Metal Biological Activity Final Report
Biological Activity of Metals
FINAL REPORT
Caroline Whalley, Juan Brown, Mark Kirby, Liam Fernand
DETR/DEFRA contract CDEP/84/5/285
June 2002
Executive Summary
The purpose of this project was to investigate associations between cadmium and phytoplankton in marine sediments in the central North Sea, and to consider the OSPAR JAMP (Joint Assessment and Monitoring Programme) recommendations for the application of biological effects methods for metals in marine waters.
The work was required to investigate:
- concerns regarding the cause of elevated concentrations of cadmium and chromium in the sandy sediments of the Dogger Bank.
- the applicability in offshore UK marine waters of the JAMP recommended cascade of biological effects techniques for metals.
An example of why it was important to carry out this work is provided by international concerns about the quality of the environment around the Dogger Bank. The QSR (1993) identified the possibility that the Dogger Bank is affected by transport of nutrients and contaminants from coastal regions. To support decisions about possible action in the case of environmental harm, we need to know more about the causes and processes that affect observations. Recent MAFF-funded work had described a summertime circulation connecting the north-east coast of England and the northern flanks of the Dogger Bank. Within these waters it was shown that there was an association between particulate cadmium and chlorophyll a concentration. Consequently, it was possible that cadmium concentrations within sediment were linked to phytoplankton production rather than any advection from the coast. Bioavailability of cadmium in Dogger sediments is of interest since dab (Limanda limanda) livers collected from the Bank have shown relatively high cadmium concentrations in the past (CEFAS, 1998).
The principal findings of the project were
- Of several benthic species considered, Echinocardium cordatum showed most promise for metallothionein induction of in-situ species, although further work is required to better understand MT dynamics in this organism.
- To test whether phytoplankton deposition affected cadmium concentrations in the sediment, surface material from sediment cores collected at the same sites in summer 2000 and winter 2001 in the Dogger region was analysed. As all cadmium concentrations were below the detection limit (0.05 mg kg–1) identification of a link between chlorophyll a and cadmium in sediments was not possible.
- Sediment cores from a transect across the bank during 1999 showed elevated concentrations of cadmium (up to 0.34 mg kg-1 ) and chromium (up to 78 mg kg1) from 5 cm and 10 cm depths on top of the Bank, but not in the surface (top 1 cm) sediment layer. Concentrations were not elevated in the finer sediments either side of the Bank.
- Evidence for direct transport of contaminants via the seasonal jet-like circulation from coastal waters was not found. Elevated concentrations of cadmium in deeper sediments were present across the whole of the Bank.
Contents
Executive summary
Table of contents 3
List of figures 5
1) Introduction and background to the work
1.1:Introduction 7
1.2 : Context of work on the Dogger Bank 7
1.3 :References 13
2)The JAMP Cascade of Biological Effects Methods for Metals - Literature review
2.1:Introduction14
2.2:Lysosomal Stability14
2.2.1Introduction 14
2.2.2Analytical Methods 15
2.2.3Field Applications and Practicalities16
2.2.4Influences on the Biomarker17
2.2.5Discussion - Lysosomal stability 18
2.3:Oxidative Stress 19
2.3.1Introduction 19
2.3.2Analytical Methods 19
2.3.3Field Applications and Practicalities19
2.3.4Influences on the Biomarker20
2.3.5Discussion - Oxidative stress20
2.4:Metallothionein 21
2.4.1Introduction 21
2.4.2Analytical Methods 21
2.4.3Field Applications and Practicalities 23
2.4.4Influences on the Biomarker24
2.4.5Discussion – Metallothionein26
2.5:-Amino Levulinic Acid Dehydratase (ALA-D) 27
2.5.1Introduction 27
2.5.2Analytical Methods 27
2.5.3Field Applications and Practicalities28
2.5.4Influences on the Biomarker29
2.5.5Discussion - ALA-D30
2.6:Conclusions and Recommendations 31
2.7: References32
3)Investigation into metallothionein induction in benthic
organisms collected between Tyne/Tees and western Dogger Bank.
