cefas contract report me5403 – module 3 - defra, uk
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Cefas contract report ME5403 – Module 3
Project ME5403 - Applied Science to support the
licensing of dredging, disposal, renewables and general
construction and associated monitoring under FEPA,
CPA and the future Marine Act
Module 3 - A marine toxicity assessment of selected
tracer dyes using standard bioassays.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page i
Cefas Document Control
Title: A marine toxicity assessment of selected
tracer dyes using standard bioassays.
Submitted to: Cathal Linnane
Date submitted: 31 March 2011
Project Manager: Sonia Kirby
Report compiled by: Freya Goodsir
Quality control by: Mark Kirby
Approved by & date: Stuart Rogers
29 March 2011
Version: 2
Version Control History
Author Date Comment Version
Freya Goodsir 03/03/11 Draft 1
Freya Goodsir 28/03/11 Final 2
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page ii
Project ME5403 – Applied Science to support the
licensing of dredging, disposal, renewables and
general construction and associated monitoring
under FEPA, CPA and the future Marine Act
Module 3 - A marine toxicity assessment of selected tracer dyes
using standard bioassays.
Authors: Freya Goodsir, Mark Kirby, Tom Fisher, Stefan White and
Andy Smith
Issue date: 31/03/2011
Head office
Centre for Environment, Fisheries & Aquaculture Science
Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK
Tel +44 (0) 1502 56 2244 Fax +44 (0) 1502 51 3865
www.cefas.co.uk
Cefas is an executive agency of Defra
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page iii
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page iv
Executive Summary
The aim of this investigation was to develop a toxicity-based ranked list of the most
commonly used fluorescent dyes, with possible applications as marine tracers, as
underpinning evidence/support to the marine consents approval process under the Food and
Environment Protection Act (FEPA). Twelve tracer dyes were selected and prioritised using
literature and other available data sources. Toxicity tests on the dyes were carried out using
two standard bioassays using the copepod acute toxicity test (Tisbe battagliai 48 hr LC50)
and the oyster embryo development bioassay (Crassostrea gigas 24 hr EC50).
Median lethal toxicity (EC50) values were generated for twelve tracer dyes for T.battagliai
and 10 tracer dyes for C.gigas. Results showed a broad range of EC50 values across the
tracer dyes for both species. EC50 values ranged from 0.0008 to 485 mg/L for oyster and
from 0.1 to 2700 mg/L for Tisbe.
The results allowed the ranking of tracer dyes in terms of their toxicity to the two standard
test species. The three most toxic tracer dyes were, Rhodamine 6G, Rhodamine B and
Lissamine and the least toxic were 8-Hydroxypyrene-1,3,6-trisulfonic acid (Pyranine),
Rhodamine WT, Sulforhodamine G, Erioglaucine, Fluorescein. This ranked list, and
supplementary data from the literature, will enable regulatory assessors to determine which
tracer dyes pose the highest risk to the aquatic environment more comprehensively and,
consequently, strengthen the regulatory advice they provide. Furthermore the data will
inform the agreement of licence exemption criteria for tracer dye applications in the marine
environment.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page v
Table of contents
1 Introduction ................................................................................................................................... 1
2 Materials ......................................................................................................................................... 4
2.1 Chemicals and Reagents .................................................................................................... 4
3 Methods .......................................................................................................................................... 7
3.1 Preparation of tracer dyes ................................................................................................... 7
3.2 Bioassays .............................................................................................................................. 7
3.4 Statistical Analysis ............................................................................................................... 9
4 Results and Discussions .............................................................................................................. 9
5 Conclusions ................................................................................................................................. 13
6 References ................................................................................................................................... 15
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 1
1 Introduction
Materials and substances can be released into the marine environment to allow scientists
and engineers to directly track the movement of water and water transported media.
Collectively these materials are known as tracers and can vary from inert particles and
soluble fluorescent dyes to radioactive/biocidal substances and bacterial/microbial cells.
Their deployment allows for the investigation of water and sediment movement and transport
including issues such as dispersion, dilution, stratification and mixing. They are used in a
wide range of marine based projects including construction/engineering, dredging,
flood/coastal defence, leak detection and water quality studies and can be critical in
providing information to minimise the risk of, for example, sifting of marine outfalls and
disposal areas.
An earlier document was produced as part of the wider project entitled, ‘Marine Tracers: A
basic ecotoxicological review’. This review confirmed that, in general, comprehensive
information regarding the toxicity of tracers, and especially with relevance to the marine
environment (i.e. ecotoxicological data on marine species), is currently lacking. There is
even less information with regards to persistence and bioaccumulation.
