ASSESSMENT OF ANTIFOULING AGENTS
IN COASTAL ENVIRONMENTS
(ACE)
(MAS3-CT98-0178)
Final Scientific and Technical Report
Partners
Prof. J. W. Readman (Programme Coordinator)
Plymouth Marine Laboratory, Plymouth, UK
Dr. B. van Hattum
Institute for Environmental Studies, Vrije Universiteit, Amsterdam, Netherlands
Dr. D. Barcelo
CID-CSIC, Barcelona, Spain
Prof. T.A. Albanis
University of Ioannina, Greece
Dr. B. Riemann
National Environmental Research Institute, Denmark
Prof. H. Blanck
Botanical Institute, Göteborg University, Sweden
Dr. K. Gustavson
DHI Water & Environment, Denmark
Dr. J. Tronczynski
IFREMER, Centre de Nantes, France
Dr. A. Jacobson (Industrial Partner)
Rohm and Haas, Consumer and Industrial Specialties, European Laboratories, France
27th June 2002
2
Contents
Index of Tables
4
Index of Figures
5
Executive Summary
6
Forward
7
Task 1: Collection And Compilation of Information Relating to Antifouling Paint/Booster Biocide
Usage.
8
Task 2: Develop Analytical Techniques And Test Models.
16
Sub-Task 2.1 - Months 3-12
16
Sub-Task 2.2 – Months 9-30
18
Task 3: Environmental Chemical Surveys And Experiments.
21
Sub-Task 3.1 – Months 7-30
21
Sub-Task 3.2 – Months 17-30
33
Task 4: Ecotoxicological Investigations
35
Sub-Task 4.1 – Months 3-20
35
Sub-Task 4.2 – Months 17-26
37
Sub-Task 4.3 – Months 26-33
38
Task 5: Integrate Results And Evaluate Risks
46
Sub-Task 5.1 – Month 34
46
Sub-Task 5.2 – Months 21-39
46
Sub-Task 5.3 – Month 40
48
Annual Workshops
49
Initiatives for the dissemination of results
49
Other Points
53
3
Index of Tables
Page
Table 1.
Leach Rates of Antifouling Agents.
9
Table 2.
Usage of Booster Biocides. [Ingredients permitted for use on
yachts < 25 m (as of 2002)].
10
Table 3.
Physico-chemical properties, persistence and toxicity of antifouling
biocides.
13
Table 4.
Suitability of available models.
19
Table 5.
Concentrations of antifouling booster biocides measured in
European coastal waters.
23
Table 6.
Concentration ranges (min. – max) .of booster biocides (µg/kg dry
weight) in sediment samples from different countries.
25
Table 7.
Concentrations of Irgarol (ng/L) in water samples collected around
the UK coast, September 2001.
26
Table 8.
Measured concentrations of ACE substances in Swedish waters.
28
Table 9.
Measured concentrations of metabolites of ACE substances in
Swedish waters.
28
Table 10.
Concentrations of selected antifouling agents (Irgarol,
chlorothalonil, dichlofluanid) in water samples collected from
French marinas of the English Channel and the Atlantic coasts in
summer 2001.
30
Table 11.
Concentrations of Irgarol 1051 in sediment samples collected in
2001 in French marinas along the English Channel, the Atlantic
and the Mediterranean coastline.
32
Table 12.
EC50 values for the selected ACE substances.
35
Table 13.
Results from a microcosm experiment with a mixture of Sea Nine,
TBT and Irgarol 1051.
40
Table 14.
Effects of Irgarol 1051 and Sea Nine on phytoplankton in pelagic
mesocosms.
41
Table 15.
Estrogenic activity determined in French water samples using the
ER CALUX test.
43
Table 16.
Estrogenic activity determined in French sediment samples using
the ER CALUX test.
44
4
Index of Figures
Page
Fig. 1.
Location of sampling areas investigated during the ACE Project.
22
Fig. 2.
Mean concentrations of diuron in samples taken from marinas and
ports throughout Europe.
22
Fig. 3.
Mean concentrations of Irgarol 1051 in samples taken from marinas
and ports throughout Europe.
22
Fig. 4:
Sample locations visited by partner 1 (UK) during year 2001.
24
Fig. 5:
Diuron and Irgarol 1051 in sediment samples from Danish harbours
and marinas (Samples taken in 1999).
29
Fig. 6.
Concentrations of Irgarol 1051 in water samples collected during
high boating period at selected sites along the English Channel,
Atlantic and Mediterranean coasts of France.
30
Fig. 7.
Relationship between concentrations of Irgarol 1051 in water
samples and the number of berths available in marinas on the
French coastline.
31
Fig. 8.
Concentrations of Irgarol 1051 in sediment samples collected at
selected sites along the English Channel, Atlantic and
Mediterranean coasts of France.
32
Fig. 9.
Estrogenic activity in water samples (pmol EEQ.l 1) and sediment
samples (pmol EEQ .g-1) collected in marinas and coastal areas of
the French coastline.
44
Fig. 10.
Comparison of measured concentrations of Irgarol 1051 (g/L;
average values) in European coastal environments and predictions
based on the Mam Pec model.
47
5
Final Scientific and Technical Report for EU-MAST project:
ASSESSMENT OF ANTIFOULING AGENTS IN COASTAL ENVIRONMENTS
(ACE)
(MAS3-CT98-0178)
Executive Summary
National and international legislation has been introduced restricting the use of organotin biocides
in antifouling paints for marine vessels. A number of replacement biocides are being used and,
although generally based on copper metal oxides, also include organic antifoulants (“booster
biocides”) to enhance the coatings efficacy. The ACE (Assessment of Antifouling Agents in
Coastal Environments) project has completed a comprehensive environmental assessment of the
following booster biocides: Irgarol® 1051 (2-methylthio-4-tertiary-butylamino-6-cyclopropylaminos-triazine); dichlofluanid (N’–dimethyl-N-phenyl sulphamide); chlorothalonil (2,4,5,6-tetrachloro iso
phthalonitrile); SeaNine® 211/Kathon 5287 (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one); and
diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea). Methods for analysis of the biocides (and some
of their metabolites/breakdown products) were developed and applied to approximately 800 water
and sediment samples from harbours, marinas, estuaries and coastal waters in Denmark, France,
Greece, the Netherlands, Portugal, Spain, Sweden, and the United Kingdom. Of the compounds
investigated, highest mean concentrations were recorded for diuron, especially in North-western
Europe. Irgarol® 1051, although as widely distributed as diuron, was generally monitored at
lower levels with highest concentrations in the Mediterranean. Dichlofluanid, chlorothalonil and
SeaNine® 211 were sporadically encountered (albeit occasionally at high levels) primarily in the
Mediterranean. Field toxicity studies using algal communities indicate that toxicity is likely for
some of the biocides (e.g., Irgarol® 1051) at current environmental concentrations.
Determination of the abiotic and biotic half-lives for these organic antifouling agents, examined in
laboratory studies and under controlled field conditions, indicated that diruon and Irgarol® 1051
were substantially resistant to degradation whereas SeaNine® 211 had a half-life of 2 to 9 days.
A model (MAM-PEC) to predict environmental concentrations of antifouling agents in the marine
environment was validated using the data set collected during this study and can now be applied
to assess future concentrations. Results achieved during ACE have resulted in over 30 peer
reviewed publications. Discussions at the final workshop considered it inappropriate to
selectively endorse antifouling products. ACE publications, however, have already contributed to
UK Government risk assessments which last year revoked licenses for some of the antifouling
biocides.
Further information is available through the ACE web site at www.pml.ac.uk/ace.
Substantial effort has been directed towards storage of the results and relevant information
relating antifouling booster biocides. The database has been made accessible to partners via the
Internet (www.pml.ac.uk/ace).
6
Forward
Following discussions at the ACE Final Workshop, it was agreed that the majority of the detailed
scientific and technical information obtained during the first two years of work had been included
in the previous ACE Annual Reports. To stand alone, however, it was considered that this Final
Scientific and Technical Report should include cohesive summaries and appraisals of all the
information that has been achieved and that each sub-task should be updated where necessary.
Consequently, the present report provides summaries on all aspects (tasks and sub-tasks) within
the ACE Programme and provides detailed descriptions of research undertaken in Year 3. It
does not, however, repeat previous Annual Reports, so if more detailed information is required for
tasks undertaken within the first two years, readers are referred to the previous/relevant Annual
Reports.
7
Task 1: Collection and compilation of information relating to
antifouling paint/booster biocide usage.
This is critical to the programme to identify which agents are most used and
are of most concern on a National/local basis to direct methodological
/analytical, chemical surveys and ecotoxicological experiments. The
gathering of data will be centralised but it is likely that usage will be
susceptible to high geographical variability.
Compilation of data within Task 1 dominated the ACE First Year Report. This information
culminated in Task 1.6 and, following discussions at the First Annual Workshop, booster biocides
were selected for study on the basis of usage, transport, reactivity and toxicity. Availability of
analytical facilities also needed to be addressed.
The core group of compounds selected for monitoring within the ACE Programme was:





Irgarol 1051
Dichlofluanid
Chlorothalonil
SeaNine
Diuron
Zinc pyrithione was also considered to be important even though the compound is difficult to
analyse.
It was agreed that toxicity tests should focus on Irgarol 1051 and SeaNine (adequate data was
available for diuron). It was decided that endocrine disruption experiments should address the
core compounds. Again zinc pyrithione was also considered important (providing that analytical
support could be made available).
Sub-task 1.1 – months 2-6
Title
Surveys of antifouling agents and products being manufactured.
Responsible:
PML
Partners:
IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration:
5 months
Objectives:
To survey antifouling agents and products being used and marketed, including
information about the level of content of the antifouling agents in the paints and all
available information on leaching rates.
Methods:
Each laboratory will assemble details regarding the national usage of antifouling
products. To this end, literature and statistics will be collected relating to manufacture /
registration / importation of products together with information on sales and product
usage.
Deliverables:
Input to database (sub-task 1.4) and major report 1 (sub-task 1.6).
Links:
1.2, 1.4, 1.5, 1.6, 2, 3, 4, 5
8
Leach Rates
The rate of leaching of an antifouling agent from its coating is a most important variable
contributing to its environmental concentration. In our previous annual reports, negligible data
was available concerning the leach rates of booster biocides. A recent publication by Thomas
and Waldock1 has provided leach rate data for a number of antifouling agents using both the ISO
laboratory test and a field simulated flume systems. Table 1 summarizes their results.
Table 1. Leach Rates of Antifouling Agents
Release Rate (μg /cm2/day)
Biocide
Trade Name
ISO Test System
Flume System
Cuprous oxide
25-40a
18.6 +/- 6.5
TBT
1.5-4.0a
1.6
Irgarol 1051
5.0
2.6b
Diuron
3.3
0.8
Dichlofluanid
Preventol A4
0.6
1.7
Zinc Pyrithione
Zinc Omadine
3.3
-c
Sea Nine 211
DCOI
2.9
3.0
Busan
-c
0.9
Densil S
0.6
3.8
TCMTB
TCMS pyridine
a
b
c
from Thomas et al. (1999)
mean of two data points
no data available

1
Thomas KV & Waldock, MJ (1999) A Study of the effects of environmental factors on the leaching rates of
biocides from antifouling coatings in order to improve environmental risk assessment. Part 2: International
competence testing and evaluation in natural environments. Health and Safety Executive Contract Report
R51.120, Sheffield, UK
9
Usage of Antifouling Agents
Partners have performed National surveys on the usage of antifouling agents. Table 2 is an
updated summary of that which appeared in the 1999/2000 ACE Annual Report. In addition to
the booster biocides, the current regulatory status of various copper antifouling agents are
included.
Table 2. Usage of Booster Biocides
[Ingredients permitted for use on yachts < 25 m (as of 2002)]
UKa
Franceb
Greeceb
Spainb
Sweden
Denmarkc
Netha,c
Copper(1) oxide
+
+
+
+
+d
+
+
Copper thiocyanate
+
+
+d
+
+
+d
+
Cu powder
Chromium trioxide
+
Diuron
-
+
+
+
Irgarol 1051
-
+
+
+
Zinc pyrithione
+
+
+
+
Dichlofluanid
+
+
+
+
TCMTB
-
Chlorothalonil
-
+
+
TCMS pyridine
-
Sea-Nine 211
-
+
-
+
-
+
+
+e
+
+
+
+
Folpet
Total (booster biocide)
+
+
Ziram
Zineb
-
+
3
5b
7b
a
UK=United Kingdom and Neth=The Netherlands.
very limited/no approval scheme (in principle, all can be used)
c regulations currently under debate
d leach rate regulated on West coast; banned on East coast
e although approved, product not used on pleasure craft
b
10
5b
1
2
5
The information in the 1999/2000 ACE Annual Report pertaining to manufacturers, formulated
products, and use percentages are essentially unchanged. An update of the regulatory scenarios
of antifouling agents follows:
In the UK, the Health and Safety Executive has removed the amateur application use of diuron,
Irgarol 1051, TCMTB, chlorothalonil, TCMS pyridine, and Sea-Nine. The currently registered
biocides for amateur application are zinc pyrithione, dichlofluanid, and zineb. In addition,
professional application of diuron has been revoked.
In the Netherlands, current information can be obtained from the “College vor Toelating van
Bestrijdingsmiddelen” (CTB), the regulatory authority, on their web-site (httfp://
www.bib.wau.nl.ctb). A review of paint formulations containing several booster biocides is
currently in progress. Formulations containing Irgarol, diruon, dichlofluanid, and zineb are
currently approved. The expiration date for Irgarol, dichlofluanid, and zineb is 2010, whilst for
diruon, it is 2003
In Spain, Greece and France, there are very limited (or no) registration schemes and, in principle,
all booster biocides can be used.
