 , -3 HC0~ A 4.2 4.5 3.5 5.6 3.9 3.8 3.3 5.3 mol m B 4.3 4.5 3.2 4.0 2.4 3.9 3.0 5.9 -3 Ca2+ g m A 29 21 28 20 30 18 30 22 B 32 17 27 19 22 17 30 27 -3 Mg2+ A 13 11 12 11 14 9.4 11 11 g m B 13 11 12 9 14 8.6 7.2 11

+ -3 K g m A 0.66 <0.01 0.35 <0.01 1.3 <0.01 0.04 <0.0I B 0.67 <0.01 0.36 <0.01 1.0 <0.0I 0.07 <0.01

+ -3 Na A 9.6 5.2 9.5 7.6 12.0 7.6 7.2 5.4 g m B 9.5 5.4 9.0 6.2 10.6 6.6 6.6 7.4 -3 Cl" A 9.4 6.6 10.0 7.3 8.0 3.5 g m 9.2 5.5 B 9.5 6.7 9.9 6.2 10.9 6.3 6.7 5.2

Electric conducti- mS m A 23 18 20 22 24 26 20 15 vity B 23 18 19 20 21 25 20 15

63 Table35 .Surve yo nth efloatin g andsubmerge dvegetatio n inth eME Sa tth een do f theexperimenta lseason s in 1977an d 1978.Figure s representpercentage so fth etota l surfaceare aminu s theare aenclose db yth esamplin gcylinders . -no tpresen t +observe d during theexperimenta l season,bu tno t founda tth etim eo fsurve y

MESI MESI I MESII I MESI V

1977 1978 1977 1978 1977 1978 1977 1978

Floating vegetation (incl. emerging vegetation)

Duckweed (Lemnamino r+ Spirodel a A - + 50 50 <1 <1 20 70 polyrhizza) B <1 + 30 5 <1 <1 20 60

Waterstarwor t A - - <1 5 - - - - (Callitrichesp. ) B <1

Total A <1 _ 50 55 <1

Hornwort (Ceratophyllum A 100 100 50 15 5 <1 70 10 demersum) B 80 95 60 50 35 40 50 10

Pondweed (Potamogeton A <1 <1

Pondweed (Potamogeton A - + 40 15 15 50 + _ berchtoldii) B 10 + 15 <1 10 - - -

Waterthyme (Elodea A + + + 15 + 20 5 <1 nuttallii) B + + <1 15 + 40 10 <1

Waterbuttercu p A 5 (Ranunculusaquatilus ) B <1

Waterstarwor t A <1 5 (Callitrichesp. ) B - + <1 - - - - -

Ivy-leavedduckwee d A _ _ 5

A - + _ + 50 _ <1 + Oedogoniumsp . B - - - 30 5 - + -

A 100 100 100 50 70 100 100 15 Total B 100 100 100 85 90 95 100 15

64 tn rs i^ oo \o o « ui irno \o I I I I I I I I I I I I -ï 00 O CO ^D O 00 •j o o n CM CN — CS es es —

J3 •O 3 O 3 o o o e —

> <

C I ai E

o U si e. O H c

o. ca a ca 4(-J( c to 4)

S oo o •f-* «3 *J •U o u Cfl I G 0» O m •H js q — •u 0 C U-( 0 r- oo r^ oo 0) c c o> ON 3 o *~i »M u vD CO f« •aH Li JJ

65 fromME SII Ii nth eroutin ecleanin gi n 1976,whe nthe ywer eevenl ydistribute dove r thewhol esystem .A t theen do fth eexperimenta lseaso ni n197 7the yagai nwer ere ­ movedan ddi dno tre-appea r in1978 .I nNIE SI Ii n197 8a filamentou sgree nalg a coveredwall san dsubmerge dwate rplant s inbot hsubsystem sbu twa smor eabundan ti n IIB a tth een do fth eseason .

6.2.4 Inaeatiaide concentrations

Theaverag econcentration so finsecticid e inth eME Sar epresente d inTabl e36 . Alsothos econcentration swhic hwer eexceptionall yhig ho rlo war etabulated .A chec k wasmad eo nth ehorizonta lan dvertica ldistributio no fth einsecticide sove rth e subsystems.Th eresult sar epresente d inTabl e37 .The yd ono tindicat etha teithe r horizontalo rvertica lconcentratio ngradient sdi dexist . Thecalculate dhalf-live so fth eapplie d insecticides inth edifferen tsubsys ­ temsar epresente d inTabl e38 .Fo rparathio nth ehalf-live swer egenerall yo fth e ordero f3 t o4 day swit ha lowe rvalu efo rSubsyste m IIA.Th ehalf-live sfo r azinphosmethylamounte dt o7 day si n197 7an dnearl y3 day s in1978 . Ingenera la nequall ydistribute dan dfairl yconstan t insecticideconcentratio n couldb emaintained .

Table37 .Assessmen to f thehorizonta lan dvertica ldistributio no f theinsecticide s parathionan dazinphosmethy l inth emode lecosystems .Measuremen t ofth ehorizonta l distributionwa smad esi xhour safte rapplication .Vertica ldistributio nwa smeasure d alongwit h thenorma lanalysis .

Insecticide Calculated Measured Water Date cone,m gm -3 cone,m gm ~ sampled

Horizontal distribution

parathion 0.87 0.93 insidecylinde r 10Jun e '77 0.89 betweenvegetatio n 0.96 0.88 insidecylinde r 15Jul y '77 0.90 betweenvegetatio n azinphosmethyl 0.76 98 insidecylinde r 15Jul y '77 80 betweenvegetatio n 0.94 94 insidecylinde r 9Aug .'7 7 0.92 betweenvegetatio n

Vertical distribution parathion not calculated 0.40 lowerpar twate rcolum n 5Oct .'7 7 0.37 upperpar twate rcolum n 0.37 totalcolum n azinphosmethyl notcalculate d 0.26 lowerpar twate rcolum n 25Oct .'7 7 0.25 upperpar twate rcolum n 0.26 totalwate rcolum n

66 Table 38.Averag ehalf-live so fparathio nan dazinphosmethy l inth ewate rphas e ofth eoutdoo rMES .Half-live so fmor e thantwic e theaverag ehav ebee nexcluded .

Insecticide Intendle dcone . MES Year Averagehalf-l i .fe Average max. Average (mg m 3) +S.D . (hours) water temp. pH parathion 1 IA 1977 75+ 2 6 16.6 8.9 1 IIIA 1978 85+ 3 4 15.4 8.9 0.5 IVA 1978 95+ 4 0 14.7 8.4 0.2 IIA 1978 36+ 1 0 15.3 8.0 azinphosmethyl 1 IIIA 1977 168+ 5 0 16.7 8.1 1 IA 1978 68+ 3 4 15.6 9.3

6.3.5 Population assessment of zooplankton and macvofauna

A listo fillustration so fth emai nspecie sstudie di spresente d inAppendi xC . Otherspecie sfoun d inth eME Sbu tno tstudie dar eliste d inAppendi xD .Th echange s inth eabundanc eo fspecie sobserve d inrelationshi pwit hth einsecticide sapplie d aresummarize d inTabl e39 .Marke dreduction si nth eabundancie so fDaphni aspp .wer e observeddurin gth eapplication so fparathio n (0.5an d 1m gm )an dazinphosmethy l at 1m gm .Stron greduction si nabundanc ywer eals orecorde d forSimocephalu svetu - lusi nrespons et o1 m gm parathionan dt oa lesse rexten tfo r1 m gm azinphos­ methyl.Number so fChydoru ssphaericu sdecrease dmarkedl y inth epresenc eo f1 m gm parathionan dsomewha t lessa ta concentratio no f0. Sm gm .Population so fChaoboru s crystallinusrapidl ydecrease da ta concentratio no f1 m gm ~ parathionbu tno ta t 0.5m gm .Onl ya sligh tdecreas e innumber swa sobserve d forGraptoleberi swit h 1m gm " parathion.Afte rth eintroductio no fth einsecticide s theabundanc eo f Cyclopoida (naupliian dcopepodit sincluded )an do fOstracod adi dno tdecrease .Mor e detailed informationca nb ederive d fromFigs.18-33 ,whic har epu ttogethe r inAppen -

Table39 .Summar yo fth echange si nabundanc eo fzooplankto nan dmacrofaun aspecie s inrelatio n toth einsecticide s applied.(- ) noreductio n innumber s compared tob e control;(+_ )hardl yan yreductio ni nnumbers ;(+ )stron greductio n innumber sa s compared toth econtrol ,bu tstil lpresen t infai rnumbers ;(++ )non eo ronl y occasional specimenswer esampled ,whil e largenumber swer epresen t inth econtrol .

Organism Parathion Parathion Parathion Azinphosmethyl 1m gm~ 3 0.5m gm~ 3 0.2m gm~ 3 1m gm~ 3

Simocephalusvetulu s ++ - - +(+)' Daphnia spp. ++ ++ - ++ Chydorus sphaericus ++ + - - Graptoleberis testudinaria + Cyclopoida - - _ Ostracoda - Chaoborus crystallinus ++

I.+ +i n 1977,+ i n1978 .

