1. New Standards, Old Methods? Cu and Zn Speciation and Bioavailability in
Implementing New Environmental Quality Standards
HOLLY B.C. PEARSON1, SEAN COMBER1, CHARLOTTE BRAUNGARDT1, PAUL WORSFOLD1
1 School of Geography, Earth and Environmental Sciences, Plymouth University, Devon, UK
holly.pearson@plymouth.ac.uk
Environmental Quality Standards (EQS) have recently been changed for Cu and Zn
(Table 1) and take into account metal bioavailability. In an attempt to better elucidate
the relationships between element speciation, complexing ligands, and ecotoxicity,
this project requires specialist methodologies to detect and quantify the metal
species associated with organism toxicity. Investigation of these relationships will
require a sensitive analytical method that can detect labile metal species
concentrations, so that the effects of DOC from differing sources, and varying water
salinity, upon metal speciation and complexation capacity may be quantified. These
impacts will be explored with respect to estuarine organism toxicity using
ecotoxicological experiments. The data generated from this research will contribute
towards improving models used in routine monitoring of natural waters, and thus
help assess the appropriateness of the new EQS.
Introduction
References and Acknowledgements
1. WFD-UKTAG 2012. Estimation of background reference concentrations for metals in UK freshwaters Edinburgh, Scotland.
2. WFD-UKTAG 2013. Updated Recommendations on Environmental Standards. River basin Management (2015-21) Final Interim Report
(SR3 -2013).
3.Gardener, M., 1999, Chemosphere, vol. 38, no. 9, p2117-2124
4. Frenzilli, G., Nigro, M., Lyons, B. P., 2009, Mutation Research/Reviews in Mutation Research, vol. 681, iss. 1, p80-92
5. Fenech, Michael, 2008, Environmental Genomics, Human Press, 185-216
6. OECD Environment Health and Safety Publications , 2005, No. 50 , Environment Directorate , Organisation for Economic Co-Operation
and Development.
Metal Previous EQS EQS 2012 EQS 2013
Cu 5 µg/L dissolved 2.64 µg/L dissolved
where DOC ≤ 1 mg/L
3.76 µg/L bioavailable
where DOC ≤ 1 mg/L
2.64 + (2.677 x
((DOC/2)- 0.5)) µg/L
dissolved, where
DOC > 1 mg/L
3.76 + (2.677 x
((DOC/2)- 0.5)) µg/L
dissolved, where DOC
> 1 mg/L
Zn 40 µg/L total 3.4 µg/L dissolved
additional to natural
background
For consultation
Table 1 Previous and new EQS set by UK Technical Advisory
Group on the Water Framework Directive.[1], [2]
The Comet assay (Fig. 3) method assesses DNA damage to
cells following exposure of an organism to a contaminant. Cell
electrophoresis causes strand breakages in the DNA to migrate
to resemble a comet shape, with tail intensity representing more
strand breaks. Quantification is achieved by cell scoring under a
microscope. This sensitive method has been used successfully
in the past to observe genotoxic effects in a range of species [4].
The Micronucleus Assay assesses genetic damage at the
chromosome level, enabling a measure of both chromosome
loss and damage through blood or tissue analysis [5].
Considerations
 Species and method must be representative of
environment and sensitive to the metal of interest
 Method must assess relevant endpoint
 Derive a suitable dose-response curve to characterise
toxic effects fully
 Establish relationships between concentrations, exposure,
and timing and effects [6].
The research will seek to answer the following questions:
(1) How does ligand source impact metal bioavailability?
The research will determine metal speciation in a variety of natural estuarine waters influenced by different
sources of DOC, including river derived humic and fulvic acids, sewage treatment works effluents which will
include synthetic ligands, and natural biogenic sources of ligands from algal blooms.
2) What is the spatial and temporal variability in metal speciation?
The variation of metal complexation with salinity is well established (Fig. 2) however, further work is
required. The DOC/ligand sources identified above are in some cases seasonal in their inputs to the
estuarine environment and so it will be necessary to determine complexation capacities and ligand strength
over the course of at least one calendar year.
(3) How does the speciation impact on bioavailability/toxicity?
Determining the speciation of Cu and Zn alone will not allow conclusions to be drawn regarding actual
toxicity to marine organisms. A series of ecotoxicity experiments will therefore be performed to establish the
sub-lethal impacts of complexation of Cu and Zn on representative estuarine-dwelling organisms such as
Mytilus edulis (common or blue mussel).
(4) What are the regulatory implications?
The results of the research will be placed in a regulatory setting in order to provide recommendations
regarding routine compliance monitoring (e.g. analytical methods, frequency of sampling and locations) to
add support to the new EQS for Cu and Zn.
Approach
With thanks to the International Zinc Association, European Copper Institute, and Plymouth University/NERC for funding this research.
Voltammetry provides the means to determine a range of potentially
bioavailable metal species in saline waters at low limits of detection (10-11-10-10
M). In addition, metal titrations (e.g. Fig. 1) can be utilised to calculate the
concentration of natural organic ligands, their stability constant with the metal of
interest and complexing capacity (CC, e.g. Fig. 2) and the free metal ion
concentration within the sample.
With adsorptive cathodic stripping voltammetry (AdCSV), the competitive
strength of complexes formed between the metal and an added synthetic ligand
(e.g. catechol, salicylaldoxime) and natural ligands are employed to quantify the
operationally defined labile metal fraction within a specific range of stability
constants (detection window). The detection window can be adjusted to
investigate different types of natural ligands.
Methodologies
Fig. 2 Dissolved Zn and complexing
capacity of natural ligands versus
salinity[3].
y = 1.06x - 5.41
0
5
10
15
20
25
0 5 10 15 20
ip(nA)
R2 = 0.994
CuT (nM)
20
0
18
16
14
12
10
8
6
4
2
0
302010 40
Salinity
ZnandCC(µg/L)
Zn concentration
Complexing capacity for Zn
Fig. 1 CC titration: AdCSV current
response to an estuarine sample
equilibrated with increasing Cu spikes.
Fig. 3 A Comet Assay: The classic comet shape following
electrophoresis and DNA strand migration, B Cell scoring is
done under a microscope.
(‰) A B