In Sweden, there have been a few changes from the discussions presented in the 1999/2000 Ace
Annual Report.
In Denmark, diuron and Irgarol 1051 were banned for use on pleasure craft in 2000. Results from
an environmental risk analysis of Sea-Nine and zinc pyrithione in Danish waters demonstrated
that, in most cases examined, the PEC/PNEC (predicted no effect level/predicted no effect level)
ratio was less than 1, indicating an acceptable risk.
International Maritime Organization’s Marine Environmental Protection Committee (MEPC)
In October 2001, the International Maritime Organization of the United Nations approved a treaty
(prepared by their Marine Environmental Protection Committee) regulating the use of harmful
antifouling agents. This will become effective when 25 countries representing 25% of the
tonnage ratify the treaty. A mechanism for placing compounds in Annex 1 (restricted use
compounds) is included in the treaty. Currently the only compound(s) on Annex 1 are organotins
which will be completely banned as antifoulants. The treaty lists a ban on application of
organotins to ship’s hulls commencing January 2003 and, either their complete removal or
application of an impermeable sealer coat by January 2008.
Biocidal Products Directive (BPD)
The European Union has instituted the Biocidal Products Directive (BPD) for authorization of
biocidal products within the European Union. The BPD harmonizes the data requirements for
existing and new biocides within the EU. Antifouling agents are included in this directive (Product
Type 21). Any existing antifouling agent seeking registration will need to notify in 2002 and
provide a base set of data. Time requirements for submission of additional necessary data has
not yet been established; however they will be part of the 10 year plan to fully implement the
directive.
11
Sub-task 1.2 – months 2-6
Title
Assessment of geographical patterns/differences in usage.
Responsible:
PML
Partners:
IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration:
5 months
Objectives:
An assessment of geographical patterns/differences in usage.
Methods:
The information inputted to the database concerning usage etc. (see sub-task 1.1) will
be investigated and compiled to identify patterns/differences in usage within the
countries represented within ACE. During the inception workshop (workshop 1), tasks
will be allocated to laboratories to obtain and compile information on the usage of such
products in other regions of the world, to identify potential inputs from transient ships
and potential future trends in usage.
Deliverables:
Input to database (sub-task 1.4) and major report 1 (sub-task 1.6).
Links:
1.1, 1.4, 1.5, 1.6, 2, 3, 4, 5
There are notable differences in usage patterns of booster biocides on pleasure boats throughout
the European region. The most commonly used biocides are: copper-oxide, Irgarol 1051, diuron,
dichlofluanid, and zinc pyrithione. Recent national regulations, voluntary agreements, and the
expected approval of the IMO/MEPC treaty have caused a shift in the usage patterns. The usage
shift from diuron and Irgarol 1051 to zinc pyrithione and Sea-Nine (noted in the ACE 1999/2000
Annual Report) continues. For copper oxide, the regulatory situation is currently changing.
The data presented in the ACE 1999/2000 Annual Report for the UK, Netherlands, Spain,
Greece, Sweden, Denmark, and France pertaining to geographical use patterns is currently
applicable.
Sub-task 1.3 – months 2-6
Title
Survey and critical assessment of the environmental and toxicological properties.
Responsible:
GU
Partners:
VKI, NERI, IFREMER
Duration:
5 months
Objectives:
A survey and critical assessment of the environmental and toxicological properties.
Methods:
For all antifouling agents, information on environmental and toxicological properties will
be assembled (e.g. stability, partitioning coefficients, NOEC levels). The data obtained
will be screened as regards their quality according to the criteria agreed upon during
the inception workshop (workshop 1). Data meeting these criteria will be identified and
included in the database to be set up (vide infra).
Deliverables:
Input to database (sub-task 1.4) and major report 1 (sub-task 1.6).
Links:
1.1, 1.2, 1.4, 1.5, 1.6, 2, 3, 4, 5
Below is a table reproduced and updated from the ACE 1999/2000 Annual Report. The data is
predominately extracted from published literature.
12
Table 3. Physico-chemical properties, persistence and toxicity of antifouling biocides
Biocide
Solubility
Kow
Koc
Degradability
Toxicity to fish
Toxicity to
algae
-1
(mg 1 )
zinc pyrithione
Photolysis half life < 1 hr
Biodegradation 4 hr
Reported
environmental
concentrations
Ref
Not determined
Irgarol 1051
2.2-11.1
631
1240
to
3100
Photolysis half life = 273 d;
not readily biodegradable
96 h LC50 for Zebra Fish =
400 μg 1-1; 96 h LC50
Bluebell sunfish = 2900 μg 1-1
72 h EC50 = 1.4
– 2.4 μg 1-1
4 – 130 ng 1-1
1
diuron
42
631
398
Limited
photolysis;
biodegradable
Bluegill 96 h LC50 8.5 – 25
mg/l
96 h EC50 0.04
to 0.12 mg/l
13 – 1000 ng 1-1
2
dichlofluanid
1.3
5000
1100
Bluegill sunfish = 0.03 mg/l
EC50 = 16 mg
1-1
copper(1)oxide
<0.007
10 – 10 200 μg 1-1 g 1-1
(Cu2+)
1 – 8000 μg 1-1
(Cu2+)
Median of approx. 7 μg 1-1
(Cu2+) for estuaries used
by commercial and leisure
craft
4
copper(1)
5
0.5
10 – 10 200 μg 1-1 (Cu2+)
1 – 8000 μg 1-1
(Cu2+)
Median of approx. 7 μg 1-1
(Cu2+) for estuaries used
by commercial and leisure
craft
4
5
631
<300 ng-l-1
5
non
thiocyanate
Sea-Nine 211
15,000
Microcosm studies half-life
ranges from 1 hr to a few
days
1
data provided by Ciba specialities;
2 Lewis and Gardiner, 1996
LC50 50% lethality concentration; EC50 50% effect concentration
2
Acute: 2-1300 μg 1-1
Chronic: 0.6 -15 μg
3 Tomlin, 1997,
3
1-1
4 ACP, 1998
5 Jacobson2
Jacobson AH Willingham, GL (2000) Sea-nine antifoulant: an environmentally acceptable alternative to organotin antifoulants, The Science of the Total Environment 258:103-110.
13
The biological data collected for this project tends to confirm that, as a class, marine antifouling agents are
toxic to aquatic organisms. Some compounds such as Sea-Nine and zinc pyrithione, in laboratory tests,
appear to degrade rapidly. As discussed above, a report prepared by the Danish EPA indicated that SeaNine and zinc pyrithione pose an acceptable risk.
In a paper to be published shortly3, the concentration of Irgarol 1051 and its degradation product (GS
26575) and diuron and its degradation products (CPDU, DCPMU, DCPU) were all detected in surface
waters near drydocks. In addition, Irgarol 1051, GS26575, and diuron were also detected in sediments.
Preliminary results indicate that hosing the hull to wash the bottom may be a significant point source for
antifouling agents.
Sub-task 1.4 – months 5-9
Title
The development of a concise database with the information obtained
Responsible:
PML
Partners:
IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration:
5 months
Objectives:
To develop a concise database with the information obtained in sub-tasks 1.1, 1.2 and 1.3.
Methods:
The database will contain the information on the antifouling agents obtained, i.e. statistics on
usage in different regions, leaching rates, environmental and toxicological properties. (Full details
of the database and data management are provided in Section 5.2)
Deliverables:
Database and major report 1 (sub-task 1.6).
Links:
All tasks.
As described in the ACE 1999/2000 Annual Report, a database was created using Microsoft Excel. This
data base has been updated regularly throughout the project. Each partner has provided the necessary
data for importation into a final controlled-access database.
Sub-task 1.5 – months 2-9
Title
Screening of literature on analytical techniques used for the different antifouling agents.
Responsible:
CSIC
Partners:
UILIC
Duration:
8 months
Objectives:
To screen the literature on analytical techniques used for the different antifouling agents.
Methods:
A review will be made of analytical strategies for samples and information will be compiled on the
concentrations of antifouling agents in estuarine and marine waters. The results will be discussed
in the light of modern developments in analytical chemistry.
Deliverables:
Information generated to guide analyses of booster biocides. This information will be provided in
major report 1 (sub-task 1.6).
Links:
1.1, 1.4, 1.6, 2.1, 3, 4
A comprehensive review of the literature on analytical techniques for various antifouling agents was
presented in the ACE 1999/2000 Annual Report. Techniques reviewed were SPE and GC-MS, liquid-liquid
extraction and GC-MS, ELISA, immuno-sensors, SPE and LC-APCI-MS, SPME and GC-MS. The
publications generated as part of this project substantially update the analytical techniques presented in
the literature.
3
Thomas KV, McHugh M, Waldock M (2002) Antifouling paint booster biocides in UK coastal waters: inputs,
occurrence and environmental fate, The Science of the Total Environment, in press.
14
Sub-task 1.6 – months 8-10
Title
All available information relating to usage, transport, reactivity and toxicity will be assessed and
final choice of the antifouling agents on which the studies will be focused will be selected.
Responsible:
PML
Partners:
IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration:
3 months
Objectives:
To assess all available information relating to usage, transport, reactivity and toxicity leading to
the final choice of the antifouling agents on which the studies will be focused.
Methods:
The information obtained will be discussed at Workshop 2 (see Table 1) with all partners. The
different antifouling agents will be discussed in the light of their volume of production and usage
and environmental properties (e.g. persistence and toxic properties). The potential for
environmental contamination / pollution will be assessed.
A strategy will be set out for the next phase of the project selecting compounds for further
investigations based on potential problems or, on the contrary, the expectation that the
compounds will be a better choice from the environmental point of view. Decisions will be made
separately about the agents to be included in a European Coastal Survey and the compounds for
which persistence and toxic properties will be assessed. This distinction is made as, at the
present level of use, may not justify a survey to be conducted, whereas an expert judgement may
give rise to the conclusion that a formulation will (or perhaps should be recommended to) be used
in the future. The decisions will be made with the perspectives and needs of the modeling in
mind. The analytical chemistry requirements necessary in order to undertake the survey will be
established. The types of bioassays and semi-field studies will be reviewed and agreed upon.
Deliverables:
Information providing final focussing of ACE (to be summarised in Major Report 1 (sub-task 1.6)).
Links:
1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, Workshop
2
Following extensive discussions at the first Annual Workshop (1999/2000) it was decided to examine the
environmental chemistry and toxicology, as well as the environmental concentrations, of the following
antifouling agents (booster biocides):





Irgarol 1051
dichlofluanid
chlorothalonil
Sea-Nine 211
diuron
During the term of this project, metabolites of both Irgarol 1051 (2-methylthio-4-tertuary-butyl amino-striazine) and diuron (1-(3,4-dichlorphenyl)urea) were also analysed in environmental samples.
15
Task 2: Develop analytical techniques and test models
Suitably sensitive analytical techniques to measure environmental levels of selected
“booster” biocides will be developed. These will include IRGAROL 1051, 2,4,5,6tetrachloroisophthalonitrile (chlorothalonil), dichlorophenyl dimethyl urea (diuron),
dichlofuanid and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (SeaNine 211).
Techniques will be introduced within the participating analytical chemistry
laboratories where appropriate instrumentation is available. Performances will be
intercompared.
Sub-task 2.1 - months 3-12
Title
The development, testing and intercomparison of suitably sensitive analytical techniques
Responsible: CSIC
Partners:
IVM , UILIC, PML, IFREMER
Duration:
10 months
Objectives:
To develop suitably sensitive analytical techniques (and to intercompare analyses) the to measure
environmental levels of compounds considered to be of concern.
Methods:
Analytical protocols will be developed for compounds that are considered to be of concern from initial
assessments of the literature, techniques for the following compounds will be developed: IRGAROL
1051, 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil), dichlorophenyl dimethyl urea (diuron),
dichlofuanid and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (SeaNine 211). Techniques will be assigned
to the partners according to analytical capabilities and geographical relevance. Matrices for analyses will
be determined according to the predicted linear free energy distributions between environmental
compartments.
The analytical techniques developed will be distributed to the partner laboratories for further testing and
use. Where partner laboratories do not have the analytical capacity for quantifying all determinands,
samples will be provided to laboratories with the capabilities. For the other analyses, inter-laboratory
studies will be developed and run.
A rapid immunoassay protocol to measure IRGAROL 1051 will also be developed (Partner 3).
Deliverables: The analytical protocols essential to investigate contamination, degradation and ecotoxicology.
Links:
1.4, 1.5, 1.6, 3.1, 3.2, 4, 5
Summary
Several highly sensitive chromatographic methods for the analysis of the selected booster biocides and
their metabolites in environmental waters and sediments were developed. Methods were directed towards:
Irgarol 1051, its metabolite 2-methylthio-4-tert-butylamino-s-triazine; diuron and its by-products dimethyl
diuron and 1-(3,4-dichlorophenyl)urea; chlorothalonil; vinclozolin; dichlofluanid; and Sea-Nine 211.
Extractions employed on-line and off-line solid phase extraction (SPE) cartridges and disks, solid phase
micro-extraction (SPME), headspace-SPME, XAD-2 resin and liquid-liquid techniques. Sediment analyses
used an ultra-sonication extraction protocol. A comparative ELISA method was also developed for trace
level determinations.
Quantification was carried out by gas chromatography (GC) with electron capture (ECD), nitrogen
phosphorus (NPD), flame photometric (FPD) and mass spectrometric (MS) (including ion trap tandem MS)
detection. High Performance Liquid Chromatography was also used in quantification with detection using
electrospray tandem MS and atmospheric chemical ionization mass spectrometry (HPLC-ACPI-MS).
16
The recovery for the antifouling agents and their degradation products from water samples by using off-line
SPE cartridges with GC-ECD, NPD and MSD ranged from 60-111% and the determination limits for
different compounds varied between 0.2 and 0.5 ng/L.
