Pollutants and the environment Bill Indge Pollutants and

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Pollutants and the environment Bill Indge

Pollutants and the environment Bill Indge

Pollutants and the environment Vitellogenin (VTG) production in the liver Liver cell Cytoplasm Oestrogen

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

Pollutants and the environment Vitellogenin (VTG) production in the liver j Oestrogen enters the

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

Pollutants and the environment Vitellogenin (VTG) production in the liver k. The activated receptor

Pollutants and the environment Vitellogenin (VTG) production in the liver k. The activated receptor now attaches to the target gene Gene coding for VTG l The DNA is transcribed and VTG m. RNA is produced

Pollutants and the environment Vitellogenin (VTG) production in the liver m The VTG m.

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

Pollutants and the environment Vitellogenin (VTG) production in the liver n VTG is synthesised

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

Pollutants and the environment A laboratory-based investigation The title of the original paper on

Pollutants and the environment A laboratory-based investigation 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

Pollutants and the environment In a laboratory-based investigation, sexually mature fathead minnows were randomly

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]

Pollutants and the environment In a laboratory-based investigation, sexually mature fathead minnows were randomly

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. 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.

Pollutants and the environment In a laboratory-based investigation, sexually mature fathead minnows were randomly

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. 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) 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]

Pollutants and the environment Some differences between fathead minnows treated with synthetic oestrogens and

Pollutants and the environment Some differences between fathead minnows treated with synthetic oestrogens and controls Treated with synthetic oestrogen Control 1. 07 1. 10* 0. 802 0. 761 1. 55 1. 35* 2 3* Body condition factor total mass/g × 100 000 total length 3 Gonadosomatic index testis mass/g × 100 body mass/g Hepatosomatic index liver mass/g × 100 body mass/g Secondary sexual characters index * Significantly different, p<0. 01

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic index liver mass/g × 100 body mass/g * Significantly different, p<0. 01 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!

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic index liver mass/g × 100 body mass/g * Significantly different, p<0. 01 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) 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

Pollutants and the environment Treated with synthetic oestrogen Control 0. 802 0. 761 Gonadosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 0. 802 0. 761 Gonadosomatic index testis mass/g × 100 body mass/g 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]

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic index liver mass/g × 100 body mass/g * Significantly different, p<0. 01 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]

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 1. 55 1. 35* Hepatosomatic index liver mass/g × 100 body mass/g * Significantly different, p<0. 01 5(b) Use your knowledge of the way in which oestrogen affects vitellogenin production to explain the presence of the proteincontaining 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 m. RNA; VTG protein synthesised; VTG accumulates in vesicles in liver cells; [3 max]

Pollutants and the environment Treated with synthetic oestrogen Control 1. 07 1. 10* 0.

Pollutants and the environment Treated with synthetic oestrogen Control 1. 07 1. 10* 0. 802 0. 761 1. 55 1. 35* 2 3* Body condition factor total mass/g × 100 000 total length 3 Gonadosomatic index testis mass/g × 100 body mass/g Hepatosomatic index liver mass/g × 100 body mass/g Secondary sexual characters index * Significantly different, p<0. 01 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

Pollutants and the environment Treated with synthetic oestrogen Control 1. 07 1. 10* Body

Pollutants and the environment Treated with synthetic oestrogen Control 1. 07 1. 10* Body condition factor total mass/g × 100 000 total length 3 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 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

Pollutants and the environment Treated with synthetic oestrogen Control 0. 802 0. 761 Gonadosomatic

Pollutants and the environment Treated with synthetic oestrogen Control 0. 802 0. 761 Gonadosomatic index testis mass/g × 100 body mass/g The difference in gonadosomatic index is not significant/The oestrogen-treated fish have significantly lower body condition and secondary sexual characters index; * Significantly different, p<0. 01 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

Pollutants and the environment Treated with synthetic oestrogen Control Larger liver in oestrogentreated males

Pollutants and the environment Treated with synthetic oestrogen Control Larger liver in oestrogentreated males likely to be linked to high vitellogenin production; Hepatosomatic index 1. 55 1. 35* liver mass/g × 100 body mass/g * Significantly different, p<0. 01 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

Pollutants and the environment Treated with synthetic oestrogen Control Secondary sexual character important in

Pollutants and the environment Treated with synthetic oestrogen Control Secondary sexual character important in courtship/defence of territory; Secondary sexual characters index 2 3* * Significantly different, p<0. 01 6 Using only data from the table, explain why synthetic oestrogens may affect the reproductive success of male fathead minnows. (4 marks)

Pollutants and the environment A field-based investigation The title of the original paper on

Pollutants and the environment A field-based investigation 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

Pollutants and the environment The protocol Control lake (B) Experimental lake (A) 2 years

Pollutants and the environment The protocol Control lake (B) Experimental lake (A) 2 years prior to starting treatment No synthetic oestrogen added 3 years of treatment 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 m. RNA • numbers and lengths of fish

Pollutants and the environment Lake B 6000 4000 2000 Lake A 3 2 Year

Pollutants and the environment Lake B 6000 4000 2000 Lake A 3 2 Year 2003 2002 2001 2000 2003 2002 0 2001 1 2000 Concentration of VTG/ arbitrary units The concentration of VTG in whole-body homogenate samples of male fathead minnows captured during the spring of each of the years shown

6000 4000 2000 Lake B Lake A 3 2 2003 2002 2001 2000 2003

6000 4000 2000 Lake B Lake A 3 2 2003 2002 2001 2000 2003 2002 0 2001 1 2000 Concentration of VTG/ arbitrary units Pollutants and the environment 1 Explain the advantage of the break in the y-axis on this graph. 2 Explain what is meant by a whole-body homogenate. (1 mark) (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]

6000 4000 2000 Lake B Lake A 3 Explain the difference in the results

6000 4000 2000 Lake B Lake A 3 Explain the difference in the results for lakes A and B. (3 marks) 3 2 2003 2002 2001 2000 2003 2002 0 2001 1 2000 Concentration of VTG/ arbitrary units Pollutants and the environment 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]

Pollutants and the environment Numbers of fish given as a population index Mean population

Pollutants and the environment Numbers of fish given as a population index Mean population index Year Control lake (B) Experimental lake (A) 1999 490. 3 180. 0 2000 19. 1 50. 3 2001 56. 1 117. 7 2002 9. 6 0. 7 2003 20. 6 2004 51. 9 0. 1 2005 355. 9 0. 1 Addition of synthetic oestrogen

Pollutants and the environment Mean population index Year Control lake (B) Experimental lake (A)

Pollutants and the environment Mean population index Year Control lake (B) Experimental lake (A) 1999 490. 3 180. 0 2000 19. 1 50. 3 2001 56. 1 117. 7 2002 9. 6 0. 7 2003 20. 6 2004 51. 9 0. 1 2005 355. 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]

Pollutants and the environment Measuring fathead minnows Fork length 6 Fork length gives a

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

Pollutants and the environment Lake B Lake A 2000 2002 Number of fish Histogram

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

Pollutants and the environment Number of fish Histogram showing lengths of fish Lake B

Pollutants and the environment Number of fish Histogram showing lengths of fish Lake B Lake A 2000 Number of fish 2002 2004 Fork length 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)

Pollutants and the environment Number of fish Histogram showing lengths of fish Lake B

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

Pollutants and the environment 7(b) Use the data from the table to explain the

Pollutants and the environment 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; Lake A 2000 2002 Histogram shows no young fish hatch in 2002; Small number in histogram the result of few fish hatching in 2001; [3] 2004 Fork length

Pollutants and the environment Collapse of a fish population after exposure to a synthetic

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