With respect to specific tracer types the marine environment should usually be a hostile
environment for the microbial tracers that are used; therefore they should not live long out of
the tracing material that they are associated with (usually sewage). The dilution factors will
also mean the microbes are likely be at concentrations below levels of risk to marine
organisms.
Sediment mimicking and floating particle tracers are usually designed from reasonably inert
materials. They generally only offer a physical (rather than chemically) threat to marine
organisms where in extreme circumstances areas of habitat could potentially be smothered
or altered by tracer material. Therefore it is generally accepted that both microbial and
particle tracers pose little risk to organisms in the marine environment and should therefore
be generally safe for use in the marine environment under typical applications.
Radioactive and specific chemical tracers do have the potential to cause harm in the marine
environment. However, their use in tracer applications where they are purposefully added to
the marine environment (and therefore may require a FEPA assessment) is relatively rare.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 2
These types of applications are therefore best assessed on a strictly case by case basis
referring to expert judgement as necessary.
Fluorescent dyes are the most commonly used in marine tracing studies (accounting for
approximately 50% of all tracer applications dealt with by Cefas’ regulatory assessment
team between 2000-2009) and therefore have the most data available for them.
Unfortunately much of the historic data was not produced for marine species, from species
with particular economic value or from species that represent specific ecological importance
to the marine ecosystem. There is evidence to suggest that some of these chemicals can
have a degree of toxicity and that they represent a range of potential hazard (range of
toxicity). Chemical material safety data sheet (MSDS) information on dye chemicals often
lack ecotoxicity data and, if it is available, it rarely includes aquatic data and especially not
marine data.
The aim of this study was to identify gaps in knowledge and to consider how these could be
filled with the aim of strengthening and underpinning the advice that regulators provide in the
assessment of tracer applications. A clear finding of the previous review was that there was
a lack of well defined and consistent hazard data for the most widely used tracer category –
Dyes. Although often considered of low toxicity the review also established that data for dyes
suggests a wide range of toxicity levels reported. Furthermore, data was not routinely
available for marine species and there was very little use of consistent species that allowed
confident comparisons of hazard between the different dye types.
In line with the ‘generation of supplementary data’ as set out in the original project proposal it
was therefore recommended that a toxicity dataset was generated for a range of dyes
potentially used as marine tracers for the following reasons:
• Chemical dyes have a level of toxicity and potential hazard in the marine
environment. The current lack of consistently generated marine toxicity data
makes the assessment of their hazard difficult.
• A number of dyes could be applicable for certain tracer applications in the marine
environment. The generation of data that allows hazard comparability will also
allow Cefas scientists to provide sound advice on substitution issues (i.e.
suggesting the use of less hazardous products).
• The generation of a standard set of toxicity data will also allow the easy and
comparable assessment of new products as they emerge.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 3
In order to address the generation of the required toxicity data a work programme of testing
has now been conducted on 12 selected dyes. Toxicity testing has been conducted using 2
standard methods:
• Copepod acute toxicity (Tisbe battagliai 48 hr LC50) (ISO, 1999)
• Oyster embryo development (Crassostrea gigas 24 hr EC50) (Thain, 1991)
These selections of tests were considered appropriate because:
• Cefas have extensive experience in their conduct and have full accreditation
(under MCerts and GLP) and relevant Quality Assurance checks in place.
• The methods are internationally accepted and are used in hazard assessment for
a number of statutory schemes.
• They are representative of the main categories of commercial groups that would
be under threat from tracer use (e.g. Crustacea and Mollusca).
The aim of this study was to generate a ranked list of tracer dyes based on their toxicity in
these two standard tests and literature data where appropriate/available. A ranked list will
allow those tracer dyes posing the greatest hazard in the marine environment to be
identified. This information, in conjunction with the contextual use of the dye (i.e. volumes to
be released, hydrodynamic character of the area, proximity of sensitive resources and
environments etc.), will enable regulatory assessors to assess uses and potential risk with
greater confidence. Furthermore, where dye types are interchangeable in terms of their use
the information will also provide potential substitutes for consideration. Ultimately the toxicity
information generated in this project will inform decisions pertaining to licence exemption
criteria for tracer dye applications.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 4
2 Materials
2.1 Chemicals and Reagents
A literature review as described above (Marine Tracers: A basic ecotoxicological review) was
carried out to collate information on the most common types of tracers currently used in the
marine environment that require regulation under the Food and Environment Protection Act
(FEPA 1985) . The aim of the review was to highlight gaps in knowledge that could be filled
to underpin the advisory process supplied by Cefas. The greatest proportion of tracer type
applications that required assessment by Cefas from 2000 to 2009 fell into the category of
‘tracer dyes’ (Figure 1), Further to this, the breakdown of tracer applications supplied by the
Regulatory Advisory Team were used to highlight most important tracer dyes currently
applied for use (Figure 2). The tracer dyes specified in figure 2 represent numbers of
applications, it is not clear whether these tracers were all used in the marine environment
during this time. Both sources of information were taken into account when selecting tracer
dyes for the generation of toxicity data.