67 Table40 .Surve yo fth edifferen tfigure swhic hsho wth edevelopmen to fth e zooplanktonspecie si nth eMES .

Species MESI MESI I MES III MESI V

1977 1978 1977 1978 1977 1978 1977 1978

Siraocephalusvetulu s 18a 19a 18b 19b 18c 19c 18d 19d Daphniaspp . 21a 22a - 22b - - 21b 22c Chydorus sphaericus 23a 24a 23b 24b 23c 24c 23d 24d Graptoleberis testudinaria 25a 26a 25b 26b 25c 26c 25d 26d Peracanthatruncat a - - 27a 27c - - 27b 27d Cyclopoida 28a 29a 28b 29b 28c 29c 28d 29d Ostracoda 30a 31a 30b 31b 30c 31c 30d 31d Chaoborus crystallinus 32a 33a 32b 33b 32c 33c 32d 33d

dixE (seeTabl e 40). Inthes efigure sth enumber so fspecimen scollecte dpe rsampl e areplotte dversu stim efo reac hspecie san dfo reac hsubsystem .Curve so fcorrespon ­ dingsubsystem sar edraw ni nth esam egraph ,wit ha soli d linefo rth eA subsystem s anda broke nlin efo rth eB subsystems .Thes ecurve swil lb ediscusse d indetai li n Section6.4.4 . Withregar dt othe ,mainl ybenthic ,macrofauna ,th enumber s inth esample swer e sosmal ltha tn oattemp twa smad et opresen tthes edat agraphically .Therefor ethes e numbersar esumme dpe rsubsyste mpe ryea ran dpresente d inTabl e41 .

6.4 DISCUSSION

6.4.1 Fhysico-ohemioal measurements

TheME Sprove d tob esufficientl y larget okee pdiurna l temperaturefluctuation s ofth ewate rwithi na narro wrange .Th ehighes twate rtemperatur eeve rrecorde di n theME Swa s27. 5° Co nJun e30 ,197 6a tth een do fa ver ywar mday . Thep Hvalue srecorde dwer erathe rhig hcompare dwit hthos eo fmos tsurfac e watersi nth eNetherlands ,bu tthi si sno tabnorma l inditche swit ha nabundanc eo f higherwate rplant s (seefo rinstanc eBeltman ,1976) .Th eeve nhighe rvalue srecorde d lateri nth eda y (Table33 )ma yhav efavoure dhydrolysi so fth eapplie dinsecticides . Althoughth emeasure dconcentration so fdissolve doxyge nwer egenerall yhigh ,wit h theexceptio no fsom eperiod si nME SI Ii n 1978 (Fig.17b),i tcanno tb eexclude dtha t nearth ebotto moxyge nconcentration soccasionall ywer es olo wa st ob eunfavourabl e forbotto mdwellin gorganisms . Forth eothe rphysico-chemica lparameters ,a ssummarize d inTabl e34 ,th evalue s measured inth eME Sfal lwithi nth erange sgenerall yencountere d insurfac ewater s inth eNetherland s (e.g.Werkgroe pBiologisch eKaterbeoordeling , 1977).Ther ear e howevera fe wexceptions ,notabl ypotassium ,orthophosphat ean dchlorid econten tan d hardnesso fwater .Th epotassiu mcontent ,whic h inth eME Swa susuall yunde r 1g m , islo wi nrelatio nt oth erang eo f 1-15g m normallyrecorde d insurfac ewater si n

68 Table41 .Number s ofmacrofaun asample d togetherwit h thezooplankto ndurin gth e experimental seasons in 1977an d1978 .

Organism MESI MESI I MES III MESI V

1977 1978 1977 1978 1977 1978 1977 1978

Tricladida A 0 6 0 7 1 7 2 14 B 0 4 2 7 3 21 2 14

Hirudinea A 0 3 5 4 3 0 4 3 B 0 4 3 1 7 5 4 7

Asellussp . A 8 12 99 34 20 64 11 26 B 6 14 61 28 41 122 6 26

Ephemeroptera A 2 6 0 0 0 0 35 0 B 6 15 1 7 12 1 29 16

Odonata A 0 6 0 0 0 0 0 0 B 0 2 0 2 0 0 0 0

Oligochaeta A 0 0 0 1 4 0 0 0 B 0 1 0 0 13 0 0 0

Chaoborussp . A 40 522 286 593 259 142 112 262 B 230 379 283 811 260 464 83 389

Heteroptera A 1 0 13 13 2 0 0 0 B 2 3 2 17 0 I 0 2

Coleoptera A 0 3 1 4 2 3 0 4 B 0 0 2 0 0 2 0 0

Gastropoda A 37 23 49 37 16 92 20 61 B 32 24 54 20 11 55 14 63

Hydracarina A 16 19 40 85 42 33 83 49 B 30 21 59 113 41 25 36 65

theNetherlands .Sinc epotassiu m isa macronutrien tfo rwate rplant si tma yhav ebee n limiting insom eo fth eML Sfo rsom eperiods .Th esam eca nb esai do fth eorthophos ­ phateconcentratio nespeciall y inME SIII .A s fara sth eio nconten to fth ewate ri n theME Si sconcerne d thiswate rca nb eregarde da soligo-ioni c(<2 0m Sm ~) o rbeta - mesoioni c (20-50m Sm ~ ).Suc hlo wvalue sar ecomparativel yrar ei nDutc hsurfac e waters. Inth eME Sthe ywer ea resul to fth elo wchlorid econtent .I ngenera lth e chlorideconten to fDutc hsurfac ewater srange sfro m20-30 0g m ~ .Consisten twit h thelo wchlorid econten t isth elo welectri cconductivit yo f15-2 5m Sm ,th enorma l rangeo fDutc hfreshwater sbein g25-10 0m Sm .Th ehardnes so fth ewate r inth eME S withvalue sgenerall y farbelo w8 0g m (asCaO )wa sa littlelo wcompare dwit h valuesove r 100g m formos tDutc hsurfac ewaters . Finallyi tca nb esai dtha tth edat aobtaine d forNH .an d XOZconcentration s werelo wan dver yunlikel yt orepresen ttoxi cconcentrations .

69 treatmentwa sstoppe d thepopulatio nincrease du p toth e levelo f thecontro lpopula ­ tion.Thi s result supports thehypothesi s thatth eabsenc eo fa recover y in 1977wa s due toth eciliat ebloo m rathertha nt oa direc teffec to fazinphosmethyl .A bloo m of Chroomonas sp.simila r to that in 1977occurre d inthi ssam epon dwit h 1m gm para- thion,bu twa so fshorte rduration .N omeaningfu l differenceswer eobserve d between corresponding subsystems ineithe r theuntreate dNE S (Figs.19b,d)o r inth eNE S treatedwit h 0.2 or 0.5m gm ~ parathion.

Daphnia spp. Daphniawa sno tpresen t ineac h^E Sever yyear . InNE S Ii n 1977a popu ­ lationo fD . longispinaappeare dafte r the treatmentwit h 1m gm parathionwa s stopped andprobabl y contributed toth eterminatio no fth ealga lbloom . (Fig.21a). Nota singl e specimenwa s foundi nth econtrol .A populationo fD . longispina inth e untreatedNE S IVi n 1977showe da paralle ldevelopmen t inbot hsubsystems . After the introductiono f 1m gm ~ ofazinphosmethy l analmos t complete dis­ appearanceo fD .pule x tookplac e (Fig.22a). Inth e control thepopulatio nwa s stable throughout theexperimenta l season.Th epopulatio n inth e treatedpar to fth eNE Sdi d not recoverafte r thetreatmen twa s stopped,(fenc eunde r thecondition s inthi sNE S theDaphni a speciesconcerne d aremor esusceptibl e thanSimocephalu s vetulus. A similareffec twa s founddurin g the treatmentwit h0. 5m gm parathion (Fig.22c) Againn o recovery tookplac eafte r thetreatmen twa s stopped. Evaluation of the0. 2m gm parathion treatment inrelatio n to theDaphni apo ­ pulationwa s difficultdu et o thelat eemergenc e ofth epopulatio n inbot hsubsys ­ temso fNE S II (Fig.22b).Howeve r the facttha t thepopulatio nemerge d inspit eo f thetreatmen t indicates that thisconcentratio nprobabl ydi dno t affect thespecie s involved (D.longispin a andD .pule xcurvirostris) .Thi s isi nagreemen twit h the no-effect levelso f 0.3m gm found inth e 3-week laboratory toxicity testwit h D.magn a (Table21) .

Chydovus ephaeviaua Introductiono fparathio na ta concentratio no f 1m gm ~ in NES IA in 1977wa s followedb ya stron g reduction innumber s of thisspecie s inth e samples (Fig.23a).Afte r the treatmentwa s stopped,th epopulatio n showed a tendency torecover .Howeve rrecover ymigh thav ebee n temperedb y theonse to fa seasona l decline asca nb e read from theothe rcurve s inFig.23 . In 1978a t thissam econcen ­ tration theeffect s of 1977coul dno tb e reproduced (Fig.24c)fo rth esam e reasons asmentione d for theSimocephalu s population inthi s>ES .Howeve r asimila r sharp reduction innumber swawa s foundafte r introductiono f 0.5 mgm " parathion in1978 , although lesspronounced . Forparathio n ata concentratio no f0. 2m gm " thedi p innumber s inJul ya t first sightmigh tb e regarded asa neffec twit ha relatively long latencyperio d due toth e lowconcentratio napplied .Howeve r asimila rdi pwa sobserve d forSimo ­ cephalus (Fig.19b).Sinc e inth e latter casea complet e recovery tookplac ewithi n theperio d oftreatmen tan dn o effectwa sobserve d at thenex thighe r levelo f0.- S

72 mgm " (Fig.19d),th edi pprobabl y cannotb e attributed toparathion . It is,there ­ fore,conceivabl e that thedeclin eo f theChydoru s curve alsocanno tb e attributed toparathion ,eve n though Chydorus isprobabl ymor e susceptible tothi s insecticide thanSiraocephalus ,a sca nb esee nb ycomparin g Fig.24dwit h Fig.19d. InFig.19 d no effect ofparathio n onSiraocephalu s isseen . Without treatment similarpopulation s developed incorrespondin g subsystems (Figs.23b,d).N o effectwa sobserve d forazinphosmethy la t aconcentratio n of 1m gm ~ in 1978 (Fig.24a).Th e gradual decrease innumber s in 1977 inrelatio n to thecontro l (Fig.23c),therefor emos t likelyha s tob e attributed toth eciliat e bloom (Section6.3.3 )instea d ofbein g adirec t effect of the insecticide.

