Farley et al.: MMME Comparison of Four Modeling Approaches, File SI-3:1
Supplemental Information for
Metal Mixtures Modeling Evaluation: 2. Comparison of Four Modeling Approaches
File SI-3: Description of “Index 7” and “Index 8” Data Sets
Data and Methods Description for Index 7 and Index 8 Datasets
(see Table 2; Van Genderen et al. in this issue for the data)
Karel A.C. De Schamphelaere†,* and Colin R. Janssen†
†
Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, Jozef
Plateaustraat 22, B-9000 Gent, Belgium
E-mail: cmebane@usgs.gov; Telephone: 1-208-387-1308
Number of pages: 5
Number of tables: 0
Number of figures: 0
Farley et al.: MMME Comparison of Four Modeling Approaches, File SI-3:2
Data and methods description for index 7 and index 8 datasets (Table 2; Van Genderen et al. in this
issue)
De Schamphelaere KAC, Janssen CR
Index 7 (tests with 35 field waters – not spiked)
Stream water samples were collected from 35 field sites in the United Kingdom, 7 of which could for
the purpose of this study be designated as ‘reference’ samples (i.e. with no metal contamination,
sample ID’s 4,6,27,32-35), 1 of which could be designated as not contaminated, but too acidic for
normal algal growth, and 27 of which could be designated as contaminated with metals due to the
influence of historic mining activities. Samples were collected in acid-washed polyethylene bottles
and were kept cool during transport back to the laboratory, where they were stored cold (4°C) and
dark. Physico-chemical parameters of all water samples were measured prior to ecotoxicity testing.
This included Ca, Mg, Na, K, Fe, Al, Mn (ICP-OES); Cl, SO4, NO3 (ion chromatography), dissolved
organic carbon (DOC) and dissolved inorganic carbon (DIC) (combustion and IR spectrometry), trace
metals (ICP-MS), and Fe(II) (colorimetry). For more details on the field sites and the chemical
analyses we refer to a report of the Environment Agency (2008).
Before ecotoxicity testing, following nutrient components of the OECD algal test medium were added
to all samples (at the same concentration as described in OECD test guideline No. 201) (OECD, 2006):
NH4Cl (0.280 mM), KH2PO4 (9.19 µM), FeCl3·6H2O (0.237 µM), H3BO3 (2.99 µM), MnCl2·4H2O (2.1 µM),
Na2MoO4·2H2O (28.9 nM). After these additions, but still before ecotoxicity testing, 3-N-morpholinopropanesulfonic acid (MOPS) buffer was added to all samples with a field pH between 6 and 8 and pH
was adjusted to field pH with an appropriate amount of NaOH. No pH buffer was added to samples
with pH > 8 or < 6 (since this is outside the useful pH buffering range of MOPS). For these samples pH
was initially adjusted to field pH by adding either HCl or NaOH if necessary. The pH in all samples was
maintained within 0.2 pH units during tests by daily manual addition of HCl or NaOH if necessary.
Algae growth experiments in all samples were conducted in triplicate according to OECD guideline
No. 201 (OECD, 2006) at 25°C. Tests were initiated by introducing 104 cells ml-1 of Pseudokirchneriella
subcapitata into the samples. The algal strain used was CCAP 278/4 (Culture Collection of Algae and
Protozoa, at the Scottish Association for Marine Science, Argyll, Scotland, United Kingdom) and was
cultured in our laboratory as described in Deleebeeck et al. (2009). Growth rate (μ) in all samples
was calculated as the slope of the linear regression between loge(cell density) versus time, taking into
Farley et al.: MMME Comparison of Four Modeling Approaches, File SI-3:3
account the initial cell density and cell densities recorded by an electronic particle counter after 24,
48 and 72 hours of exposure (OECD, 2006: Deleebeeck et al., 2009).
Index 8 (single metal and quaternary mixture tests with 2 spiked field waters)
Stream water samples were collected from 2 field sites in the United Kingdom.
The two
'uncontaminated' water samples chosen for this purpose were from Sites 32 (Uldale Beck) and 33
(Scandale Beck). Samples were collected in acid-washed polyethylene bottles and were kept cool
during transport back to the laboratory, where they were stored cold (4°C) and dark. Physicochemical parameters of both water samples were measured prior to ecotoxicity testing. This included
Ca, Mg, Na, K, Fe, Al, Mn (ICP-OES); Cl, SO4, NO3 (ion chromatography), dissolved organic carbon
(DOC) and dissolved inorganic carbon (DIC) (combustion and IR spectrometry), trace metals (ICP-MS),
and Fe(II) (colorimetry). For more details on these field sites and the chemical analyses we refer to a
report of the Environment Agency (2008).
Site 33 was characterised by a pH of 8.4 and a hardness of 95 mg CaCO3 l-1. Site 32 was characterised
by a pH of 5.5 and a hardness of 5.8 mg CaCO3 l-1. Since such a low pH is at the lower tolerance
boundary of Pseudokirchneriella subcapitata, the pH of the latter sample was buffered at pH 6
through addition of MOPS (3.6 mmol l-1) and the appropriate amount of NaOH. No pH buffer was
added to the Site 33 sample. The pH in all samples (and all metal treatments, see further) was kept
within 0.2 pH units during tests by daily manual addition of HCl or NaOH if necessary. In addition,
following nutrient components of the OECD algal test medium were added to all samples (at the
same concentration as described in OECD test guideline No. 201) (OECD, 2006): NH4Cl (0.280 mM),
KH2PO4 (9.19 µM), FeCl3·6H2O (0.237 µM), H3BO3 (2.99 µM), MnCl2·4H2O (2.1 µM), Na2MoO4·2H2O
(28.9 nM).