Twelve fluorescent tracer dyes were chosen based on ease of availability and common use
on which to carry out toxicity bioassays using two key marine species: the marine
crustacean species Tisbe battagliai and the Pacific Oyster Crassostera gigas. The most
common tracer dyes were selected to be tested first, these included the Xanthene group
(Rhodamine B, Rhodamine WT, Rhodamine 6G, and Sulforhodamine B), Lissamine, 8-
Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (Pyranine), Fluorescein, Fluorescent
Brightener 28, and Eosin Y. These dyes were also selected following reference to
information from the Cefas regulatory assessment team regarding previously assessed
tracer dye applications under FEPA and appropriate information from the scientific literature
(Flury and Wai 2003). A further three fluorescent tracer dyes were selected due to their
easy availability. All but one of these Tracer dyes were purchased from Sigma-Aldrich Co as
powders with varying degrees of purity. Rhodamine WT was purchased from Tolbest Ltd
(Warrington, UK) as a 20% aqueous solution (Tolbest Ltd, personal communication). Where
possible the tracer dyes of the highest purity available were used to carry out the tests
(Table 1). Test concentrations and the subsequently calculated toxicity (EC50’s) values were
based on the actual dye content (Table 2, Figure 5).
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 5
Figure 1: Chart showing proportion of tracers licensed by the Cefas Regulatory Advisory Team from 2000-2009.
Figure 2: Chart showing the percentage breakdown by type of a total of 183 applications of tracer dyes from 2000-2009
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 6
Table 1: Selected priority tracer dyes and their basic physical and visual properties
Tracer name Synonym CAS number Solubility Product Info Appearance Use
Rhodamine B Basic Violet 10, Brilliant Pink B, Rhodamine O, Tetraethylrhodamine
81-88-9 Clear/good Dye content ~ 95% (Sigma)
Green powder/Red purple solution
To trace rates, and transportation within water
Rhodamine WT As commercial name Not Available Clear/good 20% solution www.tolbest.co.uk
Deep purple solution / Purple to red
To trace rates, and transportation within water
Rhodamine 6G Basic Red 1 989-38-8 Clear/good Dye content ~95% (Sigma)
Brown Red powder /Dark Red solution
Stain, Dye ,Indicator and probes
Eosin Yellow 2′,4′,5′,7′-Tetrabromofluorescein, Acid Red 87, Bromo acid J. TS, XL, or XX
15086-94-9 Clear to very Hazy at higher concentrations
~99% (sigma-Aldrich)
Pinky orange powder / Orange solution
Stain or Dye
Fluorescein Acid Yellow 73 2321-07-5 Clear to opaque (higher concentrations less soluble)
Dye content 95 % (Aldrich)
Maroon Powder/ green brown solution
To trace rates, and transportation within water
8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt
HPTS, Pyranine, Solvent Green 7, Trisodium 8-hydroxypyrene-1,3,6-trisulfonate
6358-69-6 Clear/good ≥97% (Aldrich) Bright yellow Green powder / Bright Green
Stain or Dye
Sulforhodamine B Acid Red 52, Sulforhodamine B monosodium salt
3520-42-1 Clear/good Dye content 75% (Aldrich)
Brown Purple powder / Red to orange solution
Stain or Dye
Sulforhodamine G Acid Red 50 5873-16-5 Clear to very Hazy at higher concentrations
Dye content 60% (Aldrich)
Red Maroon powder / Red to Maroon Solution
Stain or Dye
Fluorescent Brightener 28
Calcofluor White M2R, Tinopal UNPA-GX
4404-43-7 Clear to hazy at higher
Not Available Yellow powder/ pale yellow to clear solution
Stain or Dye
Lissamine Acid Green 50, Wool Green S 3087-16-19 Clear/good Dye content 60% (Aldrich)
Purple powder/ Blue Solution
Stain or Dye
Erioglaucine Acid Blue 9, Alphazurine FG 2650-18-2 Clear/good Dye Content 65% (Aldrich)
Purple powder/ Blue Solution
Stain or Dye
Erythrosin B 2′,4′,5′,7′-Tetraiodofluorescein disodium salt, Acid
15905-32-5 Clear/good Dye Content ≥95% (Aldrich)
Orange pink powder/ Red to pinky orange solution
Stain or Dye
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 7
3 Methods
3.1 Preparation of tracer dyes
Stock solutions of test dyes were prepared using filtered aerated seawater (0.2 µm). Serial
dilutions were then prepared from the main stock to produce the test concentration range.
