Chironomid assemblage changes and deformity prevalence in lakes impacted by uranium
mining in northern Saskatchewan, Canada
Vanessa Bourne
Supervisor: Dr. Brian Cumming
Committee Member: Dr. Shelley Arnott
An undergrad thesis submitted to the School of Environmental Studies on partial
fulfillment of the requirements for ENSC 502
Queen’s University
Kingston, Ontario, Canada
March 2014
1
ABSTRACT
Uranium mining has the potential to have impacts on surrounding ecosystems, particularly on
the aquatic organisms found in lakes near mining activities. The objective of this study was to
use paleolimnological techniques to investigate whether changes occurred in chironomid
assemblages or the deformities in head capsules increased in two lakes (I-7 and I-9) impacted by
increases in potentially toxic elements associated with mining activities at the McClean Uranium
mine. Elemental concentrations of U, As, Mo and Se (excluding Se in I-9) have significantly
increased both lake sediments since the introduction of the mine in the late 1960s.
Concentrations of Mo differ between Lake I-7 (400 µg g-1) and Lake I-9 (9 ~25 µg g-1) with both
increasing several fold following the mine opening. In Lake I-7, Corynocera ambigua and
Zalutchia zaluticola decreased from pre-mining to active-mining conditions while
Psectrocladius (monospectrocladius) and Polypedilum nubifer increased. Corynocera ambigua
dominated the species assemblage (60%) in the pre-mining time period and reduced to minute
amounts during active-mining. In Lake I-9, Corynocera ambigua increased slightly in the postmining time period but there were only minute changes in other taxa between pre-mining and
active-mining time periods. Deformities in chironomid head capsules remained low in both lakes
and did not change following the initiation of mining actives. There was insufficient literature
pertaining to the ability for Corynocera ambigua, Polypedilum nubifer and Psectrocladius
(monospectrocladius) to withstand toxic environments except for Polybedilum nubifer which has
been previously recorded to demonstrate resistance to the effects of other metals. The stronger
change in chironomid assemblage Lake I-7 could have been observed due to the difference in
Mo concentration, geographical placement from mining facility and surface area of lake.
Multiple stressors could be impacting chironomid assemblage change, including changes in
climate. Temperature data of the area showed a probable increase in average annual air
2
temperature between pre-mining and active- mining time periods. More research is needed to
attribute changes in chironomid assemblage changes to increases in elemental concentrations,
including the need for reference sites away from mining activities.
3
ACKNOWLEDGEMENTS
This thesis would not have been possible without the dedicated support of my supervisor,
Dr. Brian Cumming. I am grateful for his guidance and encouragement throughout the research
process. I would also like to thank Moumita Karmakar for her continual support and guidance
throughout this project. Her mentorship helped in forming my knowledge and skills used in
developing my research trajectory. Additionally, I’d like to thank Kathleen Laird for her
thoughts and direction throughout my research project. I would also like to thank Dr.Shelley
Arnott who kindly served as my committee member. I am grateful to have had the opportunity to
work with P.E.A.R.L, as well as Rebecca Hansford and Zara Jennings. Their enthusiasm towards
research has been a positive influence on my academic development. I look forward to pursing
graduate studies in this field because of their collective commitments to student success.
4
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………………… 1
ACKNOWLEDGEMENTS……………………………………………………………………… 3
TABLE OF CONTENTS …….………………………………………………………………….. 4
LIST OF FIGURES ……………………………………………………………………………... 5
LIST OF TABLES ………………………………………………………………………………. 6
INTRODUCTION AND LITERATURE REVIEW ……………………………………………. 7
SITE DESCRIPTION……………………………………………………………………………13
METHODS ……………………………………………………………………………….……. 15
RESULTS ……………………………………………………………………………………… 18
DISCUSSION ………………………………………………………………………………….. 21
LITERATURE CITED ………………………………………………………………………….27
APPENDIX……………………………………………………………………………………....31
SUMMARY ……………………………………………………………………………………. 40
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LIST OF FIGURES
Figure 1. Location of the McClean mining facility in the eastern margin of the Athabasca basin,
800 km north of Saskatoon.
Figure 2. Concentrations of U, As, Mo and Se in sediment intervals in dated sediment cores
from lakes I-7 and I-9.
Figure 3. Relative abundance of common chironomid taxa (>4% in at least one level) in the
sediment core from Lake I-7, order by PSA axis one species scores.
Figure 4. MDS ordination plots based on changes in the relative abundance of chironomid taxa
in the two study lakes, I-7 and I-9.
Figure 5. Changes in the species evenness in the sediment cores from the two study lakes.
Figure 6. Relative abundance of the common chironomid taxa (4% in at least one level) in the
sediment core from Lake I-9, order by PSA axis one species scores.
Figure 7. Mean annual temperature of sites in close proximity to the McClean mine study sites.
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LIST OF TABLES
Table 1. Physical and chemical characteristics of Lake I-7 and Lake I-9.
Table 2. The percent contribution of taxa from Lake I-7 and I-9 to the species assemblage
difference between previously defined pre-mining and active-mining time periods.
7
INTRODUCTION AND LITERATURE REVIEW
Human actions have the potential to effect aquatic environments through the release of toxic
elements from mining activities (Larson and Stone 2011, Salonen et al. 2006, Schindler 2006).
Although, metals and other elements usually carry a negative connotation to their involvement in
the environment, some are required in small concentrations for physiological processes and may
also be tolerated in low concentrations (Smol 2008). When released in high concentrations a
number of elements can alter physical, chemical and biological systems (Smol 2008). They can
naturally enter aquatic systems as inflow when water flows over or through the surrounding
geology (Schindler 2001). Concentrations of elements can increase due to human actions,
causing them to surpass biological thresholds that may be detrimental (Smol 2008).Uranium
mining can result in multiple metals and elements being introduced in high concentrations to the
surrounding environment (Laird et al. submitted, Larson and Stone 2011, Muscatello and Janz
2009, Overall and Parry 2004, Pyle et al. 2001).
The northern Saskatchewan region is an ecologically important area of boreal forests,
wetlands, rivers and lakes. The rich uranium and mineral deposits have attracted the interest of
many mining companies, leading the area to have some of the top producing uranium mines in
the world (Muscatello and Janz 2009, Pyle et al. 2001). Some of these mines include Rabbit
Lake, McClean Lake Uranium mine and Key Lake Uranium mine (Pyle et al. 2001). Although,
management tactics have improved, the effects of uranium mining on the environment are still an
active topic of concern (Laird et al. submitted).