3.1 : Introduction 38
3.2 : Methods 38
3.3 : Analytical quality control 40
3.4 : Results40
3.4.1 Metals in sediments 40
3.4.2 Metallothionein results41
3.5 : Discussion 43
3.6 : Conclusions44
3.7 :References 44
4)Marine phytoplankton – a mechanism for transferring
dissolved cadmium to sediments – Literature review
4.1 : Summary 45
4.2 : Introduction 45
4.3: Nutrients in seawater46
4.4 : Plankton dynamics 46
4.5 : Toxicity of cadmium to phytoplankton 48
4.6 : Cadmium as a nutrient 48
4.7 : Cadmium uptake by phytoplankton 49
4.8 : Trophic transfer and sedimentation 51
4.9 : Metal flux by plankton blooms 53
4.10 : Production in the Dogger Bank region 53
4.11 : Cadmium in sediments from the Dogger Bank region 55
4.12 : Cadmium in benthos from the Dogger Bank region 55
4.13: Conclusions 55
4.14 : References 56
5)Investigations into cadmium and chlorophyll a
concentrations in sediments from the Dogger Bank region
5.1 : Introduction 60
5.2 : Practical work 60
5.3 : Analytical Quality Control 63
5.4 : Results63
5.4.1 : Seasonal sediment metal concentrations 63
5.4.2 : Discussion of results from seasonal sediment 71 sampling
5.4.3 : Dissolved cadmium in waters off the north east 74 coast and from the Dogger dogleg transect
5.4.4 : Metals in cored sediments from the Dogger Bank 74 dogleg, 1999
5.4.5 : Considering possible pathways for cadmium to 84 reach sediments at the Bank.
5.6 : Discussion85
5.7 : Further work86
5.8 : References87
Annex 1 : Preparation of biological samples 88
Annex 2 : Analytical procedures for metallothionein and protein 89analyses
Annex 3 : Collection, preparation and analysis of sediment samples 92
Annex 4 : Recoveries from certified reference materials (CRMs)93
Annex 5 : Sediment metal and chlorophyll concentrations, Dogger 94Bank 2000-01
List of figures
Fig 1.1Outline of project structure 8
Fig 1.2Cadmium concentrations in sediments (JMG survey) 9
Fig 1.3Cadmium concentrations in sediments (Dogger Bank survey, 11
1994-95)
Fig 1.4aCadmium vs chlorophyll a in suspended particulates from the12
Western North Sea and Dogger Bank, summer 1998
Fig 1.4bCadmium vs chlorophyll a in suspended particulates from the12
Dogger Bank (DB) and Tyne Tees regions, summer 1999
Fig 3.1Sampling locations39
Fig 3.2aMT induction in Asterias rubens42
Fig 3.2bMT induction in Echinocardium cordatum42
Fig 4.1Diagrammatic representation of phytoplankton succession 47sequence and associated physical-chemical changes (Lewis, 1979)
Fig 5.1aScanfish section August 200061
Fig 5.1bScanfish section Dogger dogleg (1999)62
Fig 5.2Comparison of recovery from sediment CRMs (BCSS-1 and 64
MESS-2)
Fig 5.3aAl in surface sediment, Dogger transect 2000-0166
Fig 5.3bFe in surface sediment, Dogger transect 2000-0166
Fig 5.3cCr in surface sediment, Dogger transect 2000-0167
Fig 5.3dMn in surface sediment, Dogger transect 2000-0167
Fig 5.3ePb in surface sediment, Dogger transect 2000-0168
Fig 5.3fZn in surface sediment, Dogger transect 2000-0168
Fig 5.4aCr, Pb, Zn vs aluminium in sediments, Dogger transect 69
2000-01
Fig 5.4bMn vs aluminium in sediments, Dogger transect 2000-0169
Fig 5.4cFe, Li vs aluminium in sediments, Dogger transect 2000-0169
Fig 5.5aMetals (Cr, Pb, Zn) vs iron in sediments, Dogger transect 70
2000-01
Fig 5.5bManganese vs iron in sediments, Dogger transect 2000-0170
Fig 5.5cMetals (Al, Li) vs iron in sediments, Dogger transect 2000-0170
Fig 5.6aChlorophyll a in Dogger transect surface sediments – 72
summer 2000
Fig 5.6bChlorophyll a in Dogger transect surface sediments – 72
winter 2001
Fig 5.7aTotal pigment in Dogger transect surface sediments – 73
summer 2000
Fig 5.7bTotal pigment in Dogger transect surface sediments – 73
winter 2001
Fig 5.8aDissolved Cd along Dogger dogleg transect 199975
Fig 5.8bDissolved Cd along Tyne Tees transect 199975