Initial tests, or range finders, were carried out on both Tisbe battagliai and Crassosterea
gigas. The range finder tests covered a concentration range of 0.001 to 1000 mg/L, where
needed this was increased to 4000mg/L for tracer dye 8-Hydroxypyrene-1,3,6-trisulfonic acid
trisodium salt (Pyranine). The observations allowed for a narrower range to be achieved for
more focussed definitive studies. Definitive studies results are outlined in section 4.
3.2 Bioassays
Tisbe battagliai
The T.battagliai bioassay followed the guidelines in ISO (1999) ISO 14669 Water Quality -
Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea), with no
further modifications. These bioassays were carried out in 12 well polystyrene plates, each
well contained 5mls of test solution (Figure 3) and 5 T.battagliai copepodites (approximately
4-6 days old). (Figure 4a) There were 4 replicates per concentration and the test was carried
out over a period of 48 hours. The Tisbe were observed using a binocular microscope and
mortality recorded.
Crassostrea gigas
The oyster embryo bioassay (OEB) followed the method outlined in Thain JE, “Biological
effects of contaminants: Oyster (Crassostrea gigas) embryo bioassay, Proceedings,
Techniques in Marine Environmental Sciences”, International Council for the Exploration of
the Sea, 11, pp. 1-12, 1991, with no further modifications. These bioassays were carried out
in 12 well polystyrene plates, each well contained 4mls of tracer test solution and
approximately 50 oyster embryo per ml (Figure 3). The oyster embryos were observed using
a high powered binocular microscope and normal and abnormal embryos were scored.
(Figure 4b)
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 8
Figure 3: 12 well polystyrene plates containing fluorescent tracer dyes
Figure 4a: T.battagliai viewed under microscope
Figure 4b: C. gigas normal D shells viewed under microscope
3.3 Quality assurance
Water quality checks for temperature, salinity, dissolved oxygen and pH were carried out at
the start and the end of each test to ensure that physical parameters remained within the
acceptable range, as set out in the test protocols, for each bioassay. All parameters were
within acceptable limits, e.g. temperature (24°C ± 2°C), salinity (32 ± 2 ‰), dissolved oxygen
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 9
(≥80%) and pH (7.8-8.2) for C.gigas and temperature (21°C ± 3°C), salinity (32 ± 2 ‰),
dissolved oxygen (≥80%) and pH (7.8-8.2) for T.battagliai.
A 48 h Zinc reference study was carried for quality assurance purposes alongside the tracer
dyes to test the sensitivity of the species population for validity of the study. This test uses
several concentrations of Zinc sulphate 0 (control), 0.1, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
for , T.battagliai and 0 (control), 0.022, 0.046, 0.1, 0.22, 0.46, 1.0 and 2.0 mg/L for C.gigas.
A test is considered valid if the mortality ranges fall with the expected limits, i.e no more than
10% mortality for T.battagliai and no more than 40% abnormal embryos for C.gigas.
3.4 Statistical Analysis
EC50 values (50% effect) and their confidence limits were calculated for Tisbe mortality and
oyster embryo development using Toxcalc statistical programme version 5 (Tidepool
Scientific Software version 5, USA). The results were tabulated and are outlined in section 4
of this report.
4 Results and Discussions
Results
All T.battagliai bioassays were successful in the determination of a definitive EC50 value as
outlined in table 2. The 12 selected tracer dyes exhibited a broad range of EC50 values
ranging from 0.1mg/l for Rhodamine 6G, being the most toxic, to 2703.1mg/l for 8-
Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, being the least toxic (Table 2), (Figure
5).
