Pesticide ecotoxicology of lobster larvae and post larvae: Do the twain ever meet?

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Pesticide ecotoxicology of lobster larvae and
post larvae Do the twain ever meet?

• Wayne Fairchild1, Paula Jackman2
• Ken Doe2,7, Jacqueline Arsenault1, Kadra
  Benhalima1, Art Cook2, Mélanie Thibodeau2, Jamie
  Aubé2, Stéphan Reebs3, Dounia Daoud4, Martin
  Mallet4, Benoit Bruneau5, Denis Chabot5, Megan
  Bauer6, Spencer Greenwood6, Andrea Locke1, Mark
  Hanson1 and Michel Comeau1

1 Fisheries and Oceans Canada, Gulf Fisheries
Centre, Moncton, NB 2 Environment Canada, Science
and Technology Branch, Moncton, NB 3 Département
de biologie, Université de Moncton, Moncton, NB 4
Homarus Inc., Shediac, NB 5 Fisheries and Oceans
Canada, Mont-Joli, QC 6 AVC, Lobster Science
Centre, Charlottetown, PEI 7 Retired, Growing
garlic, Mount Uniac, NS
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Acknowledgements

• Fishing Industry through Homarus Inc.
• Fisheries and Oceans Canada
• Environment Canada
• U de Moncton
• Marine Centre, (Shippagan, NB)
• Atlantic Cancer Research Institute
• And many colleagues and students

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Oh, East is East, and West is West, and never
the twain shall meet From the The Ballad of
East and West, by Rudyard Kiplingor a nice
quick summary of the hopes of our regulatory
regime and risk assessment process
Coastal Habitat we have a lot of this in
Atlantic Canada
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Todays Talk Outline

• Objectives Why the concern?
• Background on pesticides/contaminants
• What we know about these things?
• What we don't know?
• What have we being working on?
• Surely this is all academic, I mean really?
• Future directions and research questions
• Some thoughts and conclusions

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Little lobsters - Objective

• Investigate the potential for pesticides to
  interfere with the nearshore/estuarine early life
  stage development of American lobster (Homarus
  americanus).
• Particular emphasis will be placed on high hazard
  pesticides and pesticides with modes of action
  that may act at very low concentrations with only
  transitory exposures.
• Given the short length of most PEI freshwater
  systems, there is a close link between events in
  rivers and the adjacent salt water.

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Why a concern to fishing industry?

• There was a huge die off of lobster in Long
  Island Sound that was co-incident with the
  arrival of West Nile virus
• To combat West Nile mosquitoes, an insecticide
  spray program was initiated around New York
  (methoprene and others) Was there a link?
• Nonylphenol was also found in samples and
  implicated
• Some of the recent workshops are now wondering if
  a synthetic pyrethroid insecticide could have
  been a contributor (very potent to FW aquatic
  invertebrates)
• These events have received wide media and
  industry attention
• Can contaminants be a factor (causal, added
  stress) in the incidence of lobster shell disease?

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What we know about these things?

• contaminants are in many effluent streams
  (municipal, industrial, agricultural)
• Contaminants are mostly below regulated
  concentrations for regulated endpoints
• pesticides are in river runoff, particularly on
  PEI
• new industrial developments, new crops or use
  patterns, brings new contaminants (or
  concentrations) to the region

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What we don't know about?

• Nanoparticles (novel behaviours, fresh to salt
  transition?)
• Pyrethroid pesticides, safer but more toxic
  (effects at very low concentrations)
• New chemicals (all classes, 1000s per year)
• Particle bound contaminants (fate and effects)
• Drugs, personal care products, endocrine
  disrupting compounds (usually low concentrations,
  but potential high potency)

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Contaminant exposure

• Spray Drift, direct and indirect
• Runoff, especially storm events
• Water, fields to streams to estuaries to ?
• Particles, from fields or in natural water
• Sediments, settling or estuarine processes
• Food, from all sources
• Plus interactions with environment for all

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• Figure 3 Transfer of individual larvae into
  Mason jars.

• Figure 4 Lab set-up for Chronic and Acute
  exposure experiments

Environment Canada, Moncton, NB
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Acute Tests 48-Hour LC50 (as µg/L active
ingredient)
Pesticide Stage I Stage IV
Azinphosmethyl (Guthion) 3.16 (1.0010.0)
Chlorpyrifos (Lorsban) 0.35 (0.27-0.46)
Diflubenzuron (Dimilin) gt 1000 gt 1000 µg/L
Endosulfan (Thiodan) 2.51 (1.73-3.64) 3.98 (2.576.16)
Hexazinone (Velpar) gt 1000 gt 1000 µg/L
Methamidiphos (Monitor) gt 1000
Tebufenozide (Confirm) gt 1000 gt 1000
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Pyrethroid formulations of deltamethrin

• AMERICAN LOBSTER Homarus americanus
• Stage III lobster larvae had acute 96-hr LC50
  values between 3.74 to 4.92 ng/L (n 3). Results
  for the two formulations over three trials were
  within a range of 1.2 ng/L (NOTE units are
  1000x lower that previous table)
• Stage IV (post larvae) had 96-hr LC50 of 28.2
  ng/L.
• Stage III lobster larvae given a 1-hr pulse
  exposure followed by 16 days in clean water had
  an LC50 of 36.5 ng/L
• Chronic exposure of stage III larvae for 16 days
  had an LC50 of 4.45 ng/L
• This pesticide is released into sea water after
  sea lice treatment, and is in streams from
  agricultural areas