Graptoleberis testudinaria Neither parathion nor azinphosmethyl did affect the populations of thisspecie s (Figs.25a,can d Figs.26a-d). Population developments incorrespondin g subsystems alsower equit e similar (Figs.25c,d).

Peraaantha truncata The results of the insecticide-treatedME S are inconclusive as fara s this species isconcerned ,becaus e thepopulation s started todevelo p late (Figs.27c,d)o rwer e almost completely absent during theexperimenta l season aswa s thecas e inME S IIan d IV in 1977an d inNE S III in 1978.Simila r population develop­ ments wereobserve d incorrespondin g subsystems of theuntreate d MES (Fig.27a).

Cyclopoida Cyclopoida didno t seem toexperienc e anydeleteriou s effect from the insecticide concentrations applied in theMES-experiments . This is inagreemen t with literature data (Section 2.3.3). Lüdemann andNeuman n (1962)foun d no effect on

Cyclopsstrenuu s at 1g m " parathion ina test period of2 4hours .No-effec t levels •I of6 other organophosphorousinsecticide s for thisspecie s ranged from 5t o 8g m -3 Baytexwa s foundno t tocaus emortalit y inMicrocyclop s bicolor atconcentration s up to0. 5 g m"3. All corresponding subsystems showed a similar population development over the seasonan d levelswer e equal inmos t cases. Inth eME S treatedwit h parathion atcon ­ centration levels of 1an d 0.5 mgm thenumber s inth e treated subsystemswer e even higher thani nth econtrols .A tentative explanation of thisphenomeno nmigh t be that thesuppl y of food for theherbivorou s part of theCyclopoid a population relatively increased becauseo f thereductio n inth epopulation s of theCladocera .Als o areduc ­ tion inprédatio npressur e by the larvae ofChaboru s crystallinus asa result of its population declinema y have contributed (Figs.32a,33c).Th edecreas e innumber s of Cyclopoida at theen d of 1977 inME S IA (Fig.28a)coul d thenb e attributed toa re ­ duced availability of food as a result of the recovery ofSimocephalu s (Fig.19a)an d theemergenc e ofDaphni a longispina (Fig.21a). Such events didno t occur ineithe r MES IIIAo r IVA in 1978an d thismigh t explain why in theseME S thenumber s ofCyclo ­ poida remained abovecontro l levels evenafte r the treatment was stopped.

73 Ostraaoda Noeffec twa s found foreithe r insecticide used,whic h resulti si nlin e withth eliteratur e data (Section 2.3.3).Hardl y anymortalit ywa s observed inChlamy - dotheca arcuata aftera 48-hou r exposuret o2 g m ~ fenthion (Khudaira& Ruber,1974) . Baytexwa s foundt ocaus e3 0an d10 0pe rcen tmortalit yi nCypridopsi s viduaa tcon ­ centrationso f1 an d2g m ,respectively ,i na 24-hou r test. Population developments among differentME Swer e also similarbu tth epattern s differed between the twoyear s (Figs.30,31).

Chaoborus orystallinus Bothi n197 7an d197 8 applicationo fparathio na t1 m gm resulted ina reductio ni nth enumber so fChaoboru s larvae (Figs.32a,33c). Lateri n theperio do ftreatmen tth elarva ewer e almost invariably foundi nth efin e fraction ofth ezooplankto nsamples ,whic h indicated that theywer enewl y hatched ones. Inal l otherML Sth epopulation so fcorrespondin g subsystems developed similarly,whic h shows thatparathio na t0. 5an d0. 2m gm andazinphosmethy la t1 m gm " didno taffec tth e larvaei nth eMLS .Thes e resultsar ei naccordanc ewit hth elaborator y toxicity data obtained inthi s study (Section 4.4.4).I nal l otherME S thepopulatio n developments betweencorrespondin g subsystemswer e similar.

Other species Asellus spp.,Gastropod a (snails)an dHydracarin a (watennites)di dno t seemt ob eaffecte db yan yo fth einsecticid e concentrations.Althoug h Cloeondipte - rumwa sno t collectedi nan ysampl e fromth esubsystem s treatedwit h parathioni n 1978,o rwit h azinphosmethyl in1977 ,n oconclusion sca nb edraw n from this result, becauseb yfa rth emos t larvae sampledi nth ecorrespondin g control subsystems were collected afterth etreatmen twa sstopped .Th elowe r totalnumber sfo rChaoboru s crys­ tallinusi nME S IA (1977)an dII A(1978 )correspon d withth elowe rnumber s inth e treatment period (Figs.32aan d33c ,respectively) .Fo rth eothe r species eventh e totalnumber swer eto osmal lt odra wan ydefinit e conclusions.Ciliat aan dRotatori a specieswer e oftenpresen ti nth esample si nlarg enumbers ,bu twer eno tcounte dfo r lacko fmanpower .However ,als ofo rthes eorganism s thepopulatio n development seemed roughly similari ncorrespondin g subsystems. From thenumber so forganism s collected during theautum n cleaning inNovembe r 1977an d1978 ,n oconclusion s canb edraw n aboutan yeffec to finsecticid e treatment. They onlyprovid ea momentar y surveyo fth enumber so fmacrofaun a present inth eMLS . A table listingth eorganism s collecteda tth etim eo fth ecleanin g ispresente di n AppendixF .

6.4.5 Similarities and differences betueen corresponding subsystems

Corresponding subsystemso fth euntreate dML Sshowe da simila r developmentfo r nearly allphysico-chemica l andbiologica l parameters.Onl y thep Hi nÎ>E SI Ii n 1977 largelydiffere d betweenSubsystem sA an dB (Fig.14b) . Such similarities were also observed betweencorrespondin g subsystemso f?>E Stha twer e treated with insecticide,

74 for thoseparameter swhic hwer eno taffecte d by the insecticide treatment.O n the other hand differenceswer eobserve d inparameter swhic hwer eno t likely tob eaffec ­ tedb y theapplie d insecticides.On eexampl e is thecompletel y different vegetation inME S IIIAcompare dwit h that in HIB in 1978.Thi s differencemigh t have been causedb y theciliat ebloom ;a t the time theME Swa s subdivided no submerged plants werevisibl e atal lan d itmigh t havebee n that the systemha dno t yet takenit s final courseo fdevelopment .Subdivisio n then resulted inth edivergen t development of thevegetatio n inth e subsystems.Suc hdissimila r development might be averted infutur e studiesb ypostponin g thesubdivisio nunti l thevegetatio n has grown toa substantially largerbiomass .Sinc e inME Sa suse d in this study the vegetationpro ­ bably isa majo r conditioning parameter for the zooplankton,a later subdivisionma y alsopromot e similar development of thepopulatio n of thedifferen t zooplankton species. The limitednumbe r ofME S didno tpermi t astatistica l evaluation of theresults . Nevertheless thechange s in zooplanktondevelopmen t in the treated subsystems com­ paredwit h thoseo f thecontrols ,coul d inmos t casesb e attributed to theinsectici ­ de treatment onth ebasi s ofcircumstantia l evidence.Suc hevidenc e isbase d on the observed general similarity indevelopmen tbetwee ncorrespondin g subsystems ando n the rapid andclea r changes inpopulatio ndevelopment ,i nrespons e tosuc h treatment.

6.5 CONCLUSIONS

The results show that^E So f the typeuse d inth epresen t study largelymee t the requirements set inSectio n6.1 . - Several trophic and otherrelationship s arepresen t in theMES .Th edifferen t spe­ cies ofplants ,zooplankto nan dmacrofaun a that takepar t in these relationships are coninonan dwidesprea d inth eNetherland s (Werkgroep Biologische Katerbeoordeling,1977; De Lange,1976;Notenboom-Ra m et al.,1976) . -Th eME Sca nb ekep t intact fora perio d ofa t least 3-4 yearswithou t any apparent sign of long-term deterioration. It therefore seems thaton eo f themai n problems in model ecosystem research,namel y thegenerall y short lifetimeo f the systems,doe s notpla y a role inthi s type ofMES . -Th eME Sprove d tob e largeenoug h tomaintai n adivers e and equilibrated zooplank­ tonan dmacrofaun a community and topermi t anadequat e biological and chemical sampling programme.However ,wit h theexceptio n ofChaoboru s larvae,n o adequate monitoring of themacrofaun a community could be accomplished with the sampling method inthi sstudy . -Als o theME Sprovide d thepossibilit y todistinguis h betweennatura l phenomena and those induced by the insecticide treatments even though the limited numbers of MESdi dno tpermi t astatistica l evaluation.Th eexperiment s showed thatunde rnear - -natural conditions O.Sm gm parathionan d 1m gm J parathion and azinphosmethyl canstrongl y reducepopulation s ofCladocer a fora t least as long as theseconcentra -

75 tionsar emaintained . Populations ofChaoboru s crystallinus are strongly reduced by 1m gm parathion. Inth eME Sexperiment s thepopulation s sometimes recovered after the treatmentwa s stopped andsometime s didnot .Bu t it isno t clearwha t thismean s for field situations.Sometime sphytoplankto nbloom s occurredwhic h seemed tob e caused by theabsenc eo fa nadequat e herbivorous zooplanktonpopulatio n due toa reduction inth enumber so fCladocera .Cyclopoid aan dOstracod awer eno t sensitive to eitherparathio n orazinphosmethy l inth econcentration s applied.Althoug h no detailed conclusions canb emad e aboutothe rmacrofaun a thanChaoborus ,i tca nb e concluded that therewer en o severe effects.Parathio na t 0.2m gm most likelyrepresent s a no toxic effect level foral l species studied.T owha t extent blooms ofciliate s and filamentous algae aret ob e attributed to the insecticides applied cannotb e assessed from thepresen t data.No r canan ydefinit e conclusionb emad e as towhethe r orno t thesmal l dimensions of theNE Shav epromote d theseblooms .

