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(n

A) R2 = 0.994

Cu T (nM)

20

0

1816

14121086420

302010 40

Salinity

Zn a

nd C

C (

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