The ecotoxicity testing occurred in two phases. In a first test phase, we wanted to determine the
single metal toxicity of five metals (Cu, Zn, Ni, Cd, Pb) in both waters. Each metal was spiked as its
chloride salt at six different concentrations into both test waters. Solutions were left to equilibrate
for 24 hours at 25°C. Dissolved metal concentrations (filtered, 0.45 µm) at test initiation were
measured using ICP-MS. In a first run, tests with Cu, Zn and Ni (n = 6, 2 waters x 3 metals) were
conducted simultaneously; in a second run, tests with Cd and Pb (n=4, 2 water x 2 metals) were run
simultaneously. All tests were conducted according to OECD guideline No. 201 (OECD, 2006) in a test
chamber at 25°C. Tests were initiated by introducing 104 cells ml-1 of Pseudokirchneriella subcapitata
into the test solutions. The algal strain used was CCAP 278/4 (Culture Collection of Algae and
Farley et al.: MMME Comparison of Four Modeling Approaches, File SI-3:4
Protozoa, at the Scottish Association for Marine Science, Argyll, Scotland, United Kingdom) and was
cultured in our laboratory as described in Deleebeeck et al. (2009). Growth rate (μ) in all samples
was calculated as the slope of the linear regression between loge(cell density) versus time, taking into
account the initial cell density and cell densities recorded by an electronic particle counter after 24,
48 and 72 hours of exposure (OECD, 2006: Deleebeeck et al., 2009). As such, growth rates (µ) were
calculated for the entire 72-hour exposure period, but also for the first 48-hour period. Since there
were indications of non-exponential growth between the 48th and the 72nd hour of exposure, only
the 48-hour growth rates were used for further data treatment. This decision was in line with the
OECD criteria for a valid test (OECD, 2006). Concentration response curves were fitted to the data of
the individual metals exposure using the logistic equation:
μ[Me] = μ0 / {1 + ( [Me] / EC50Me )β }
(Eq. 1)
where μ[Me] = the estimated growth rate at metal concentration [Me], μ0 = the mean growth rate in
the unspiked control sample, [Me] = the metal concentration, EC50Me = the median effective
concentration, and β= the slope of the concentration response curve. This was not performed for Pb
because of precipitation of Pb, likely in the form of Pb5(PO4)3Cl (chloropyromorphite) due to
phosphate additions to the test solutions to ensure algal growth. This left less than 5% of the Pb in
the dissolved form, making the test results with this metal unreliable. For this reason, Pb was also
omitted in the second test phase (single + mixed metal exposure). An alternative form of equation 1
(see De Schamphelaere and Janssen, 2004) was used to estimate the EC10. Parameter estimation
was carried out with the Levenberg-Marquardt method in Statistica 6.0 software (Statsoft, Tulsa, OK).
The results of this first test phase were then used to design the mixture toxicity experiment
conducted in the second phase. In this experiment, next to the toxicity of 6 quaternary (Cu, Zn, Ni,
Cd) metal mixture treatments (in both field waters, n=2x6=12), each metal was also simultaneously
tested individually for its toxic effect. The estimated EC10 of each metal (from test phase 1) was used
to choose the concentrations to be tested. Next to a control, individual metals were tested at
concentrations of EC10/2, EC10, 2×EC10, 4×EC10, 8×EC10 and 16×EC10, and quaternary mixtures
(four metals together) of Zn, Cu, Ni and Cd were tested in which each metal was present at
concentrations of EC10/8, EC10/4, EC10/2, EC10, 2×EC10 and 4×EC10. This resulted in a total number
of 31 treatments that were investigated for each field water. Methods for preparation of the media
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and treatments, analyses, and toxicity testing were exactly the same as in the first test phase.
Growth rates were calculated for the first 48-hour period of the exposure.
References cited:
Deleebeeck NME, De Schamphelaere KAC, Janssen CR. 2009. Effects of Mg2+ and H+ on the toxicity of
Ni2+ to the unicellular green alga Pseudokirchneriella subcapitata: Model development and validation
with surface waters. Sci Total Environ 407:1901-1914.
De Schamphelaere KAC, Janssen CR. Bioavailability and chronic toxicity of zinc to juvenile rainbow
trout (Oncorhynchus mykiss): comparison with other fish species and development of a biotic ligand
model. Environl Sci Technol 38:6201–9.
Environmental Agency. 2008. Environmental Quality Standards for trace metals in the aquatic
environment. Science Report – SC030194. ISBN: 978-1-84432-887-1. Environment Agency,
Almondsbury, Bristol, United Kingdom, 177p.
OECD. 2006. Guidelines for testing of chemicals. Guideline 201: Freshwater Alga and Cyanobacteria,
growth inhibition test. Adopted, 23 March 2006. Organisation for Economic Co-operation and
Development, Paris, France, p.