76 7 Comparison of the MES-experiments with the laboratory toxicity trials

Ingenera lther ewa sa goo dagreemen tbetwee nth eresult so fth e>>ES-experiment s andthos eobtaine d inth elaborator ytoxicit ytests .I norde rt oillustrat ethi s point,th eeffect so npopulation so fsevera lspecie si nth ef^ES ,whic hwer eascribe d toth einsecticid etreatment ,wer ecompare dwit hth esub-acut e laboratorytoxicit y trials.Fo rthi spurpos eprediction sbase do nth emaximu mconcentration scausin gn o effecti nth elaborator ytest s('no-effec tconcentrations* )wer ecompare dwit hth e outcomeo fth eMES-experiments .Fo rthes eprediction s the3-wee kno-effec tconcentra ­ tionsobtaine dwit hDaphni amagn awer econsidere d tob erepresentativ eno tonl yfo r allDaphniida e (e.g.Daphni aspp. ,Simocephalus )bu tals ofo rth eChydorida e (e.g. Chydorus,Graptoleberis ) (Table 42). ForDaphni aspp .th eobserve d effectsfull ycom ­ pliedwit hth epredictions.Discrepancie sbetwee npredicte dan dobserve deffect sfo r O.Sm gm ~ parathionwit hregar dt oS .vetulus ,G .testudinari aan dC .crystallinu s onlyreflec tmino rquantitativ edifferences ;th epredicte deffect soccu ra t 1m gm . Theprediction swit hregar d toa neffec to f1 m gm azinphosmethylo nC .sphaericu s

Table42 .Predicte d andobserve d effectso fparathio nan d azinphosmethyl inth e MESuse d inth epresen tstudy .Firs tsig nrepresent spredicte d effects (Chapter7) , second signgive s theobserve deffects . (+)effect ,(- ) noeffect ,(± )dubiou seffect ,(o )dat ainconclusive .

Species Paraith :io n Azinphosmethyl

0.2 mg m-3 0.5m gm -3 1m gm~ 3 1m gm~ 3

Daphniaspp . -/- +/ + +/+ +/+

Simocephalus -/- +/ - +/+ +/+ vetulus

Chydorus -/- +/+ +/+ +/- sphaericus

Graptoleberis -/- +/- +/± +/ - testudinaria

Asellus -/o -/o -/o +/o aquaticus

Chaoborus -/- +/- +/+ -/- crystallinus

Cloeon -/o +/o +/o -/o dipterum

77 and G.testudinari a didno tcom e true.However ,th equestio n remains whether aslight ­ lyhighe r concentration ofthi s insecticidewoul d haveproduce d thepredicte d effect. ForA .aquaticu s andC .dipteru m theresult so fth e^ES-experiment swer e inconclusive. From theoveral l good agreementbetwee n theresult s fromth e laboratory tests and theMES-experiment son eshoul dno tdra wth econclusio n thati ngenera l simple laboratory testsca nprovid e sufficient information forth eecologica l hazard assess­ ment forothe r groupso fcompound s aswell .Fo rothe r compounds the comparability ofresult s observed forth eorganophosphoru s insecticides testedi nthi sstud yma y notexist .Bu tmor e important,an dthi sals oapplie s toparathio n andazinphosmethyl : the results ofth eMES-experiment s have theadvantag eo fbein gobtaine d undernear - natural environmental conditions andthu sprovid ea mor e solid basis forextrapolatio n to field situations. On thebasi so fth epresen t study itis ,therefore ,recommende d that thepresen t tentative limit ('voorlopigegrenswaarde' ) forth econcentratio n of Cholinesterase inhibitors insurfac ewater s inth eNetherland s shouldb e lowered from 1m gm " (Ministerieva nVerkee r enWaterstaat , 1975)t o0. 1m gm ~ paraoxon equivalents.Fo r comparison:th ecriteri a forfreshwate r andmarin e aquatic lifea sse tb yth eU S Environmental ProtectionAgenc y (1976)fo rparathion ,azinphosmethy l (Guthion)an d malathionar e0.04 ,0.01 ,an d0. 1m gm ,respectively .Thes e criteria arebase do n the9 6hou rLC ™ values ofsevera l specieso fmicrocrustacean smultiplie d bya narbi ­ traryapplicatio n factoro f0.1 .Th eresult so fth epresen t study suggest thatfo r organophosphorus insecticidesno-effec t levels forfiel d situations might becalcu ­ lated from 48hou rEC ™ valuesb ymultiplicatio n bya factor0.1 . 8 Conclusions and recommendations

-Th eresult s obtainedi nth eMES-experiment s show thatwit ha limite dnumbe ro f systems,i nspit eo fth eimplici t statistical limitations, invaluable information canb eobtaine d aboutth evulnerabilit yo fa rang eo fspecie swithi na comple xeco ­ logical setting. -Th eresult so fth epresen t studysugges t thatfo rorganophosphoru s insecticides no-effect levelsfo rfiel d situationsmigh tb ecalculate d from 48-hourEC ™ values forDaphni amagn aStrau sb ymultiplicatio nb ya facto r0.1 . -I ti srecommende d that thepresen t tentative limit ('voorlopigegrenswaarde' )fo r theconcentratio no fCholinesteras e inhibitorsi nsurfac ewater si nth eNetherland s shouldb elowere d from1 m gm (Ministerieva n Verkeere nKaterstaat , 197S)t o 0.1m gm " paraoxonequivalents .

Finally,i ti sm yopinio n that,becaus eo fou rsmal lan dfragmentar y knowledge ofth etota l complexityo frelationship si nfreshwate recosystems ,freshwate r quality criteriawit h regardt otoxi c substances shouldb eteste di nmode l ecosystemso fa relativelyhig hnatura l complexityan dunde rnear-natura l testcondition s before they come into force (seeals o Golterman,1976).Th etyp eo fHE Suse di nth epresen t study couldb edevelope d intoa valuabl emode lfo rsuc ha purpose .

79 Summary

A number oforganophosphorou sinsecticide s arecontinuousl y present ascontami ­ nants insurfac ewater s inth eNetherlands .Simila r findingshav ebee n reported from otherEuropea n countries.Th e levels reported canb e considered as relatively high, because theyar eo f the sameorde ra s thelevel s found tob e toxic tosevera l aquatic organismsunde r laboratoryconditions . Itwa s the aimo f thepresen t study toasses s thehazard s of long-term exposure offreshwate recosystem s toorganophosphoru s insecticides,wit h specialemphasi s on themos t susceptible groups.Sinc e sucha nassessmen t cannotb e basedo n laboratory toxicity dataalone ,a nattemp twa smad e todevelo pa testmode l incorporating some ofth emajo r environmental variables tonarro w down the riskmargi n inth eextra ­ polation of laboratory data to field situations and toenabl e amor e realistic hazard assessment. After the introduction inChapte r 1,th e literature on the toxicology oforgano ­ phosphorus insecticides inrelatio n to freshwater ecosystems is surveyed inChapte r 2. Inthi schapte r the following subjects arecovered : theenvironmenta l fate, including volatilization,distributio n over theecosystems , (photo)chemica l andmicrobia l breakdown,an d the toxicity toaquati corganisms .Fro m the literature data thecon ­ clusion isdraw n that theconcentration s inth e surfacewater s in theNetherland s may represent a toxicological risk forCladocer a (waterfleas)an d certain aquatic insects. Chapter 3give s the analytical-chemical methods asapplie d inthi s study,fo r themeasuremen t oforganophosphoru s insecticides. InChapte r 4detaile d information on the laboratory toxicity tests isgiven .Th e insecticides testedwer eparathion ,azinphosmethyl ,diazinon ,malathio nan d parathion- methyl.Thes e compoundswer e tested instati c 48-hour and 21-day toxicity trials on Daphniamagna , (water flea),Asellu s aquaticus (water hoglouse),Cloeo ndipteru m (mayfly larva),an dChaoboru s crystallinus (phantommidg e larva).O f these species onlyDaphni a was reared inth e laboratory.Al l other test specieswer e collected in the fieldo r fromoutdoo rmode l ecosystems (i\ES). Inadditio ncomparativ e testswer e carried out inwhic hal l five insecticideswer e tested simultaneously oneac hspecies . Toxicity rangeswer e assessed forHerpobdell a octoculata (leech),Dugesi a lugubris (flatworm),Notonect a sp.an dSigar a sp. (water bugs),an d Culexsp . (mosquito larva). A summaryo fth e tests conducted and themajo r testcondition s ispresente d in Table 19.I tca nb econclude d from thecomparativ e tests thatparathio n isth emos t toxic of the five compounds tested (Table 27). Parathion-methyl appears tob e by far the least toxic forth e insect larvae.Th e toxicityo f theothe r compounds doesno t