EC50 values for the oyster embryo bioassay (OEB) were obtained for 10 of the test tracer
dyes as outlined in table 2. The results for Eosin Y, Fluorescein, Rhodamine WT and
Sulforhodamine B were determined from definitive tests (with a more defined range etc),
whereas the results for other 6 tracers were determined only from the sighting shot test with
a broader range of test concentrations (denoted by an asterisk in table 2). Full definitive tests
were not finalised for all the dyes in the OEB tests, due to the unavailability of high quality
oysters during all of the testing phase, but the sighting shot results allowed their toxicity to be
ranked appropriately for the purposes of this study.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 10
Overall the OEB was a more sensitive test, producing EC50 values that were routinely
substantially lower than that of T.battagliai, apart from tracer Rhodamine B and Lissamine.
(Table 2) Results revealed the most toxic dye in both tests, with LC50 values below 1mg/l,
was Rhodamine 6G. The least toxic was tracer 8-Hydroxypyrene-1,3,6-trisulfonic acid
(Pyranine), again reflected in both test species. (Figure 5)
The T.battagliai assay results demonstrate that Rhodamine 6G, Rhodamine B and
Lissamine are the three most toxic tracers out of the 12 selected for these test. This is
consistent with results from previous studies, which indicated that Rhodamine B and
Rhodamine 6G are toxic and, in addition, may have potential carcinogenetic and genotoxic
properties (Material safety data sheets, Sigma-Aldrich Ltd), Furthermore, Behrens et al
(2001) recommended against the use of these dyes in aquatic environments. For C. gigas
the three most toxic tracers dyes are shown to be Rhodamine 6G, Eosin Y and Fluorescent
brightener 28.
The 5 dyes ranked the least toxic of the 12 were Fluorescein, Erioglaucine, Sulforhodamine
G, Rhodamine WT and 8-Hydroxypyrene-1,3,6-trisulfonic acid (Pryanine) (Table 2). Even
though data are not available for C.gigas for Erioglaucine, the results show that these dyes
are ranked similarly with both tests, even though the OEB EC50 values were consistently
lower. (Figure 5)
Behrens et al (2001) indicated that certain tracer dyes are toxicologically safe and could be
used in the marine environment, these included Uranine (Fluorescein), Sulforhodamine G,
Eosine, Pryanine and Tinopal CBS-x Tinopal ABP. Several of these dyes, including
Fluorescein, Sulforhodamine G and Pyranine are included in the above least toxic tracers
demonstrated by our results.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 11
Table 2: Toxicity values and rankings for a range of tracer dyes. EC50 values and 95% confidence limits are provided for the T.
battagliai acute bioassay and the C. gigas oyster embryo bioassay.
Tisbe battagliai Crassostrea gigas
Tracer Dye Rank EC50 mg/L 95% Confidence limits Rank EC50 mg/L 95% Confidence limits
Rhodamine 6G 1 0.1 0.1-0.2 1 *0.0008 0.0005-0.0382
Rhodamine B 2 1.9 1.4-2.7 6 *7.5 4.4-46.5
Lissamine 3 2.0 1.4-2.8 5 *4.7 2.8-16.2
Erythrosin B 4 14.6 11.6-18.4 − − −
Eosin Y 5 28.3 16.9-41.5 2 0.31 0.04-0.42
Sulphordamine B 6 32.8 22.0-49.1 4 2.30 2.0-3.0
Fluorescent Brightener 28
7 63.75 47.17-86.04 3 *0.92 0.08-3.39
Erioglaucine 9 145.1 115.8-181.8 − − −
Fluorescein 8 160.7 128.6-200.7 7 28.00 12.0-34.5
Sulforhodamine G 10 171.4 123.8-240.7 8 *44.7 0-138.1
Rhodamine WT 11 422.3 282.6-631.1 9 118.20 98.2-131.7
8-Hydroxypyrene-1,3,6-trisulfonic acid
12 2703.1 2579.6-832.5 10 *485.37 264.6-562.4
*Data generated using range finder results and not true definitives − No test carried out for these tracers
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 12
Figure 5: Chart showing a comparison of toxicity (EC50 values) for a range of tracer dyes
using the Tisbe battagliai and Crassostrea gigas (OEB) bioassays.
