Integration of Embryonic Zebrafish and Passive Sampling Device Extracts to Explore Mixture Toxicity

Transcription

1 Integration of Embryonic Zebrafish and Passive Sampling Device Extracts to Explore Mixture Toxicity Margaret M. Corvi 1 R.L. Tanguay 2 K. A. Anderson 2 1 BioResource Research 2 Environmental and Molecular Toxicology Oregon State University 1

2 What are the environmental concentrations available to biota? Integration: Questions Are levels and mixtures of chemical compounds found in our rivers, lakes, and streams of concern? 2

3 What are the environmental concentrations available to biota? Are levels and mixtures of chemical compounds found in our rivers, lakes, and streams of concern? This research demonstrates a proof of concept integration of two tools. A brief application of this integration is the evaluation toxicity of pesticide mixtures. Exploiting Integration 3

4 Passive Sampling Device (PSD) Applications Environmental Sampler Deployed at contaminated sites Freely dissolved compounds Simple and durable 4

5 Passive Sampling Device (PSD) Properties Low density polyethylene tubing Sequesters hydrophobic and semi-polar contaminants Pore size of PSD similar to biological membrane 1,2 1) Huckins, J. et al. (26) 2) Anderson et al (28) 5

6 Passive Sampling Device (PSD) Lay-flat tubing microns X 2.5 cm wide Pre-cleaned with 2 hexanes dialyses Tubing was constructed Final dimensions L = 1 cm Storage Amber jars at -2 C until dialysis Extract: 2 separate dialysis 4 hour (h) and 2 h in hexanes Concentration to 2 ml 6

7 PSD tubing extract Hexanes Concentrated Passive Sampling Device (PSD) Extract Applications Analysis Bioassays 7

8 Embryonic Zebrafish (Danio rerio) Model Properties Rapid assessment High fecundity Vertebrate model Developmental model Transparent Multiple-endpoint assessment Less expensive than other in vivo assessments 1 hpf 72 hpf 8

9 Embryonic Zebrafish Exposures Assessments Glass-coated 96 well plate One embryo per well Volume for exposure 1µL Dimethyl sulfoxide (DMSO) Highest concentration of DMSO for exposures was 1% 1,2 Control fish were exposed to fish water (FW) only Tropical 5 D line 1) Hallare, A et al. (26) 2) Usenko, et al. (26) 9

10 Embryonic Zebrafish Exposures Assessments Embryonic Zebrafish Exposure Exposure: pre-organogenesis Embryos were dechorionated DMSO was used as vehicle Assessments 24 embryos per group (n) 3 hpf Mortality 126 hpf Morphology and mortality 1

11 Mortality Embryonic Zebrafish Observations Multiple Endpoints Morphological defect 1 (location) Heart (HE) Yolk sac (YSD) Snout (Snt); Jaw (J) Tail (T) Body Axis (Ax) Control: 126 hpf J Eye (E) Fin (Fc) ; (Fp) Ax E Otic (Ot) Notochord (Not) Body proportion (St) HE 1) Kimmel et al Treatment: 126 hpf YSD 11

12 Versus: Mortality Only Assessment Mortality : One common measure of toxicity is LC5 Definition LC5 Standardized test typically 96 hours measuring the concentration (mg/l of water) of a chemical that will lead to mortality in half of the sample Also called median lethal concentration 12

13 Hypothesis 1 Blank passive sampling device extracts will not cause abnormal development in embryonic zebrafish. Expose embryos to blank extracts 13

14 Experimental Design: Blank Extracts 14

15 Blank PSD Extracts % Mortality 2 1 Fish water PSD Blank DMSO % Heart Edema 2 1 % Yolk Sac Defect 2 1 Treatment % Curved Axis 2 1 Treatment Assessment 126 hours post fertilization FW, PSD Blk, DMSO; n= 17, 15, 5* *DMSO data provided by Anderson Lab 15

16 Conclusion 1: Zebrafish embryos exposed to blank PSD extracts showed no significant abnormal responses above fish water control. 16