80 varymarkedl y from species to species.O f the species tested only inth e range-finding tests,th eparathio nEC ™ values for the insects,viz .Cule x sp.,Notonect a sp.an d Sigara sp.ar e of the sameorde ra s forCloeo ndipteru man dChaoboru s crystallinus. Theconclusio n canb edraw n thatchroni c exposure toconcentration s of theorde r of tenths ofm gm " of organophosphorus insecticides may endanger the survival and the reproductive potential ofCladocer a and aquatic insects. Chapter 5describe s the studyo f the rateo fdissipatio n of the fiveorganophos ­ phorus insecticides fromth ewate rphas e of three freshwater aquaria.Aquariu m 1an d 2wer e providedwit h a bottom layer of sand andver y finemu d andplante d with Elodea densa.Aquariu m 3wa sno tprovide dwit heithe rbotto mmateria l orwate rplant s so that the influenceo f sediment andplant so n thedissipatio n rate could be evaluated. The five insecticides were applied to theaquari a ata concentration of 1m g m each.On e dm aliquotswer ewithdraw n from the aquaria after 1,20 ,50 ,90 ,152 ,216 , 355,52 5an d 1005hour s afterdosin g andafte rextractio n analysed by gas-liquid chromatography. The half-lives ofth einsecticide s were calculated,assumin g a first order reaction (Table 28). Thepresenc eo fplant s andbotto mmateria l resulted ina meaningful increase indissipatio n rate forazinphosmethyl ,parathion,parathion-methy l anddiazino nbu tno t formalathion . Inbot h the aquariawit hplant s and sediment the ordero fth e half-lives for the five insecticides isth e same:diazino n >azinphosme ­ thyl >parathio n >parathion-methy l >malathion .O n thebasi so fth e resultso f this experiment acalculatio n could bemad e of the frequency bywhic h the two insecticides parathion and azinphosmethyl had tob eapplie d to theMES . InChapte r 6 a report isgive no f theMES-studies . InSectio n6. 1 theus e of >ES in toxicological research isbriefl ydiscussed . It isstate d that long-term eco­ logical effects ofchemical s on freshwater ecosystems canonl yb e studied ifsevera l requirements,whic h cannot possibly beme to na laboratory scale,ar e fullfilled. For that reasonoutdoo rME Swer e designed to study theeffect so fparathio n and azinphosmethyl undernear-natura l conditions.Fou rplasti c containerso f 3x 1x 1 meterwer e dug into the soilo na plo tnea r the laboratory (Fig.10).Subsequentl y the containerswer eprovide dwit h bottommaterial ,water ,wate rplant s and aquatic inver­ tebrates.Al l four containerswer e divided into twosubsystem swit h acrosswis e water­ proof partition at thestar t of theexperimenta l season,whic husuall y lasted from May toOctober .Thereafte r the sheetswer e removed inorde rt o reunite the subsystems and restorepre-experimenta lconditions . During theexperimenta l period in 1977tw oNE Swer e treatedwit h 1m gm para­ thion and azinphosmethyl,respectively,whil en o insecticide was applied toth e other NES. In 1978al l fourME Swer e treatedwit h parathion (1,0. 5 and 0.2 mgm )o r azinphosmethyl (1m gm " ).I nal l thetreate dMES ,on e subsystem received the insec­ ticide treatmentwhil e thecorrespondin g subsystem served ascontrol .Durin g the experimental periods anextensiv emonitorin g programme was carried outwhic h consisted ofphysico-chemica l measurements,vegetatio n assessment,insecticid e analyses and population assessment of zooplanktonan dmacrofauna .Th epurpos e of this programme

81 was tose ewhethe r orno tdifference s could be observed between thedevelopmen t of theparameter s incorrespondin g subsystems and ifso ,whic h ofthes e differences could beattribute d to insecticide treatments. The results aredescribe d anddiscusse d inSectio n 6.3 and 6.4,respectively , while conclusions arepresente d inSectio n6.5 . Itwa s found that corresponding sub­ systems of theuntreate dME S showed a similardevelopmen t fornearl y allphysico - chemical andbiologica l parameters.Suc hsimilitarie swer e alsoobserve d between corresponding subsystems ofME S thatwer e treatedwit h insecticide,fo rthos epara ­ meterswhic hwer eno taffecte d by the insecticide treatment.O n theothe r handdif ­ ferenceswer e observed inparameter swhic hwer eno t likely tob e affected by the applied insecticides.However ,thes e lastdissimilaritie smigh t be averted in future studies bypostponin g thesubdivisio nunti l thevegetatio n as themajo r conditioning parameterha s grown toa substantially larger biomass. Although thelimite d numbero fME Sdi dno tpermi ta statistical evaluation of the results,th e changes in zooplanktondevelopmen t inth e treated subsystems compared with thoseo f thecontrol s couldnevertheles s inmos t casesb e attributed to thein ­ secticide treatmento nth ebasi so fcircumstantia l evidence.A survey of thesechange s in zooplanktondevelopmen t isgive n inTabl e 39.Marke d reductions inth e abundance ofDaphni a spp.wer e observed during theapplication s ofparathio n (0.5an d 1ni gm ~ ) and azinphosmethyl (1m gm ).Stron g reductions inabundanc ewer e recorded for Simocephalusvetulu s in response to 1m gm parathion and toa lessier extent for 1m gm azinphosmethyl.Number so fChydoru s sphaericus decreasedmarkedl y in the presenceo f 1m gm parathion and somewhat lessa t aconcentratio n of0. 5 mgm . Populations ofChaoboru s crystallinus rapidlydecrease d ata concentration of1 mgm parathionbu tno ta t 0.5m gm .Onl ya slightdecreas e innumber swa s obser­ ved forGraptoleberi swit h 1m gm parathion.Afte r the introduction of theinsecti ­ cides theabundanc e ofCyclopoid a (naupliian d copepodits included)an d of Ostracoda did notdecrease .Sometime sphytoplankto nbloom s occurredwhic h seemed tob e caused by theabsenc e ofa nadequat e herbivorous zooplanktonpopulatio ndu e toa reduction of thenumber so fCladocera .Althoug h nodetaile d conclusions canb emad e about macrofauna other thanChaoborus ,i tca nb econclude d that therewer e no severeef ­ fects.Th eNE Sprove d tob e suitable forproducin g invaluable information about the vulnerability fororganophosphorou sinsecticide s fora rangeo fspecie s ina complex ecological setting. InChapte r 7a compariso n ismad ebetwee n theoutcom eo f the^ES-experiment s and the resultso f the laboratory toxicity trials. Ingenera l therewa s a good agreement as isillustrate d inTabl e42 . InChapte r 8th econclusion s andrecommendation s aregiven .

82 Samenvatting

Eenaanta l organofosforinsekticideni svoortduren d als ontreiniging aanwezigi n Nederlandse oppervlaktewateren. Soortgelijke bevindingen zijngemel dui t andere Europese landen.D egerapporteerd e niveaus kunnen als tamelijkhoo gworde nbeschouwd , wanneerme ni naanmerkin g neemtda tz eva ndezelfd egrootte-ord e zijnal sd eniveau s dieonde r laboratoriumomstandigheden schadelijk zijngebleke nvoo rdivers ewater ­ organismen. Doelva nd eonderhavig e studiewa see nschattin gt emake nva nd erisico' sva n langdurigeblootstellin gva nzoetwater-ecosysteme naa norganofosforinsekticiden , waarbijd enadru kla go pd evoo rdez e stoffengevoeligst e groepen.Aangezie nhe t nietmogelij ki see ndergelijk e risicoschatting uitsluitend tebasere no ptoxici - teitsgegevensverkrege ni nhe t laboratorium,wer dee n poging ondernomeno mee n test- modelt eontwikkele nwaari nee naanta lva nd ebelangrijkst emilieuvariabele n konden wordenverenigd .Di tteineind ed erisicomarge ,di ebi jd eextrapolati eva n labora- toriumgegevensnaa rveldsituatie s optreedt,t everkleine ne nd emogelijkhei d teschep ­ penvoo ree nrealistische r risicoschatting. Nad einleidin gi nhoofdstu k 1,word ti nhoofdstu k2 ee n overzicht gegevenva n de literatuurbetreffend ed etoxicologi eva norganofosforinsekticide n inrelati eto t zoetwater-ecosystemen. Indi thoofdstu kworde nd evolgend e onderwerpen behandeld: het lotva nd esto fi nhe tmilieu ,inclusie fvervluchtiging ,verdelin g overhe teco ­ systeem, (foto)chemisch ee nmicrobiël eafbraa ke nd etoxicitei tvoo rwaterorganismen . Uitd eliteratuurgegeven s wordtd econclusi egetrokke nda td econcentraties ,zoal s aangetroffeni nhe tNederlands e oppervlaktewater,ee ntoxicologisc h risicokunne n vormenvoo rCladocer a (watervlooien)e nbepaald ei nhe twate r levende insekten. Hoofdstuk3 geef td eanalytisc h ehemischemethode n die zijntoegepas tvoo rhe t metenva n organofosforinsekticiden. Inhoofdstu k4 word tuitvoeri g ingegaano pd etoxiciteitstoetse n inhe t labora­ torium.D etoetse nwerde nuitgevoer dme td einsekticide nparathion ,azinfosmethyl , diazinon,malathio ne nparathion-methyl .Dez e stoffenwerde ngetoets ti nstatisch e 48-uur-e n21-dagen-toetse ne nuitgevoer dme td eproefdiere n Daphniamagn a (watervlo), Asellus aquaticus (waterpissebed),Cloeo ndipten m (eendagsvlieglarve)e nChaoboru s crystallinus (pluimmuglarve).Va ndez e soortenwer d alleenDaphni ai nhe t laboratorium gekweekt.All e andere toetsorganismenwerde ni nhe tvel dverzamel do fui td ebuiten ­ gelegenmodelecosysteme n (MES)gehaald . Daarnaastwerde ne rvergelijkend e toxiciteits­ toetsenuitgevoerd ,waari n allevij f insekticiden simultaano pelk e soortwerde n uit­ getest.Voo rHerpobdell a octoculata (bloedzuiger),Dugesi a lugubris (platworm),