Uranium mining has been correlated with increased concentrations of Arsenic (As),
Cadmium (Cd), Chromium (Cr), Copper (Cu), Iron (Fe), Molybdenum (Mo), Selenium (Se),
8
Vanadium (V) and other elements (Mucatello and Janz 2008, Pyle et al. 2001). Numerous
studies have investigated the impacts of uranium mining on aquatic environments (Laird et al.
submitted, Laird et al. in preparation, Mucatello and Janz 2008, Pyle et al. 2001, Schindler
2001). In impacted sites near multiple uranium mines in northern Saskatchewan (Key Lake,
McClean Lake, Rabbit Lake Uranium Mine etc.) there have been elements detected in the
surrounding aquatic environment that has showed strong correlation with mortality of larval
fatheaded minnows and bioaccumulation of Se (Muscatello and Janz 2008, Pyle et al. 2001).
The McClean uranium mine is located in the eastern margin of the Athabasca basin, 800km
north of Saskatoon and has an ore body that has been estimated to be ~ 37 million pounds of
U3O8 (Laird et al. submitted). The mine consists of 980 hectares of two mineral leases and nine
mineral claims covering an area of 3,148 ha (Laird et al. submitted). This mine includes the Sue
A, B, C and E, the McClean North, the JEB deposits, and other prospects. To date almost 50million pounds of U3O8 have been processed at the McClean Lake mining facility. Uranium
mining has the potential to have a large impact on the surrounding ecosystems, particularly on
the aquatic organisms found in surrounding lakes. The McClean Lake operation has achieved an
ISO 14001 certification based on an international set of standards for maintaining environmental
management systems (Areva website at us.areva.com). Extensive monitoring for regular
sampling of air, water, land, plants and animals are followed, and the McClean mine is one of the
most advanced uranium mine in the world. It is currently going through a 150 million dollar
expansion project and will soon commence production on secure sources of ore for the next 30
years (Areva website at us.areva.com).
Baseline conditions and natural variability of the environment is challenging to determine in
aquatic ecosystem studies (Smol 1992). Comparing current conditions to historical conditions
can assist in determining magnitude of change in an ecosystem (Smol 1992). Long-term
9
environmental monitoring is not always readily available, making it challenging to compare
impacted environments to prior conditions (Smol 2008). Paleolimnology techniques can use
indirect proxy methods to trace back to these unrecorded time periods (Smol 1992).
Lake ecosystems are often subjected to multi-stressors, such as eutrophication, climate
change, catchment deforestation and fish manipulations, the effects of which can be difficult to
disentangle (Smol 2008, Smol 2010). Climate change can be linked to changes in biological
communities by several mechanisms including increased average temperatures, increased
abundance of high temperature days and change in precipitation patterns (Wetzel 2001). Lake
ecosystems are sensitive to changes in climate and can be documented through proxies, such a
chironomid and cladocera taxa (Battarbee et al. 2002). Climate change can induce increases or
reductions in biodiversity and taxa assemblages due to variation in pH, DOC etc. (Jeziorski et al.
2013, Smol 2008, Wetzel 2001). It can have synergistic effects with other human stressors such
as deforestation, land erosion and release of pollutants (Schindler 2001). The combined
consequences of climate change and metal toxicity can affect how metal pollutants interact with
aquatic biota (Jeziorski et al. 2013, Schindler 2001, Smol 2008).
A study by Laird et al. (submitted) suggests that five lakes adjacent to the McClean Lake
uranium mine showed significantly higher levels of heavy metals and other elements compared
to five reference lakes. The elements found at elevated levels in most impact lakes include:
uranium, arsenic, molybdenum and selenium. In four out of the five impacted lakes, arsenic,
molybdenum and selenium, showed significant increase above the lowest effect level. Arsenic
and vanadium were present in elevated levels in pre-1960 samples in both reference and impact
lakes inferring that these were naturally elevated in the area.
10
A study was conducted to determine the ecological effect of the increased metals and
other elements in lakes surrounding the McClean Uranium mine. Paleolimnological techniques
used diatoms as an ecological proxy on 5 lakes with increased elemental concentrations
associated with mining in comparison to changes in diatom species composition over the same
time period in 5 reference lakes. The mining impacted lakes had significant changes in diatom
assemblages in three of the five lakes between pre-mining and active-mining time periods (Laird
et al. in preparation). The reference lakes were also assessed for changes in diatom species
composition over similar time frames. The result showed significant changes in four of the five
reference lakes. Overall, these results suggest small changes in diatom assemblages in both the
mine-influenced and reference lakes, suggestion that an overriding regional signal may be
responsible for the observed changes, and that the increases in elements in the lakes near mining
have cause no or minimal changes in diatom assemblages.
Paleo indicators, are used to reconstruct past environments which can provide both direct
and indirect inferences to lake ecosystem conditions (Smol 2008). Aquatic and terrestrial
organisms with diverse and abundant species assemblage can be used as paleo indicators (Wetzel
2001). To be used as an indicator the morphological remains of these taxa must preserve in
sediments for extended periods of time allowing for them to be identified (Wetzel 2001). Some
reliable paleo indicators include chironomids, diatoms and scaled chrysophytes (Smol 2008).
Chironomids are non-biting midges that spend their larval stages in benthic lake
environments (Vermeulen 1995). Each taxa has specific ecological requirements which indicate
certain ecological conditions (Hofmann 1968). Chironomid head capsules preserve in lake
sediments for extended periods of time due to being made of chitin (Brooks et al. 2007).
Chironomids are considered to be good paleoecological indicators because of their high
11
abundance in most aquatic systems, identifiable to the genus level (over 1400 valid species occur
in Europe), and generally diverse taxonomic assemblages (Brooks et al 2007, Swansburg et al.
2009).
Chironomids have been used as an indicator of environments with increased metal
concentrations in multiple studies (Ilyashuk et al. 2003, Swansburg et al. 2009, Vermeulen
1995). Chironomid larvae live in benthic environments and often feed on detritus and algae
associated with this environment (Vermeulen 1995). This brings them in close contact with
metals and other elements than can be introduced to the sediments during mining activities
(Brooks et al. 2007, Quinlan and Smol 2000, Vermeulen 1995). The presence of metals and
other elements has been known to produce reduced total abundance and species richness leading
to changes in species dominance to chironomid taxa (Iluashuk et al. 2003).
