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
Acknowledgements
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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
“Oh, East is East,
and West is West,
and never the twain
shall meet”
From the The Ballad of
East and West, by
Rudyard Kipling
or … 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
Today’s Talk Outline
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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
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.
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?
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
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)
Contaminant exposure
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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
• Figure 3: Transfer of
individual larvae into
Mason jars.
• Figure 4: Lab set-up
for Chronic and Acute
exposure experiments
Environment Canada, Moncton, NB
Acute Tests 48-Hour LC50
(as µg/L active ingredient)
Pesticide
Stage I
Stage IV
Azinphosmethyl (Guthion)
3.16 (1.00–10.0)
Chlorpyrifos (Lorsban)
0.35 (0.27-0.46)
Diflubenzuron (Dimilin)
> 1000
> 1000 µg/L
Endosulfan (Thiodan)
2.51 (1.73-3.64)
3.98 (2.57–6.16)
Hexazinone (Velpar)
> 1000
> 1000 µg/L
Methamidiphos (Monitor)
Tebufenozide (Confirm)
> 1000
> 1000
> 1000
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
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 (Fcell), B (B-cell), L (Lumen), V (Vacuole), Ep (Epithelium). Magnification = 40 X
Larval Behaviour
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 (< Maximum MR= MMR)
• Metabolic scope – absolute MS: difference between SMR and
MMR. Energy available to grow, digest food, support locomotion
etc.
Experimental setup - metabolism
• Larvae obtained from Coastal
Zones Research InstituteHomarus 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
Results : Metabolic scope
Significant difference t2,14 = -2.63 (p = 0.0160)
Genomics - Sample Preparation
• Sample preservation
-Trizol
• RNA extraction
-Phenol Chloriform
• Amplification & labeling
-Cy3 & Cy5
• Microarray hybridization
Microarray
Cluster Analysis
Down Regulation
0.01
0.1
0
0.03
0.3
Up Regulation
1.0
0.01
0.1
0
0.03
0.3
1.0
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.
Mayflies on Weather Station
Doppler Radar – Lake Erie
De http://seagrant.psu.edu/publications/fs/Mayfly_12-2003.pdf
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
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
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.
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.
• …
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
Questions?
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).