The recoveries of the antifouling agents using SPME fibers (followed by GC-ECD and MSD quantification)
ranged from 70-124 % and the determination limits for different compounds varied between 5 and 50 ng/L.
The relative recoveries by using on- and off-line SPE with HPLC-ACPI-MS ranged from 76-96 % and the
detection limit was at the part-per-trillion level.
The recovery for antifouling agents from sediment samples using ultrasonic extraction with HPLC-APCIMS ranged from 54-109%, and the determination limits for different compounds varied between 0.2 and
1.6 µg/Kg dry sediment.
Sensitive enzyme-linked immunosorbent assays (ELISAs) were developed for the determination of Irgarol
1051. The dynamic ranges of the assays were between 30 and 200 ng/L, and the limit of detection was
16ng/L.
Publications generated:
Barceló, D. (1999) Sample handling and analysis of pesticides and their transformation products in water matrices by
liquid chromatographic techniques. Elsevier Science BV, pp 155-207.
Castillo, M and Barceló, D. (1999) Identification of polar toxicants in industrial wastewaters using toxicity-based
fractionation with liquid chromatography/mass spectrometry. Analytical Chemistry, Vol. 71, Number 17 pp 3769-3776
Ferrer, I and Barceló, D. (1999) Simultaneous determination of antifouling herbicides in marina water by on-line soldphase extraction followed by liquid chromatography-mass spectrometry. Journal of Chromatography A, 854 pp 197206
Ferrer, I, Thurman, EM and Barceló, D. (2000) First LC/MS Determination of Cyanazine amide, Cyanazine Acid, and
Cyanazine in Groundwater Samples. Environmental Science and Technology, Vol 34, No 4, pp 714-718.
Lampropoulou DA, Konstantinou, IK and Albanis, TA. “Determination of fungicides and antifouling compounds in
natural waters using SPME techniques and gas chromatography coupled with electron capture and mass
spectrometric detection”, 5th International Conference on Environmental Pollution, Thessaloniki 28-30 August 2000
Lampropoulou, DA, Konstantinou, IK and Albanis, TA (2000), “Determination of antifouling compounds in natural
waters using solid phase microextraction (SPME) and gas chromatography coupled with electron capture and mass
spectrometric detection”, submitted to J. Chromatography
Martinez, K, Ferrer, I and Barceló, D. Part-per-trillion level determination of antifouling pesticides and their by-products
in seawater samples by off-line sold Phase Extraction followed by HPLC-APCI-MS.
Martnez, K, Ferrer, I and Barceló, D. (2000) Part-per-trillion level determination of antifouling pesticides and their
byproducts in seawater samples by off-line solid-phase extraction followed by high-performance liquid
chromatography-atmospheric pressure chemical ionization mass spectrometry. Journal of Chromatography A, Vol.
879, pp 27-37.
Peñuela GA and Barceló, D. (2000) Comparative photo-degradation study of atrazine and deethylatrazine in water
samples containing titanium dioxide/hydrogen peroxide and ferric chloride/hydrogen peroxide. Journal of A.O.A.C.
Intl., 83 pp 53-60
Peñuela GA, Ferrer, I and Barceló, D. (2000) Identification of new photodegradation byproducts of the antifouling
agent Irgarol in seawater samples. Intern.J. Environ. Anal. Chem (in press)
17
Sub-task 2.2 – months 9-30
Title
Implementation of models capable of predicting concentrations and effects for different scenarios.
Responsible:
IVM
Partners:
PML
Duration:
21 months
Objectives:
Implementation of models capable of predicting transport, reactivity, concentrations and
effects in model situations for different scenario’s for usage (utilising the most effective models available
from EXAMS II, Delwag/Charon, EQC and Jackson0Baar Modd)
Methods:
Two of the partners within this project (IVM and PML) currently have proven models which,
with adjustment, are admirably suited to address this sub-task. A study (financed by the European
Paintmakers Association CEPE) is presently being carried out at IVM to compare and evaluate a number
of existing computer models for the prediction of antifoulant levels in the aquatic environment. Among the
models currently available are: ECoS (Plymouth Marine Laboratory), EXAMS II (US-EPA), Delwaq/Charon
(Delft Hydraulics), EQC (Environmental Modelling Centre Canada) and the Jacobson-Bauer model (Rohm
& Haas company). Based on the outcome an improved model will be developed at Delft Hydraulics; its
completion is scheduled for autumn 1998. The results will be used during the course of this project.
Deliverables:
3).
Links:
An evaluation of models to predict the environmental behaviour of biocides (major report
1.4, 1.6, 3, 4, 5
Many complex and interacting processes that can be of a biological, chemical or physical nature determine
the chemical fate of antifoulants in the environment. Especially in energy rich marine environments, the
hydrodynamic transport and mixing processes of water masses tend to have a major impact. For
compounds with a high affinity for particulate matter or sediment, such as TBT, sediment-transport
phenomena will be of dominant importance. Stable dissolved compounds, such as some of the modern
booster biocides, are likely to be affected most by river discharges or tidal currents. In specific marine
environments with low exchange rates or pseudo-stagnant conditions, the chemical and biological
processes will become more important.
An evaluation was made of the suitability of currently available chemical-fate models to predict
environmental concentrations of antifoulants (Table 4). Most of the current generic risk-assessment
models, such as EUSES (ECB, 1997) and the fugacity based EQC screening models (Mackay et al.,
1996), do not account for the complex hydrodynamic processes in coastal environments and include only a
limited number of emission, transport and chemical fate pathways required for a reliable assessment of the
fate of antifouling products. This includes e.g. factors related to characteristics of the paint matrix (binding
of biocide, leaching, erosion, life-time), shipping related factors (e.g. size, loading, speed, intensity, season
- in the case of yachting marinas), factors such as temperature, salinity or pH dependent characteristics
and processes (biodegradation, speciation, sorption), and hydrological characteristics of typical marine
environments. Only some of the more sophisticated generic chemical equilibrium models, such as QWASI
(Ling et al., 1993), TOXFATE (Halfon and Allan, 1995), ECOS (Harris et al., 1993), EXAMs (Willingham
and Jacobson, 1996) and DELWAQ (Delft Hydraulics, 1994) are capable of a more comprehensive
treatment of the subtle hydrodynamic, physico-chemical and biological processes and interactions, but
require skilled personnel to run the models or to derive estimates for model-input parameters. From the
models specially developed for antifouling agents, the Mam-Pec (Van Hattum et al., 1999) and the REMA
model (HSE, 1999) meet the requirements of handling both hydrodynamics and chemical fate processes
properly. The REMA model is limited to typical estuaries in the UK and does not allow a flexible handling of
shipping emissions. It is based on the QWASI model, but does not allow an estimation of the
hydrodynamic exchange processes. The Mam-Pec model, developed for the European Paintmakers
Asociation (CEPE-AWG, 1999), is based on the 2D-grid based DELWAQ water quality modelling
environment in combination with the SILTHAR model (Delft Hydraulics, 1995) for estimation of mixing and
transport processes. It was selected because of its ability to cope with the large differences in
hydrodynamics and shipping characteristics among marinas across Europe.
18
C/f
Nr. of Media
Hydro Dynamics
Emiss. from ships
Skills required
Effect of S,T,pH
Generic
Dimensions
Steady state / Dynamic
Table 4. Suitability of available models.
EUSES
c
4
-
-
low
-
g
1D
s
EQC-based models
f
>4
-
-
low
-
g
1D
s
QWASI
f
>4
+
(+)
high
(+)
l
2D
s/d
TOXFATE
f
>4
+
(+)
high
(+)
l
2D
s/d
EXAMS
c
>4
+
+
high
+
g
3D
s/d
ECOS
c
>4
+
+
high
(+)
g
2D
s/d
DELWAQ
c
>3
+
+
high
+
g
3D
s/d
Johnson and Luttik '94
c
3
-
(+)
low
-
g
1D
s
Baur and Jacobson '96
c
3
-
+
low
-
g
1D
s
Mam-Pec
c
4
+
+
low
(+)
g
2D
s
REMA
f
4
(+)
(+)
low
-
l
2D
s
Generic models
Models for antifoulants
Explanation: C/f: concentration or fugacity based model; Nr. of Media: number of abiotic main and subcompartments included (water column, sediment, air, soil, particulate matter); Hydrodynamics: ability to
cope with more complex marine hydrodynamic features; Emiss. from Ships: ability to allow for typical
emission patterns. Generic: generic model (g) or location specific (l). - : missing; + : option is available;
(+): option is partly available.
19
Modelling References
Baur, D., A. Jacobson (1996). Modelling of marine antifoulants. Rohm and Haas Company, European Laboratories,
Valbonne (France) and Research Laboratories, Spring House (PA, USA), p. 1-12 (manuscript).
CEPE-AWG (1999). Utilisation of more 'environmentally friendly' antifouling products. EC project No
96/559/3040/DEB/E2. Phase 1 - final report. CEPE Antifouling Working Group, Brussels. July 1999
Cowan, C.E., D. Mackay, T.C.J. Feijtel. D. van de Meent, A. Di Guardo, J. Davies and N. Mackay (1995). The multimedia fate model: a vital tool for predicting the fate of chemicals. SETAC Press, Pensacola (Fl).
Delft Hydraulics (1994). DELWAQ 4.0: Technical Reference Manual. Delft Hydraulics, Delft, Netherlands
Delft Hydraulics (1995). Silthar - a mathematical programme. Delft Hydraulics, Delft, Netherlands
ECB (1997). EUSES - The European union system for the evaluation of substances. Joint Research Centre European
Commission Environment Institute, European Chemicals Bureau, Ispra (Italy).
Halfon, E. and R.J. Allan (1995). Modelling the fate of PCBs and Mirex in aquatic ecosystems using the TOXFATE
model. Environmental International 21, p. 557-569.
Harris, J.R.W., R.N. Gorley, C.A. Bartlett (1993). ECOS version 2 user manual - an estuarine simulation shell.
Plymouth Marine Laboratory, Plymouth, UK.
Hattum, B. van, A.C. Baart, J.G. Boon, R.J.C.A. Steen and F.Ariese (1999). Computer model to generate predicted
environmental concentrations (PECs) for antifouling products in the marine environment. IVM-E99/15, Institute for
Environmental Studies, Amsterdam,
HSE (1999). REMA – regulatory environmental modelling of antifoulants. Biocides and Pesticides Assessment Unit,
Health and Safety Executive, London, UK.
Johnson, A., R. Luttik (1994). Risk assessment of antifoulants - position paper. Paper nr. 1994-05-03. Paper presented
at the 7th meeting of the Ad Hoc Group of Experts of Non-Agricultural Pesticides, 16-18 May 1994. National
Chemicals Inspectorate, Sweden; National Institute for Public Health and the Environment, Netherlands.
Ling, H., M. Diamond, D. Mackay (1993). Application of the QWASI fugacity/equivalence model to assessing sources
and fate of contaminants in Hamilton Harbour. J. Great Lakes Research 19, p. 582-602
Mackay, D. et al. (1996). Assessment of chemical fate in the environment using evaluative, regional and local scale
models: illustrative application to chlorobenzene and linear alkylbenzene sulfonates. Environ. Toxicol. Chem. 15, p.
1638-1648.
Steen, R.J.C.A., J. Jacobsen, F. Ariese, A.G.M. van Hattum, and A. Jacobson (2002). Monitoring of the marine
antifoulant 4,5-dichlor-2-n-octyl-4-isothiazolin-3-one (DCOI) in a Danish harbour. Environmental Science and
Technology (submitted)
Willingham, G.L. and A.H. Jacobson (1996). Designing an environmentally safe marine antifoulant. In: De Vito, S.C
and R.L. Garrett (eds). Designing safer chemicals - Green chemistry for pollutant prevention. American Chemical
Society, Washington DC. ACS Symposium Series 640, p. 225-233.
20
Task 3: Environmental chemical surveys and experiments
Sub-task 3.1 – months 7-30
Title:
Assessment of the extent of contamination of European coastlines through chemical surveys of relevant
areas
Responsible: PML
Partners:
IVM, UILIC, IFREMER, CSIC, GU, VKI, NERI
Duration:
23 months
Objectives:
To assess the extent of antifouling agent contamination of European coastlines
Methods:
Once installed and tested, analyses will commence on environmental samples for the antifouling agents
listed in Sub-task 2.1. Areas previously identified as those potentially subject to most contamination will
be targeted for assessment. ‘Good geographical coverage’ will also, however, be incorporated as a
prerequisite in survey design. A critical feature relating to the potential for pollution by antifouling agents
is the dissipation of the compounds from marinas and harbours. Is accepted that toxic concentrations are
likely to exist in the direct proximity to the vessels, and the primary concern is that coastal environments
adjacent to port facilities will be impacted (as was the case for TBT). As part of the surveys undertaken,
intensive investigations will be performed at the most contaminated locations to investigate dissipation.
Samples will be exchanged between partners in order to ensure that a full data set is generated for each
area. The survey data produced by individual partners will be compiled to provide a Europe-wide
assessment of coastal contamination with the antifouling agents in question.
Deliverables: Maps depicting the extent of contamination of European coastlines by the selected booster biocides.
Links:
1.3, 1.4, 1.6, 2.2, 3.1, 3.2, 4.3, 5
Summary
Approximately 800 water samples (and sediments from some areas) have been collected from the areas
shown in Fig. 1. These have included marinas, harbours, estuaries and coastal waters and cover diverse
European coastal systems. Results from analyses are summarised in Tables 5 and 6.
Results indicate that of the major booster biocides, highest mean concentrations of diuron are
encountered. The distribution of this compound is shown in Fig. 2 and indicates highest levels in North
Western Europe. Irgarol 1051 tends to be present at lower mean concentrations than diuron, although for
this compound, Mediterranean coastal environments are most contaminated (Fig. 3). Chlorothalonil,
dichlofluanid and Seanine were sporadically encountered (primarily in the Mediterranean). In isolated
cases, however, high concentrations of these were recorded.
