Leif Norrgren. Department of Biomedicine and Veterinary Public Health Faculty of Veterinary Medicine and Animal Health SLU, Uppsala, Sweden

Transcription

1 Fish Models for Ecotoxicology Leif Norrgren Department of Biomedicine and Veterinary Public Health Faculty of Veterinary Medicine and Animal Health SLU, Uppsala, Sweden

2 Acknowledgements Patrik Andersson Anders Bignert Poul Bjerregaard Gunnar Carlsson Henrik Holbech Anna Norman Halden Gitte Petersen Mats Tysklind Stefan Örn

3 1970 Apical lesions Bone abnormalities Tumours 2011 Disturbed gonad differentiation and maturation Offspring mortality

4 Reproductive disorders in wild fish Fish with both eggs and sperms Small and immature ovaries Degenerated eggs within ovaries Mortality of larvae M74 Regulation and legislation! Baltic proper Baltic Salmon Perch Pike Map: Fish pictures.

5 250 HBCD in Guillemot egg, ng/g lipid w. HBCD, ng/g lw, guillemot egg n(tot)=177,n(yrs)=32 Average annual increase ca 3%/yr Кайра pia :58, HBCDUb Chemical analysis at ITM, SU

6 PFOS, ng/g ww, guillemot egg Кайра 1200 Average annual increase ca 9%/yr Data source: ITM, Katrin Holmstrom :31, pfosu2 Chemical analysis at ITM, SU

7 A Few Examples of Models Fish Embryo Toxicity Assays Fish Screening Assays Fish Sexual Developmental Assays Fish Full Life Cycle Assays Mesocosms Assays

8 A model species in the testing of chemicals suggested by OECD Offspring continuously and at high numbers Embryonic period rapid Chorion is transparent Short life cycle Reproduction ~ 4 months Reproduction > 4 years Hatching ~ 3 days Hatching ~ 6 months

9 Routes of exposure to chemicals From water via skin and gills From food and ingested water via the gastrointestinal tract From female to oocytes within the ovary - maternal transfer Ovary containing oocytes at different maturation stages From water via the chorion (egg shell) Spawned egg containing embryo

10 Fish Embryo Toxicity Assay Reproduction Embryo Larvae Adult Juvenile

11 The fish embryo assay Regulatory risk assessments of chemicals and drugs Assess effluents (oil/gas productions, domestic effluents, industrial effluents, runoff water from dumping sites and similar potentially toxic waters. Alternative to vivo models (RRR).

12 Studied parameters: Lethal: Coagulation No extension of tail No somites No heartbeats Sublethal: Spontaneous movement Eye development Pigmentation Circulation Oedema Heart frequency Hatching time 1h 24h 48h

13 Beats per minute Heart rate - Musk ketone ** ** ** ** ** 20 0 K Concentration (µg/l)

14 Heart arrythmia in zebrafish 18 of 23 drugs causing heart arrythmia in humans were positive in a zebrafish embryo assay after immersion The remaining 5 drugs were administered by nanoinjection into the yolk-sac resulting in that another 4 were positive (Milan et al 2003, Circulation )

15 (rörelser/minut) Doramectin (antiparasitic) Rörelser 24hpf 6 5 Movements / minute *** 1 0 Is ok 0,10 0,22 0,46 1,0 2,2 4,6 10 Concentration (mg/l)

16 Lakvatten A - Kläckningstid (hpf) Hatching time (hpf) *** *** *** 80 ** IsoK HSK 0,5 1,3 3, Spädning (%)

17 Conclusively, the zebrafish embryo assay Is a simple in vitro assay for screening of chemicals causing general effects, developmental disturbances and teratogenicity Dechorination may increase bioavailability and sensitivity Nonoinjection may reflect maternal transfer Introduction of a metabolic system means that metabolites can be assessed

18 Fish screening assay Reproduction Embryo Larvae Adult Juvenile

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20 THE YOLK PROTEIN-VITELLOGENIN Environmental factors Hypothalamus GnRH Pituitary GtH-I Ovary E2 VTG Egg yolk precursor Liver

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24 Fish Sexual Developmental Assay Reproduction Embryo Larvae Adult Juvenile

25 Phenotypic sex in fish is dynamic and sex is determined by many factors in the environment This has been used for decades in aquaculture to produce all male or all female populations Some species change sex normally Intersex is a common phenomen in many species

26 % 17 -estradiol * * * Control 4 ng E2/l 8 ng E2/l 14 ng E2/l 24 ng E2/l 54 ng E2/l 96 ng E2/l 259 ng E2/l Females Males Intersex Undifferentiated

27 THE YOLK PROTEIN-VITELLOGENIN Environmental factors Hypothalamus GnRH Pituitary GtH-I Ovary E2 VTG Egg yolk precursor Liver

28 Vtg ng/g fish 17 -ethinylestradiol E D C A A B control 30 <0.6 ng/l ng/l ng/l 31 9,9 ng/l ng/l EE2 concentrations

29 % 17 -ethinylestradiol Males Females 100 Undifferentiated * * * 90 * 80 * control <0,6 ng/l ,5 ng/l ,8 ng/l ,9 ng/l ng/l 16 EE2 concentrations

30 Methyltestosterone males females intersex 100 * * * * * 90 * % control ng/l 42 ng/l ng/l ng/l ng/l ng/l 4 MT concentrations