The presence of toxic metals and other element have been found to increase
concentrations of chironomid deformities (Swansburg et al. 2009). These are sub lethal effects
that can be seen as early warning signs for environmental degradation by toxins. Chironomid
deformities are morphological configurations that departs from the norm (Swansburg et al.
2009). They have been observed in mouthparts (mentum, ligula, mandibles and maxillary palps)
of chironomids in metal and element enriched environments (Swansburg et al. 2009).
Hypothesis
Prior studies confirmed that there is a positive correlation of metal and element
concentration within lakes that have mining activity in northern Saskatchewan (Laird et al.
submitted). This study will investigate if changes in chironomid assemblages occur due to
increased concentrations of potentially toxic elements in dated sediment cores from two lakes
adjacent to uranium mining activites. A higher abundance of metal tolerant species is expected.
12
The proxy could also show deformities due to the metal toxicity (Diggins and Stewart 1998). It is
expected that the number of deformities would rise following the increases in metal
concentrations in the sediment associated with mining. More specifically, the objective of this
study is to assess whether there was detectable change in assemblage composition of non-biting
midges (chironomids) with the onset of mining activities in comparison to pre-mining
background conditions.
13
SITE DESCRIPTION
The lakes that were chosen for this study are located near the McClean open pit uranium
mine, approximately 800 km north of Saskatoon (Figure 1). The geology of the area consists of
old Precambrian gneisses overlain by flat lying metamorphosed sandstone and conglomerates of
the Athabasca group (Areva website at us.areva.com). It is located in a continental subarctic
climate region where only 3 months have temperatures above 10°C but there is an average
temperature of -4°C. Mean annual precipitation is ~546mm, of which 65% is rain (Donahue et
al. 2000).
Sediment cores from two lakes adjacent to the McClean Uranium mine were taken to
assess changes in chironomid assemblages. These two lakes were selected from the five lakes
studied by Laird et al. (submitted). The rational for choosing these lakes (Lakes I-7 and I-9) was
that the concentration of potentially toxic metals and other elements were the highest
concentrations in cores from of the five impact lakes.
Lakes I-7 and I-9 have similar biological and chemical characteristics (Table 1). They are
relatively low in total phosphorus (TP) and total nitrogen (TN) causing them to fall in the
oligotrophic to slightly mesotrophic range (Laird et al. submitted). These lakes exhibit pH
values in the circumneutral range and have relatively high buffer capacities (Laird et al.
submitted). Lake I-7 is located adjacent to the southern mining operation at the Sue-McClean
deposit and is downstream from the effluent management and treatment system. Lake I-9 is
located adjacent the McClean open pit mining operation.
Lowest effective levels (LEL) measurements refer to the concentrations of elements in
the sediment, at which 95% of the benthic taxa can be present (Figure 2) (Deckere et al. 2011).
14
Uranium concentrations in both cores do not exceed LEL of sediment guidelines yet in Lake I-9
(11.5 µg.g-1) it is slightly higher than Lake I-7 (Laird et al. submitted). The background
conditions of As is above LEL and increased in both Lakes I-7 and I-9 in the active-mining time
period (Laird et al. submitted).The pre-mining concentrations of Mo were mostly above LELs
yet both impacted lakes had a significant increase during the active-mining time period (Laird et
al. submitted). Lake I-7 was 18.5x more concentrated than the initial Mo concentration during
the active mining time period, ranging from ~27 µg.g-1 to a maximum of ~480-500 µg.g-1(Laird
et al. submitted). In Lake I-9, the Mo sediment concentration only increased by ~4x above
background conditions but still exceeded LEL sediment guidelines (Laird et al. submitted). The
selenium concentrations significantly increase in active-mining compared to pre-mining in Lake
I-7 by about 1.5 times the background conditions and are above LEL (Laird et al. submitted).
Background conditions in Lake I-9 of selenium were above LEL then decreased with the
addition of the mine (Laird et al. submitted).
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METHODS
Sediment cores for each lake were taken using a Glew gravity core and were subsectioned into 0.25cm intervals. Core chronologies were obtained by 210Pb dating using gamma
spectroscopy (Laird et al. submitted). Based on the core chronologies, samples were selected in
two different periods for analysis of chironomid assemblages: Pre-mining (pre-1960s); and after
the onset of uranium mining (post-1970). The pre-mining and post-mining time periods were
selected based on the regional onset of the start of uranium mining activities in this region (Laird
et al. submitted). The chironomid assemblages, were assessed in terms of assemblage
composition (percent relative abundance, and concentration), as well as the presence of headcapsule deformities.
Data Collection
The sediment samples were processed for chironomid analysis using standard procedures
(Walker 2001). Sediment was deflocculated by adding 0.15g of dried sediment to 4 grams of 5%
KOH by heating for approximately 10 minutes at 200 ̊C. The sample was run through 100-µm
sieve under running tap water and the remains of chironomid head capsules maintained on the
sieve were then collected. The chironomid head capsules were picked with fine forceps under
20X magnification using a Bogorov counting tray. Chironomid head capsules were placed on
coverslips and mounted on slides using Entellan®. A minimum abundance of 50 head capsules
was picked for each sample (Quinlan and Smol 2000). Head capsules were identified using
Wiederholm (1983) and the fossil key of Brooks et al. (2007). Deformities in head capsules
were assessed during identification by observing for morphological abnormalities, more
16
specifically observing loss genuine segments and/or presence of questionable segments,
reduction in length of antenna, and the fusion of apex with the basal segment (Warwick 1991).
Statistical Analysis
Chironomid counts were expressed as percent relative abundance of the common taxa
(present in at least 4% in at least one sample). The dominant pattern of variation within
chironomid assemblage in each lake is summarized by Principal Component Analysis (PCA)
using a computer program CANOCO (ter Braak and Smilauer, 1998). The chironomid
distributions in the cores visualized using using the computer program Tilia version 2.0.2 (Grim,
1987), and the species were arranged according to centered PCA axis-1 scores. Assemblage
zones in the core were defined by a constrained cluster analysis using Euclidian distance as a
measure of dissimilarities with the program CONISS (Grim, 1987). Species diversity measures
in this study included species richness and evenness. Species richness was represented by the
total number of taxa. Hill N2 is a metric of species evenness (Hill, 1973). Both total number of
taxa N and Hill’s N2 were standardized by rarefaction using R, version 3.0.1 (Oksanen et al.,
2010) and plotted using Origin ver. 6.1 (Origin, 2000).