Measurable concentrations of the degradation products of Irgarol 1051 and diuron were also encountered,
albeit at lower levels than those of the parent compounds.
(Detailed reports on this sub-task were provided in the ACE 2000/2001 Annual Report.)
21
Fig. 1. Location of sampling areas (indicated by squares) investigated during the ACE Project.
Fig. 2. Mean concentrations (ng/L) of diuron in samples taken from marinas and ports.
Fig. 3. Mean concentrations (ng/L) of Irgarol 1051 in samples taken from marinas and ports.
22
Table 5.
Concentrations (ng/L) of antifouling booster biocides measured in European coastal waters.
Country
Sweden
Denmark
Netherlands
UK
France
Spain
Greece
Site
Description
No. of samples
analysed
Irgarol
1051
Diuron
Dichlofluanid
Chlorothalonil
Seanine
Marinas
10
range
mean
median
2 – 364
61
16
<1 - 35
5
3
<1
<1
<1
<1
<1
<1
<1 - 3
<1
0
Ports
8
range
mean
median
<1 – 6
2
1
<1 - 3
1
0
<1
<1
<1
<1
<1
<1
<1 - 1
<1
<1
Coastal
19
range
mean
median
<1 – 36
<1 - 7
2
2
<1
<1
<1
<1
<1
<1
<1
<1
<1
0
Marinas
21
range
mean
median
4-9 37 - 174
2
27
0
0
n/a
n/a
n/a
Ports
3
range
mean
median
<1 – 68 <1 - 628
23
209
0
0
n/a
n/a
n/a
Marinas
26
range
<1 – 87
n/a
n/a
n/a
mean
median
20
17
<1 1129
328
233
<1 – 39 <1 - 282
4
51
0
19
n/a
n/a
n/a
Coastal
12
range
mean
median
Marinas
168
range
mean
median
<1 – 621 <1 - 685 <1 - 390
52
62
8
19
<1
<1
<1 - 30
1
<1
<1
<1
<1
Ports
47
range
mean
median
<1 – 208 <1 - 110
10
27
4
20
<1 - 26
1
<1
<1 - 20
1
<1
<1
<1
<1
Estuaries
64
range
mean
median
<1 – 47 <1 - 438
9
43
7
20
<1 - 40
1
<1
<1
<1
<1
<1
<1
<1
Coastal
49
range
mean
median
<1 – 92 <1 - 465
6
23
2
7
<1 - 7
1
<1
<1 - 26
1
<1
<1
<1
<1
English channel Marinas
3
range
mean
median
6 – 23
15
17
n/a
<1
<1
<1
8 - 11
9
9
n/a
Atlantic coast Marinas
14
range
mean
median
3 – 491
55
18
n/a
<1
<1
<1
<1
<1
<1
n/a
Atlantic Coastal
19
range
mean
median
<1 – 21
5
2
n/a
<1
<1
<1
<1
<1
<1
n/a
Meditteranean Marinas
18
range
mean
median
11 – 244
67
33
n/a
<1
<1
<1
<1 - 27
9
6
n/a
Meditteranean Coastal
32
range
mean
median
<1 – 11
1
1
n/a
<1
<1
<1
<1 - 2
1
<1
n/a
Marinas
112
range
<1 – 670
<1
<1 - 3700
mean
median
80
40
<1 - <1 - 760
2190
190
30
80
<1
<1
<1
110
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
30 – 323 <1 - 240
100
90
80
60
Ports
11
range
mean
median
Marinas
58
range
mean
median
<1 – 90
18
15
n/a <1 - 284
61
38
<1 - 63
16
16
<1
<1
<1
Ports
27
range
mean
median
<1 - 24
6
<1
n/a
<1 - 35
10
11
Detected
<1
<1
23
<1 - 88
25
<1
From the summarised data, it is clear that the assessment of the extent of contamination of
European coastal waters by booster biocides has progressed well, and that the participating
countries have obtained substantial data. This data has been entered into the database according
to the format of the reporting forms included in the ACE 2000-2001 Annual Report. A copy of the
database has been made accessible to partners via the Internet (www.pml.ac.uk/ace).
During the final year, some work within this sub-task continued.
finalised in Denmark, Sweden, France and the UK.
Surveys and analyses were

Shipping lanes in Sweden and Denmark were monitored (Brofjorden and Øresund).

An assessment of the extent of contamination of French coastlines was continued by
analyses of water and sediment samples collected during summer 2001 at selected
harbours along the English Channel, Atlantic Ocean and Mediterranean coasts.

A coastal survey was undertaken in the UK in summer/autumn 2001 to extend coverage.
Results from this research, together with appropriate summaries follow within this Section.
Publications on the subject:
To date, 11 publications have been generated relating to this sub-task. These are listed in the
following National reports within this Section.
24
Table 6. Concentration ranges (min. – max.) of booster biocides (µg.kg-1 dry weight) in sediment samples from different countries.
Country
Sampling sites
UK
The Netherlands
Site description
Number
samples
of Irgarol
1051
Irgarol
metabolite
Diuron
Dichlofluanid
Chlorothalonil
SeaNine
Marinas
Harbours
Estuaries
Coastal waters
NO SAMPLES
Spain
Marinas
15
2-88
nd-15
15-136
nd-10
na
nd-2
Greece
Marinas
Harbours
59
21
10-690
3-19
nd
nd
nd
nd
9-195
8-90
8-165
9-56
nd
nd
Marinas
Harbours
Estuaries
3
7
2
3.1-23
nd-10
1.9-4.5
na
na
na
3.4-12
1-8.5
3.4-5.0
na
na
na
na
na
na
na
na
na
3
2.8-5.8
na
na
na
na
na
2
3
17.9-20.6
15.1-29.5
na
na
na
na
na
na
na
na
na
na
Sweden
Denmark
France
NO SAMPLES
Mediterranean
Marinas
coastline
English Channel Marinas
Atlantic coast
Marinas
na : not analysed, nd : not detected (below detection limit)
Partner 1: Plymouth Marine Laboratory, UK
UK results were summarised in the ACE 2000-2001 Annual Report. Monitoring, however,
continued during 2001 and new data were produced. Samples were collected in July and
September 2001 at selected marinas (locations shown in Fig. 4). Concentrations recorded are
shown in Table 2. Detailed results have been included in the ACE-database.
5°W
0°
60°N
55°N
50°N
Fig. 4: Sample locations for partner 1 (UK) during year 2001. Red dots are for samples
collected inside marinas, blue dots are for samples collected outside marinas.
Table 7.
Concentrations of Irgarol (ng.l-1) in water samples collected around the UK coast, September 2001.
Station sampled
Irgarol 1051
Aberystwyth
Boston
Boston Dock
Brighton
Glasson Marina
Glasson Outer
Holyhead
Kingston Upon Hull
Lowestoft
Newcastle Inside
Newcastle Outside
Ramsgate Inner
Ramsgate Outer
Sutton
Swansea
Watchet
Waveney Dock
Weymouth
Weymouth Beach
Whitehaven Marina
Whitehaven Outer Loch
<1
<1
<1
47
220
26
12
390
16
54
27
54
13
86
62
<1
4
290
7
<1
<1
26
Partner 2: Institute for Environmental Studies, Vrije Universiteit, Amsterdam, Netherlands
Results from the research will be published in the following paper:
Lamoree, M.H, C.P. Swart, A. van der Horst and B. van Hattum (2002). Determination of diuron and the
novel antifouling paint biocide Irgarol 1051 in Dutch marinas and coastal waters. J. Chrom. (A). In
press.
Partner 3: Consejo Superior de Investigaciones Cientificas (CSIC), Department of
Environmental Chemistry, Barcelona, Spain
Results from the research have/are being published in the following papers:
Penuela, G.A., Ferrer, I., Barcelo, D., Identification of new photodegradation byproducts of the antifouling
agent Irgarol in seawater samples. Intern.J. Environ. Anal. Chem 78 (2000) 25-40
Martinez, K., Ferrer, I., Hernando, M.D., Fernandez-Alba, A.R., Marce, R.M., Borrull, F. and Barcelo, D.
(2002) Occurrence of antifouling biocides in the Spanish Mediterranean marine environment.
Environmental Technology, in press.
Karell Martinez and Damia Barcelo, Determination of antifouling pesticides and degradation products in
marine sediments by means of ultrasonic extraction and HPLC-APCI-MS, Fresenius J. Anal. Chem.,
(in press)
D.A.Azevedo, S. Lacorte, P. Viana and D. Barcelo, Analysis of priority pesticides and phenols in
Portuguese river water by Liquid Chromatography – Mass Spectrometry, Chromatographia 53 (3/4)
pp 113-118 (2001)
Partner 4 University of Ioannina (IULIC), Laboratory of Industrial Chemistry, Ioannina,
Greece
Detailed descriptions of the analyses are given in the following publications:
D. Lampropoulou, I. Konstantinou and T. Albanis, Determination of fungicides in natural waters using solidphase microextraction and Gas Chromatography coupled with Electron Capture and Mass
Spectrometric Detection, Journal of Chromatography, Vol. 893, 2000, 143-156.
V. Sakkas, D. Lampropoulou, I. Konstantinou and T. Albanis, Simultaneous determination of Antifouling Paint
Booster Biocides in Greek Ports and Marinas by means of solid phase extraction and gas
chromatography, Marine Pollution Bulletin. (submitted).
D. Lampropoulou,V. Sakkas, I. Konstantinou and T. Albanis, Antifouling Paint Booster Biocides contamination
in Greek marine sediments, Chemosphere (submitted).
V. Sakkas, I. Konstantinou and T. Albanis, Photodegradation of Antifouling biocides in natural waters Water
Research (in preparation).
Partner 5: Goteborg University (GU), Department of Plant Physiology, Goteborg, Sweden
The legal use of antifouling compounds on the Swedish west coast is restricted to Irgarol 1051
and copper on pleasure craft, although the illegal use has been hypothesised. In the Baltic, only
“non-toxic” paints are allowed. On commercial vessels, TBT is still expected to be the main
component, although Sea-Nine is allowed. In the investigated coastal areas, levels of TBT were
generally low whilst concentrations of Irgarol 1051 and diuron varied with the periodically
intensive pleasure craft traffic. Sea-Nine was not found in coastal areas. In harbours and marinas
TBT, Irgarol 1051, Sea-Nine and diuron were detected and occasionally reached high levels.
Results are summarised in Tables 8 and 9.
27
Table 8.
Measured concentrations of ACE-substances in Swedish waters. All concentrations in ng L -1
Fiskebäcks
kil
Irgarol
min
Irgarol
max
Diuron
min
Diuron
max
SeaNine
min
SeaNine
max
TBT
min
TBT
max
Seasonal
Spot
Type
2
363.6
0
35
0
2.7
1.9
7.2
x
marina
0.3
6.8
0
5.5
0
0
0.6
1.2
x
coastal
x
Marina
Kalvhagefjorden
Bonden
0
36
0
6.8
0
0
-
-
Lysekil
0.04
5.6
0
2.9
0
0.6
0.3
0.7
x
harbour
coastal
Brofjorden
0
3.5
0
2.4
0
1.5
1.5
5.2
x
harbour
Table 9.
Measured concentrations of metabolites of ACE-substances in Swedish waters.
All concentrations in ng L-1
Irgarol
met.(214) min
Irgarol
met.(214) max
Irgarol
met.(198) min
Irgarol
met.(198) max
Seasonal
Spot
Type
Fiskebäckski
l marina
0
47,819
0
12,478
x
marina
Kalvhagefjorden
0.3
1.9
0
0
x
coastal
Bonden
0
4.5
0
34.4
x
coastal
Lysekil
0
2.0
0
0
x
harbour
Brofjorden
0
4.5
0
695.2
x
harbour
Partner 6: Institute for the Water Environment (VKI) (which recently merged with DHIInstitute for Water and Environment), Horsholm, Denmark.
During 2000-2001, water and sediment samples from Danish harbours, marinas and open areas
were were collected and analysed for antifouling compounds (diuron, Irgarol 1051, atrazine,
simazine, copper, TBT, DBT and MBT). Data for sediments from marinas and harbours, and the
results for the water-samples and sediments from open areas are included in the ACE-database.
Highest concentrations of diuron and Irgarol 1051 were found in sediments from marinas. Hot
spots for TBT were in sediments from oil terminals, industrial harbours and shipyards. However,
high concentrations of TBT were generally found in all types of harbour, probably because some
vessels using the harbours and marinas still use TBT based antifoulings. Also, concentrations in
the sediment reflect contamination over several years. This is rendered more pertinent where
Danish authorities have not given permission for dredging of sediment.
Data for the antifouling concentrations measured in the Danish environment are summarised in
Figure 5.
28
Diuron and Irgarol in Danish harbours and marinas.
25
µg/kg dw
20
15
10
5
å
Sv
(I)
an
em
øl
le
(M
)
Få
bo
rg
M
(M
ar
)
se
lis
bo
rg
(M
Sø
)
nd
er
bo
rg
(M
)
(I)
(I)
Diuron
Åb
en
r
Ko
ld
in
g
Ve
jle
(I)
O
de
ns
e
sh
.(
I)
(F
)
Fr
ed
er
ik
År
hu
s
År
hu
s
(O
)
0
Irgarol
Fig. 5: Diuron and Irgarol 1051 in sediments from Danish harbours and marinas (1999).
(Oil harbours (O), Fishery harbours (F), Industry Harbours (I) and Marinas (M).
More information is given in the following publications:
Jensen, Gustavson & Petersen. Concentrations of organic pollutants and metals in sediments from Danish
waters. Danish EPA. In press.
