Pollutants and the environment Bill Indge

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

• Oestrogen
• enters the nucleus and binds with the oestrogen
• receptor

Oestrogen receptor
Ribosome
Gene coding for VTG
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Vitellogenin (VTG) production in the liver

• The DNA is transcribed and VTG mRNA is produced

• The activated receptor now attaches to the
  target gene

Gene coding for VTG
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Vitellogenin (VTG) production in the liver

• The VTG mRNA migrates to the ribosomes in the
  cytoplasm

Gene coding for VTG
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Vitellogenin (VTG) production in the liver

• VTG is synthesised and secreted from the liver

Gene coding for VTG
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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 ß-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
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• 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
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• 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.
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• 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
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Some differences between fathead minnows treated
with synthetic oestrogens and controls
Treated with synthetic oestrogen Control
Body condition factor total mass/g 100 000 total length3 1.07 1.10
Gonadosomatic index testis mass/g 100 body mass/g 0.802 0.761
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Secondary sexual characters index 2 3
Significantly different, plt0.01
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Treated with synthetic oestrogen Control
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Significantly different, plt0.01
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 dont attempt the question, you cannot
  gain credit.
• Show your working you can gain marks even if
  the answer is wrong.
• Use some common sensethe liver wont weigh more
  than the fish!

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Treated with synthetic oestrogen Control
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Significantly different, plt0.01
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
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Treated with synthetic oestrogen Control
Gonadosomatic index testis mass/g 100 body mass/g 0.802 0.761
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
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Treated with synthetic oestrogen Control
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Significantly different, plt0.01
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
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Treated with synthetic oestrogen Control
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Significantly different, plt0.01
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
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Treated with synthetic oestrogen Control
Body condition factor total mass/g 100 000 total length3 1.07 1.10
Gonadosomatic index testis mass/g 100 body mass/g 0.802 0.761
Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35
Secondary sexual characters index 2 3
Significantly different, plt0.01
6 Using only data from the table, explain why
synthetic oestrogens may affect the
reproductive success of male fathead minnows.
(4 marks)
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Treated with synthetic oestrogen Control
Body condition factor total mass/g 100 000 total length3 1.07 1.10



Mating success is likely to depend on body
condition/Males with poorer body condition likely
to have lower mating success
Significantly different, plt0.01
6 Using only data from the table, explain why
synthetic oestrogens may affect the
reproductive success of male fathead minnows.
(4 marks)
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Treated with synthetic oestrogen Control

Gonadosomatic index testis mass/g 100 body mass/g 0.802 0.761


The difference in gonadosomatic index is not
significant/The oestrogen-treated fish have
significantly lower body condition and secondary
sexual characters index
Significantly different, plt0.01
6 Using only data from the table, explain why
synthetic oestrogens may affect the
reproductive success of male fathead minnows.
(4 marks)
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Treated with synthetic oestrogen Control


Hepatosomatic index liver mass/g 100 body mass/g 1.55 1.35

Larger liver in oestrogen-treated males likely to
be linked to high vitellogenin production
Significantly different, plt0.01
6 Using only data from the table, explain why
synthetic oestrogens may affect the
reproductive success of male fathead minnows.
(4 marks)
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Treated with synthetic oestrogen Control



Secondary sexual characters index 2 3
Secondary sexual character important in
courtship/defence of territory
Significantly different, plt0.01
6 Using only data from the table, explain why
synthetic oestrogens may affect the
reproductive success of male fathead minnows.
(4 marks)
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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
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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

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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
6000 4000 2000
Concentration of VTG/ arbitrary units
3 2 1 0
2000 2001 2002 2003
2000 2001 2002 2003
Year
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6000 4000 2000
Concentration of VTG/ arbitrary units
Lake B
Lake A
3 2 1 0
2000 2001 2002 2003
2000 2001 2002 2003
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
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6000 4000 2000
Concentration of VTG/ arbitrary units
Lake B
Lake A
3 2 1 0
3 Explain the difference in the results for
lakes A and B. (3 marks)
2000 2001 2002 2003
2000 2001 2002 2003
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
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Numbers of fish given as a population index
Year Mean 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 2.6
2004 51.9 0.1
2005 355.9 0.1
Addition of synthetic oestrogen
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Year Mean 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 2.6
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
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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
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Histogram showing lengths of fish
Number of fish
Lake B
Lake A
2000
Number of fish
2002
Number of fish
2004
Fork length
Fork length
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Histogram showing lengths of fish
Lake B
Lake A
Number of fish
2000
Number of fish
2002
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)
Number of fish
2004
Fork length
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Histogram showing lengths of fish
Lake B
Lake A
Number of fish
2000
Number of fish
2002
7(a) The histogram is bimodal/contains two peaks,
suggesting two classes of fish. 1
Number of fish
2004
Fork length
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7(b) Use the data from the table to explain the
results for 2002 in lake A. (3 marks) 
Lake A
2000
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
2002
2004
Fork length
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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