To assess if the chironomid assemblages were significantly different between the prior
defined periods (pre-mining and active-mining), an analysis of similarity (ANOSIM) test was
performed using PRIMER 6.0 (Clarke, 1993). ANOSIM, a non-parametric test, was run on
common chironomid taxa to assess if within group similarities are greater than between group
similarities. Non-metric multidimensional scaling (nMDS) was performed on both
untransformed and square-root transformed data to assess the separation in common chironomid
assemblages in the pre-mining and active-mining time periods. To identify taxa that are primarily
17
responsible for differences between the defined time periods, a similarity percentage (SIMPER)
test was used.
Climate Records
To assess if a recorded change in temperature has occurred over the last century in
northern Saskatchewan (a possible confounding environmental variable for chironomid
assemblages), trends from the three closest weather stations to the McClean mine were assessed.
Environment Canada’s National Climate Data of annual mean surface air temperature (2012)
was used. The closest stations were Collin’s Bay Station (58.18°N,-103.70°W), Brochet Station
(57.88°N, -101.68°W) and Whitesand Dam Weather Station (56.23°N,-103.15°W).
18
RESULTS
Lake: I-7
A total of 1355.5 chironomid head capsules were identified in the core from Lake I-7 over
the past 200 years, with 21 groups achieved at an abundance of 4% for each sample (Figure 3).
The total number of head capsules counted varied from 71.5 to 163 with no obvious trends over
the core length. The data was rarified to 71.5 head capsules. A constrained cluster analysis
(CONISS) was used to divide the core into periods in three different groups. 1) pre-mining; 2)
end of pre mining and beginning of mining 3) active-mining. The PCA ordination shows an
approximately linear decrease in site score over time. The most noticeable change was in the
Corynoccera ambigua (C.ambigua) which achieved over 60% relative abundance prior to mining
activity which declined to trace amounts by the top of the core. Similarily, Zalutschia
zalutscicola was found to be more abundant in the pre-mining intervals and absent in activemining period. Polypedilum nubifer type and Psectrocladius (Monosectrocladius) both showed
an increase in abundance during active-mining.
ANOSIM analysis indicated a significant difference between the pre-mining and activemining periods (Figure 4). The MDS ordination plots of both transformed (stress= 0.05) and
untransformed (stress= 0.08) data (Figure 4). I-7 showed clear separation between pre-1960s and
post-1960s, time periods with the clearest separation on the transformed species data. The taxa
that contributed to this separation varied between the non-transformed and square root
transformed data (Table 2). The evenness increased in the post mining time period compared to
pre mining while the N value was variable throughout the core (Figure 5). There were no
deformities recorded in I-7 samples.
Lake: I-9
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A total of 874 chironomids were identified in the core from Lake I-9 over the past 200 years,
with 25 groups achieved at an abundance of 4% for each sample (Figure 6). The total number of
head capsules counted varied from 48 to 101.5 with no obvious trends over the length of the
core. A constrained cluster analysis (CONISS) was used to divide the core into periods in three
different groups. 1) pre-mining; 2) end of pre mining and beginning of mining 3) active-mining.
The PCA ordination shows an approximately linear decrease in site score over time. The most
noticeable change was in the Corynoccera ambigua which was found to be more abundant in the
pre-mining intervals and absent in active-mining periods.
ANOSIM analysis indicated a significant difference between the pre-mining and activemining periods (Figure 5). The MDS ordination plots of both transformed (stress= 0.18) and
untransformed (stress= 0.18) data (Figure 4). The chironomids assemblages in the core form
Lake I-9 showed slight separation between pre-1960s and post-1960s, time period while the
transformed species did not have a clear separation. The taxa that contributed to this separation
varied between the non-transformed and transformed data (Table 2). The evenness decreased in
the active-mining time period compared to pre-mining while the N value was variable
throughout the core (Figure 6). There were no deformities recorded in I-9 samples.
Lake Comparison
Lakes I-7 and I-9 both have a significant division between pre-mining and active-mining
time periods although I-7 has a greater difference between both groups. The lakes have a
difference in response of evenness between the two time periods. Both lakes did not show any
deformed head capsules. The lakes both had a consistent number of total head capsules per a
time interval and CONISS displayed similar grouping results in both lakes.
Climate Records
20
The air temperature data from Brochet and Sand Dam showed a significant increase
while at Collin’s Bay it was not significant (Figure 7). There was a high correlation between the
Collin’s Bay and Sand Dam data (0.95) and a much lower correlation with the Brochet and Sand
Dam data (0.57).
21
DISCUSSION
The main objective of this study was to investigate if recent changes in elements in lake
sediment caused changes in the composition of chironomid assemblages. Many studies have
demonstrated assemblage changes, abundance changes and morphological changes in the
presence of mining (Brooks et al. 2005, Mucatello and Janz 2008, Pyle et al. 2001, Schindler
2000, Smol 2008).