Jensen & Gustavson. Contamination of sediments by organic pollutants in Danish marinas and harbours.
Danish EPA. In press.
Partner 8: IFREMER – France
The study of the extent of contamination of the French coastlines by booster biocide compounds
was pursued in 2001 through:

Determination of the concentrations of antifouling compounds in water samples
collected in July 2001 in marinas located along the English Channel (Cherbourg,
Saint Malo, Saint Quai Portrieux) and the Atlantic coast (Brest, La Rochelle,
Arcachon). These sites have been selected because of their high pleasure boat
activity (1000 to 3200 berths). Sampling was undertaken during high boating activity
(July 2001).

Determination of the concentrations of Irgarol 1051 in sediment samples collected (in
July 2001) from the marinas mentioned above and additionally from marinas on the
Mediterranean coast during Autumn 2001 (September/November).
The results give information about regional differences in water contamination by antifouling
compounds along the French coastlines.
The results from the analyses of water samples are presented in Table 10 and Fig. 6. The
compounds identified were chlorothalonil and Irgarol 1051. Dichlofluanid was not detected in any
of the samples. Dissolved Irgarol 1051 was quantified in all samples at levels between 5.4 ng.l-1
and 491.1 ng.l-1. Concentrations of dissolved chlorothalonil were between undetectable levels
along the Atlantic coast to 10.9 ng.l-1 at Saint Malo (Britanny).
29
Table 10. Concentration (ng.l-1) of selected antifouling agents (Irgarol 1051, chlorothalonil,
dichlofluanid) in water samples collected from French marinas of the English Channel and
the Atlantic coasts during summer 2001.
Limit of Detection (LOD) = 0.8 ng.l-1 for chlorothalonil and dichlofluanid.
Location
Sampling
date
Salinity
Location
type
Number Irgarol
of berths ng.l-1
Chlorothalonil Dichlofluanid
ng.l-1
ng.l-1
Cherbourg
05/07/2001
30.57
Marina
1200
16.6
8.5
< 0.8
Saint Malo
06/07/2001
34.23
Marina
1200
22.7
10.9
< 0.8
Saint
Quai
05/07/2001
Portrieux
34.35
Marina
1000
5.4
7.7
< 0.8
Brest
04/07/2001
34.32
Marina
1300
32.6
< 0.8
< 0.8
La Rochelle
02/07/2001
33.56
Marina
3200
491.1
< 0.8
< 0.8
Arcachon
03/07/2001
30.75
Marina
2300
73.3
< 0.8
< 0.8
600
WATER SAMPLES
Atlantic Coast
ng.l
-1
400
English Channel
Mediterranean Coast
200
0
Cherbourg
(July 2001)
St Quai
St Malo
Portrieux (July 2001)
(July 2001)
Brest (July
2001)
La
Arcachon
Rochelle (July 2001)
(July 2001)
Marseille
(Aug. 2000)
Saint
St Raphael
Mandrier (Aug. 2000)
(Sept. 2000)
Sampling Location and dates
Fig. 6: Concentrations (ng.l-1) of Irgarol 1051 in water samples collected during high
boating period at selected sites along the English Channel, Atlantic and Mediterranean
coasts of France.
30
The highest concentrations of Irgarol 1051 were found in the largest marinas with the highest
pleasure boat densities. No statistically significant relationship, however, was found between
concentrations and the number of berths (Fig. 7). The correlation would probably improve should
we have used the number of boats present instead of the number of berths available in marinas.
500
400
y = 0.1128x - 51.194
R2 = 0.5207
Irgarol (ng.l-1)
300
200
100
0
0
500
1000
1500
2000
Number of berths
2500
3000
3500
Fig. 7. Relationship between concentrations of Irgarol 1051 (ng.l-1) in water samples and
the number of berths available in marinas on the French coast.
Results from the analyses of Irgarol 1051 in sediments are presented in Table 11 and Fig. 8.
Concentrations of Irgarol 1051 in sediment samples collected in July 2001 from the English
Channel and Atlantic sites were between 15.1 ng.g-1 dry weight (d.w.) and 29.5 ng.g-1 d.w.
Sediment samples collected at three Mediterranean sites in September and November 2001 were
less contaminated (concentrations between 2.8 ng.g-1 (d.w.) and 5.8 ng.g-1 (d.w.)). This might be
influenced by the difference in the sampling period and composition of the sediments.
31
Table 11.
-1
Concentrations (ng.g dry weight) of Irgarol 1051 in sediment samples collected (during
2001) from marinas along the English Channel, Atlantic and Mediterranean coasts of
France.
Location
Sampling
date
Location
type
Number
of berths
Irgarol
Cherbourg
05/07/2001
Marina
1200
17.9
Saint Malo
06/07/2001
Marina
1200
20.6
Brest
04/07/2001
Marina
1300
15.1
La Rochelle
02/07/2001
Marina
3200
21.5
Arcachon
03/07/2001
Marina
2300
29.5
Marseille
22/11/2001
Marina
3000
2.8
Saint Mandrier
19/09/2001
Marina
630
5.3
St Raphael
14/11/2001
Marina
1550
5.8
Atlantic Coast
ng.g-1
SEDIMENT SAMPLES
30
20
-1
ng.g dry weight
English Channel
Mediterranean Coast
10
0
Cherbourg St Malo (July
(July 2001)
2001)
Brest (July
2001)
La Rochelle
(July 2001)
Arcachon
(July 2001)
Marseille
(Nov. 2001)
St mandrier
(Sept. 2001)
St Raphael
(Nov. 2001)
Sampling location and dates
Fig. 8. Concentrations (ng.g-1 dry weight) of Irgarol 1051 in sediment samples collected at
selected sites along the English Channel, Atlantic and Mediterranean coasts of
France.
32
Sub-task 3.2 – months 17-30
Title
Laboratory studies to assess the degree of physical and chemical degradation of the
antifouling agents..
Responsible:
UILIC
Partners:
CSIC, PML
Duration:
13 months
Objectives:
Laboratory studies to assess the degree of physical and chemical degradation of the
antifouling agents
Methods:
In the literature, few data are available regarding the persistence of antifouling agents,
some of which are conflicting. In this project, a consistent set of degradation studies
will be carried out so as to obtain reliable, comparable information for compounds of
interest.
Deliverables:
Information concerning degradation/dissipation of selected booster biocides (to be
summarised in major report 5).
Links:
1.4, 1.5, 1.6, 2.1, 2.2, 3.1, 4, 5
Summary
The photochemical degradation of Irgarol 1051, chlorothalonil, diclofluanid and sea nine 211 have
been studied in different natural waters (sea, river and lake) as well as in distilled water under
natural and simulated solar irradiation. The effect of dissolved organic matter (DOM) such as
humic and fulvic substances on the photodegradation rate was also studied under simulated
sunlight. The addition of DOM in distilled water was shown to greatly increase the rate of
degradation, however a decrease was observed in natural waters.
The photodegradation of Irgarol 1051 proceeds via a pseudo-first-order reaction in all cases, with
half-lives ranging from 2 to 1432 hrs. In natural and humic enhanced waters, Irgarol 1051
photodegradation gave rise to its dealkylated derivative, demonstrating that the transformation of
Irgarol 1051 depends on the constitution of the irradiated media and especially on the DOM
concentration and type. The byproducts identified by GC-MS were: 2-methylsulfonyl-4terbutylamino-6-cyclopropylamino-s-triazine,
2-hydroxy-4-terbutylamino-6-cyclopropylamino-striazine, 2-methylthio-4-terbutylamino-6-ethylamino -s-triazine, 2-methylsulfonyl-4-terbutylamino6-amino-s-triazine and diaminohydroxy-s-triazine.
The presence of DOM enhanced the photodegradation of chlorothalonil with the exception of
seawater. The photodegradation proceeds via a pseudo first order reaction in all cases. Half-lives
ranged from 1 to 48 h. In natural and humic rich waters, chlorothalonil photodegradation gave rise
to two different intermediates not found in a distilled water control, demonstrating that the
transformation of chlorothalonil depends on the constitution of the irradiated media and especially
the DOM. The byproducts identified were chloro-1,3-dicyanobenzene, dichloro-1,3dicyanobenzene, trichloro-1,3-dicyanobenzene and benzamide.
It was also found that the photodegradation of dichlofluanid proceeds via a first-order reaction in
all cases. The presence of various concentrations of DOM, however, inhibited the photolysis
reaction. Kinetic experiments (monitored with GC-ECD) indicated half-lives varying between 8
and 83 hrs. The major photo-decomposition products identified by GC-MS were
dichlorofluoromethane, aniline, and DMSA. Based on this byproduct identification, a possible
degradation pathway has been proposed for the photolysis of dichlofluanid in aqueous media.
33
Kinetic photodegradation experiments for sea nine 211 (monitored with GC-ECD) provided halflives (t1/2) varying between 6 and 433 hrs. Irradiation of the aqueous sea-nine 211 solutions gave
rise to a great number of transformation products that were isolated by means of SPE using SDB
extraction disks. Six of these products (n-octyl acetamide, n-octyl oxamic acid, n-octaldehyde, 4,5
dichloro-3-(n-octyl) thiazole, octanenitrile and n-octyl isocyanate) were tentatively identified using
GC-MS. Based on this byproduct identification, a possible degradation pathway has been
proposed for the decomposition of sea-nine 211 in aqueous media.
Degradation experiments involving the main antifouling agents quantified in samples taken from
the N.E. coast of Spain were conducted in order to determine the persistence of the compounds
in the marine environment. Seawater samples spiked with antifouling compounds (diuron, Irgarol
1051 and sea nine) were exposed to simulated natural environmental conditions. The more
persistent antifouling compounds were found to be diuron and Irgarol 1051. Even after 240 days,
concentrations in the water remained at one third of the original level. (Moreover, the degradation
products of Irgarol 1051 and diuron have been quantified at significant concentrations in
environmental samples from marinas and harbours.) Sea nine was found to degrade very
rapidly. In the first 11 days, its aqueous concentration decreased 6 fold and the compound could
not be detected after 87 days.
Further toxicity/degradation experiments are described under sub-task 4.3.
The following publications describe results:
I.K. Konstantinou, T. Sakellarides, V.A. Sakkas, and T.A. Albanis. Photocatalytic degradation of selected striazine herbicides and organophosphorus insecticides over aqueous TiO 2 suspensions.
Environmental Science and Technology, 2001, 35, 398-405.
D. Larsen, I. Wagner, I and K. Gustavson. Degradation of Sea-Nine in coastal water. (submitted).
K. Martinez and D. Barcelo. (2002) Determination of antifouling pesticides and degradation products in
marine sediments by means of ultrasonic extraction and HPLC-APCI-MS. Fresenius J. Anal.
Chem., (in press).
K. Martnez, I. Ferrer and D Barceló. (2000) Part-per-trillion level determination of antifouling pesticides and
their by-products in seawater samples by off-line solid-phase extraction followed by highperformance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.
Journal of Chromatography A, 879, 27-37.
K. Martinez, I. Ferrer, M.D. Hernando, A.R. Fernandez-Alba, R.M. Marce, F. Borrull and D. Barcelo. (2002)
Occurrence of antifouling biocides in the Spanish Mediterranean marine environment.
Environmental Technology, in press.
G.A. Penuela, I. Ferrer and D. Barcelo. (2000) Identification of new photodegradation byproducts of the
antifouling agent Irgarol in seawater samples. Intern.J. Environ. Anal. Chem., 78, 25-40
V. Sakkas, I, Konstantinou and T. Albanis. Photodegradation of antifouling biocides in natural waters.
Submitted to Water Research.
V.A. Sakkas, I.K. Konstantinou and T.A. Albanis. Photodegradation of antifouling fungicides in water under
simulated sunlight.
Presented at: 5th International Congress on Environmental Pollution,
Thessaloniki, Greece, 28 August - 1 September 2000; 1st European Congress on Pesticides and
Related Micropollutants in the Environment, Ioannina, 5 - 8 October 2000 and 3rd International
Conference of Balkan Environmental Association (B.E.N.A.), Bucharest - Romania, 23 - 26
November, 2000.
V.A. Sakkas, I.K. Konstantinou and T.A. Albanis. Photodegradation of selected antifouling booster biocides
in various natural waters under environmental conditions. 2 nd World Water Congress, Berlin Germany, 15 - 19 October, 2001.
V.A. Sakkas, I.K. Konstantinou and T.A. Albanis. Photodegradation of antifouling fungicides in water under
simulated solar light. 7th Conference on Environmental Science and Technology. Aegean
University, Ermoupolis, Syros, 3 - 6 September, 2001.
34
Task 4: Ecotoxicological Investigations
Sub-task 4.1 – months 3-20
Title
Bioassays to investigate toxic effects of the selected antifouling agents
Responsible:
GU
Partners:
VKI, NERI, PML
Duration:
18 months
Objectives:
Effects studies (bioassays) to investigate toxic effects.
Methods:
Bioassays to be conducted on Irgarol and SeaNine. These will include:

Short-term toxicity of antifouling agents to microbial activity in periphyton and plankton.

Experimental ecosystem studies of effects on microbial communities of antifouling agents

Ecosystem studies of effects on microbial communities by antifouling agents (TBT, Irgarol, SeaNine)
around selected harbours.
Deliverables:
An assessment of the toxicity of the “most-used” biocides. These results will form the
basis of major report 6.
Links:
1.3, 1.4, 1.6, 2.2, 3.1, 3.2, 4.3, 5
Summary
The toxicity of all six ACE-substances (Irgarol 1051, diuron, Sea-Nine, chlorothalonil,
dichlofluanid and zinc pyrithione) to phytoplankton communities were tested by the Göteborg
group. Data for periphyton were already available for three of the substances and the remaining
three were tested. The method used involved measuring photosynthetic incorporation of
radioactive carbon. Results established EC50-values for the ACE substances on phytoplankton
and periphyton (see Table 12).