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Histological effects of nonylphenol and
pesticides on cuticle and hepatopacreatic cells.
Legend Cuticle Ep (Epicuticle), Ex
(Exocuticle), En (Endocuticle), ML (Membraneous
layer), Ct (Connective tissue). Hepatopancreas
N (Nuclei), F (F-cell), B (B-cell), L (Lumen), V
(Vacuole), Ep (Epithelium). Magnification 40 X
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Larval Behaviour
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Introduction Metabolism

• Metabolic rate most fundamental biological rate
  as it represents the rate of energy uptake,
  transformation, and allocation. (Brown et al.
  2004)
• Standard metabolic Rate (SMR) energy required to
  maintain basic biological functions independent
  of activity, digestion or costs of physiological
  stressors.
• Active metabolic rate (AMR) energy required to
  perform specific levels of activity (lt Maximum
  MR MMR)
• Metabolic scope absolute MS difference between
  SMR and MMR. Energy available to grow, digest
  food, support locomotion etc.

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Experimental setup - metabolism

• Larvae obtained from Coastal Zones Research
  Institute-Homarus Inc. hatchery
• Acute Exposure stage V-VI- 0.1 µg L-1- 20 C,
  96-hr
• One individual per jar (1 L) to avoid
  cannibalism, no renewal
• Daily feeding of live artemia, frozen artemia,
  and dry food
• Water quality verified daily

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Results Metabolic scope
Significant difference t2,14 -2.63 (p 0.0160)
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Genomics - Sample Preparation

• Sample preservation-Trizol
• RNA extraction-Phenol Chloriform
• Amplification labeling-Cy3 Cy5
• Microarray hybridization

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Microarray
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Cluster Analysis
Down Regulation
Up Regulation
0.01 0.1 0 0.03 0.3 1.0
0.01 0.1 0 0.03 0.3 1.0
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Bugs as food?

• Lobster larvae and insects in neuston
• Insect sampling on land and sea
• Is there another great circle of life lesson in
  the potential for land based sources to affect
  the near shore marine environment?
• Insects are eaten by lobster larvae
• After everything, this still surprises me

Contents of a neuston sample from the
Northumberland Strait. Biota of terrestrial
origin sorted to left in pan, marine origin to
the right in pan and in sieve.
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Mayflies on Weather Station Doppler Radar Lake
Erie
De http//seagrant.psu.edu/publications/fs/Mayfly_
12-2003.pdf
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Surely this is all Academic?

• Leight et al 2005, grass shrimp population
  monitoring, golf and agriculture, South Carolina,
  BMP and IPM more shrimp
• Hartwell 2011, Chesapeake Bay, mysid relative
  pesticide toxicity (esp. pyrethroids) and crab
  harvest, total pesticide application there in
  range of PEI totals
• Baldwin et al 2009, Chinook salmon population
  modelling, population effects from sublethal
  pesticide effects, including growth

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Little lobster - Conclusions

• Toxicological screening of pesticides of concern
  in our region has generated a number of
  significant results for biological effects on
  lobster larvae
• In our limited screening we have seen compounds
  that affect survival, moulting, timing of
  development, histology and behaviour
• The high end of test concentrations for most
  chemicals are above the expected environmental
  concentrations, effects on lobster larvae running
  down into the low µg/L (ppb) range and below 1
  µg/L are a concern
• There is potential for movement of these
  chemicals into coastal waters, though the
  pathways and likelihood of such an occurrence
  remain to be determined

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Little lobster Conclusions (cont.)

• While there is nothing definitive to indicate a
  link between lobster population levels, and
  contaminants in the environment, there is
  certainly enough information now available to ask
  good questions (which ironically?)
• Microarray analyses are being conducted to study
  the gene expression changes that occur following
  exposures of lobster larvae to nonylphenol and
  pesticides.
• The intent is to correlate these changes with
  effects on survival, growth, moulting, cell
  histology and behaviour.
• A library of contaminant response in the
  laboratory will be developed to test against
  field collected larvae to verify exposure.

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What the research opportunities/needs are?

• Linking contaminants and biological effects in
  real world context. Field studies? Lab-field
  studies? Linked to biological monitoring?
  Mixtures?
• Below regulated concentrations, but does this
  mean no biological effects?
• Fate of current use contaminants in the
  environment. Are contaminants accumulating in
  unexpected reservoirs?
• Fate of some degradation resistant pesticides
  (ex endosulfan). Are these in decline and gone
  as much as we would like to think?
• Estuaries are great chemistry experiments in
  progress.

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Some thoughts, and conclusions?

• Nutrients and sediments, can be good surrogates
  for occurrence of some groups of contaminants,
  integration of projects on these topics might
  provide good synergies
• Biological effects approaches may provide an
  indication of differences in ecosystem health
  that may go non-detected with a chemical
  measurement approach

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Sagittal section of the stereomicroscopy
micrographs showing the general view of internal
anatomy with the positions the organs of stage IV
larval lobster (E Eye, G Gill, H Heart, He
Hepatopancreas, M Muscle, CS Cardiac stomach).
Questions?