Martina Duft, Ulrike SchulteOehlmann, Michaela Tillmann, Lennart Weltje and Jrg Oehlmann

1
Marine pollution of organotin compounds and
their biological impact

• Martina Duft, Ulrike Schulte-Oehlmann, Michaela
  Tillmann, Lennart Weltje and Jörg Oehlmann

J.W. Goethe University Frankfurt Department of
Ecology and Evolution Ecotoxicology
Siesmayerstrasse 70, D-60323 Frankfurt am
MainGermany
www.bio.uni-frankfurt.de/ee
2
Contents

• Organotin compounds tributyltin (TBT) and
  triphenyltin (TPT) as xeno-androgens
• Prosobranch snails as invertebrate models for
  endocrine disruption studies imposex and
  intersex
• Effects of organotins laboratory experiments
• Field studies on organotin pollution biological
  effect monitoring in France, Ireland and Germany
• Estrogenic responses in snails
• Conclusions

3
Organotin compounds
TBT
TPT

• Examples tributyltin (TBT) and triphenyltin
  (TPT)

• degrade only slowly in the environment
• strongly adsorb to suspended particles and
  sediments in aquatic systems
• show a high persistency
• reveal substantial bioaccumulation potential

4
Tributyltin (TBT)
TBT

• since the 1970s mainly used asactive biocidal
  compound in antifouling paints on ship hulls,
  harbour and offshore installations
• also used as biocide in wood preservatives,
  textiles, dispersion paints, agricultural
  pesticides and as UV stabiliser in plastics
• exhibits the highest toxicity of all organotins
  (acute toxicity, teratogenicity, immunotoxicity)
• one of the most toxic xenobiotics ever produced
  and deliberately introduced into the environment

5
Triphenyltin (TPT)
TPT

• since the early 1960s use as broad-spectrum
  agricultural fungicide to combat a rangeof
  fungal diseases in various crops, particularly
  potato blight
• mainly as triphenyltinhydroxide (fentinhydroxide)
  and triphenyltinacetate in Brestan, Brestanid and
  Du-Ter
• was also used in antifouling paints on ships,
  mostly in combination with TBT

6
Environmental concentrations in sediments
TBT
TPT

• TBT maxima µg as Sn/kg
• in freshwater sediments 3,920
• in marine sediments 360,000
• in harbour sediments 340,000
• TPT
• in freshwater sediments 309
• in marine sediments 1,860
• in sewage sludge 3,400
• in harbour sediments 5,500

7
Organotin compounds in sediments
TBT
TPT

• since January 2003 complete ban of TBT in all
  antifouling paints in the EU
• but still high environmental concentrations,
  especially in harbour sediments and along main
  shipping routes
• disposal of dredged harbour material into the sea
  is still common in many countries and leads to a
  hazard for marine biocoenoses
• analytical measurement is time-consuming,
  expensive, complicated and ends at the detection
  limit
• bioindication represents a more economical and
  more relevant method

8
Bioindication

• For centuries, organisms are used for the
  evaluation and assessment of environmental
  quality. Examples

Galmei plants (e.g. Thlaspi caerulescens)denote
metalores
Canaries warn for toxic mine gases
Lichens reflect air pollution (example Berlin
1993/94)
9
What is endocrine disruption ?

• Definition of the EU commission (1998) Exogenic
  compounds which negatively affect the health of
  an intact organism or its offspring by
  interference with its endocrine function.
• Endocrine disruptors (EDCs) are environmental
  chemicalswhich directly or indirectlyinfluence
  the hormonal systemand may be active at low
  concentrations.

