1. 00000
Implications of acute copper and nickel metal toxicity
in the Hyalella azteca species complex
Jessica Leung1, Jonathan D. S. Witt1, Warren Norwood2, D. George Dixon1
1Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1
2Aquatic Contaminants Research Division, Environment Canada, Burlington, Ontario L7R 4A6
Introduction
Hyalella azteca has been used in toxicity tests since the mid-
1980s. However, genetic studies revealed that H. azteca is
actually a cryptic species complex, a group of genetically
distinct species that are morphologically similar, but has been
erroneously grouped together [1]. Witt and Wellborn, (in
preparation) discovered that there are 86 provisional species
or clades within the H. azteca species complex.
Experimental Design
Test animals and DNA Sequencing
• Clades 1 and 8 were identified using DNA barcoding: an
established method for species identification that uses the
mitochondrial gene, cytochrome c oxidase I (mtCOI)
Toxicity Tests
• Clades 1 and 8 were exposed to increasing Cu and Ni
concentrations for 14 days
• A total of 12 experiments were performed (2 clades×2
metals×3 repeats)
• LC50s were calculated using the saturation-based mortality
model [4] (see red and blue model lines on plots)
Clade
Ni LC50
(nmol/L)
± r2
1 (o) 1980c 493 0.84
8 (×) 3550d 626 0.87
References
[1] Witt J, Hebert P. 2000. Cryptic species diversity and evolution in the amphipod genus
Hyalella within central glaciated North America: a molecular phylogenetic approach. Can J
Fish Aquat Sci 57:687-698.
[2] Major K, Soucek D, Giordano R, Wetzel M, Soto-Adames F. 2013. The common
ecotoxicology laboratory strain of Hyalella azteca is genetically distinct from most wild
strains sampled in eastern North America. Environmental Toxicology and Chemistry
32:2637-2647.
[3] Weston DP, Poynton HC, Wellborn GA, Lydy MJ, Blalock BJ, Sepulveda MS, Colbourne
JK. 2013. Multiple origins of pyrethroid insecticide resistance across the species complex of
a nontarget aquatic crustacean, Hyalella azteca. Proc Natl Acad Sci USA 110:16532-16537.
[4] Norwood WP, Borgmann U, Dixon DG. 2007. Chronic toxicity of arsenic, cobalt,
chromium and manganese to Hyalella azteca in relation to exposure and bioaccumulation.
Environmental Pollution 147:262-272.
[5] Soucek DJ, Dickinson A, Major KM, McEwen AR. 2013. Effect of test duration and
feeding on relative sensitivity of genetically distinct clades of Hyalella azteca. Ecotoxicology
22:1359-1366.
Are LC50s for clades 1 and 8
different when exposed to Ni?
YES, the Ni LC50s for clades 1
and 8 are significantly different.
Clade 8 is 1.8 times more
tolerant to Ni than clade 1.
Discussion and Conclusion
This study examined whether two members of the H. azteca
cryptic species complex have different responses.
• Clades 1 and 8 had different sensitivities to Cu and Ni.
Generally speaking, clade 8 was two times more tolerant
than clade 1 to either Cu or Ni, based on LC50s
• This observation is consistent with other toxicity research
on members of the H. azteca cryptic species complex [3,5]
• Since the clades used in this study were representative of
the H. azteca that had been sequenced in 17 laboratories
[2,3], the results presented provides evidence that Hyalella
utilized in toxicology work should be genetically identified
• Evidence of different sensitivities to contaminants among
members of the H. azteca species complex has important
implications for biomonitoring programs and protocols in
ecotoxicology laboratories [3,5]
Clade 6
Clade 3
Of the 86 clades
surveyed in the wild, only
two clades (clades 1 and
8) have been genetically
identified in 17 toxicity
test laboratories [2,3].
Using different clades of
the H. azteca species
complex may confound
toxicity tests.
Objective
To test whether clades 1
and 8 have different Cu
and Ni LC50s
Experimental vessels in toxicity tests
Cu
Ni
Are LC50s for clades 1 and 8
different when exposed to Cu?
YES, the Cu LC50s for clades 1
and 8 are significantly different.
Clade 8 is 2.6 times more
tolerant to Cu than clade 1.
Clade
Cu LC50
(nmol/L) ± r2
1 (o) 491a 68.3 0.90
8 (×) 1260b 262 0.70
Ni in Solution (nmol/L)
MortalityRate0.25
1-8
1-9
1-7
1-2
1-3
1-4
1-5
1-6
1-1
8-5
8-3
8-4
8-6
8-2
8-1
Mesohyalella
100
100
100
0.02
Clade 1
Clade 8
DNA Sequencing Results
How genetically different are
clades 1 and 8?
Clades 1 and 8 are very different.
They exhibit 26% nucleotide
sequence divergence at the
mtCOI gene
10 100 1000 10000
0.0
0.5
1.0
1.5
10 100 1000 10000
0.0
0.5
1.0
1.5
14 day Cu and Ni Toxicity Tests Results
Acknowledgments
10 100 1000
Cu in Solution (nmol/L)
0.0
0.5
1.0
1.5
10 100 1000
0.0
0.5
1.0
1.5
MortalityRate0.25