Fig 5.9aDissolved Cd vs chlorophyll a in Dogger dogleg transect 76
1999
Fig 5.9bDissolved Cd vs chlorophyll a in Tyne/Tees transect 199976
Fig 5.10aDissolved Cd vs Cd in suspended particulates in Dogger 77
dogleg transect 1999
Fig 5.10bDissolved Cd vs Cd in suspended particulates in Tyne/Tees 77
transect 1999
Fig 5.11aDogger dogleg 1999 core data – Aluminium79
Fig 5.11bDogger dogleg 1999 core data – Iron79
Fig 5.11cDogger dogleg 1999 core data – Lithium80
Fig 5.11dDogger dogleg 1999 core data – Manganese80
Fig 5.11eDogger dogleg 1999 core data – Cadmium81
Fig 5.11fDogger dogleg 1999 core data – Zinc81
Fig 5.11gDogger dogleg 1999 core data – Chromium82
Fig 5.11hDogger dogleg 1999 core data – Lead82
Fig 5.12aCr vs Al in cored sediment Dogger dogleg 199983
Fig 5.12bPb vs Al in cored sediment Dogger dogleg 199983
Fig 5.12cCr vs Mn in cored sediment Dogger dogleg 199983
Fig 5.12dCd vs Fe in cored sediment Dogger dogleg 199983
1) Introduction and Background to the work
1.1: Introduction
This project has two main themes (fig 1.1). One was to investigate the series of biological effects methods for metals, suggested by the OSPAR Commission as part of JAMP in 1998 (OSPAR, 1998), for their effectiveness in examining metal contamination effects in biota in marine waters. The other was to further investigate the anomalous cadmium concentrations found in some sediments from the Dogger Bank, following previous MAFF funded work (AE1214) which suggested a possible link between phytoplankton and cadmium concentrations within the water column. A further impetus to examine the Dogger Bank anomaly arose following the discovery of a seasonal (May – October) jet-like circulation from the north east English coast and skirting the northern side of the Dogger Bank (Brown et al, 1999, 2001).
The main aims of the project were:
- To perform a literature review focused on the practical application of the series of linked analytical and biological effects techniques for metals suggested by OSPAR.
- Using those biological effects techniques identified in the literature review as being robust and cost effective, to assess the biological activity of metals at a selection of offshore sites.
- To investigate anomalous cadmium concentrations in sediments from the Dogger Bank, and test whether increased concentrations of contaminants there may result from natural, in situ processes.
- To explore whether contaminant concentrations in sediment vary with the seasonal cycle of phytoplankton production and if the spatial variability is related to the distribution of primary production.
The two parts of the project proceeded in parallel, and are reported in different chapters in this report. Literature reviews were produced for both strands and are presented in chapters 2 and 4.
This work has benefited by being associated with other MAFF/DEFRA-funded projects, notably AE1214, AE1219, AE1225 and BEQUALM.
1.2 :Context of work on the Dogger Bank
Interest in metals in sediments at the Dogger Bank – particularly cadmium and chromium – was heightened in the early 1990s when the JMG survey (Rowlatt and Lovell, 1994a and b) found indications of some elevated concentrations in the sandy sediments. Such concentrations were unexpected because sands do not normally accumulate contaminants. Figure 1.2 shows cadmium concentrations in sediments collected from the region during the JMG survey. Concern was raised given the existence of industry on the North east English coast which discharged cadmium and chromium, although at the time there was no evidence for a pathway for the
Fig 1.1
fig 1.2
(mapinfo plot not available in e-version)
contaminants out to the Bank. Dab collected under the NMP from the western Dogger and the Tyne/Tees regions showed relatively high concentrations of cadmium in liver in samples collected during 1994 (CEFAS, 1998).