This study has enabled the toxicity of all the main tracer dyes likely to be used in UK waters
to be comparatively assessed using bioassays with two important marine species. These
species are surrogates of the primary commercially exploited species, crustaceans and
molluscs, and as such provide an important indication of the potential hazard of each dye if
released into the marine environment. It is anticipated that the toxicity ranking in table 2 will
be incorporated into updated tracer application assessment protocols to strengthen decision
making and will enable assessors to suggest more environmentally acceptable alternatives
for some marine applications. The data will also be used in a review of exemptions for tracer
usage under the new marine licensing regime. Finally, the approach used here, comprising a
dual toxicity test approach, will be suggested as a means by which future applications for
new, or unknown, dye usages in the marine environment can be assessed and compared to
existing data.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 13
5 Conclusions
Fluorescent dyes are the most commonly used in marine tracing studies and therefore have
the most environmental data available for them. However, there is very limited historic
toxicity data available for relevant marine species, i.e. from species that represent a specific
ecological importance/value to the marine ecosystem. The aim of this study was to produce
a ranked list of tracer dyes based on their toxicity using two standard tests and literature
data where available.
The main findings/conclusions of the study were:
• The most toxic tracer dye overall was Rhodamine 6G, with EC50 values below 1mg/L
for both test species. The least toxic was tracer dye 8-Hydroxypyrene-1,3,6-trisulfonic
acid (Pyranine) again reflected in both test species.
• Previous studies have also indicated that Rhodamine B and Rhodamine 6G tracer
dyes are toxic with potential carcinogenetic and genotoxic properties and are
therefore not recommended for the use in aquatic environments (Behrens et al,
2001). Our Results demonstrated EC50 values under 10mg/L for both these dyes
along with Lissamine for both test species. This shows that these tracer dyes are
potentially highly toxic to the marine environment.
• Previous studies have also indicated that several tracer dyes are toxicologically safe
and could potentially be used in the marine environment, these include Uranine
(Fluorescein), Sulforhodamine G, Eosine, Pyranine and Tinopal CBS-x Tinopal ABP
as outlined in , ‘Marine Tracers: A basic ecotoxicological review’. Our results showed
lower levels of toxicity for Fluorescein, Sulforhodamine G and (8-Hydroxypyrene-
1,3,6-trisulfonic acid) Pryanine for both test species, however, it is difficult to rule out
whether these are considered to be safe for the marine environment.
• Overall C.gigas was a more sensitive test species, showing considerably lower EC50
values than that of T. battagliai for most dyes apart from tracer Rhodamine B and
Lissamine. It may be advisable therefore to consider these lower EC50 values when
considering a threshold and exemption limits.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 14
• It is unknown whether any tracer dye is completely safe that can be used for
applications in the marine environment, and that toxicology of the tracer dyes may
change considerably with species, however future research may provide solutions for
safer alternatives.
A marine toxicity assessment of selected tracer dyes using standard bioassays. Page 15
6 References
Behrens H, Beims U, Dieter H, Dietze G, Eikmann T, Grummt, T., Hanisch, H.,Henseling, H.,
Käß, W., Kerndorff, H., Leibundgut, C., Müller-Wagner, U., Rönnefahrt, I., Scharenberg, B.,
Schleyer, R., Schloz, W and Tilkes ,F. 2001. Toxicological and ecotoxicological assessment
of water tracers. Hydrogeology Journal 9:321-325
Flury, M., and N. N. Wai (2003), Dyes as tracers for vadose zone hydrology, Rev. Geophys.,
41(1), 1002, doi:10.1029/2001RG000109.
ISO, 1999. ISO 14669:1999(E) Water quality -- Determination of acute lethal toxicity to
marine copepods (Copepoda, Crustacea)
Marine Tracers: A Basic Ecotoxicological Review. Stefan White, Mark Kirby, Kins Leonard
and Chris Vivian (July, 2010)
SCA (2007). The Direct Toxicity Assessment of Aqueous environmental samples using the
marine copepod Tisbe battagliai’ (2007).
SCA (2007). The Direct Toxicity Assessment of Aqueous environmental samples using the
oyster (Crassotrea gigas) embryo-larval development test’.
SOP 1579 (2010). A 24 hr Oyster embryo-larval development test using the species
Crassostrea gigas.
SOP 1575 (2010). A 48 hr acute assay using early stage marine harpacticoid copepods of
the species Tisbe battagliai.
Thain JE, “Biological effects of contaminants: Oyster (Crassostrea gigas) embryo bioassay,
Proceedings, Techniques in Marine Environmental Sciences”, International Council for the
Exploration of the Sea, 11, pp. 1-12, 1991.
http://www.ices.dk/pubs/times/times11/TIMES11.pdf
© Crown copyright 2010
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Tel +44 (0) 1502 56 2244
Fax +44 (0) 1502 51 3865
Web www.cefas.co.uk
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