Chironomid Assemblage Change
The taxa that were emphasized from the stratigraphy as changing in percent abundance
between previously defined periods were also identified by SIMPER as having the highest mean
difference between pre-mining and active mining conditions. In this study, C.ambigua was
present in cores from both lakes. The domination of this taxon in the pre-mining assemblage in I7 then reduction in active-mining assemblage is characteristic of this taxon (Brodersen and
Lindegaard 1999). In I-9, it failed to dominate the assemblage and occurred only in the activemining time period (Brodersen and Lindegaard 1999). C.ambigua is characterized as a cold
stenothermous which is present in glacial and sub- glacial regions however it has also been found
warm (~20°C) Danish lakes (Brodersen and Lindegaard 1999). In North America, it is
commonly abundant in Alaska, Yukon, Northwest Territories and adjacent regions, but
essentially absent farther north, east and south (below 60°N) (Barely et al. 2006). From this it
can be concluded that the distribution is not temperature dependent, therefore it is not the main
driver of its occurrence in I-7 and I-9 (Barely et al. 2006, Ilyashuk et al. 2013). A study
conducted on 41 Danish lakes, found C.ambigua in concentrations ranging from 0.5% to 25% of
the assemblage with multiple environmental variables (chlorophyll- a, TN, pH, Alkalinity,
surface area, volume, mean depth, max depth and secchi depth) (Brodersen and Lindegaard
22
1999). C. ambigua diverges from other chironomids in several characteristics including often
occurring in high amounts and unusual structure of mentum indicating a specialized feeding
strategy (Brodersen and Lindegaard 1999). Charophyte algae have been commonly found when
C.ambigua is dominant in an assemblage (Brodersen and Lindegaard 1999). This type of algae
often covers the lake bottom and prefer oligo-mesotrophic calcareous clear-water lakes
(Brodersen and Lindegaard 1999. In this study, Z. zalutschicola was present in the pre-mining
and disappears post-mining of I-7 while in I-9 it was present in both time intervals. This taxa is a
temperate chironomid that has mainly been found in lakes below the tree line (Ilyashuk et al.
2013). Z. zalutschicola can indicate the presence of high POC and PON conditions leading to its
appearance in humic lakes (Porinchu and Cwynar 2000).
Psectrocladius (monopsectrocladius) became abundant in the active-mining time period
in Lake I-7. Psectrocladius tend to be associated with acidified sites, however due to the
circumneutral nature of both lakes studied, lake acidification was not considered a reasonable
factor in assemblage change. Psectrocladius (monopsectrocladius) has been correlated with low
TN and TP systems (Brodersen and Anderson 2002). Lastly, their assemblage can be effected by
the presence of fish as they are preferred by some planktivorous fish (Henrikson and Oscarson
1985). Polypedilum nubifer became abundant during the same time as Psectrocladius
(Monopsectrocladius). Polypedilum nubifer is characteristic of warm and shallow lakes
(Ilyashuk et al. 2013, Eggermont and Heiri 2011).
Ability to Withstand Toxic Environments
There has been little research completed on the ability for C.ambigua, Z.zalutcicola and
Psectrocladius (Monospectrocladius) to withstand sediments. With high concentrations of
potentially toxic elements. In a study by Bhattacharyay et al. (2005), they concluded that
23
Polypedilum nubifer may have a strong ability to withstand toxic environments as it increased in
abundance and showed minimal deformities (compared to other chironomid taxa) in the
Damodar River in India. This river had high sediment concentrations of zinc, copper, mercury
and cadmium (Bhattacharyay et al. 2005). Due to the increase in Polypedilum nubifer in the
active-mining time period of Lake I-7, it may be able to withstand the metals found in Lake I-7
as it did in the Damodar River.
Species Evenness
Species evenness in stressed environments is widely considered to decrease overtime due
to less taxa being able to survive in strenuous conditions (Azrina et al. 2006, Ilyashuk et al.
2013, Wetzel 2001). This would allow for chironomids with favorable characteristic for this new
environment to thrive while others would decrease. Species evenness (N2 values) of Lake I-7
became more even between pre-mining and active-mining time periods while in Lake I-9
evenness decreased. The pattern seen in Lake I-7 could be due to the change in species
abundance of C.ambigua. Since this taxa originally dominated the assemblage, the reduction in
C.ambigua may have allowed for more resources to be available for other taxa therefore
increasing the evenness of chironomids in I-7. In Lake I-9, the decrease in evenness expressed
from pre-mining to active-mining time periods could be due to metals and other elements in the
sediment creating a harsh environment. This would allow for less chironomid taxa to be able to
survive, causing the ones that can survive to take over the assemblage.
Deformities
Deformities were not present or very rare in the species assemblage of lakes I-7 and I-9.
The uranium concentrations were not above LEL which could have led to the absence of
deformities (Laird et al. 2013). In other studies, chironomid deformities have been found in lakes
24
with As and Se in the lake sediments (Martinez et al. 2001, Martinez et al. 2006, Warwick
1991). Arsenic may have led to deformities in other studies while not in this study because of
having lower concentrations of As and Se in lake sediments from lakes I-7 and I-9, have high As
background conditions or due to relationships with other metals present in the sediments
(CCME). It also could be due to the bioavailability of the metals as this effects the ability of the
chironomid to take up the metal (CCME).
Response of I-7 compared to I-9
Ordination analysis of the sediment samples indicated that the core from I-7 had greater
separation between the pre-mining and active-mining time periods than the core from Lake I-9.
This was supported by the change observed in the Lake I-7 and I-9 stratigraphies.
The difference between the reaction of lakes I-7 and I-9 to uranium mining could have
been induced by multiple factors: difference in the metal concentrationsm, as well as the
physical-chemical characteristics of the two lakes. I-7 has a surface area of 117.2 ha while the
surface area of I-9 is 60.1 ha. This can influence the amount of contaminant from the air that can
settle into the lake system. The geographical positioned of Lake I-7 and I-9 could impact the
effect of mining pressures as each lake is closer to different parts of the mine. Lake I-7 is located
adjacent to the southern mining operation at the Sue-McClean deposit and is downstream from
the effluent management and treatment system while I-9 is located adjacent the McClean open
pit mining operation.
In Lake I-7, the concentrations of Mo surpass 400 µg g-1 while in I-9 Mo reached ~25 µg
g-1 (Figure 2). Mo is a trace metal that is necessary for life at low concentrations, however when
it occurs in access in the environment it has the potentially to be toxic (Xu et al. 2013). This
difference in Mo concentration could be having an effect on chironomids and be responsible for
25
the assemblage shift in I-7 but not in I-9. Although Mo is a non-bioaccumulative element, it has
the potential to settle in the sediment from the water column and be bioavailable to organisms
(Regoli et al. 2012, CCME). A study by Pyle et al. (2001), determined that increases in
molybdenum concentration from uranium mining can impact flathead minnow larva. There is
evidence that other metals and elements (Cd, Al, Mn, Ni, Zn and Cu) can be absorbed through
the body surface of a chironomid yet there has been limited research conducted on the ability of
Mo to be absorbed (Krantserg and Stokes 1988). In other animals, elevated levels of Mo have
caused reduction in the animal’s ability to take up copper leading to molybdenosis (Dickman and
Rygiel 1995).