Marbuse The fusion ofSalmakis and
Hermaphroditos(early 16th century)
10
Suspected endocrine disrupting compounds
(from Oehlmann Markert, 1997)
11
Working mechanisms of EDCs

• EDCs can act
• directly by binding to the receptors as
• agonists - acting like natural hormones (e.g.
  ethinylestradiol, nonylphenol)
• antagonists - blocking receptors for natural
  hormones(e.g. tamoxifen, TCB-77, p,p'-DDE)
• indirectly by influencing biosynthesis,
  metabolism, elimination and/or bioavailability of
  natural hormones Example
• inhibition of the aromatase - blocking the
  transformation of androgens to estrogens

12
Evidence in vertebrates

• Malformations in sexual organs. Examples
• Hemi- and minipenes in alligators in
  Florida,Lake Apopka - causedby a DDT accident

• Feminisation in male fish (ovotestes) in sewage
  polluted surface waters in North America and
  Europe

• Masculinisation (arrhenoidy) in
  female carp downstream of pulp mills

• Masculinisation of female fathead minnows
  downstream of animal farms

13
Invertebrates a source of diversity

• Hormonal regulation of biological processes is a
  common characteristic in all animal phyla.
• Invertebrates provide
• key species for ecosystem structure and
  functioning
• approximately 30 different phyla
• more than 95 of all animal species
• huge diversity of life histories with e.g.
  larval stages, pupation, metamorphosis, diapause

14
First effects of TBT in oysters
TBT

• since 1960 use of TBT-based antifouling paints
  since 1970 also on leisure boats/yachts
• 1979 first observation of deleterious effects
  caused by TBT in oysters at Arcachon (France)
  which led to massive economical damage
• missing fall of larvae - reproductive failure
• shell deformations (formation of chambers and
  "balling")

deformed shell
normal shell
15
Prosobranchs as proposed models

• A number of international expert workshops,
  including the EDIETA workshop, proposed
  prosobranch snails as promising models for ED
  research.
• Rationale TBT effects in molluscs as the best
  documented example for ED in wildlife.

16
Effects of organotins - endpoints
TBT
TPT

• Effects on reproduction in various prosobranch
  snails
• imposex
• intersex
• reduction of fecundity (embryo production)

17
Imposex

• acronym for superimposed sex additional
  formation of male sex characteristics (penis
  and/or vas deferens) in females of
  gono-choristic prosobranchs, resulting in
  sterility
• is induced by TBT from antifouling paints at
  concentrations below 1.5 ng as Sn/L, but also by
  TPT (at least in some species), natural and
  synthetic androgens
• is known for more than 160 species worldwide

18
Responses I virilisation and sterility

• Imposex is a gradual virilisation of females in
  response to ambient TBT and TPT concentrations.
• There are six different stages of imposex
  expression. Females are sterilised at the imposex
  stages 5 and 6.

Hydrobia ulvae, stage 0
Hydrobia ulvae, stage 5
19
Response II sex change

• Ultimately, a protogyne sex change (from female
  to male) can be induced on the level of the gonad

spermatogenesis
ripe oocyte
disintegrating oocytes
20
Measurement of imposex intensities

• Imposex development scheme for Nucella lapillus
  (only most frequent stages considered)
  

• Vas deferens sequence index (VDS index,
  VDSI) mean value of imposex stages in a sample

21
Intersex

• means a modification or supplanting of female by
  male sexual characteristics (e.g. in the
  periwinkle Littorina littorea)
• gradual transformation of the female pallial
  tract towards a male morphological structure
• is induced by TBT at concentrations gt 5.0 ng Sn/L
• measured as Intersex Index (ISI) mean value of
  allintersex stages in a sample

22
Response functional sterilisation

• Intersex stage 2 first stage of functional
  sterilisation - oocytes and capsular material
  from the female gland complex leak into the
  mantle cavity so that the female can no longer
  produce intact egg capsules.

