NICKEL TOLERANCE OF PHYTOPLANKTON ISOLATED FROM A
RECOVERING LAKE NEAR SUDBURY CANADA
D. G. Woodfine1, M. Havas2 and J. Acreman3
1 Canadian Environmental Modelling Centre, Trent University, Peterborough, On, Canada K9J 7B8, dwoodfine@trentu.ca
2 Environmental Studies Department, Trent University, Peterborough, On, Canada K9J 7B8
3 University of Toronto Culture Collection, Botany Dept. University of Toronto, Toronto, On, M5S 3B2
The current study compared “free”metal tolerance to Ni of three species of phytoplankton, Urosolenia eriensis, Cosmarium minimum and Senedesmus acutus isolated from Alice Lake, Sudbury, Canada in an attempt to determine if Ni tolerance was related to presence of these species in the lake. Alice Lake was subjected to 60 years of Ni and Cu pollution from both aerial inputs and runoff from slag piles from a nearby Ni-Cu smelter. After the closure of the smelter and building of the super stack in 1972 the air quality improved immediately and water quality began to improve by the late 1970s and early 1980s. Phytoplankton, which had been all but absent from the lake, had recovered by the mid 1980s. Tolerance was measured in defined media allowing for the modeling of speciation, “free” Ni, using GEOCHEM-PC (ver. 2.) Nickel tolerance (Effective Concentration causing a 50% reduction in growth = EC50) in a defined medium was compared with ambient lake water concentrations and the presence of each species from the mid 1980s to the mid 1990s. Results from these comparisons suggest that there are different mechanisms of tolerance, unique to each species. The diatom U. eriensis, showed high Ni tolerance (EC50 = 1664 ± 70FgL-1), which was consistent with its dominance of the phytoplankton assemblage in 1985-86 and 1995. The second species C. minimum (EC50 = 298 ± 2FgL-1), the dominant phytoplankton in the lake during 1992, showed an intermediate tolerance but appeared to “lose” tolerance during isolation and culturing. The third species S. acutus had a much lower tolerance (EC50 = 120 ± 20FgL-1) and therefore should not have been able to survive in the lake at the Ni concentration recorded. However, this species was present in the lake in all years sampled. Intraspecies comparison with two other S. acutus strains reveals that this species is “plastic” in its tolerance to Ni. These results emphasize the difficulties with using laboratory trace metal tolerance to interpret ecological field observations.
Different phytoplankton species are expected
to vary in their ability to tolerate Ni and their ability to survive in a Ni
contaminated lake. Tolerance may be a
major factor in the “success” of a particular species within a contaminated
lake system. Measurement of tolerance
of lake isolates may provide information to help interpret the ecology within
the system.
For toxicity experiments using Ni
(or other trace metals) the concept of metal speciation may be very important
(Price and Morel 1995, Campbell and Tessier 1996). Specifically, the amount of
“free” metal versus total metal must be considered (de Filipps and Palghy
1994). The “free” metal is considered
to be the metal that is not complexed with other constituents in the water
(Price and Morel 1995) and is believed to be the most biologically
relevant. The pH of the medium is also
important to speciation as it can effect the complexation of trace metals
(Stokes and Campbell 1985, Price and Morel 1995). Standardized methodologies for experiments, which determine
effective concentration that limit growth (EC) allows comparisons to made
between different species or strains.
The EC values can be compared and analysed statistically. Better comparisons between different media
can be made when the EC values are calculated using speciation modelling and
are presented as “free” metal EC concentrations.
The current study determined the “free” Ni EC50 for three
different phytoplankton isolates from Alice Lake in batch culture tests. Water
quality in Alice Lake has improved since the closure of the Coniston smelter
and the concurrent building of the super stack (381m) at Copper Cliff, Canada,
in 1972 (Hutchinson and Havas 1986, Woodfine and Havas 1995). The species Cosmarium minimum was dominant in the
lake in 1992, Urosolenia eriensis was
dominant in 1995 and Scenedesmus acutus
was present in the lake during both years.
These algae belong to three different genera and it was hypothesised
that differences in tolerance to Ni might help explain the change in dominance
from 1985-86 to 1992 and in 1995. S. acutus was included in these tests in
the hope that it would provide a "benchmark" between years and
because previous work had been conducted on the tolerance of the Boucher Lake
strain of this species which could be used for comparison.
METHODS
Study
site and isolation
Alice Lake (27 ha, maximum depth 14 m) located < 1.5 km from the
Coniston smelting complexes near Sudbury, Canada (46E28' N 80E52'W). The uninhabited watershed consists of areas
of exposed Precambrian Shield bed-rock.