Climate Change as an Alternate Stressor
Multiple stressors on aquatic environments can have additive, synergistic or antagonistic
effects (Smol 2008). The implications of climate change on aquatic system are dependent on
multiple biological and chemical factors such as lake surface area, depth, pH etc. (Smol 2008).
For example, climate change can reduced ice cover while warmer temperature can increase the
time period of thermal stratification which may enhance deep-water anoxia (Smol 2008). This
can change internal nutrient loading and distribution and assemblage of biota in the lake (Smol
2008).
Annual mean air temperature data indicates that the air temperature near lakes I-7 and I-9
increased between pre-mining and active-mining time periods. This increase was inferred due to
the high correlation factor between the Collin’s Bay weather station and the longer climate
record from Whitesand Dam recording.
In this study, the main change in assemblage was the by C. ambigua which has a wide
temperature availability lending towards temperature not being a driving factor in the change
26
(Barely et al. 2006). Z. zalutchicola is characteristic of temperate environments but has been
shown to be dependent on nutrient level rather than temperature. Polypedilum nubifer is
characteristic of warm temperature lakes which may support why Lake I-7 increased in the post
mining assemblage. Due to the ability for climate and other stressors to affect chironomid
species assemblage it is recommended that a reference lake is compared to the impacted lakes
investigated in this study.
27
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31
APPENDIX
Figure 1. The McClean mining facility located in the eastern margin of the Athabasca basin, 800
km north of Saskatoon. This facility has the northern McClean Lake open pit mine, which is in
close proximity to impact Lake 9. The Sue McClean deposit, which also has an effluent
management system, is in close proximity to impact Lake 7.
32
A)
B)
Figure 2. Sediment concentrations of potentially toxic elements in dated sediment cores from a)
Lake I-7 and b) Lake I-9. Two time periods were chosen within each sediment cored to
investigate if changes in chironomid assemblages could be related to changes in sediment
element contaminants. The green section indicates the pre-mining time period (before 1960)
while the red section indicates the active-mining time period (after 1970). The solid black stars
indicate metals in concentrations above lowest effective levels (LEL) (Laird et al. submitted).
Total Sum of Squares
33
Figure 3. Relative abundance of common chironomid taxa (4% in at least one level), in a 210Pbdated sediment core from Lake I-7. The chironomid taxa are arranged according to the axis-1
species scores of a PCA ordination. Total head capsules counted at each interval are also shown.
A depth- constrained clustering technique (CONISS) was used to help define major zones of
similar chironomid assemblages within the sediment core (Zone A and Zone B).
34
Figure 4. Non-metric multi-dimensional scaling (nMDS) ordination plot of site scores based on
the relative abundance of common chironomid taxa in the sediment cores from Lake I-7 and I-9.
nMDS plots were run on both transformed and square-root species data. Blue represents the premining time period while green represents the active-mining time period.
35
A)
B)
Figure 5. Changes in A) N values B) Hill’s N2 values are displayed according to the depth of the
core. Red indicates the active-mining time period while green indicated the pre-mining time
period.
Total Sum of Squares
36
Figure 6. Relative abundance of common chironomid taxa (4% in at least one level), in a 210Pbdated sediment core from Lake I-9. The chironomid taxa are arranged according to the axis-1
species scores of a PCA ordination. Total head capsules counted at each interval are also shown.
A depth- constrained clustering technique (CONISS) was used to help define major zones of
similar chironomid assemblages within the sediment core (Zone A and Zone B).
37
Figure 7. Mean annual temperature from climate stations in close proximity to the McClean
mine study sites; Brochet (173km), Collin’s Bay (13 km) and Whitesand Dam (230 km). The
solid line indicates the linear trend, with the p-values indicating the significance of the trends.
1
Table 1. Physical and chemical characteristics of the two lakes studied from Laird et al. submitted.
Lake
Longitude (W)
Latitude (N)
Elevation
(m)
Surface
area (ha)
pH
Alk
mg/L
DOC
mg/L
I-9
I-7
103° 49' 32.5"
103° 51' 57.6"
58° 19' 57.2"
58° 15' 46.8"
448.7
447.6
60.1
117.2
7.0
7.1
8.4
10.9
2.4
3.4
TP
TN
µg/L µg/L
9
10
200
260
Depth of
Coring (m)
10 m
3.5 m
Table 2. The percent contribution of taxa from lakes I-7 and I-9 to the species assemblage
difference between prior defined periods. The highlighted taxa are the four highest percent
contributors in each lake. The transformed data were square root transformed.
Taxa Present
Corynocera ambigua
% Contribution of Difference Between Pre-mining and PostMining
I-7
I-9
NonNonTransformed
Transformed
Transformed
Transformed
29.04
12.30
6.59
7.17
Tanytarsus lugens
8.54
5.57
6.09
3.25
Tanytarsus pallidicornis
6.87
5.06
4.55
4.80
Tanytarsini
Polypedilum nubifer
5.16
5.02
7.29
5.78
-
-
Zalutschia zalutschicola
Procladius
Ablabesmyia
Tanytarsus mendax
Psectrocladius
(monopsectrocladius)
Cladotanytarsus mancus
Cladopelma
Dicrotendipes nervosus
Tanytarsus lactescents
Parakiefferiella
Chironomus anthracinus
Cricotopus orthocaldius
Heterotrissocladius marcidus
Microtendipes pedellus
Pagastiella
Cryptochironomus
Stictochironomus
Parakiefferiellia type A
Stempellinella zavrelia
Tanypodinae
Sergentia
Tanytarsus chinyensis
Rheotanytarsus
Synorthocladius
Stempellinella
Chrionimini
4.56
3.82
3.70
3.52
3.39
8.49
3.91
5.17
3.74
5.88
2.97
4.57
3.91
-
4.89
4.63
3.74
3.03
3.00
2.89
2.66
2.38
2.37
2.26
-
3.47
3.48
4.13
4.78
3.98
4.13
3.69
3.62
3.18
-
3.25
5.27
3.52
5.52
4.18
6.65
6.34
4.29
3.44
2.91
2.70
2.58
-
3.08
4.73
4.69
5.38
3.43
4.73
6.28
4.01
3.60
4.13
4.02
3.88
3.43
2.90
-
1
SUMMARY
1. Chironomids were used as a proxy in a paleolimnological studies to indicate effects of
mining on aquatic organisms. This approach is beneficial due to allowing for comparison
to pre-mining conditions.