31 * * * * * * * Female Male Intersex Undifferentiated 60 % Control < 5 ng/l ng/l 23 ng/l 117 ng/l 67 trenbolone trenbolone trenbolone trenbolone 2779 ng/l trenbolone

32 Prochloraz; aromatase inhibitor

33 Fish full life cycle test Reproduction Embryo Larvae Adult Juvenile

34 BRFs, PBDDs and PCBs; Mixture studies High octanol-water partition coefficient (K ow ) Tend to adsorb to particulate matter, and may accumulate in organisms that serve as food for fish Maternal transfer have been shown for hydrophobic chemicals Exposure via feed and maternal transfer may be important routes for BFRs, PCBs and PBDDs

35 BFR MIXTURE The BFRs were selected to cover the range of physicochemical properties -The BFRs have different molecular structures and properties, such as varying degree of hydrophobicity. -The BFRs include those produced in highest volumes, and BFRs with high environmental relevance, such as tetrabromobisphenol A, hexabromocyclododecane (HBCD), and different brominated diphenyl ethers (BDEs) -equimolar mixture of the 11 BFRs

36 O O O BDE 28 BDE 183 BDE 209 HBCD HO HO OH OH O O O TBBPA TBBPA OHEE TBBPA DBPE O HO 2sty 246Ph TBECH HxBz

37 Conc (nmol/g lw) Concentrations in eggs after 35 days exposure (Hd and Ld) of females HBCD BDE 28 BDE 183 TBBPA DBPE TBECH BDE Ph TBBPA

38 Hatching success % Atretic follicles Dietary exposure of adult zebrafish to a mixture of brominated flame retardants (BFRs) ** ** 0.5 Eight of the eleven BFRs were detected in females and eggs Control LD MD HD ** Oocyte degeneration Reduced hatching 0 Control LD MD HD Nominal BFR mixture feed concentrations : 0 (control), 1 (LD), 10 (MD), 100 (HD) nmol of each BFR per g dry weight

39 Conclusions BFRs can be transferred from female to the progeny BFRs can affect female fish reproductive physiology BFRs can affect early life-stage development, both after parental and water exposure

40 PBDD Baltic Sea MIXURE -A mixture of 10 PBDDs was designed to reflect the relative proportions of PBDDs found in Baltic Sea biota, so that the most abundant congeners in environmental samples were in highest proportions in the mixture. -TBDD, which has been detected only in a few fish samples from the Baltic Sea at very low concentrations, was tested due to its high potency in other test systems and used as a positive control.

41 ominated Dioxins Studied PBDDs Chemical name Abbreviation Baltic Sea mixture 1- monobromodibenzo-p-dioxin 1-MBDD 2,7/2,8- dibromodibenzo-p-dioxin 27/28-DBDD 1,3,7- tribromodibenzo-p-dioxin 137-TrBDD 1,3,8- tribromodibenzo-p-dioxin 138-TrBDD 2,3,7- tribromodibenzo-p-dioxin 237-TrBDD 1,3,6,8- tetrabromodibenzo-p-dioxin 1368-TeBDD 1,3,7,9- tetrabromodibenzo-p-dioxin 1379-TeBDD 1,2,4,7/1,2,4,8- tetrabromodibenzo-p-dioxin 1247/1248-TeBDD 1,2,3,7- tetrabromodibenzo-p-dioxin 1237-TeBDD 1,2,3,8- tetrabromodibenzo-p-dioxin 1238-TeBDD Positive control 2,3,7,8-tetrabromodibenzo-p-dioxin TBDD

42 Dietary exposure of adult zebrafish to a Baltic Sea mixture or to TBDD Unfertilized and abnormal 5 hpf (%) Results All PBDDs were found in females & eggs Mix & TBDD affected early development Mix & TBDD affected genes & proteins regulated via the dioxin receptor pre-exposure exp week 8-9 * * 0 Control Mix-L Mix-M Mix-H TBDD-L

43 Dietary exposure of adult fish to TBDD III I II Reduced spawning success Control Reduced vitellogenin gene expression Atr Altered ovarian morphology TBDD-L (3 ng/g feed) TBDD-M (30 ng/g feed )

44 Conclusions PBDDs are maternally transferred to eggs PBDDs act via same mechanism as the chlorinated dioxins PBDDs cause effects on fish reproduction and development similar to those of chlorinated dioxins

45 Mesocosm studies have the advantage compared to laboratory approaches that it maintains a natural community under close to natural conditions, taking into account relevant aspects from the real world such as indirect effects, biological compensation and recovery, and ecosystem perspectives.

46 Combinations

47 Some conclusions and future challenges Development of models to meet for instance REACH, trade restrictions and risk assessment New technologies i.e. genexpression, -omics, behaviour and novel biomarkers will add alternatives to estimate chemical risks, but never forget basics such as pathology and physiology.

48 Envirovet Network of environmental health scientist/educators from USA and the nine countries bordering the Baltic Sea (Denmark, Estonia, Finland, Germany, Latvia, Lithuania, Poland, Sweden, and Russia), was founded in 2001 on an initiative of the College of Veterinary Medicine, University of Illinois and the Centre for Reproductive Biology in Uppsala, SLU,Sweden

49 Thanks for your attention

50 Conclusively, the PCB mixture Accumulated in a dose related manner After 13 weeks of exposure body, liver and ovary weights were lower in exposed fish The PCB mixture induced EROD activity Egg production was reduced in all groups exposed to PCBs The survival time of larvae from the HD group was 7 days compared with 14 days in the control