83 Notonecta sp.e nSigar asp . (waterwantsen)e nCule x sp. (steekmuglarve) werden 'range-finding' toetsenuitgevoerd .Ee n overzichtva n deze toetsene nva nd ebelang ­ rijkste toetsomstandigheden isgegeve n intabe l 19.Ui t de resultatenva nd everge - ' lijkende toetsenka nworde n geconcludeerd datparathio nhe tmees t toxisch isva n de vijfgetoetst everbindinge n (tabel 27).Parathion-methy lblijk tverui t demins tgif ­ tige te zijnvoo rd e insektelarven.D eoverig e stoffenverschille n pertoetsorganis - me onderling niet duidelijk intoxiciteit .D eECr n-waardenva nparathio nvoo r dein - sektelarvenva nCule x sp.,Notonect a sp.e nSigar a sp.zij nva ndezelfd egrootte - ordeal svoo rd e larvenva nCloeo ndipteru m enChaoboru s crystallinus. Geconcludeerd kanworden ,da t chronischeblootstellin g aanconcentratie s van tiendenva nm gm van organofosforinsekticiden devoortplantin g enoverlevin g voor cladoceren en inhe t water levende insekten ingevaa rka nbrengen . Inhoofdstu k 5word the t onderzoekbeschreve nnaa rd e snelheidwaarme e de vijf organofosforinsekticiden verdwijnenui t dewaterfas eva ndri e zoetwateraquaria. Aquarium 1e n 2werde nvoorzie nva nee nbodemlaa g van zand en fijnsli b enbeplan t metElode adensa . Inaquariu m 3ontbrake n dezeelemente n teneinde de invloed van sediment enplante n opd everdwijnsnelhei d tekunne nbepalen .D evij f insekticiden werden elk inee n concentratieva n 1m gm " ind e aquariagedoseerd .N a 1,20 ,50 ,90 , 3 152,216 ,355 ,52 5e n 1005uu rwer d telkens 1d m waterui td e aquaria geëxtraheerd engaschromatografisc hgeanalyseerd . De halfwaardetijdenva nd e insekticidenwerde n berekend opbasi sva nee neerste-orde-reacti e (tabel 28).D e aanwezigheid van planten enbodemmateriaa l resulteerde inee nbelangrijk e toename ind everdwijnsnelhei d van azinfosmethyl,parathion ,parathion-methy l endiazinon ,maa rnie t indi eva nmala - thion. Inbeid eaquari ame t planten enbodemmateriaa l was devolgord e vanhalfwaarde ­ tijdendezelfde :diazino n >azinfosmethy l >parathio n >parathion-methy l >malathion . Opbasi sva nd e resultatenva ndi texperimen t kanworde nbereken dme twelk e frequentie parathion enazinfosmethy l ind eME S zoudenmoete nworde n gedoseerd. Inhoofdstu k 6word tversla g gedaanva nhe t onderzoekme t deMES . Inparagraa f 6.1 wordthe tgebrui kva nME S inhe t toxologisch onderzoek kortbediscussieerd .Ge ­ steldword tda t lange-termijneffectenva nchemicalië n op zoetwater-ecosystemen slechtsda nkunne nworde nbestudeerd ,wannee rword t voldaan aanee naanta l eisen welkewaarschijnlij k niet op laboratoriumschaal kunnenworde n gerealiseerd. Om die redenwerde nME S ontworpenwelk ebuite nware n gesitueerd endi ediende n terbestude ­ ringva nd eeffecte nva nparathio n enazinfosmethy l onder zonatuurlij k mogelijke omstandigheden.Hierto ewerde nvie rplasti cbakke nva n3 m x 1mx lmingegrave n op een terreindich tbi jhe t laboratorium (fig. 10).Vervolgen swerde nd ebakke n voorzien vanbodemmateriaal ,water ,waterplante n en zoetwater-evertebraten.All e vier debak ­ kenwerde n bijaanvan g vand eperiod e vanproefnemingen ,welk emeesta l duurde vanme i totoktober ,doo rmidde lva nee nwaterdich t schotverdeel d intwe e subsystemen. Na afloopva ndi eperiod ewerde n de schottenwee rverwijder d teneinde de subsystemen te herenigene n terugkeer tebewerkstellige n naar omstandigheden vergelijkbaarme t die vanvóó rd eproefnemingen .

84 Gedurended eexperimenteerperiod e in197 7werde ntwe eME Sbehandel dme t 1m gm vanrespectievelij kparathio ne nazinfosmethyl ,terwij l ind eander eME Sgee nbehan ­ delingme tinsekticid ewer dtoegepast .I n197 8werde nall evie rd eME Sbehandel dme t -3 -3 parathion(1 ,0, 5e n0, 2m gm )o fazinfosmethy l (1m gm ).Bi jall ebehandeld e MESwer dhe tinsekticid etoegedien d aanéé nsubsysteem ,terwij ld ecorresponderend e helftal scontrol efungeerde .Gedurend ed eproefneminge nwer dee nuitgebrei dbemon ­ steringsprogrammauitgevoerd ,omvattend e fysisch-chemischebepalingen ,vegetatie - opnamen,insekticide-analyse se ntellinge nva n zoöplanktone nmacrofauna .Doe lva n ditprogramm awa st ezie no fe rverschille nzoude nzij nwaa rt eneme ni nd eontwikke ­ lingva nd eparameter sva ncorresponderend e subsystemene nz oja ,welk eva ndez e verschillen zoudenkunne nworde ntoegeschreve naa nd ebehandelin gme tinsekticide . Deresultate nworde nbeschreve ne nbediscussieer d inrespectievelij kparagraa f 6.3e n6.4 ,terwij ld econclusie sworde ngeformuleer d inparagraa f6.5 .E rwer dge ­ vondenda tvrijwe lall efysisch-chemisch ee nbiologisch eparameter si nd ecorrespon ­ derendesubsysteme nva nd eonbehandeld eME S zicho pdezelfd ewijz eontwikkelden . Soortgelijkeovereenkomste nwerde noo kwaargenome ntusse ncorresponderend esubsyste ­ menva nME Swelk eme t insekticidewerde nbehandeld ,voo rdi eparameter swaarva nee n beïnvloeding doorhe tinsekticid enie twaarschijnlij kwas .Anderzijd svertoonde nsom ­ migeva ndergelijk eparameter sjuis twe lverschillen .Dez everschille nechte rzoude n intoekomstig eproefneminge nkunne nworde nvoorkome ndoo rd etweedelin gva nd eME S uitt estelle nto td evegetatie ,al sd ebelangrijkst econditionerend eparameter ,zic h zouhebbe nontwikkel d totee naanzienlij kgroter ebiomassa . Ofschoonhe tbeperkt eaanta lME Sgee nstatistisch eevaluati eva nd eresultate n toestond,ware ne rtoc hvoldoend eaanwijzinge no md everanderinge ni nd eontwikkelin g vanhe tzoöplankto ni nd ebehandeld esubsystemen ,i nvergelijkin g totdi e ind econ ­ troles,i nd emeest egevalle nto et ekunne nschrijve naa nd ebehandelin gme tinsek ­ ticide.Ee noverzich tva ndez everanderinge n ind eontwikkelin g vanhe tzoöplankto n ist evinde ni ntabe l39 .E rwer dee nsterk eafnam eva nd eomvan gva nd epopulatie s vanDaphni aspp .waargenome ngedurend ed ebehandelin gme tparathio n (0,5e n1 m gm ) enazinfosmethy l (1m gm ~ ).Ster kverminderd eaantalle nwerde noo kgesignaleer d voorSimocephalu svetulu sbi jee nconcentrati eva n 1m gm parathione ni nminder e matebi jd edoserin gme t 1m gm azinfosmethyl.D eaantalle nva nChydoru ssphaericu s daaldenster kbi jee nconcentrati eva n 1m gm parathione ni niet sminder emat ebi j 0,5m gm .Populatie sva nChaoboru scrystallinu sname nsne li nomvan ga fbi j 1m gm parathion,maa rnie tbi j0, 5m gm"' 3.Slecht see n lichtedalin g inaantalle nwer dwaar ­ genomenvoo rGraptoleberi stestudinari abi j 1m gm parathion.Voo rd eCyclopoid a (inclusiefnaupli ie ncopepodieten )e nOstracod awer dgee nafnam eva nd epopulatie - omvangwaargenome nal sreacti eo pd e insekticiden. Inenkel egevalle ntra dalgenbloe i op,welke ,naa rhe tscheen ,wer dveroorzaak tdoo rd eafwezighei dva nvoldoend eherbi ­ voorzoöplankto ntengevolg eva nee nafnam eva nd ecladoceren-populatie .Ofschoo ngee n gedetailleerdegevolgtrekkinge nkunne nworde ngemaak tte naanzie nva nander emacro - faunasoortenda nChaoborus ,ka ntoc hworde ngestel dda tzic hgee ningrijpend eeffecte n

85 hebbenvoorgedaan . Inhoofdstu k 7worde n de resultatenva nd eMES-experimente nvergeleke nme t die vand e toxiciteitstoetsen inhe t laboratorium. Overhe t algemeen stemdend e resulta­ tengoe dovereen ,zoal sval t af te lezenui t tabel42 . Inhoofdstu k 8worde n de conclusies enaanbevelinge n gegeven.Dez e luiden: -D e resultatenverkrege nme t deMES-experimente n tonenaa nda tme t eenbeperk t aan­ talsystemen ,i nweerwi lva nd edaaraa n inherente statistische beperkingen,zee r waardevolle informatie kanworde nverkrege n omtrent dekwetsbaarhei d vanee n reeks organismen inee ncomplex e ecologische context. - De resultatenva nd eonderhavig e studie suggererenda tvoo r organofosforinsektici- denno-effect-concentratie s voorveldsituatie s zoudenkunne nworde n afgeleid van 48-uurEC ™ waardenvoo rDaphni amagn a Straus,doo rdez ewaarde n tedele ndoo r10 . -Aanbevole nwordt ,d evoorlopig e grenswaardevoo r de concentratieva n cholinesterase- remmersi nNederlands e oppervlaktewateren teverlage nva n 1m gm (IndicatiefMeer ­ jarenprogramma 1975-1979va nhe tMinisteri e vanVerkee r enWaterstaat ) naar 0.1 mg m paraoxon-equivalenten.