The internationally-based ICES/IOC Bremerhaven workshop examined a transect from the German Bight to the south eastern Dogger Bank. Results in cadmium concentrations in sediments and biota were varied (Cofino et al 1992). Sediments (<63µm) showed decreasing Cd concentrations with increasing distance from the coast. However, Cd concentrations in Aphrodite aculeata showed maximum values offshore, and those in dab liver and Pagurus bernhardus increased with increasing distance from the coast. Highest metallothionein (MT) concentrations were found in dab collected near the Dogger Bank, in contrast to that expected which was closer to the coast where sediment Cd concentrations were highest (Hylland et al, 1992). The elevated Cd and MT concentrations at offshore stations were thought to imply metal inputs in addition to those from the Weser and Elbe estuaries considered in the project.
Following the JMG survey, a project jointly funded by MAFF and DoE examined conditions at the Dogger in greater depth. This included a detailed sediments and benthos survey across the Bank region (Whalley et al 1997). This study confirmed that there were some elevated cadmium concentrations in the sediments (fig 1.3), but found less evidence for elevated chromium concentrations. It was noticeable that the “hot” spots did not occur in exactly the same locations as had been found during the JMG survey, prompting the comment that they “were consistently variable” (Rowlatt, pers. comm.). Examination of replicate grab samples, performed by D Lovell during the JMG survey, showed that while reproducibility of the entire sediment sampling and analysis procedure in samples was generally good, those for Cd in samples from the Dogger were considerably more variable. For example, Cd in 6 replicate grabs from the Dogger Bank (#775) showed a mean concentration of 74 µg kg-1, 95% confidence interval (CI) of 80%, while for German Bight samples (#781), the concentration was 36 µg kg-1 with 95% CI 30%. Reproducibility was much better for other metals, typically being 5-20%.
More recently, Brown et al (1999, 2001) discovered that seasonal stratification in the deeper waters (~40m) off the north east coast leads to a transport pathway during the spring-summer months. The pathway runs from the coast and skirts past the northern flank of the Dogger Bank. As part of this MAFF-funded investigation (AE1225), we examined the possibility that the pathway might transport particulate contaminants offshore. To this end, we collected 100l water samples at three depths from the water column (~6m below the surface, at the thermocline 20-30m depth, and ~6m above the seabed) using a CTD. The samples were filtered, retaining the particulate material for total digestion (HF) and metals analysis. Other analyses upon samples collected on the same CTD “dip” included chlorophyll a, which is used as a proxy for the phytoplankton content. Particulate samples were analysed for total metals content, which allowed normalisation for the clay content.
While normalisation for clays generally works well for most metals, it does not usually produce strong associations with cadmium. However, an unexpected relationship was found between the cadmium in the suspended load and chlorophyll a (fig 1.4). A cursory examination of the literature revealed evidence to suggest that
Fig 1.3
(mapinfo plot not available in e-version)
Fig 1.4
such a relationship might indeed occur, and if it did, this could have significant consequences upon our understanding of cadmium cycling in marine waters. At this stage, MLIS - DETR funded the current project.
1.3 : References
Brown J, Hill AE, Fernand L and Horsburgh KJ. (1999) “Observations of a Seasonal Jet-like Circulation at the Central North Sea Cold Pool Margin” Est Coast Shelf Sci 48 (3) 343-355
Brown J, Fernand L, Horsburgh KJ, Hill AE. and Read JW. (2001). “Paralytic shellfish poisoning on the east coast of the UK in relation to seasonal density-driven circulation” J Plankton Res23 105-116
CEFAS. (1998) Monitoring and surveillance of non-radioactive contaminants in the aquatic environment 1995 and 1996. CEFAS, Lowestoft. AEMR 51 ISSN 0142 2499
Cofino, WP, Smedes F, de Jong SA, Abarnou, A, Boon JP, Oostingh I, Davies IM, Klungsoyr J, Wilhelmsen S, Law RJ, Whinnett JA, Schmidt D and Wilson S. (1992) "The chemistry programme" Mar Ecol Prog Ser91 (1-3) 47-56.