Alice Lake has experienced extensive pollution inputs in the form of
emissions from the local smelter and two other smelting complexes in the
Sudbury area (<20km). More complete descriptions of the lake and watershed
are available elsewhere Hutchinson and Havas 1986,
Woodfine and Havas 1995).
Lake water samples were collected
and then stored at 4EC. A technique of micropipetting
and serial washings was used to isolate single cells. All species were grown in a defined growth medium. Cosmarium
minimum and Scenedesmus acutus
were grown in Bold's Basal Medium (BBM) while Urosolenia eriensis was grown in M169 + Si. The cultures were kept in 125mL or 250mL
pyrex acid washed Erlenmeyer flasks in an environmental chamber at 25EC using a 16:8 hour,
light:dark, photoperiod under warm and cool white fluorescence lights (approx.
600 Fmolm-2S-1). The contents of the flasks were swirled
daily. The duration of exponential
growth was used as the time for experiments.
Estimating
Cell Density
Standard curves were calculated for
both S. acutus and C. minimum by using a dilution series of
a stock culture. Actual cell counts of
the dilution series were performed using an Improved Neabaur Haemocytometer.
Counts were made using standard technique and triplicate counts (Schoen 1988).
Cell density in the flasks was determined using a spectrophotometer at a wavelength
of 665nm. Experimental cell densities were then measured using absorbance. For U. eriensis each 50mL sample was
centrifuged at 2000rpm for 10 minutes and 40mL of supernatant was decanted. The
pellet was then resuspended in the remaining 10ml by gentle shaking. Absorbance
was then measured from the concentrated sample.
Nickel
Tolerance Experiments
The condition for growth (i.e.
temperature light, light:dark period) were the same as those for the
maintenance cultures. Experiments were also carried out in acid washed 125mL
Erlenmeyer flasks to which 50mL of test medium was added. The pH of the medium + metal was adjusted to
the required pH (7.0 for mM169 + Si and 6.8 for mBBM) using 0.1 M NaOH or 0.1M
HCl. At least 3 replicates were
prepared for each concentration used in any one experiment. To each autoclaved flask a measured amount
of inoculum was added using aseptic technique in a laminar flow hood. Flasks were randomly placed on trays in the
growth chamber and the contents were swirled by hand daily. After a set period of time (corresponding to
the transfer period) the flasks were removed and the cell density of each
replicate was estimated using the absorbance at 665nm.
Modelling
Chemical Speciation
The modelling of chemical speciation
for the media was performed using the computer speciation program GEOCHEM-PC
(Ver.2.0).
RESULTS AND DISCUSSION
The Ni tolerance measured as EC50
for all three species are shown in Table 1.
The order of EC30 concentrations for Ni was U. eriensis >C. minimum> S. actutus. There was a dramatic difference among
species in their EC50 for Ni.
This result was unanticipated because all three species were present in
the lake in 1995 and as such it was believed they would have similar tolerance
to Ni. The Ni concentration in Alice
Lake water varies over the seasons and between years however, a concentration
of total Ni below 0.6 mgL-1 was not recorded during the period the
study (1992 and 1995). The values for
the maximum concentration with observable growth for S. acutus and C. minimum
are both less than the free Ni minimum recorded for the lake.
Table 1: The EC30 and EC50 measured as
"free" Ni concentrations for Alice Lake isolates of Cosmarium minimum, Scenedesmus actus and Urosolenia
eriensis and estimated for the 1972 Boucher Lake S. acutus and 1972 UTEX S.acutus
(Stokes et al. 1973).