Tenslotte spreekt deauteu r als zijnmenin g uitdat ,i nverban dme t onzebeperk ­ tee nfragmentarisch e kennisva nhe t complexe geheel van relaties in zoetwater-eco­ systemennorme nme tbetrekkin g tottoxisch e stoffen inhe t zoetwater,alvoren s van kracht teworden ,eers t zoudenmoete nworde ngetoets t inmodelecosysteme nme t een relatiefgrot emat eva nnatuurlijk e complexiteit enonde r zonatuurlij k mogelijke omstandigheden. Naar zijnmenin g zoudend eME S gebruikt ind e onderhavige studie tot eenwaardevo lmode l vooree ndergelijk e toetsing kunnenworde n ontwikkeld.

86 AppendixA .Lis to f theimportan t crustaceanplankto n inth efreshwater so fth e Netherlands. (AfterGulati ,1978) .

Phylum:Arthropod a class :Crustace a I. Subclass:Malacostrac a division:Pericarid a order :Mysidacea e (Neomysis integer) order :Isopod a (Asellus spp) order :Amphipod a (Gammarus spp)

II. Subclass:Branchiopod a 1.order :Anostrac a (Chirooephalus sp) 2.order :Phyllopod a 1.suborder :Notostrac a (Lepidurus spp, Triops sp) 2.suborder :Conchostrac a (brackishwate rforms ) 3.suborder :Cladocer a 1.Haplopod a (Leptodora kindtii) 2.Eucladocer a 1.superfamil y Sidoidea (Sida sp, Diaphanosoma sp) 2.superfamil yDaphnoide a 1famil yChydorida e (Alona spp, Graptoleberis sp, Alonella spp, Peracantha sp, Pleuroxus spp, Chydorus spp) 2famil yMacrothricida e (Iliocryptus sp) 3famil yBosminida e (Bosmina spp) 4 familyDaphniida e (Daphnia spp, Simooephalus spp, Ceriodaphnia spp)

III.Subclass :Maxillopoda . 1.order :Copepod a 1.suborder :Cyclopoid a 2.suborder :Calanoid a 3.suborder :Harpactoid a

IV. Subclass:Ostracod a AppendixB .Numbe ro fmicrocrustacean s inth ewate rsample s takenfro m thetes t aquariafo rinsecticid eanalysi sa tdifferen t timesafte r theinsecticide swer e introduced.

Time Simocephalus Chydorus Graptoleberis Cyclopoida (hours) vetulus sphaericus testudinaria

Aquarium1 0 3 155 - 75 88 - 55 20 2 11 1 40 50 2 3 - 93 90 1 5 1 139 152 5 2 28 203 216 5 4 23 88 355 24 3 26 224 525 46 5 25 140 1002 14 - 11 59

Aquarium2 0 16 206 10 298 1 6 205 10 178 20 5 32 7 246 50 1 - 13 285 90 - 2 4 155 152 21 30l 4551 216 2 355 - 1 12 242 525 13 2 5 90 1002 35 - 22 118

Aquarium3 0 - 998 86 31 1 1 1058 82 42 20 - 17 12 30 50 - - 4 25 90 - - 7 31 152 - - 6 31 216 - - 2 29 355 - - 4 12 525 1 - 4 7 1002 - - 3 13

*Th e2 sample swer eaccidentl ypu ttogether .

Theresult so fth ezooplankto ncounting sd ono tpermi tmor e thansom epreliminar y conclusions:- Cyclopoid ad ono tsee mt eb eaffected ;- population s ofChydoru s sphaericusar eheavil y reduced innumbers ;- population so fGraptoleberi s testudinaria dono tsee mt ob eaffecte d inth eaquari awit hsedimen tan dplants .However ,number s inth eaquariu mwithou t thesematter swer eclearl yreduced .

88 AppendixC .Mai naquati c invertebrates studied inth eMES-experiments .Permissio nt o reproducefigure so grante d by theFranckh'sche nVerlagshandlung ,KOSMOS-Verlag , Stuttgart,fro mStreble/Krauter ,Da sLebe ni mWassertropfen ,1976 . ofro mH.C .Redeke , Hydrobiologieva nNederlan d -D ezoet ewateren .Backhuy s& Meesters ,Amsterdam ,1975 .

ODaphniamagn a DDaphniapule x CDaphnia longispina <6ram <4m m <2.5m m

CSimocephalusvetulu s CGraptoleberistestudinari a CPeracanthatruncat a <3m m <0.7m m <0.65m m

"Chaoboruscrystallinu s <14m m

^Copepod 1-4m m cOstracod0.5-1. 2m m nAsellusaquaticu s (Macrocyclopsalbidus ) (Cypridopsisvidua ) <20m m 89 AppendixD .Lis to faquati c zooplanktonan dmacrofaun a species collected fromth e MESdurin g 1976-1978.

Trioladida Cladoaera - Chydoridae Dendrocoelumlacteu m Alonaguttat a Dugesia lugubris Alonella (exiqua?) Planariatorv a Chydorus sphaericus Polycelisnigr a Graptoleberis testudinaria P.tenui s Peracantha truncata Pleuroxusaduncu s Ciliata Spirostomumambiguu m Ostracoda Stentorpolymorphe s CandonaCandid a Cypridopsisvidu a Rotatoria Notodromasmonach a Brachionussp . Keratellaquadrat a Copepoda - Cyolopoida Lecanesp . Acanthocyclopsvernali s Polyarthrasp . Cyclopsfurcife r C.strenuu s Mollusca Eucyclopssperatu s Macrocyclopsalbidu s Bythinia tentaculata Lymneaperegr a L.stagnali s Copepoda - Harpaotoida Physa fontinalis Isopoda Planorbiscorneu s P.planorbi s Asellusaquaticu s P.vorte x Proasellusmeridianu s Valvatacristat a Ephemeroptera Hirudinea Cloeondipteru m Glossiphoniacomplanat a G.heteroclit a Odonata - Zyg optera Helobdellastagnali s Dipt era Herpobdellaoctoculat a H.testace a Ceratopogonidae Culexsp . Oligochaeta Chaoborus crystallinus Chironomussp . Chaetogastersp . Lumbriculusvariegatu s Coleoptera Stylarialacustri s Tubifexsp . Ilibiussp . Haliplusspp . Helephorusguttatu s Cladooera - Daphniidae Hydroporus erythrocephalus Ceriodaphniaquadrangul a H.planu s Daphniapule xcurvirostri s Laccophilusminutu s Daphniapule xpule x Simocephalusvetulu s Heteroptera Corixapunctat a Gerrislacustri s Notonectaglauc a Sigara lateralis Sigarastriat a

90 Appendix E. Figs 18-33.

(N+1) organisms 10*, -PARATHION 1 mgm"

2 10 H io1H

MAY ' JUNE ' JULY ' AUG. ' SEPT. '

Figure 18.Number so fSimocephalu svetulu s present inth ezooplankto nsample sfro m MESI/I V (Figs,a/d ,respectively )i n 1977.Perio do f insecticide treatment isindi ­ catedb yvertica l lines.Shade dbar s indicateperiod so falga lbloom ,dotte dbar s ofciliat ebloom . treatedsubsyste m (exceptME S IIan d IV) control.

91 (N*1)organisms AZINPHOSMETHYL -, 1 mgm-3

1U - -PARATHION 0.2 mgm-3 ^

102-

1 Ir 10 - 1/

•x—'l L L_ . ! l^u^L ^2 " 1 T'

MAY JUNE JULY AUG. SEPT. OCT.

Figure 19.Number so fSimocephalu svetulu s present in thezooplankto nsample sfro m MESI/I V(Figs ,a/d ,respectively )i n 1978.Perio d ofinsecticid e treatment isindi ­ catedb yvertica l lines.Shade dbar sindicat eperiod so falga lbloom ,dotte dbar s ofciliat ebloom . treatedsubsyste m control.

92 cone,azinphosmethy l( m gm )

13 27T4 MAY JUNE JULY AUG. SEPT. OCT. Figure 20.Number so fSimocephalu spresen t inth ezooplankto nsample s fromME S Ii n 1978.Perio do finsecticid e treatment isindicate d byvertica l lines.Inser tshow s thepatter no f theconcentratio no fazinphosmethy l inth ewate rphase . treated subsystem control.