23
Working mechanisms of TBT and TPT
TBT
TPT
pregnenolone
3ß-hydroxysteroid-DH
progesterone
17a-hydroxylase
17a-hydroxyprogesterone
C17,20-lyase
CYP19 aromatase
estrone
17ß-estradiol
24
TBT - Laboratory experiments
TBT

• various prosobranch snails were tested
• Nucella lapillus (marine)
• Marisa cornuarietis (freshwater)
• Nassarius reticulatus (marine) - new sediment
  biotest
• Potamopyrgus antipodarum (freshwater) - new
  sediment biotest

25
Nassarius reticulatus

• The netted whelk
• carrion feeder
• lives burrowed in coastalfine sediments
• up to 40 mm shell height
• exposed to 750 g freshsediment, covered with 10
  L artificial sea water
• test duration 4 weeks
• endpoints
• imposex induction
• uterotrophic assay

26
Effects of TBT in sediments - imposex
Concentration-response relationship for
sediments- Nassarius reticulatus -
TBT
27
Sensitivity of N. reticulatus to TBT

• LOEC (lowest observed effect concentration)

TBT
10 µg TBT-Sn/kg (dry wt)

• Assessment criteria for field sediments
  (according to EU Water Framework Directive
  WFD)

28
Potamopyrgus antipodarum

• New Zealand mudsnail
• detritus feeder
• up to 5 mm shell height
• lives burrowed in freshwater and estuarine fine
  sediments
• exposed to 50 g freshsediment, covered with 1 L
  artificial water
• test duration 4 to 8 weeks
• endpoints
• mortality
• embryo production

embryos
29
Effects of TBT in sediments - mortality

• Concentration-response relationship for
  sediments - Potamopyrgus antipodarum
  -

TBT
TBT µg as Sn/kg dry weight
after 4 weeks
after 8 weeks
30
Effects of TBT - embryo production

• Concentration-response relationship for
  sediments - Potamopyrgus antipodarum
  -

TBT
after 4 weeks
31
Sensitivity of P. antipodarum to TBT

• LOEC (lowest observed effect concentration)

TBT
10 µg TBT-Sn/kg (dry wt)

• effect concentrations for the most sensitive
  parameter (embryo production, after 4
  weeks)
• EC10 0.98 µg TBT-Sn/kg (dry wt)
• EC50 45.8 µg TBT-Sn/kg (dry wt)

32
Effects of TPT

• imposex induction
• in the freshwater ramshorn snail Marisa
  cornuarietis (EC10 4 months 12.3 ng TPT-Sn/L)
• but not in the marine species Nucella lapillus
  and Nassarius reticulatus
• reduced fecundity (EC10 4 months 5.6 ng
  TPT-Sn/L), total spawning inhibition at ? 163 ng
  TPT-Sn/L
• impairment of spermatogenesis (in M. cornuarietis
  and N. reticulatus) and oogenesis (only N.
  reticulatus)

TPT
33
Effects of TPT on P. antipodarum

• Concentration-response relationship for
  sediments - Potamopyrgus antipodarum
  -

TPT
after 8 weeks
34
Sensitivity of P. antipodarum to TPT

• LOEC (lowest observed effect concentration)

TPT
10 µg TPT-Sn/kg (dry wt)

• effect concentrations for the most sensitive
  parameter (embryo production, after 4
  weeks)
• EC10 0.05 µg TPT-Sn/kg (dry wt)
• EC50 23.6 µg TPT-Sn/kg (dry wt)

35
Effects on nematodes

• TBTsignificant inhibition of reproduction in the
  sediment biotest with the roundworm
  Caenorhabditis elegansLOEC 30 µg TBT as Sn/kg
  EC10 3 µg TBT as Sn/kg
• TPTsignificant inhibition of growth, fertility
  and reproduction in the same sediment
  biotestLOEC 5 µg TPT as Sn/kg
• other studies show effects of TBT on nematode
  community structure in marine sediments

TBT
TPT
36
Effects on chironomids

• TBTmortality and significant inhibition of
  larval development in the sediment biotest with
  the non-biting midge Chironomus ripariusLOEC
  250 µg TBT as Sn/kg EC10 43 µg TBT as Sn/kg
• TPTmortality and significant inhibition of
  larval development in the same sediment
  biotestLOEC 5 µg TPT as Sn/kg