|
Species (year) |
UTTC #* |
Source |
Duration (days) |
EC50 ± s.e (mgL-1) |
Highest concentration with measurable growth (mgL-1) |
|
Urosolenia eriensis 1995 |
338 |
Alice Lake |
10 |
1.664 ± 0.07a |
2.34 |
|
Cosmarium minimum 1992 |
350 |
Alice Lake |
10 |
0.298 ± 0.002b |
0.38 |
|
Scenedesmus acutus 1992 |
282 |
Alice Lake |
7 |
0.12 ± 0.02c |
0.57 |
|
Scenedesmus acutus 1972 |
10 |
Boucher Lake |
11 |
0.512* |
1.43 |
|
Scenedesmus acutus 1972 |
6 |
U. Texas C.C. |
11 |
0.205* |
0.24 |
Note: values not sharing the same superscript
are significantly different form each other (p<0.05) * indicate insufficient
data for statistical analysis
This suggests that
there may be other constituents in the water of Alice Lake that mitigate the
toxicity of the Ni present. Modelling
of the speciation of Ni in mBBM reveals that over 93% is free Ni. This compares to a mean of 90% in Alice
Lake. Therefore it would be assumed
that the toxicity in mBBM should be similar to that in Alice Lake water. This was probably not the case as a
mechanism of competitive exclusion of Ni by the presence of other divalent
cations may reduce the toxicity of Ni in Alice Lake. The subject of competitive exclusion has been reviewed by
Campbell and Tessier (1996). The
concentrations of Ca and Mg in Alice Lake water were much higher than in the
mBBM (25X and 10X respectively) and as such would support the competitive
exclusion concept. A loss of
tolerance through culturing may also be a plausible explanation as C. minimum and S. acutus did not show growth at concentrations of Ni similar to
those recorded for lake water may be a slight "loss" of tolerance
through culturing. This species of Scenedesmus has been observed to
dominate phytoplankton assemblages in limnocorral studies where trace metals
were added (Welbourn pers. comm.). This
suggests that either S. acutus as a
species has an innate ability to tolerate elevated concentrations of metals
(i.e. has "built in" tolerance mechanism(s)) or that it is sufficiently
plastic enough that some individuals (mutants?) are able to reproduce at
elevated concentrations of trace metals.
These individuals are then able to out compete other species. In a similar manner there may be some
"loss" of tolerance because the metal "stress" is removed
during the culturing process. This
would imply an energetic cost of fitness (tolerance). There is some evidence to support this hypothesis from work
conducted on the 1972 Boucher Lake isolate.
After more than 20 years of culturing its' tolerance to Ni has decreased
(Welbourn pers. comm.).
There
may also be some question as to the ecological relevance of EC50
values as an ecologically meaningful metric.
A better indicator may be the actual concentration that a species is able
to tolerate and still maintain measurable growth. Indeed, this may be a better predictor of a species presence in a
lake’s phytoplankton assemblage. There
may be many species of phytoplankton in Alice Lake (and other metal
contaminated lakes) that are only able to maintain very low cell densities of
viable populations. These populations
may be able to survive in refugia either as resting stages (i.e. spores, cysts
etc.) or as mature cells in areas such as sediments or sediment pore water.
Then when water quality (i.e. abiotic conditions) improve or change they regain
a presence in the assemblage. As such these small populations may act as
inoculum to the whole lake. This is
tolerance through avoidance in space or time.
The seasonal nature of Ni concentration in Alice Lake may allow for some
less tolerant species to survive during different time(s) of the year when the
concentration of Ni in the water is lowest. Support for small but viable
populations comes from the observation that the EC50 for S. acutus was lower than for the other
two species, however, the concentration with measurable growth was close to
actual concentration of Ni in Alice Lake.
Also the phytoplankton enumeration and seasonal analysis demonstrated
that although present in Alice Lake S.
acutus makes up only a minor component of the total assemblage present in
the lake at any time during the year (Woodfine1998). In contrast to the other species Urosolenia eriensis has an EC50 greater than the highest
recorded total Ni concentration for the lake.
This suggests that this species would be able to survive in the lake at
even higher concentrations of Ni.
During 1995, the phytoplankton assemblage in Alice Lake was dominated by
U. eriensis and this species also
dominated the lake phytoplankton during 1985 and 1986 (Havas et al. 1995). Interestingly the Ni concentrations in the lake water were
similar in all years. This suggests
that the switch in dominance in Alice Lake from U. eriensis in 1986 to C.
minimum in 1992 and back to U. eriensis
in 1995 was probably not due just to greater Ni tolerance. This species has been observed to dominate
other metal contaminated (Cu-Pb-Zn) lake system (Deniseger et al. 1986). These authors
suggest that it may be metal tolerance combined with an unpalatable growth form
(i.e. a large frustule with long spines on the ends), which allows it to
succeed in these environments.
The
ability of U. eriensis to grow in
over 2 mgL-1 "Free" Ni is remarkable and is one of the
highest levels of tolerance recorded for a defined media.
CONCLUSIONS
The three species
isolated from Alice Lake differ in their ability to tolerate Ni, however, it
appears that these differences in tolerance alone cannot account for the
changes in dominance observed over the past 10 years. The diatom U. eriensis showed
a remarkable ability to tolerate Ni in a defined medium. It appears the mechanism of tolerance
probably differs between the three species. Intraspecies comparison of S. acutus with two other strains reveals that this species is
“plastic” in its tolerance to Ni. Other factors such as nutrient uptake, predation and susceptibility to
infection may also prove to be important to the success of each species in
Alice Lake. These
results emphasize the difficulties with using laboratory trace metal tolerance
to interpret ecological field observations.
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