1. Pollutants and the environment Bill Indge

2. Pollutants and the environment Liver cell Cytoplasm Nucleus Vitellogenin (VTG) production in the liver Oestrogen receptor Gene coding for VTG Ribosome

3. Pollutants and the environment Vitellogenin (VTG) production in the liver Oestrogen receptor Gene coding for VTG Ribosome  Oestrogen enters the nucleus and binds with the oestrogen receptor

4. Pollutants and the environment Vitellogenin (VTG) production in the liver Gene coding for VTG  The activated receptor now attaches to the target gene The DNA is transcribed and VTG mRNA is produced

5. Pollutants and the environment Vitellogenin (VTG) production in the liver Gene coding for VTG  The VTG mRNA migrates to the ribosomes in the cytoplasm

6. Pollutants and the environment Vitellogenin (VTG) production in the liver Gene coding for VTG VTG is synthesised and secreted from the liver

7. Pollutants and the environment The title of the original paper on which this exercise is based is: Fathead minnow and bluegill sunfish life-stage responses to 17 β-estradiol exposure in outdoor mesocosms Sarah M. Elliott, Richard L. Kiesling, Zachary G. Jorgenson, Daniel C. Rearick, Heiko L. Schoenfuss, Kim T. Fredricks and Mark P. Gaikowsk A laboratory-based investigation

8. Pollutants and the environment In a laboratory-based investigation, sexually mature fathead minnows were randomly divided into two groups — an experimental group and a control group. A solution of synthetic oestrogen was added to the tank containing the experimental group of fish. The resulting concentration of oestrogen reflected environmental concentrations. A control solution was added to the tank containing the control group. 1 Explain why it was important that the fish were randomly divided into the two groups.(2 marks) 1 The idea of preventing conscious choice; This might influence results;[2]

9. Pollutants and the environment 2 A control solution was added to the tank containing the control group of fish. What would you expect to be in this control solution? Explain your answer.(2 marks) Think carefully about the nature of the control that is appropriate in this investigation. With the information provided, ‘nothing’ would be an inappropriate response. ‘Water’ might be a standard answer but it makes assumptions about the composition of the synthetic oestrogen solution. In a laboratory-based investigation, sexually mature fathead minnows were randomly divided into two groups — an experimental group and a control group. A solution of synthetic oestrogen was added to the tank containing the experimental group of fish. The resulting concentration of oestrogen reflected environmental concentrations. A control solution was added to the tank containing the control group.

10. Pollutants and the environment 2 Everything that was added to the fish in the experimental tank other than the synthetic oestrogen; This ensures that any difference would result from the oestrogen/ would not be the result of other substances in the solution;[2] In a laboratory-based investigation, sexually mature fathead minnows were randomly divided into two groups — an experimental group and a control group. A solution of synthetic oestrogen was added to the tank containing the experimental group of fish. The resulting concentration of oestrogen reflected environmental concentrations. A control solution was added to the tank containing the control group. 2 A control solution was added to the tank containing the control group of fish. What would you expect to be in this control solution? Explain your answer.(2 marks)

11. Pollutants and the environment Treated with synthetic oestrogenControl Body condition factor total mass/g × 100 000 total length 3 1.071.10* Gonadosomatic index testis mass/g × 100 body mass/g 0.8020.761 Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* Secondary sexual characters index 23* Some differences between fathead minnows treated with synthetic oestrogens and controls * Significantly different, p<0.01

12. Pollutants and the environment Treated with synthetic oestrogenControl Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* 3 One of the control fish had a hepatosomatic index of 1.35. The body mass of this fish was 2.73 g. Calculate the mass of its liver. Show your working.(2 marks) Remember If you don’t attempt the question, you cannot gain credit. Show your working: you can gain marks even if the answer is wrong. Use some common sense…the liver won’t weigh more than the fish! * Significantly different, p<0.01

13. Pollutants and the environment 3 2 marks for correct answer of 0.04 g; 1 mark for incorrect answer but clearly showing that the calculation is based on the formula:hepatosomatic index = liver mass × 100; body mass 3 One of the control fish had a hepatosomatic index of 1.35. The body mass of this fish was 2.73 g. Calculate the mass of its liver. Show your working.(2 marks) Treated with synthetic oestrogenControl Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* * Significantly different, p<0.01

14. Pollutants and the environment Treated with synthetic oestrogenControl Gonadosomatic index testis mass/g × 100 body mass/g 0.8020.761 4 The testis mass is given as an index. Explain why it was given as an index, not the actual mass.(2 marks) 4 Allows results to be compared/standardised/body size to be taken into consideration; Because testis mass will vary with body mass/larger fish will have larger testes;[2]

15. Pollutants and the environment 5 The scientists who carried out this investigation reported that large protein-containing vesicles had accumulated in the cytoplasm of the liver cells in a significant number of the experimental group of fish. (a) Explain how the data in the table support this observation. (2 marks) 5(a) The experimental/oestrogen-treated group have a (significantly) higher hepatosomatic index; (Larger liver) caused by the protein-containing vesicles; [2] Treated with synthetic oestrogenControl Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* * Significantly different, p<0.01

16. Pollutants and the environment 5(b) Use your knowledge of the way in which oestrogen affects vitellogenin production to explain the presence of the protein- containing vesicles in the liver cells of the experimental group of fish. (3 marks) 5(b) Binds with oestrogen receptors present in liver cells; Stimulates transcription of VTG mRNA; VTG protein synthesised; VTG accumulates in vesicles in liver cells;[3 max] Treated with synthetic oestrogen Control Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* * Significantly different, p<0.01

17. Pollutants and the environment * Significantly different, p<0.01 Treated with synthetic oestrogenControl Body condition factor total mass/g × 100 000 total length 3 1.071.10* Gonadosomatic index testis mass/g × 100 body mass/g 0.8020.761 Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* Secondary sexual characters index 23* 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