TBT
TPT
37
Sensitivity of biomonitors
TBT

• Sensitivity to TBT of various prosobranch snails

38
Biological TBT effect monitoring I

• Example Northern Brittany, France(biomonitor
  Nucella lapillus biomarker imposex intensities)

TBT
39
Temporal trend monitoring for TBT

• Example Northern Brittany, France (biomonitor
  Nucella lapillus biomarker imposex intensities)

TBT
40
Biological TBT effect monitoring II

• Example Ireland (1993) (biomonitor Nucella
  lapillus biomarker imposex intensities)

TBT
41
VDSI and sterility of females
TBT
42
TBT in sea water and VDSI
TBT
43
Biological TBT effect monitoring III

• Example ISI in Germany (1994/95) (biomonitor
  Littorina littorea biomarker intersex
  intensities)

TBT
44
Androgenic activity of river sediments I
River Elbe, Germany - Nassarius reticulatus
imposex assay
45
Androgenic activity of river sediments II
Nassarius reticulatus imposex assay
46
Reproductive toxicity of river sediments
River Elbe, Germany - Potamopyrgus antipodarum
sediment assay
47
Lessons from TBT and TPT
TBT
TPT

• The example of organotins and their effect on
  molluscs
• demonstrates that endocrine disrupting chemicals
  (EDCs) may impact different levels of biological
  integration, affecting also the survival of
  populations in the field.
• indicates that androgenic compounds can
  havedeleterious impact on wildlife.
• provides evidence that vertebrate-type steroids
  play an important functional role in
  prosobranchs.
• The latter conclusion motivated further
  investigations with xeno-estrogens in prosobranch
  snails.

48
Xeno-estrogen effects in prosobranchs

• Xeno-estrogens such as bisphenol A (BPA) and
  octylphenol (OP) induce a complex syndrome of
  alterations in female marine and freshwater
  prosobranchs, referred to as "superfemales"
• formation of additional female organs (e.g.
  second vagina)
• enlargement of accessory pallial sex glands
• gross malformations of the pallial oviduct
  section, resulting in an increased female
  mortality
• massive stimulation of oocyte and egg clutch
  production.

49
Examples of superfemales I

• Formation of additional female organs (second
  vagina)

rectum
oviduct
normal vagina
additional vagina
anus
50
Examples of superfemales II

• Hypertrophy of accessory pallial sex glands

51
Examples of superfemales III

• Gross malformations of the pallial oviduct
  section

52
Conclusions

• A variety of benthic invertebrates is strongly
  affected even by low concentrations of organotin
  compounds in aquatic systems.
• Molluscs and especially prosobranch snails
  provide striking examples for endocrine mediated
  effects of environmental chemicals.
• Extensive surveys of coastal sediments in France,
  Germany and Ireland indicate that there is still
  a threat for sensitive marine organisms.
• Due to the persistence of organotin compounds in
  the environment, a continuing impact on aquatic
  wildlife has to be expected.

53
Many thanks .!!!!

• to the following members of staff at IHI, Zittau
• Constanze Stark, Ulrike Schneider and Simone
  Ziebart from the Ecotoxicology lab,
• Dr. Jacco van Doornmalen, Dr. Siegfried
  Korhammer, Heike Heidenreich, Anke Laufer and
  Gerlinde Liepelt for chemical analyses,
• to Dr. Jean Bachmann, Dr. Matthias Oetken and
  Gabi Elter in the Frankfurt lab,
• to numerous students in Zittau and Frankfurt,
• for funding our research
• Federal Environmental Agency Berlin (UBA),
  project codes 102 40 303/01, 297 65001/04 and 299
  24 275,
• ARGE Elbe, project code W 25/00.