2. This study investigated whether there was detectable change in chironomid assemblages
to metals that is consistent with the increase in metals in sediment cores nether the
McClean Lake Uranium mine.
3. Chironomid abundance stayed relatively constant in both lakes. In lake I-7, the species
assemblage was split into pre-mining and post mining groups while in I-9 there was little
evidence of change in species composition.
4. Corynocera ambigua has the ability to dominate an assemblage. This has occurred in
many studies in multiple environments.
5. Chironomid deformities did not increase in frequency between pre-mining and activemining periods, and only occurred at a low frequency throughout the cores from the two
study lakes.
6. Lake I-7 differs from I-9 in the size of lake surface area, geographical positioning and Mo
concentrations, all of which could be contributing factors in the chironomid species
assemblage change observed in lake I-7 rather than I-9.
7. An increase of mean annual air temperature was inferred from weather stations. This
increase in temperature could have led to changes in chironomid assemblage.
8. A reference lake is recommended to be added to the study to attempt to control for
climate change variables.
Chironomid Full Head Capsule Counts
Lake I-7 Raw Counts
Chironominae
Chironomus
anthracinus
Chironomus early
instar
Cladopelma
Cryptochironomus
Dicrotendipes
nervosus
Glyptotendipes
severini
Microtendipes
pedellus
Pagastiella
Polypedilum nubifer
Polypedilum
Stictochironomus
Sergentia coracina
Glyptotendipes
pallens
Einfeldia
Paratendipes
nudisquama
Phaenospectra
Einfeldia dissidens
Sergentia
Endochironomus
Paratendipes
0
1
0
2
0
13151719212313.25
15.25
17.25
19.25
21.25
23.25
2
0.5
0.5
1
1
1
1
2
2
2.5
4
2
1
0
3
1
0
1
2
3
5
5
1
1
1
1
3
1
0
2.5
0.5
0
6
1
0
7.5
0
0
2.5
0
0
1
1
0
8.5
1.5
0
4
1
0
4
1
0
5.5
0
7
1.5
5
5
4
4
2.5
2
5
4
1.5
0
1
0
0
0
0
2
0
0
0
0
0
2
2
5
6.5
1
1
0
1
1
2
0
0
2
0
2
5
1
1
0
3.5
0
5
2
1
0
1
1
6
0
1
0
5
0
0
0
0.5
0
1
1
0
1
0
0
2
1
0
0
0
0
2.5
3
0
0
0
0
0
0
3
2
0
0
2
0
1
0
0
0
0
1
0
0
1
0
0
0
1
1
0
0
2
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
2
2.5
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0-0.5
2-2.25
4-4.25
6-6.25
1010.25
8-8.25
1
Paracladopelma
Gillotia
Phaenopsectra
flauipes
Microchironomus
Chironomous
plumosus
Orthocladiinae
Psectrocladius
Parakiefferiella
triquetra
Parakiefferiella
Corynoneura type A
Heterotrissocladius
Psectrocladius
(mono)
Cricotopus
Heterotrissocladius
marcidus
Chaetocladius
Cricotopus
Orthocladius
Cricotopus
(isocladius)
Psectrocladius
Psectrocladius
Epiocladius
eukiefferiella
limnophyes
paralimnophytes
Zalutschia
zalutschicola
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
0
1
0
0.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
1.5
1
0
0
0
0
0
0
0
2
0
0
0
0
0
1
0
0
0
0
0
0
1.5
0
0
1
0
2
2.5
1
2
2.5
0
0
0
2
2
1
2
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
1
1
0
0
0
1.5
0
0
2.5
1
0
0
0
1
0
2.5
0
1
1
0
0
1
0
1
0
5.5
0
1.5
2
1
1
0
6
0
3
0
0
0
5.5
0
0
0
0
0
0
0
1
0
2
0
0
0
1
0
2
1
0
0
0
0
5.5
0
0
0
1
0
0.5
0
2
0
1
0
1
0
1
0
1
3
5
3
2
1.5
2
0.5
0.5
1
3
2
0
1
0
1
0
0
0
0
1
0
2
0
0
0
0
0
0
0
1
0
0
1
1
0
2
0
0.5
2
0
0
0
0
0
0
0
1.5
0.5
0
0.5
2.5
0
1
1
0
0
1
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
0
0
3.5
1.5
3.5
6.5
6
1.5
2
Psectrocladius
(Monospectracladius)
septentriocalis
Synorthocaldius
Corynoneura (worn)
Nanocladius
Orthocladina (worn)
Tanytarsinii
Cladotanytarsus
mancus
Tanyarsus mendax
Tanytarsus pallicornis
Tanytarsus lugens
Stempellinella
zarvrelia
Tanytarsus
glabrescens
Corynocera ambigua
Tanytarsus
lactescents
Stempellina
Microspectra
Tanytarsus
Tanytarsus
chinyensis
Stempellina Zavrelia
long spare