(N+1)organisms

MAY JUNE JULY AUG. SEPT. Figure 21.Number so fDaphni a longispina present inth ezooplankto nsample sfro m MESI (Fig.a )an dME S IV(Fig.b )i n 1977.Perio do f treatment isindicate d byverti ­ cal lines.Horizonta lba r indicatesa perio do falga lbloom . treatedsubsyste m (exceptME S IV) control.

93 (N+1)organisms

-AZINPHOSMETHYL - 1 mgm-3

A. y- •_

•PARATHIO N 0.2 mgm-3-. 10'

10 V /\ ts ó V. ^^- 103-!

-PARATHION 0.5mgm-3-i

10' V r~—. v. 10' ^-

1 -^ A /f\ MAY JUNE JULY AUG. SEPT. OCT.

Figure 22.Number so fDaphni a longispinaan dD .pule xpresen t inth ezooplankto n samplesfro mME S I,I Ian d IV (Figs,a/c ,respectively )i n 1978.Fo rME SI Inumber s ofD .pule xcurvirostri s are included inD . longispinanumbers .Perio d oftreatmen t isindicate db yvertica l lines.Shade dbar s indicateperiod so falga lbloom ,dotte d barso fciliat ebloom . treatedsubsyste m control.

94 (N+i)organisms I PARATHION i mgm-3

101 C -AZINPHOSMETHYL S\ tmgm"3 / '"\ * 2 \ .' \ 10 - V \ \ Arf 'X A \

101- '1 A. / / '«. 1\ 1 l/\J \J / [^ -'..<; S.*;'; 1 1 • • i I

MAY JUNE JULY AUG. SEPT.

Figure23 .Number so fChydoru ssphaericu spresen t inth ezooplankto nsample sfro m MESI/I V(Fig .a/d ,respectively )i n 1977.Perio do f treatment isindicate db yver ­ tical lines.Shade dbar sindicat eperiod so falga lbloom ,dotte dbar so fciliat e bloom. treated subsystem (exceptME S IIan d IV) control.

95 (N+1)organisms l—AZINPHOSMETHYL 1 rngm"3

10J ' ,"•3v V rPARATHION 0.2mgrrr

10" r~-" pPARATHION 1 mgrTT?^=-^'

10

IO'H

^%\$4iRej3\&&:ï>M!*m V//W/W/M'////A

PARATHION Q5mgm-3 —| 10'

10

' MAY ' JUNE ' JULY ' AUG. ' SEPT ' OCT.

Figure 24.Number s ofChydoru s sphaericus present inth ezooplankto n samples fromME S I/IV (Figs,a/d ,respectively ) in 1978. Period of treatment isindicate d byvertica l lines. Shaded bars indicate periods ofalga l bloom, dotted bars ofciliat e bloom. treated subsystem control.

96 (NM)organisms

PARATHION 1mgm- 3 — A/

10"-| NO INSECTICIDE TREATMENT

2 10 - Av A VA J»* A^

!01- 1' II Jl '1 • ill l I

NO INSECTICIDE TREATMENT

' MAY ' JUNE ' JULY ' AUG. ' SEPT. ' OCT. Figure 25.Number s ofGraptoleberi s testudinaria present inth ezooplankto n samples fromMES I/I V (Figs,a/d ,respectively )i n 1977. Period of treatment isindicate d by vertical lines. Shaded bars indicate periods ofalga l bloom, dotted barso f ciliate bloom. treated subsystem (exceptME SII ,IV ) control.

97 (N+1)organisms

MAY JUNE JULY AUG. SEPT. OCT. Figure 26.Number so fGraptoleberi s testudinariapresen t inth ezooplankto nsample s fromME SI/I V(Figs ,a/d ,respectively )i n 1978.Perio do f treatment isindicate d byvertica l lines.Shade dbar s indicateperiod so falga lbloom ,dotte d barso f ciliatebloom . treatedsubsyste m control.

98 (N+Dorganisms io3

10:

PARATHION 0.5mg m -3.

10 ,y\ \ io1H A '

MAY JUNE ' JULY ' AUG. ' SEPT. ' OCT.

Figure 27.Number s of Peracantha truncata present in the zooplankton samples from MES II, IV (Figs. a,b, respectively) in 1977 and MES II, IV (Figs. c,d, respectively) in 1978. Period of treatment is indicated by vertical lines. Shaded bars indicate periods of algal bloom. treated subsystem (except MES II, IV in 1977) control.

99 (N+1) organisms 10"-,

IO1-T t>'>i>v^^yAV/,''y.^-\y/////^v///ï r

104n

NO INSECTICIDE TREATMENT io3H

io2i

NO INSECTICIDE TREATMENT

MAY ' JUNE ' JULY ' AUG. ' SEPT ' OCT.

Figure28 .Number so fCyclopoid a(al lstages )presen ti nth ezooplankto nsample sfro m MESI/I V (Figs,a/d ,respectively )i n1977 .Perio do ftreatmen ti sindicate db y vertical lines.Shade dbar sindicat eperiod so falga lbloom ,dotte dbar so fciliat e bloom. treatedsubsyste m (exceptME SII ,IV ) control.

100 (N+ 1)organism s 104n rAZINPHOSMETHYL 1mgrrr 3

MAY JUNE ' JULY ' AUG. SEPT. OCT. Figure 29.Number so fCyclopoid a (allstages )presen ti nth ezooplankto nsample sfro m MESI/I V (Figs,a/d ,respectively )i n 1978.Perio do f treatmenti sindicate db y vertical lines.Shade dbar sindicat eperiod so falga lbloom ,dotte dbar so fciliat e bloom. treatedsubsyste m control.

101 (NM) organisms 10: PARATHION imgm"

io2H i v / \ ioH J

"":.; i

toS rAZINPHOSMETHYL Imgm'

MAY 'JUNE' JUL Y 'AUG. 'SEPT. 'OCT .

Figure 30.Number so fOstracod apresen t inth ezooplankto nsample s fromME SI/I V (Figsa/d ,respectively )i n 1977.Perio do ftreatmen t isindicate d byvertica llines . Shadedbar sindicat eperiod so falga lbloom ,dotte dbar so fciliat ebloom . treated subsystem (exceptME SII ,IV ) control.

102 (N+1) organisms io3

rAZINPHOSMETHYL1 mgm" 3

103n

•PARATHIO N O.Smgm-l^

MAY JUNE JULY 'AUG .' SEPT.'OCT . Figure 31.Number so fOstracod a present inth ezooplankto nsample s fromME SI/I V (Figs,a/d ,respectively )i n 1978.Perio do ftreatmen t isindicate d byvertica l lines.Shade dbar s indicateperiod so falga lbloom ,dotte dbar so fciliat ebloom . — treated subsystem control.

103 (N+1)organisms 102-i |—PARATHION 1 mgm"3- a.

10 ./ . /

I v_yC_> wWfr 7ZZZZZÊSfc2.

102 NO INSECTICIDE TREATMENT

r-AZINPHOSMETHYL 1 mgm-3.

MAY JUNE ' JULY ' AUG. SEPT. OCT. Figure 32.Number so fChaoboru s crystallinuspresen ti nth ezooplankto nsample sfro m MESI/I V (Figs,a/d ,respectively )i n1977 .Perio do ftreatmen ti sindicate db y vertical lines.Shade dbar s indicateperiod so falga lbloom ,dotte dbar so fciliat e bloom. treatedsubsyste m (exceptME SII ,IV ) control.

104 (N*1)organism s -AZINPHOSMETHYL1 mgm- 3

b 101 -PARATHION 0.2mgm-3 "*55v '\--7< 1 v 10 - h /T m. 1 - ' 1 r i—,— —. i

Figure 33.Number s ofChaoboru s crystallinus present inth ezooplankto n samples from MES I/IV (Figs,a/d ,respectively ) in1978 .Perio d of treatment isindicate d byver ­ tical lines. Shaded bars indicate periods ofalga l bloom, dotted bars of ciliate bloom. treated subsystem control.

105 Appendix F. Number ofmacrofaun a collected from the water plants that were removed from theME S inNovembe r 1977 and 1978.

Organisms Sub MES I MES II MES III MES IV sys­ tem 1977 1978 1977 1978 1977 1978 1977 1978

Trialadida A 15 99 28 218 159 26 53 64 B 4 58 18 323 216 18 42 56

Dendrocoelum lacteum A 4 63 17 167 30 4 9 33 B 2 42 9 291 55 1 4 17

Dugesia lugub iris A 11 36 2 41 129 22 5 4 B 2 16 1 117 161 17 5 5

Polycelis spp A - - 9 10 - - 39 27 B 8 5 33 34

Hirudinea A 141 26 127 123 161 38 5 71 B 57 27 68 131 195 54 6 64

Herpobdella s pp. A 119 19 97 88 36 30 54 69 B 49 12 46 123 126 50 25 59

Glossiphonia spp. A 11 7 10 3 14 2 2 B 1 4 5 5 6 1 1 5

Helobdella stagnalis A 11 20 24 18 6 - B 7 9 17 28 3 -

Crustacea A 243 54 664 11 367 38 176 20 B 60 28 412 1 1039 84 188 164

Asellus sp. A 200 54 430 II 307 38 122 20 >4ram B 58 28 410 1 633 84 163 164 Asellus sp. A 43 234 60 54 <4 mm B 2 2 406 25

Oligochaeta Lumbriculus A - - 142 - - variegatus B — — 134 _

Mollusca A 42 11 163 232 92 448 130 64 B 91 1 111 67 30 111 127 42

Gastropoda A 42 11 163 232 63 441 130 64 B 91 1 111 65 29 109 127 41

Bivalvia A - 29 47 - B - - 2 1 2 1

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