cefas contract report me5403 – module 3 - defra, uk

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.


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.


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.


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).


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


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


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


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


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)


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


(≥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.


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.


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


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.


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.


• 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.


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

About us Cefas is a multi-disciplinary scientific research and

consultancy centre providing a comprehensive range

of services in fisheries management, environmental

monitoring and assessment, and aquaculture to a large

number of clients worldwide.

We have more than 500 staff based in 2 laboratories,

our own ocean-going research vessel, and over 100 years

of fisheries experience.

We have a long and successful track record in

delivering high-quality services to clients in a confidential

and impartial manner.

(www.cefas.co.uk)

Cefas Technology Limited (CTL) is a wholly owned

subsidiary of Cefas specialising in the application of Cefas

technology to specific customer needs in a cost-effective

and focussed manner.

CTL systems and services are developed by teams that

are experienced in fisheries, environmental management

and aquaculture, and in working closely with clients to

ensure that their needs are fully met.

(www.cefastechnology.co.uk)

Customer focus With our unique facilities and our breadth of expertise in

environmental and fisheries management, we can rapidly put

together a multi-disciplinary team of experienced specialists,

fully supported by our comprehensive in-house resources.

Our existing customers are drawn from a broad spectrum

with wide ranging interests. Clients include:

• international and UK government departments

• the European Commission

• the World Bank

• Food and Agriculture Organisation of the United Nations

(FAO)

• oil, water, chemical, pharmaceutical, agro-chemical,

aggregate and marine industries

• non-governmental and environmental organisations

• regulators and enforcement agencies

• local authorities and other public bodies

We also work successfully in partnership with other

organisations, operate in international consortia and have

several joint ventures commercialising our intellectual

property

.

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

Web www.cefas.co.uk

printed on paper made from a minimum 75% de-inked post-consumer waste

cefas contract report me5403 – module 3 - defra, uk

Documents