17 Hypothesis 2 Pesticide spiked PSD extracts will induce concentrationdependent increase of abnormal morphology in zebrafish embryos. Comprehensive pesticide mixture (14 compounds) Incidence of effects EZ Metric (EZM) to evaluate toxicity of mixture Evaluated pesticide mixture without PSD extract 17

18 Experimental Design: Spiked Extracts 18

19 Pesticide Mixture Fourteen pesticides Chlorpyrifos Fenitrothion Dimethoate Lindane p,p-ddt; p,p- DDE, p,p- DDD Dicofol Dieldrin Stereoisomers: α, β endosulfan Propanil Fipronil Cypermethrin Organophosphates Organochlorines Other Pesticides General use herbicide Modern insecticide Pyrethroid 19

20 Embryonic Zebrafish Metric (EZM) Score EZM Score evaluates group toxicity Non-biologically ranked sub-lethal endpoints Example: The sum of 12 effects for a group of 24 embryos at 126 hpf would score 21.6 on EZM scale Mortality Notochord Mortality Notochord Heart Brain Yolk Sac Body (stubby) Circulation Eye Jaw Tail Somite Caudal Fin Pectoral Fin Snout Body Axis Otic 3 hpf 126 hpf 2

21 Pest Mix Comprehensive (14 compounds) EZM Score Pesticide Mixture r Pest Mix PSD Pest Mix FW r r r r r r r Concentration (µm) 6 Mix+ PSD Mix Assessment 126 hours post fertilization FW; Pest Mix PSD, Pest Mix exposures n= 17; 3, 3 r = significantly different than control 21

22 Pesticide Mixture: Endpoint Incidence % Mortality 1 Pest Mix PSD Pest Mix Fish Water 5 % Heart Edema 1 5 % Yolk Sac Defect Concentration (µm) % Curved Axis Concentration (µm) 22

23 Conclusions 2 EZM score was for control (FW) and all concentrations of Pest mix greater than 6.4 µm were significantly different. EZM score was not significantly different for the 4 µm Pest mixture with PSD when compared to the Pest mix at 4 µm and 43 µm. 23

24 Hypothesis 3 Mixture subsets of pesticide spiked PSD extracts will induce abnormal morphology in zebrafish embryos. Individual pesticides found in mixture subsets will elicit the same toxic response as the corresponding partial mixture at the same molar concentration. Organochlorine group v. dieldrin Organophosphate group v. fenitrothion EZ Metric (EZM) to evaluate toxicity of mixture Incidence (%) 24

25 Partial Mixtures and Reasoning OC mix Dicofol, dieldrin, DDT, lindane, endosulfan (α; β) OP mix Chlorpyrifos, Dimethoate, Fenitrothion Individual compounds Fenitrothion Dieldrin Group mixtures Similar characteristics Individual pesticides were present in group mixtures 25

26 Organochlorine mix: EZM Scores and Mortailty OC Mix v. Dieldrin EZ Metric Score 24 Dieldrin OC Mix FW 12 r r, s Concentration (um) r % Mortality r Concentration (µm) Assessment 126 hours post fertilization r = significantly different than control FW, OC Mix, Dieldrin; n= 17, 3, 2 (16) s = significantly different than 21µM dieldrin 26

27 Organochlorine mixture: Incidence OC Mix v. Dieldrin % Curved Axis 1 Fish Water Dieldrin OC Mix Concentration (µm) %Yolk Sac Defect 1 5 r Concentration (µm) r r Assessment 126 hours post fertilization FW, OC Mix, Dieldrin; n= 17, 3, 2* (16 embryos) r = significantly different than control 27

28 Organophosphate mix: EZM Scores and Mortality OP Mix v Fenitrothion EZ Metric Score 24 Fenitrothion OP Mix FW vs FW avg 12 r % Mortality Concentration (µm) Concentration (µm) Assessment 126 hours post fertilization FW, OC Mix, Dieldrin; n= 17, 3, 2* (16 embryos) r = significantly different than control 28