18. Pollutants and the environment Treated with synthetic oestrogen Control Body condition factor total mass/g × 100 000 total length 3 1.071.10* 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks) Mating success is likely to depend on body condition/Males with poorer body condition likely to have lower mating success; * Significantly different, p<0.01

19. Pollutants and the environment Treated with synthetic oestrogen Control Gonadosomatic index testis mass/g × 100 body mass/g 0.8020.761 The difference in gonadosomatic index is not significant/The oestrogen- treated fish have significantly lower body condition and secondary sexual characters index; 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks) * Significantly different, p<0.01

20. Pollutants and the environment Treated with synthetic oestrogen Control Hepatosomatic index liver mass/g × 100 body mass/g 1.551.35* Larger liver in oestrogen- treated males likely to be linked to high vitellogenin production; 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks) * Significantly different, p<0.01

21. Pollutants and the environment Treated with synthetic oestrogen Control Secondary sexual characters index 23* Secondary sexual character important in courtship/defence of territory; 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks) * Significantly different, p<0.01

22. Pollutants and the environment The title of the original paper on which this exercise is based is: Collapse of a fish population after exposure to a synthetic estrogen Karen A. Kidd, Paul J. Blanchfield, Kenneth H. Mills, Vince P. Palace, Robert E. Evans, James M. Lazorchak and Robert W. Flick A field-based investigation

23. Pollutants and the environment The protocol Control lake (B)Experimental lake (A) 2 years prior to starting treatment 3 years of treatment No synthetic oestrogen added Regular addition of synthetic oestrogen 2 years after treatment stopped Measurements collected from fathead minnows from both lakes of concentration of the egg protein vitellogenin (VTG) concentration of vitellogenin mRNA numbers and lengths of fish

24. Pollutants and the environment 32103210 6000 4000 2000 Concentration of VTG/ arbitrary units 2000200120022003 2000200120022003 The concentration of VTG in whole-body homogenate samples of male fathead minnows captured during the spring of each of the years shown Lake B Lake A Year

25. Pollutants and the environment 32103210 6000 4000 2000 Concentration of VTG/ arbitrary units 2000200120022003 2000200120022003 Lake BLake A 1 Explain the advantage of the break in the y-axis on this graph.(1 mark) 2 Explain what is meant by a whole-body homogenate.(2 marks) 1 Allows a wide range of values/high and low values to be plotted/read accurately;[1] 2 In homogenate the cells have been mechanically disrupted; In this case the whole fish/all tissues homogenised;[2]

26. Pollutants and the environment 32103210 6000 4000 2000 Concentration of VTG/ arbitrary units 2000200120022003 2000200120022003 Lake BLake A 3 Explain the difference in the results for lakes A and B. (3 marks) 3 Lake B and lake A in 2000 and 2001 show natural variation/small differences may not be significant; Synthetic oestrogen added to lake A in 2002; Stimulates secretion of VTG by liver; Carried by blood to all organs (so homogenate concentration increases);[max 3]

27. Pollutants and the environment Year Mean population index Control lake (B)Experimental lake (A) 1999490.3180.0 2000 19.1 50.3 2001 56.1117.7 2002 9.6 0.7 2003 20.6 2.6 2004 51.9 0.1 2005355.9 0.1 Addition of synthetic oestrogen Numbers of fish given as a population index

28. Pollutants and the environment Year Mean population index Control lake (B)Experimental lake (A) 1999490.3180.0 2000 19.1 50.3 2001 56.1117.7 2002 9.6 0.7 2003 20.6 2.6 2004 51.9 0.1 2005355.9 0.1 4 Explain why the figures were given as the population index and not as the total population. (1 mark) 5 Describe the results shown in the table. (3 marks) 4 Difficulty of obtaining an accurate/reliable count; [1] 5 Variable population in lake B, and in lake A before treatment; In 2002/year after treatment large drop in population of lake A; Population in lake A after treatment stays low/does not recover; [3]

29. Pollutants and the environment Fork length Total length Measuring fathead minnows 6 Fork length gives a more reliable measurement of the length of the fish than total length. Suggest why. (1 mark) 6 The ends of the tail may be worn/two lobes of tail fin may be different lengths;[1]

30. Pollutants and the environment Fork length Lake BLake A Number of fish Histogram showing lengths of fish 2000 2002 2004

31. Pollutants and the environment Fork length Lake B Lake A 2000 2002 2004 Number of fish Histogram showing lengths of fish 7(a) Few fathead minnows live beyond 2 years. In any given year there are young fish that hatched that year and mature fish that hatched in the previous year. Explain how the histograms support this information.(1 mark)

32. Pollutants and the environment Fork length Lake B Lake A Number of fish Histogram showing lengths of fish 7(a) The histogram is bimodal/contains two peaks, suggesting two classes of fish.[1] 2000 2002 2004

33. Pollutants and the environment Fork length Lake A 7(b) Use the data from the table to explain the results for 2002 in lake A.(3 marks) 7(b) The table shows the large decrease in population the year after the addition of synthetic oestrogen; Histogram shows no young fish hatch in 2002; Small number in histogram the result of few fish hatching in 2001; [3] 2000 2002 2004

34. Pollutants and the environment Collapse of a fish population after exposure to a synthetic estrogen Karen A. Kidd, Paul J. Blanchfield, Kenneth H. Mills, Vince P. Palace, Robert E. Evans, James M. Lazorchak and Robert W. Flick But also… Feminising males means extinction Fish devastated by sex-changing chemicals in municipal wastewater Because of oestrogen, Canadian fish are too ‘confused’ to mate Birth control for fish