Table 12. EC50 values for the selected ACE-substances. Ranges depict extreme values.
Compound
EC50 Phytoplankton
EC50 Periphyton
Irgarol 1051
2-3 nM1
4.7-5.5 nM2
Diuron
9-12 nM1
15-23 nM3
Sea-Nine
25-28 nM1
200-800 nM4
Chorothalonil
76-187 nM1
6203-17579 nM5
Dichlofluanid
23-36 nM1
1.4-734 nM5
Zinc pyrithione
9-30 nM1
37-84 nM5
1)Eriksson
2001 2) Dahl & Blanck
3)
Molander & Blanck
35
4)
Arrhenius et al. 1999
5)
Preliminary data
Short-term toxicity of antifouling agents to periphyton and plankton (PML & GU)
Inhibition of photosynthesis in natural phytoplankton samples (GU)
A laboratory study of the effects of the six ACE substances (Irgarol 1051, chlorothalonil, SeaNine, diuron, dichlofluanid and zinc pyrithione) on phytoplankton samples from the Swedish
coastal area was made during summer 2000. Acute effects were measure using two different
methods: incorporation of radiolabelled carbon dioxide and variable in vivo fluorescence (PAM
fluorometry). Data for periphyton were already available for some of the substances and efforts
were made to complete the list in summer 2002. Measurements of photosynthetic incorporation
established EC50-values for the ACE substances on phytoplankton (already shown in Table 12).
PAM measurements indicated an immediate decrease in photosynthetic yield at EC 50
concentrations for the two photosystem II inhibitors Irgarol 1051 and diuron. Compounds with
other modes of action showed a gradually emerging inhibition of photosynthesis over time.
Consequently, for Sea-Nine, chlorothalonil, dichlofluanid and zinc pyrithione, short-term
measurements of photosynthesis is likely to underestimate the toxicity to the algae.
Flow cytometry and pigment analyses of natural phytoplankton communities exposed to
Irgarol 1051 (PML)
Following exposure of natural phytoplanktonic communities to Irgarol 1051 (2-methylthio-4tertiary-butylamino-6-cyclopropylamino-s-triazine), flow cytometry analyses revealed that
approximately half of the phytoplankton are killed at concentrations as low as 100 ng/L. High
performance liquid chromatographic analyses of pigments extracted from the phytoplankton
demonstrated that 19'-hexanoyloxyfucoxanthin was selectively lost. This carotenoid is specific to
the prymnesiophytes which are key constituents of phytoplanktonic communities within temperate
marine waters.
Selective reductions in this compound were recorded at Irgarol 1051
concentrations as low as 40 ng/L. Concentrations substantially exceeding this toxic threshold
have been reported under Sub-task 3.1. A manuscript is in preparation.
36
Sub-task 4.2 – months 17-26
Title
Bioassays to investigate the endocrine disrupting characteristics of the antifouling agents.
Responsible:
IVM
Partners:
IFREMER
Duration:
10 months
Objectives:
To investigate the endocrine disrupting characteristics of the antifouling agents
(including IRGAROL 1051, Maneb and Ziram) using bioassays.
Methods:
In this sub-task, the antifouling agents will be tested with bioassays which
indicate estrogenic activity, e.g. the yeast screen assay and an estrogenic assay based on a
mammalian breast cancer celline.
Deliverables: Information to judge the endocrine disrupting potential of the booster biocides (to
be summarised in major report 7).
Links:
1.3, 1.4, 1.6, 2.2, 3.1, 3.2, 4.3, 5
Summary
The selection of compounds to be included in the endocrine disruption experiments was
discussed and adopted at the 1st annual workshop in Barcelona (Feb. 2000). Based on an
evaluation of potentially suitable test systems, it was decided to apply the ER-CALUX assay (IVM
and IFREMER). The ER-CALUX (Estrogen Responsive – Chemically Activated Luciferase
eXpression) assay is used for the determination of the (anti)estrogenicity of pure compounds and
complex environmental mixtures. The assay comprises a genetically modified T47D human
breast cancer cell-line, incorporating the firefly luciferase gene coupled to estrogen responsive
elements as a reporter gene for the presence of estrogenicity. By addition of the appropriate
substrate for luciferase, light is emitted. The amount of light produced is proportional to the
amount of ligand-ER biding, which can be related to estradiol equivalents (EEQs) (Legler et al.
1999). The ER-CALUX experiments have been completed. None of the antifoulants selected
(Irgarol 1051, SeaNine, chlorothalonil, diuron, dichlofluanid, maneb and ziram) showed an
estrogenic response in the ER-CALUX assay. Diuron, dichlofluanid and Irgarol 1051 were shown
to have a limited anti-estrogenic potency. However, levels in the marine environment are
generally 1000 times less than the levels that may cause such an anti-estrogenic response in invitro systems.
Full details were provided in the ACE 2000/2001 Annual Report.
37
Sub-task 4.3 – months 26-33
Title
Investigate whether effects observed under laboratory conditions occur under (semi-)
field conditions.
Responsible:
GU
Partners:
VKI, NERI, IFREMER
Duration:
8 months
Objectives:
To investigate the effects observed in laboratory under (semi-) field conditions.
Methods:
The bioassays tested in the above sub-tasks will predict effects. The validity of
these predictions will be checked with a limited number of tests under semi-field conditions.
Deliverables:
report 8).
Links:
An assessment of environmental damage through booster biocide usage (major
1.3, 1.4, 1.6, 2.2, 3, 4.1, 4.2, 5
Summary
In field experiments on phytoplankton from Sweden’s busiest oil harbour (Brofjorden) and the
strait of Öresund between Sweden and Denmark, Pollution Induced Community Tolerance (PICT)
to TBT and Sea-Nine could be detected. Tolerance to Irgarol 1051 could be detected specifically
in Brofjorden and also in field experiments in a marina on the Swedish west coast. The tolerances
found indicate that the present use of antifouling agents are causing a selection pressure on algal
communities and are thus damaging their structure.
In a mesocosm study performed on the Swedish west coast, No Effect Concentrations (NEC)
could be calculated for Pollution Induced Community Tolerance, reflecting structural changes in
the communities at 0.2 nM for Irgarol 1051 and 0.7 nM for Sea-Nine (nominal concentrations).
An experiment with mixtures of Sea-Nine, TBT and Irgarol 1051 has been made in cooperation
with the EU-project PREDICT. NOEC values, where each component is present in concentrations
lower than their individual NOEC, show that additive effects can be anticipated in mixtures of the
three antifoulants. Concentrations of TBT and Irgarol 1051 in the mixture at LOEC are below
concentrations found on the Swedish west coast.
To extend the toxicity studies undertaken by the UK partner, during 2001 natural populations of
phytoplankton were incubated as batch cultures under static conditions. Fv/Fm was measured on
a daily basis using fast repetition rate fluorometry. Preliminary data have been obtained for
diuron, Irgarol 1051, zinc pyrithione and Sea-Nine. Additional studies on natural populations were
undertaken to investigate toxicity and tolerance of different populations to Irgarol 1051 and
diuron. Preliminary data did not indicate that populations consistently exposed to antifouling
agents developed significant tolerance.
Work concerning the determination of endocrine disrupting potency was performed on extracts of
water and sediment samples collected in French marinas and coastal areas. The oestrogenic
activity of the extracts was determined using the ER-CALUX assay. Water samples from the
Mediterranean coast showed lower oestrogenic activity than samples from the Atlantic or the
English Channel. The results show higher estrogenic activity in Mediterranean water samples
collected inside marinas (6.10-3 to 0.15 Estradiol Equivalents –EEQ- /liter of water sample)
compared to coastal Mediterranean waters (from undetectable levels to 8.4.10 -4 EEQ/liter of
sample). Oestrogenic activity in sediment samples was between undetectable levels to 1.6.10 -1
EEQ/g, except for a sample collected in Marseille, where activity was 5 times higher.
38
Ecosystem studies of effects on communities by antifouling agents around selected
harbours (GU, DHI Water&Environment (VKI), CID-CSIC)
Pleasure craft marina
A field study of Irgarol 1051 effects on periphyton communities in the Fiskebäckskil area on the
Swedish west coast was started in 1999 and was also conducted in summer 2000. Effects were
estimated according to the PICT-concept (Blanck, et al. 1988), in this case measuring increased
tolerance to Irgarol 1051. A method using the incorporation of radiolabelled carbon was used to
measure the tolerance of periphyton. An increase in PICT indicates structural changes in the
microalgal community where less tolerant species decrease and more tolerant species dominate.
Close to the marina, where the concentrations of Irgarol 1051 were highest, a strong increase in
community tolerance (PICT) was found. (Analyses of water samples from the investigated sites
were made by the CID-CSIC group in Spain.) The tolerance observed in summer 2000 was of
the same magnitude as observed 1999, and like 1999, only occurred in late summer. Earlier
studies had failed to find any significant increase in tolerance to Irgarol 1051(Blanck & Dahl,
unpublished data). Tolerance towards PSII-inhibitors such as atrazine and Irgarol 1051 involves
alterations to the conservative D1-protein which seems to require a long time and constant
exposure to develop. The results of this study were presented as a poster at the 2001 SETAC
conference in Madrid and a manuscript is currently under preparation. Efforts are being made to
isolate the altered gene from tolerant algal communities in order to couple the community
response to a tolerance mechanism. The strategy involves PCR-technology on DNA samples
taken from tolerant sites. Hopes are that the Göteborg group will be able to sequence the altered
gene.
Oil harbour and major shipping lane
Field studies on the effects of Irgarol 1051, Sea-Nine and TBT on phytoplankton and bacteria
communities in Sweden’s busiest oil harbour (Brofjorden) and one of the world’s busiest shipping
lanes (Öresund) between Sweden and Denmark, were made in January, 2002. Effects were
estimated using the aforementioned PICT-concept (Blanck, et al. 1988), measuring increased
tolerance to Irgarol 1051, Sea-Nine or TBT on phytoplankton and bacteria communities from
stations in Öresund and just outside Brofjorden. As could be expected from the amount of
commercial traffic in Öresund and Brofjorden, community tolerance (PICT) towards TBT and SeaNine could be found. Communities from one of the sampling sites in Brofjorden also showed
tolerance to Irgarol 1051. This indicates that the present usage of antifouling agents is causing a
selection pressure on algal communities and is thus damaging their structure. Results from this
study will be put together and a manuscript will be prepared during 2002.
39
Marine periphyton microcosms (GU, NERI, CID-CSIC)
A microcosm study on the effects of mixtures of Sea-Nine, TBT and Irgarol 1051, focusing on
marine periphyton communities, was made during summer 2000 in cooperation with the EUfunded project PREDICT. Analytical chemistry was performed by the CSIC and NERI groups.
Evaluation and modelling of the mixture toxicities were made by PREDICT scientists. Results
(Table 13) indicate additive effects for the mixture (when each component is present at
concentrations lower than their individual NOECs). Results from this study will be compiled for
publication in 2002.
Table 13. Results from microcosm experiment with a mixture of Sea-Nine, TBT and Irgarol.
All concentrations are nanomolar.
Value
NOEC
TBT
0.21-0.23
Irgarol
0.07-0.11
Sea-Nine
0.01-0.18
LOEC
0.05
0.08
0.03
Marine plankton mesocosms (GU, DHI Water&Environment (VKI), NERI & CID-CSIC)
A joint mesocosm study focusing on plankton communities was conducted in August 2000.
Irgarol 1051 and Sea-Nine were assessed in parallel experiments. A novel mesocosm design
was used employing submerged plastic enclosures of 150 L each. Two parallel systems of 12
bags each were used for the experiment using triplicate controls. The bags were filled with
seawater, filtered to avoid uneven grazing pressure on the replicated communities. Effects were
measured on species composition, photosynthetic activity, biomass, pigment profiles, induced
community tolerance, bacterial production, bacterial community composition and bacterial
motility. Egg production and hatching rate from a selected zooplankton species (Acartia sp.)
feeding on plankton extracted from the bags were also measured. Water samples for chemical
analyses were taken at regular intervals. All exposure levels given are nominal concentrations
immediately after addition of toxicants at the start of the experiments.
Effects on phytoplankton (GU & NERI)
Results are summarised in Table 14.
Phytoplankton biomass quickly decreased after the addition of Sea-Nine. At low concentrations of
Sea-Nine (0.32-10 nM), biomass quickly recovered (<1d) whilst at higher concentrations,
recovery was slower. For Irgarol 1051, the decrease in biomass was slower than for Sea-Nine (12 d). At low concentrations (0.032-0.1 nM) there was even an initial increase in biomass
(measured as in vitro chlorophyll a concentration).
A shift in species composition (with sensitive species being eliminated and replaced by more
tolerant species) could be seen for both substances. This was confirmed by short-term PICT
toxicity tests which showed increased community tolerance for Irgarol 1051 and Sea-Nine. Sizefractionated PICT (conducted by the NERI partners) to complement the PICT short-term toxicity
tests showed similar patterns in both size-fractions for Sea-Nine. For Irgarol 1051, however, there
were considerable discrepancies between whole community PICT and Size-Fractionated PICT.
Finally, communities subjected to Irgarol 1051 showed a decrease in photosynthetic efficiency
while no such trend could be seen for Sea-Nine. (GU)
40
Table 14. Effects of Irgarol 1051 and Sea-Nine on phytoplankton in pelagic mesocosms.
Exposure levels are nominal nanomolar concentrations.