29 Conclusions 3 Increasing concentration of dieldrin spiked PSD did induce an increase in EZM score in exposed zebrafish embryos. Dieldrin treatments 13 and 21 µm were different than control. Analysis of multiple endpoints confirmed that the incidence of yolk sac defect at 126 hpf was higher than control for dieldrin at the two concentrations EZM score for OC mix exposure and dieldrin exposure at 21 µm was significantly different. Analysis of multiple endpoints showed that incidence of mortality, heart edema, and yolk sac defect were different for OC mix when compared to control 29

30 Conclusions 3 (cont.) Increasing concentration of fenitrothion spiked PSD did not induce an increase in EZM score for exposed embryos. No difference between control and highest and lowest concentrations Analysis of multiple endpoints revealed no difference in % incidence among three fenitrothion treatments 4, 7, 18 µm OP mix EZM score was different than control EZM score. OP Mix exposures produced an increased incidence of yolk sac defect 3

31 Hypothesis 4 Individual pesticides spiked PSD extracts will induce different sub-lethal effects in embryonic zebrafish. Individual pesticide exposures will result in EZM scores that are different than control. EZ Metric (EZM) Incidence (%) 31

32 Individual pesticides Individual compounds Cypermethrin Propanil Fipronil Individual compounds Toxicity Compounds were in comprehensive mixture 32

33 Cypermethrin, Propanil, Fipronil Individual Pesticides EZM Score 24 Fish Water Cypermethrin Propanil Fipronil 12 r r Concentration (µm) Assessment 126 hours post fertilization FW, Fipronil, Cypermethrin, Propanil; n= 17, 3, 3, 3 r = significantly different than control 33

34 Cypermethrin, Propanil, Fipronil % Mortality % Yolk Sac Defect 8 Fish water Fipronil Cypermethrin Propanil r r Treatment % Curved Axis % Heart Edema r r r Treatment 34

35 Conclusion 4 Toxicity (EZM Score) of cypermethrin and fipronil exposures were different than control. EZM Score of propanil was not different when compared to control Cypermethrin, fipronil, and propanil did not produce a difference in the incidence of mortality for 3 and 126 hpf, eye, tail, snout deformations when compared to control. 35

36 Conclusion 4 (cont.) Fipronil exposures had an increased incidence of heart edema, yolk sac defect, stubby body, curved axis when compared to control. Propanil exposures were not different than control for nine endpoints analyzed. Cypermethrin exposures had an increased incidence of heart edema and yolk sac defect when compared to control. 36

37 Hypothesis 5 Partial mixtures and individual pesticides exposures do not induce the same EZM scores as pesticide mixtures at similar molar concentrations. 37

38 EZ Metric Score comparison EZM Score Pest Mix PSD Pest Mix FW EZM Score Fenitrothion OP Mix FW vs FW avg f EZM Score 24 Fish Water Cypermethrin Propanil Fipronil 12 s s EZM Score 24 Dieldrin OC Mix FW 12 b Concentration (µm) Concentration (um) s = different than 4.2 µm Pest mix f = different than 13 µm Pest mix b = different than 2 µm Pest mix not all concentrations of dieldrin, fenitrothion were analyzed 38

39 Conclusions 5 Exposures to propanil and OC mix do not produce a different EZM scores when compared to their respective molar concentration of the comprehensive pesticide mixture. Exposures to cypermethrin and fipronil resulted in a higher EZM scores than their respective pesticide mixture concentration. Exposures to fenitrothion and OP mix resulted in lower EZM scoring than the pesticide mixture at the same molar concentration 39

40 Further research Investigate toxicity of other compounds in the mixture Investigate toxicity based on mode of action Integrate PSD and embryonic zebrafish with field studies Quantify amounts of compounds taken up by the embryo with and without chorion 4

41 Big picture conclusion This study indicates that the coupling of PSD extracts and in vivo toxicological assessments is realistic. Mixture toxicity is complex. Mixes can produce synergistic, additive or antagonist effects. 41

42 Acknowledgements Environmental and Molecular Toxicology Kim Anderson Robert Tanguay Laboratory group members: Sarah Allan Wendy Hillwalker Jane La Du Lucas Quarles Lisa Truong Bioresource Research Wanda Crannell Kate Field Students : Dawn Merrill Other support Josh Drescher Other faculty and students at Oregon State University. 42

43 Thank you Questions.? 43