Rheotanytarsus
Microspectra
contracta
Tanypodinae
Procladius
0
0
0
0
0
5.5
0
0
0
0
0
10
0
0
0
0
0
10.5
0
0
0
0
0
12.5
0
0
0
0
0
12.5
0
0
0
0
0
11.5
0
0
0
0
0
10.5
1
1
0
0
0
7
0
0
2
1
1
16.5
0
0
0
0
0
21.5
0
2
0
0
0
15.5
0
0
0
0
0
14.5
2
11.5
7
14
0
1
7
6
6
2
11
13
3
1
10
16
7
0
10
7.5
3
2
12
13.5
3
1
8
6
4
1
8
6.5
2
5
3
2.5
4
2
1.5
5.5
2.5
6
4
6
2
1
4
3
2.5
2
3
1
1
0
1
1
0
0
1
0
3
1
2
2
1
9.5
0
20
4
13
3
22
2
21.5
0
12
1
42.5
3
48
0
56.5
2
32
1
0
1
0
1
2
0
0
0
0
1
1
2
2.5
0
0
2
0.5
1
0
0
0.5
0
0
2
2
0
0
3
0
1
0
3
3
0
0
2
0
0
0
7
2
0
0
2
0
1
0
0
0
1
0
2
1
2
0
0
0
2
0
0
0
0
0
0
0
1
2
1
1
0
3
0
1
0
0
1
1
0
1
0.5
1
0
0
0
0
2
0
2
3
0
1
10
0
3
4.5
0
2
2
0
2
8
0
2
0
0.5
1
2
0
5
4
0
0
5
0
5.5
9
0
0
3
3
Ablabesmyia
Diamesinae
Pseudochironomi
Unidentifiable
Total Head Capsules
Lake I-9 Raw Counts
Chironomini
Chironomus anthracinus
Sergentia
Microtendipes pedellus
Stictochironomus
Dicrotendipes nervosus
Einfeldia
Cladopelma lateralis
Endochironomus
Cryptochironomus
Paracladopelma
Paratendipes
Polypedilum nubifer
Tribelos
Glypotendipes pallens
Pagastiella
Chironomini Early instar
Endochironomus
Orthocladiinae
4.5
0
0
3
111
0
0.5
1
2
1
1.5
0
0
0
0
0
0
0
0
0
0
0
0
0
1
3
1
0
5
71.5
1
0.5
0
0
1
1.5
2
1
0
0
0
0
0
0
0
0
0
0
0
1.5
4
0
0
5
115.5
2
1.5
1
3
1
8.5
1.5
1
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
4
129.5
3
1.5
2
1
0
9
2.5
1.5
0
1
0.5
0
0
0
0
0
0
0
0
2
6
0
0
4
109.5
4
1.5
0
3.5
0
2
2.5
0
0
0
0
1
0
0
0
0
0
1
1
2.5
1
0
0
1.5
122
5
1
0
2
0
4
5
0
0.5
0
0
0
0
0
0
0
0
0
0
2
3
0
0
3.5
87
10
1.5
1
1
0
1
1
1
0
0
0
1
0
0
0
0
0
0
0
2
0
0
0
3
74.5
12
2.5
1
1
3.5
9.5
2
0
0
0
0.5
0
0
0
0
0
0
0
0
1
1
0
0
6.5
141
14
4.5
2
2.5
0
4.5
4
0
1.5
0
0
1
1
1
0
0
0
0
0
0
1
1
2
4.5
137
16
4
3
2.5
1
5
1.5
0
1
0
0
0
0
1
1
1
0
0
0
3.5
1
0
0
5.5
163
18
2
2
0
0
4
1
1
0
1
0
0
0
0
0
1
1.5
0
0
2
0
2
0
2.5
94
20
1.5
4
2.5
0
0
3.5
0
1
0
0
0
0
1
0
0
0
0
0
0.5
4
Heterotrisscladius
marcidus
Zalutschia zalutshicola
Cricotopus orthocladius
Corynoneura
Eukiefferiella
Parakiefferiella
Parakiefferiella triquettra
Parakiefferiella
bathophila
Parakiefferiella type A
Psectrocladius
(Monospectrocladius)
Psectrocladius
(Psectrocladius)
Zalutschia
Synorthocladius
Cricotopus (isocladius)
Rheocricotopus
Hydrobaenus
Psectrocladius
Paracladius
Tanytarsini
Tanyatarsus lactescens
Cladotanytarsus mancus
Corynocera ambigua
Tanytarsus lugens
Tanytarsus pallidicornis
Tanytarsus chinyensis
Stempellinella Zavrelia
Tanytarsus mendax
Tanytarsus glabrescens
2.5
3
4
1
1
0
0
4
2.5
4.5
0
0.5
0
0
4
4
2.5
1
0.5
0
0
10
2.5
3
0
0
1.5
0
3.5
4
1
1
1
0
0
8
1.5
2
1
0.5
0
1
3
3
0
1
0
1.5
0
8
2
1.5
1
2.5
0
0
5.5
3.5
3
1
2
1
0
5
3.5
6.5
0
1
0
0
3.5
4.5
0.5
2
0.5
0
0
4
2
0
1
1
0.5
0
0
0.5
0
6
0
6
0
1
0
3.5
1
0
0
0
0
0
0
9
1
3.5
2
0
0
5
0
1.5
0
0
0
4
0
0
1
0.5
0
1.5
0
0
0
0
0
0
0
0
5.5
1.5
0.5
4
4.5
2
1
0.5
0
0
2
0.5
0.5
2
0
0
0
0
8.5
1
1
3.5
3.5
0.5
2
1
2.5
0
1.5
0
0
0
1
0
0
0
9.5
2
0
3.5
3
1
2
3
4
0.5
0
0
2
0
0
0
0
0
11
8
0
4
2.5
5
0
0
4
1.5
2
0
1
0
0
0
0
1
6
6
0
2.5
9.5
5
0
4.5
3
1
2.5
0
1
0
0
0
0
0
4
0
0
2
5
3.5
2
1
2
0
0
0
0
0
0
1
0
0
5.5
2
1.5
0
6.5
2
2
1
1
1
0
0
0
0
0
0
2
0
8.5
5
1
0.5
7
0
1
4.5
2
1
0
0
2
0
0
0
0
1
6
4.5
2.5
2
3.5
1.5
2
7.5
1
1
1.5
0
0
0
0
0
1.5
0
3
6
3
0
5
1
0
0.5
0
1
0
0
0
0
1
0
0
0
7
0
2
0
2.5
2
0
2
1.5
1
2
0
3.5
0
0
0
0.5
0
4
2.5
0
1
7
0
0
3
0
1.5
5
Microspectra
Rheotanytarsus
Stempellinella
Tanypodinae
Procladius
Ablabesmyia
Labrundinia
Diamesinae
Unidentifiable
Full Head capsules
0
0
0
0
1
0
0
0
8.5
48
0.5
3.5
0
1
1
1
0
0
5.5
67.5
0
0
0
1
0
0
0
0
7.5
77
0
1
2
2.5
2.5
0
0
1
7
93
0
0
0
1.5
1
0
0
1
6.5
80.5
0
1
0.5
1
0
0
0
0
5
64
0
0
0
2
1
1
1
0
3.5
50
0
1.5
0
2.5
1
1
0
0
7
81.5
0
1
1
4
6.5
1
0
0
5.5
101.5
1
0.5
1.5
3
3
0
0
0
3
80
0
2
2.5
2.5
0
1
0
1
4.5
61
0
1.5
0
2
5.5
1
0
0
6
70