Endpoint
Measured parameter
NEC
Sea-Nine
NEC
Irgarol
1051
Community structure
Species abundance Day 5
Species abundance Day 7
Pigments (cyanobakteria) Day 7
Pigments (Diatoms) Day 7
Short-term toxicity
5.6
45.4
0.7
0.4
0.1
0.1
0.9
0.2
Short-term toxicity, Sizefractionated, >10 µm
Short-term toxicity, Size-fractionated,
2 –10 µm
In vitro fluorescence
Incorporation of radiolabelled carbon
in short-term test controls
1.0
-
1.6
-
12.2
-
0.02
Pollution Induced Community
Tolerance (PICT)
Biomass (chl a)
Photosynthetic efficiency
Effects on bacterioplankton (DHI Water&Environment (VKI), NERI)
Initially the biomass, activity and motility of the bacteria in the mesocosms were distinctly
reduced. Motility was reduced by up to 70%. However, the bacteria recovered after a few days to
approximately 10-20% lower than the motility in the control. Increased community tolerance of the
bacteria was found at nominal concentrations higher than 3.2 nM SeaNine. In addition, the effects
on the composition and diversity of the bacterial community were evaluated by means of a DGGE
fingerprint technique and the community’s ability to utilise 95 different carbon sources. Indirect
effects of Irgarol 1051 on the bacteria communities have yet to be analysed.
Effects on zooplankton (NERI)
Toxicity of water in the mesocosms spiked with SeaNine was evaluated using bioassays with
Acartia. Egg-production and mortality of Acartia transferred to mesocosm water was followed
over 48 hours in 1-litre vessels. Water from mesocosms with the highest concentrations of
SeaNine had a distinct effect on both egg-production and mortality of the Acartia. The bioassays
indicate a decline in the toxicity of the water with time.
Fates of toxicants (NERI, DHI Water&Environment (VKI), CSIC)
The concentrations of Irgarol 1051 and SeaNine were followed throughout the 12-day mesocosm
experiments to investigate degradation/removal. In parallel, the toxicity of the water was tested in
bioassays with phytoplankton. The half–life of SeaNine was about 1.6 days and results indicate
that the metabolite(s) was not toxic to phytoplankton.
41
Microcosm studies of the effects (and degradation of) SeaNine on natural phytoplankton
communities (DHI Water&Environment (VKI))
The environmental risk of SeaNine is considered by Shade et al. (1993) to be relatively low due to
its very rapid degradation when released from the hulls of ships. But the sparsity of effects data
under realistic conditions, and in response to actual (measured) concentrations of SeaNine,
highlights the need for further investigations about the fate and effects of SeaNine in the
environment. Effects on productivity, biomass and succession of species in phytoplankton
communities have been monitored. A microcosm experiment was performed with single additions
of 0, 3.2, 10, 32 and 100 nM of SeaNine and was monitored over a period of sixteen days.
Increased tolerance for SeaNine was observed in the phytoplankton communities exposed to the
concentrations of 32 and 100 nM SeaNine. The half-life of the compound was estimated to be
about 5 days. The tolerance was maintained for a period of 16 days even though the SeaNine
was degraded during this period. The cause(s) for the persistent tolerance will be discussed in
relation to the degradation of SeaNine and structural changes in the phytoplankton community in
a proposed publication.
Bacterial degradation of Sea-Nine in coastal water: analysed by chemical analysis and
bioassays for toxicity. (DHI Water&Environment (VKI))
SeaNine is marketed as a biocide with effects on a broad spectrum of fouling organisms. It is
claimed that SeaNine is rapidly degrades to harmless derivatives when it is released from ships
hulls. But the analyses of the biodegradation of DCOI (the active component in SeaNine) have
some limitations. They have been based on either chemical analysis, which only determine the
disappearance of the parent compound in natural water or toxicity tests with single species, which
do not examine whether DCOI and/or its metabolites cause the toxicity. Biodegradation of DCOI
was investigated in eutrophic coastal water under realistic conditions for 27 days. Concentrations
of SeaNine were analysed using solid phase extraction and GC-MS. The decline in the toxicity of
the water was evaluated using sub-samples of the experimental waters in bioassays for toxicity
on phytoplankton. The degradation experiment was performed in darkness with GF/C filtered
coastal water at concentrations of 10, 32 and 100 nM DCOI. A half-life of 9 days was calculated
from the experiment. Also, DCOI does not seem to affect the number, activity and diversity of
bacteria.
Endocrine Disruption Investigations (IFREMER)
The estrogenic activity of water and sediment sample extracts has been investigated using the
ER-CALUX bioassay. Tests were performed using a BioDetection System (BDS, The
Netherlands). The ER-CALUX (Estrogen Responsive – Chemically Activated Luciferase
eXpression) assay is used for the determination of the (anti)estrogenicity of pure compounds and
complex environmental mixtures. The assay comprises a genetically modified T47D human
breast cancer cell-line, incorporating the firefly luciferase gene coupled to estrogen responsive
elements as a reporter gene for the presence of estrogenicity. By addition of the appropriate
substrate for luciferase, light is emitted. The amount of light produced is proportional to the
amount of ligand-ER binding, which can be related to estradiol equivalents (EEQs).
Water samples collected from several marinas and coastal areas along the Atlantic and the
Mediterranean coast of France were tested for estrogenic activity using the ER-CALUX assay.
Samples of ca.100 liters were collected from the English Channel (Saint Malo, Saint Quai
Portrieux), the Atlantic coast (Brest and Arcachon) and the Mediterranean coast (Nice, St
Raphael, Marseille and Port Cogolin/Port Grimaud). One sample was taken from the open sea
between Toulon and Corsica. Samples were extracted using XAD-2 resin and were concentrated
42
in iso-octane. Concentrated extracts were diluted in 50 µl DMSO for the ER-CALUX test, and
were subsequently subjected to 10-fold dilutions as appropriate (depending on the cytotoxicity of
the samples). Three replicates were used for each sample. A procedural blank was also
processed and showed no estrogenic activity.
Results for the samples are summarised in Table 15 and Figure 9.
Table 15. Estrogenic activity determined in water samples using the ER-CALUX test.
Results expressed in pmol estradiol equivalents (EEQ) / liter of water sample.
Sample location
English Channel
Atlantic
Mediterranean
Saint Malo
Saint
Quai
Portrieux
Brest
Arcachon
Marseille
Cogolin
/
Grimaud
St Raphael
Nice
Open sea
Salinity
Marina
Marina
34.23
34.35
Sampling date
Estrogen activity
pmol EEQ/liter of
water sample
RSD
%
06/07/2001
7.7 10-2
8.0
05/07/2001
10-1
21.4
10-1
1.5
Marina
Marina
Marina
34.32
30.75
37.91
04/07/2001
03/07/2001
26/08/2000
1.1
1.5 10-1
6.0 10-3
30.0
21.4
25.5
Coastal
38.23
25/08/2000
8.4 10-4
13.8
25/08/2000
24/08/2000
18/08/2000
10-3
9.7
24.3
--
Marina
Marina
Coastal
38.17
32.56
38.26
8.0
7.5 10-3
not detectable
Higher estrogenic activities were detected in samples collected from marinas on the English
Channel and the Atlantic coasts when compared to samples from the Mediterranean. Samples
collected from inside marinas (Marseille, St Raphael and Nice) showed higher activities than
samples collected from outside marinas (Cogolin/Grimaud). No estrogenic activity was recorded
in the sample collected from the open sea.
Sediment sample extracts from selected marinas were also tested using the ER-CALUX assay.
About 20 grams (dry weight) of samples were extracted using accelerated solvent extraction
(ASE) into n-hexane. Tetrabutylamonium sulphite was used to remove sulphur prior to the ERCALUX assay. Results are presented in Table 16 and Figure 9. Estrogenic activity in the
sediment samples varied between undetectable levels to 1.6 10 -1 EEQ.g-1 for most samples.
Activity in the sample collected from Marseille, however, was 5 times higher.
43
Table 16. Estrogenic activity determined in sediment samples using the ER-CALUX test.
Results expressed in pmol estradiol equivalents (EEQ) / gram (dry weight) of sediment sample.
Sample location
English Channel
Atlantic
Mediterranean
Cherbourg
Saint Malo
Brest
La Rochelle
Arcachon
Marseille (39)
Saint Mandrier
St Raphael (26)
Marina
Marina
Marina
marina
Marina
Marina
Marina
Marina
Sampling date
Estrogen activity
pmol EEQ/gram of
sediment sample
RSD
%
05/07/2001
06/07/2001
04/07/2001
02/07/2001
03/07/2001
22/11/2001
19/09/2001
14/11/2001
1.3 10-1
< 2.5 10-3
1.3 10-1
< 7.4 10-4
1.6 10-1
5.3 10-1
9.9 10-2
3.4 10-2
14.8
-7.7
-3.3
2.7
10.0
4.4
6.0E-01
4.0E-01
English
Channel
English
Channel
Atlantic Coast
Atlantic Coast
Not detected
Not detected
Mediterranean Coast
Not detected
2.0E-01
tQ
tM
al
o
(J
ul
y
20
ua
01
i(
)
Ju
ly
20
Br
01
es
)
t(
Ju
Ar
ca
ly
ch
20
01
on
M
)
ar
(J
se
ul
y
ill
20
e
St
01
(A
Ra
ug
)
ph
us
ae
t2
l
0
00
(A
ug
)
us
Ni
t
ce
20
00
(A
)
ug
Co
us
as
t2
ta
l
00
(A
O
0)
ug
pe
n
us
Se
t2
a
00
(A
0)
ug
Ch
u
er
st
bo
20
ur
00
g
)
Sa
(J
ul
in
y
tM
2
00
al
o
1)
(J
ul
y
20
Br
01
es
La
)
t(
Ju
Ro
ly
ch
20
el
01
le
Ar
)
(J
ca
ul
y
ch
20
on
01
)
M
(J
ar
ul
se
y
Sa
20
ill
e
01
in
tM
)
(N
ov
an
.2
dr
ie
00
Sa
r(
1)
in
Se
tR
pt
ap
.
20
ha
01
el
)
(N
ov
.2
00
1)
0.0E+00
Sa
in
-1
SEDIMENT SAMPLES
WATER SAMPLES
Sa
in
-1
pmol EEQ.l or pmol EEQ.g of sample
Mediterranean Coast
Sampling location and dates
Fig. 9. Estrogenic activity in water samples (pmol EEQ.l-1) and sediment samples
(pmol EEQ .g-1) collected in marinas and coastal areas in French coastline.
44
The following scientific papers related to Task 4 are in preparation:
Arrhenius et al., 2002. ”Long-term toxicity of the antifouling agent Sea-Nine on periphyton in marine
microcosms.” Manuscript to be submitted 2002
Blanck et al., 2002. ”Long-term toxicity of mixtures of three antifouling agents on periphyton communities in
marine microcosms.” Manuscript under preparation
Blanck et al., 2002. ”Development of community tolerance to Irgarol 1051 in marine periphyton after years of
coastal water contamination.” Manuscript to submit 2002
Dahllöf et al., 2002 “Degradation and toxicity of Sea-Nine in coastal water.” Manuscript in preparation
Eriksson, M., 2001 “Effects of six antifouling agents (Irgarol 1051, Sea-Nine, chlorothalonil, diuron,
dichlofluanide, zinc pyrithione) on photosynthetic activities in marine phytoplankton communities.” Master
thesis, Göteborg University.
Eriksson, M., 2002 ” Effects of six antifouling agents (Irgarol 1051, Sea-Nine, chlorothalonil, diuron,
dichlofluanide, zinc pyrithione) on photosynthetic activities in marine phytoplankton communities.”
Manuscript to be submitted 2002
Grönvall et al., 2001. ”Development of community tolerance to Irgarol 1051 in marine periphyton after years
of coastal water contamination.” Poster, SETAC Annual meeting Madrid, 2001
Grönvall et al., 2002 ”Effects of Irgarol 1051 and Sea-Nine on phytoplankton in pelagic marine mesocosms.”
Manuscript to be submitted 2002
Gustavsson et al., 2002 “Direct and indirect effects of Irgarol and Sea-Nine on bacterial communities in
marine mesocosms.” Manuscript in preparation
Henriksen et al., 2002 ”Egg production and hatching in Acartia sp feeding on Sea-Nine contaminated marine
phytoplankton.” Manuscript in preparation
Readman et al., 2002 “Flow cytometry and pigment analyses reveal the susceptibility of prymnesiophytes to
a triazine.” Submitted
Wagner, Larsen and Gustavson et al., 2002 “Effects and degradation of Sea-Nine in natural marine
phytoplankton communities.” Submitted
Larsen, Wagner and Gustavson et al., 2002 “Biodegradation of Sea-Nine in coastal water assayed by
bioassays and chemical analysis.” Submitted
45
Task 5: Integrate results and evaluate risks
Sub-task 5.1 – month 34
Title
Update/extend the database (see section 5.2).
Responsible: PML
Partners: IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration: 2 months
Objectives: Additional data generated in this project will be added to the database developed in
sub-task 1.4.
Deliverables: Updated database.
Links:
1.4, 1.6, 3.1, 3.2, 4.1, 4.2, 4.3
Results from the environmental studies have been updated in the database and summary data
generated (see Sub-task 3.1).
Sub-task 5.2 – months 21-39
Title
Use the database and models to predict concentration levels and effects in selected
European coastal zones using different scenario’s for usage.
Responsible: IVM
Partners: PML, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration: 19 months
Objectives: To use the database (sub-task 5.1) (see section 5.2) and models to predict
concentration levels (sub-task 2.2) and effects (sub-task 4.3) in selected European
coastal zones using different scenario’s for usage will be integrated to assess
environmental distributions and potential effects.
Deliverables: Models and contribution to final report.