Hylland K, Haux C and Hogstrand C (1992) “Hepatic metallothionein and heavy metals in dab Limanda limanda from the German Bight” " Mar Ecol Prog Ser91 (1-3) 89-96
OSPAR (1998) JAMP guidelines for contaminant-specific biological effects monitoring. OSPAR, London 38pp
OSPAR (1993) North Sea Quality Status Report OSPAR, London 132pp
Rowlatt S and Lovell D. (1994a) Survey of contaminants in coastal sediments. MAFF, Burnham-on-Crouch. DoE research contract PECD 7/7/358
Rowlatt S and Lovell D. (1994b) “Lead, zinc and chromium in sediments around England and Wales” Mar Poll Bull. 28 (5) 324-329
Whalley C, Rowlatt S, Jones L, Bennett M and Campbell S. (1997) Metals in sediments and benthos from the Dogger Bank, North Sea. CEFAS Burnham-on-Crouch, DoE contract CW0 301
2) The JAMP Cascade of Biological Effects Methods for Metals
- A Literature Review
2.1 : Introduction
In recent years there has been a trend towards associating biological effects measurements with chemical measurements of relevant media, such as the water or sediment in which an organism lives. This development has arisen since chemical or biological studies could be indicating a problem while being unable to identify but a causal association. For instance, biological effects data might suggest that something was causing deleterious changes in marine species, but could not be used to identify the particular cause of the problem. Meanwhile, chemical data might show the presence of contaminants but could not be used to identify any biological significance. The need to better understand the links between contaminant presence and biological effects has been highlighted in various fora, such as at OSPAR/ICES workshops.
In February 1998 the OSPAR commissions, as part of their Joint Assessment and Monitoring Programme (JAMP), issued guidelines for general biological effects monitoring and also for contaminant-specific biological effects monitoring (OSPAR, 1998). Within these guidelines a strategy was recommended for the monitoring of metal-specific biological effects, incorporating a “cascade” of stages that included the measurement of metallothionein (MT), -amino levulinic acid dehydratase (ALA-D) and a range of “antioxidant enzymes”. The JAMP guidelines also drew attention to a number of “early warning” techniques, amongst which lysosomal stability was suggested as potential monitor of metal-mediated effects.
One problem affecting the application of the suggested biological effectstechniques to sediments is that there is no commonly accepted chemical method of determining thebiologically-available metal bound to sediment. Indeed, the proportion of bioavailable metals bound to sediment may vary depending upon the organism being studied. Since study of biological effects tends to rely upon some correlation with the metal concentration, the lack of an accepted sediment digestion technique is a serious drawback for these contaminants.
This document reviews the use of the JAMP cascade techniques for metals in research and monitoring programmes. In particular, it addresses their practicalities in a field-monitoring context and their application specifically to the investigation of metal-mediated biological effects.
2.2 : Lysosomal Stability
2.2.1 : Introduction
Lysosomes are key cell organelles that play a vital role in the catabolism of cellular components, intracellular transport and the sequestration of organic and inorganic pollutants and their metabolites. It is because of their involvement in the latter, i.e. as part of an organism's defensive mechanism against pollution, that they have attracted substantial interest as potential indicators of pollutant-induced stress.
Evidence suggests that various forms of chemical and physical stress can damage or destabilise the lysosomal membrane, thus leading to the leakage of lysosomal enzymes into the cytosol. These enzymes, normally used for the breakdown of damaged internal structures or exogenous items taken in by phagocytosis, have the ability to damage cells. It is on this basis that the measurement of lysosomal stability has been given importance.
2.2.2 : Analytical Methods
The two main methods that have been deployed in marine monitoring of lysosomal perturbations focus on the stability of the lysosomal membrane. Both techniques measure membrane damage although in different ways. Comparative studies have shown that the two techniques correlate well (Lowe et al., 1995a).