Links:
2.2, 4.3, 5.1
Modelling of Contamination
A model (MAM-PEC) to predict environmental concentrations of antifouling agents in the marine
environment was validated using the data set collected during this study The Mam-Pec model,
which was developed for the European Paint-makers Association (CEPE-AWG, 1999), is based
on the 2D-grid DELWAQ water quality modelling environment in combination with the SILTHAR
model (Delft Hydraulics, 1995) for estimation of mixing and transport processes. It was selected
because of its ability to cope with the large differences in hydrodynamics and shipping
characteristics amongst marinas across Europe. Following its validation, it can now be applied to
assess future concentrations.
A preliminary figure, based on validation trials during the final ACE workshop is indicated below
(Fig. 10). Based on the main characteristics of a variety of different coastal environments
(including different ports and marinas throughout Europe), good agreement is demonstrated
between the measured and predicted concentrations.
46
Fig. 10. Comparison of measured concentrations of Irgarol 1051 (g/L; average values) in
European coastal environments and predictions based on the Mam-Pec model. Error bars
indicate the min-max range of predictions and measurements.
Publications on the subject (partly coinciding with task 2.2):
In-preparation: manuscript by IVM/WL-Delft, PML, R&H on prediction of environmental
concentrations of antifoulants in European marinas.
Van Hattum, B., A. Baart, J. Boon and J. Readman (2002). Development and application of a
model (Mam-Pec) for exposure assessment of antifouling products in the marine environment.
Env. Sci. Technol. (in prep).
ACE Summary publication.
47
Sub-task 5.3 – month 40
Title
Undertake a critical comparison of the products selected regarding environmental
impact.
Responsible: PML
Partners: IVM, CSIC, UILIC, GU, VKI, NERI, IFREMER
Duration: 2 months
Objectives: To critically compare the potential environmental impact of the products selected.
Deliverables: Assessment concerning booster biocide usage providing guidance to
environmental managers (summary in the Final Report).
Links:
5.1, 5.2
This important aspect of the project was debated carefully and thoroughly at the final workshop.
Field toxicity studies using algal communities indicate that toxicity is likely for some of the
biocides (e.g., Irgarol® 1051) at current environmental concentrations. Determination of the
abiotic and biotic half-lives for these organic antifouling agents examined in laboratory studies
and under controlled field conditions indicated that diruon and Irgarol® 1051 were substantially
resistant to degradation. Discussions at the final workshop, however, considered it inappropriate
to selectively endorse antifouling products. ACE publications, however, have already contributed
to UK Government risk assessments which last year revoked licenses for some of the antifouling
biocides.
48
Annual Workshops
Subsequent meetings/workshops will be organised (in order to plan development of databases,
analytical methodologies, surveys, experiments and models, etc., and to agree the design of the
programme) on an annual basis. All workshops will be co-ordinated by the Project Co-ordinator.
Following completion, results from the development of methods and models and of the
measurements/experiments will be discussed and integrated within these fora.
The Final Workshop to discuss results achieved during the ACE programme was held from 25 –
28 February 2002 in Plymouth, UK. Full details are provided in the Workshop Report (see ACE
Final Management Report).
Initiatives for the dissemination of results
The ACE web site (www.pml.ac.uk/ace) is constantly being updated with the information
generated and includes the database.
All publications (see listing below) fully acknowledge support through the European Commission.
Three general presentations of the programme have been made by the Co-ordinator:
Readman, JW. (2000) Recent developments in antifouling booster biocide research. To be published in
proceedings of the 1st European Conference on Pesticides and Related Organic Micropollutants in
the Environment. (Ioannina, Greece. 5 – 8 October 2000).
Readman, JW. (2000) Poster presentation with an Extended Abstract in the proceedings from the
EurOCEAN Conference. (Hamburg, Germany. 29 August – 2 September 2000)
Readman, JW, van Hattum, B, L’Amoree, M, Barcelo, D. Albanis, TA, Riemann, B, Blanck, H, Gronvall,
F, Gustavson, K, Tronczynski, J, Munschy C. & A. Jacobson (2002), Usage, Contamination and
Effects of Antifouling Booster Biocides in European Coastal Waters. Presentation to the 32nd
International Symposium on the Environment and Analytical Chemistry (ISEAC 32). 17-21 June 2002,
Plymouth, UK.
A summary abstract and publication has been compiled (see ACE Final Management Report).
An ACE Flyer was produced (see ACE Final Management Report).
49
Publications resulting from the ACE Project
Results achieved during ACE have resulted in over 30 peer-reviewed papers being published, inpress or submitted. Others are in preparation. Publications include:
Albanis, T.A., Lambropoulou, D.A., Sakkas, V.A. and Konstantinou, I.K. (2002) Antifouling paint
booster biocide contamination in Greek marine sediments. Chemosphere, in press.
Azevedo, D.A., Lacorte, S., Viana P. and Barcelo, D. (2001) Analysis of priority pesticides and
phenols in Portuguese river water by Liquid Chromatography – Mass Spectrometry.
Chromatographia, 53 (3/4), 113-118.
Barceló, D. (1999) Sample handling and analysis of pesticides and their transformation products
in water matrices by liquid chromatographic techniques. Elsevier Science BV, pp 155-207.
Castillo, M and Barceló, D. (1999) Identification of polar toxicants in industrial wastewaters using
toxicity-based fractionation with liquid chromatography/mass spectrometry.
Analytical
Chemistry, 71 (17), 3769-3776.
Eriksson, M., (2002) Effects of six antifouling agents (Irgarol 1051, Sea-Nine, chlorothalonil,
diuron, dichlofluanid, zinc pyrithione) on photosynthetic activities in marine phytoplankton
communities. Masters thesis, Göteborg University.
Ferrer, I and Barceló, D. (1999) Simultaneous determination of antifouling herbicides in marina
waters by on-line sold-phase extraction followed by liquid chromatography-mass spectrometry.
Journal of Chromatography A, 854, 197-206.
Ferrer, I, Thurman, EM and Barceló, D. (2000) First LC/MS Determination of Cyanazine amide,
Cyanazine Acid, and Cyanazine in Groundwater Samples. Environmental Science and
Technology, 34 (4), 714-718.
Jensen, Gustavson & Petersen. Concentrations of organic pollutants and metals in
sediments in Danish waters. Danish EPA, in press.
Κonstantinou, Ι.Κ., Sakellarides, T.M., Sakkas, V.A. and Albanis, T.A. (2001) Photocatalytic
degradation of selected s-triazine herbicides and organophosphorus insecticides over aqueous
TiO2 suspensions. Environmental Science and Technology, 35, 398-405.
Lampropoulou, D., Konstantinou., I. and Albanis, T. (2000) Determination of fungicides in natural
waters using solid-phase microextraction and Gas Chromatography coupled with Electron
Capture and Mass Spectrometric Detection. Journal of Chromatography, 893, 143-156.
Lambropoulou, D.A., Sakkas, V.A. and Albanis, T.A. (2002) Analysis of the antifouling biocides
Irgarol 1051 and Sea-Nine 211 in environmental water samples using Solid Phase
Microextraction (SPME) and gas chromatography. Journal of Chromatography A, in press.
Lambropoulou, D.A., Sakkas, V.A. and Albanis, T.A. (2002) Headspace solid phase
microextraction for the analysis of new antifouling agents Irgarol 1051 and sea-nine 211 in natural
waters - Application of SPME for the determination of partition coefficients to humic acids. Anal.
Chim. Acta, in press.
Lampropoulou, D., Sakkas, V., Konstantinou, I. and Albanis, T. Antifouling Paint Booster Biocide
contamination of Greek marine sediments. Chemosphere (submitted).
50
Larsen, Wagner and Gustavson et al. Biodegradation of Sea-Nine in coastal waters assayed by
bioassays and chemical analyses. Submitted.
Martinez, K.,and Barcelo, D. (2002) Determination of antifouling pesticides and degradation
products in marine sediments by means of ultrasonic extraction and HPLC-APCI-MS. Fresenius
J. Anal. Chem., (in press).
Martnez, K., Ferrer, I. and Barceló, D. (2000) Part-per-trillion level determination of antifouling
pesticides and their by-products in seawater samples by off-line solid-phase extraction followed
by high-performance liquid chromatography-atmospheric pressure chemical ionization mass
spectrometry. Journal of Chromatography A, 879, 27-37.
Martinez, K., Ferrer, I., Hernando, M.D., Fernandez-Alba, A.R., Marce, R.M., Borrull, F. and
Barcelo, D. (2002) Occurrence of antifouling biocides in the Spanish Mediterranean marine
environment. Environmental Technology, in press.
Penuela, G.A., Ferrer, I. and Barcelo, D. (2000) Identification of new photodegradation
byproducts of the antifouling agent Irgarol in seawater samples. Intern.J. Environ. Anal. Chem.,
78, 25-40
Peñuela, G.A. and Barceló, D. (1999) Comparative photodegradation study of atrazine and
desethylatrazine in water samples containing titanium dioxide/hydrogen peroxide and ferric
chloride/hydrogen peroxide. Journal of AOAC International, 83 (1), 53-60.
Readman, J.W. (2000). Assessment of antifouling agents in coastal environments. In
proceedings of EurOCEAN 2000, the European Conference on Marine Science and Ocean
Technology. (Hamburg, Germany. 29 August – 2 September 2000). European Communities. 466
– 473.
Sakkas, V.A., Konstantinou, I.K. and Albanis, T.A. (2001) Photodegradation study of the
antifouling booster biocide dichlofluanid in aqueous media by gas chromatographic techniques.
Journal of Chromatography A, 930, 135-144.
Sakkas, V.A., Konstantinou, I.K. and Albanis, T.A. (2002) Aquatic Photodegradation of the
Antifouling Booster Biocide Sea-Nine 211. Kinetics and the Influence of Organic Matter. Journal
of Chromatography A, in press.
Sakkas, V.A., Konstantinou, I.K., Lambropoulou, D.A. and Albanis, T.A. Simultaneous
Determination of Antifouling Paint Booster Biocides in Greek Ports and Marinas by Means of
Solid Phase Extraction and Gas Chromatography. Environ. Sci. Pollut. Research, (submitted).
Sakkas, V.A., Lambropoulou, D.A. and Albanis, T.A. (2002) Kinetics of chlorothalonil
photodegradation in natural and humic water. Chemosphere, in press.
Sakkas, V.A., Lambropoulou, D.A. and Albanis, T.A. (2002) Photochemical degradation of Irgarol
1051 in natural waters: influence of humic and fulvic substances on the reaction., J. Photochem
and Photobiol. A: Chem, in press.
Sakkas, V., Lampropoulou, D., Konstantinou, I. and Albanis, T. Simultaneous determination of
Antifouling Paint Booster Biocides in Greek Ports and Marinas by means of solid phase extraction
and gas chromatography. Marine Pollution Bulletin, (submitted).
Steen, R.J.C.A., Jacobson, J., Ariese, F. and Van Hattum, B. (1999). Monitoring Sea-nine 211
Antifouling agent in a Danish Harbor. IVM-E99/10, Institute for Environmental Studies,
Amsterdam.
51
Steen, R., Van der Vaart, J., Hiep, M., Van Hattum, B., Cofino, W. and Brinkman, U. Gross
Fluxes and estuarine behaviour of pesticides in the Scheldt Estuary (1995-1997). Environmental
Chemistry and Toxicology, (submitted).
Steen, R.J.C.A., Van Hattum, B. and Brinkman, U.A.T. (2000) A study on the behaviour of
pesticides and their transformation products in the Scheldt estuary using liquid chromatographyelectrospray tandem mass spectrometry. Journal of Environmental Monitoring, 2, 597-602.
Van Hattum, B., Baart, A.C., Boon, J.G., Steen, R.J.C.A. and Ariese, F. (1999). Computer model
to generate predicted environmental concentrations (PECs) for antifouling products in the marine
environment. IVM-E99/15, Institute for Environmental Studies, Amsterdam.
Wagner, Larsen and Gustavson et al. Effects and degradation of Sea-Nine in natural marine
phytoplankton communities. (submitted).
Publications in preparation:
Arrhenius et al. Long-term toxicity of the antifouling agent Sea-Nine on periphyton in marine microcosms.
Manuscript to be submitted 2002.
Blanck et al. Long-term toxicity of mixtures of three antifouling agents on periphyton communities in marine
microcosms. Manuscript in preparation.
Blanck et al. Development of community tolerance to Irgarol 1051 in marine periphyton after years of
coastal water contamination. Manuscript to be submitted in 2002.
Dahllöf et al. Degradation and toxicity of Sea-Nine in coastal water. Manuscript in preparation.
Eriksson, M. Effects of six antifouling agents (Irgarol 1051, Sea-Nine, chlorothalonil, diuron, dichlofluanid,
zinc pyrithione) on photosynthetic activities in marine phytoplankton communities. Manuscript to be
submitted in 2002.
Grönvall et al. Effects of Irgarol 1051 and Sea-Nine on phytoplankton in pelagic marine mesocosms.
Manuscript to be submitted in 2002.
Gustavson et al. Direct and indirect effects of Irgarol and Sea-Nine on bacterial communities in marine
mesocosms. Manuscript in preparation.
Henriksen et al. Egg production and hatching in Acartia sp feeding on Sea-Nine contaminated marine
phytoplankton. Manuscript in preparation.
Readman et al. Flow cytometry and pigment analyses reveal the susceptibility of prymnesiophytes to a
triazine. To be submitted in 2002.
52
Other points
Concern was expressed by the partners (at the Second Annual Workshop) that within the ACE
time-frame, research on zinc pyrithione would not be achievable owing to the difficulty in the
analysis (LC-MS) and lack of information concerning its mode of action, environmental stability
and potential for transchelation. This compound is marketed in paints produced by International
and Hempel and, whilst currently not used to a great extent, its usage is increasing rapidly.
Negligible data exists on its presence in the environment and potential for ecotoxicological
effects. Whilst attempts were made to study the compound within ACE, a detailed study was not
possible. The same is true for zineb. Following discussions with the Commission, these will
need to be addressed in a future project.
53