www.elsevier.com/locate/marpolbul

Marine Pollution Bulletin 52 (2006) 1661–1667

A practical ranking system to compare toxicity of anti-fouling paints

Jenny Karlsson *, Magnus Breitholtz, Britta Eklund

ITM, Department of Applied Environmental Science, Stockholm University, SE-106 91 Stockholm, Sweden

Abstract

The toxicity of a number of new anti-fouling paints, claimed to function by physical means and not by leakage of toxic substances,have been tested on two common organisms in the Baltic Sea, i.e., the red macro alga Ceramium tenuicorne and the copepod Nitocra

spinipes. In order to compare the toxicity between the paints a ranking system was developed based on the EC50- and LC50-values.The results showed a wide span in toxicity with the most toxic paints ranked 160 times more toxic than the ones ranked least toxic.

Also, TBT, irgarol and diuron, which have been used as active ingredients in traditional anti-fouling paints, were used to evaluate thesensitivity of the two test organisms. The results showed that the test organisms were equally sensitive to the substances as similar organ-isms in earlier studies.

In conclusion, the ranking system presented in this study permits ranking and comparison of total toxicity of complex mixtures.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Anti-fouling; Nitocra; Ceramium; Irgarol; Diuron; TBT

1. Introduction

In Environmental Risk Assessment (ERA) possibleinteractions between millions of species and tens of thou-sands of existing and new chemicals have to be considered.For economical and practical reasons, the hazard identifi-cation and dose–response assessment of ERA is based ona few functional tests and species to estimate effect con-centrations (European Commission, 2003a). The imple-mentation of REACH (Registration, Evaluation andAuthorisation of Chemicals), the new proposed Europeanchemicals legislation, will result in a number of changesin the ERA process in Europe. The most significant ofthese is that a large number of previously untested chemicalsubstances will undergo testing and risk assessment (Euro-pean Commission, 2003b). To fulfil a scientifically justifiedand resource efficient process for testing and risk assess-ment, the availability of reliable and ecologically relevant

0025-326X/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.marpolbul.2006.06.007

* Corresponding author. Tel.: +46 8 674 7279; fax: +46 8 674 7636.E-mail address: jenny.karlsson@itm.su.se (J. Karlsson).

test methods and testing strategies is also needed (Bre-itholtz et al., 2006). For individual chemical substances,REACH (and perhaps other similar legislative attempts),may begin to provide information for decisions makingwith the purpose to adequately protect the environmentfrom unwanted effects of chemicals. However, in the cur-rent approval process, only single substances are consid-ered and not the product as a whole.

Anti-fouling paints are complex mixtures that tradition-ally contain toxic substances, which during the last 30 yearshave caused increasing attention as they have shown tocause adverse effects in non-target organisms (Alzieuet al., 1986; Bryan et al., 1986; Konstantinou and Albanis,2004). Common biocides used in anti-fouling paints aree.g., tributyltin (TBT) and copper. Due to undesirableeffects in e.g., oysters (decline in populations, shell malfor-mations) and gastropods (imposex) the use of TBT inpaints is now restricted (Alzieu et al., 1986; Bryan et al.,1986). Different copper compounds mainly replaced TBTas the active component in anti-fouling products (Dahland Blanck, 1996). However, several algal species showedtolerance to copper and to achieve protection against these

1662 J. Karlsson et al. / Marine Pollution Bulletin 52 (2006) 1661–1667

tolerant species, a number of so-called booster biocides,e.g., zinc pyrithione, zineb, irgarol and diuron, were intro-duced together with copper to enhance prevention of foul-ing (Voulvoulis et al., 2002).

The two most commonly used booster biocides are irga-rol 1051 and diuron (Konstantinou and Albanis, 2004).Consequently, elevated concentrations of both irgaroland diuron have been detected all over the world in areaswith intense boat traffic, particularly in marinas and har-bours (Dahl and Blanck, 1996; Voulvoulis et al., 2000;Haglund et al., 2001; Thomas et al., 2001, 2002; Okamuraet al., 2003). Also, laboratory studies with irgarol and diu-ron have shown negative effects on aquatic organisms atvery low concentrations (Okamura et al., 2000a; Fernan-dez-Alba et al., 2002). Because of the high concentrations incoastal waters and negative impact on the aquatic life somecountries, e.g., Sweden, UK and Denmark, have bannedthe use of these two anti-fouling agents (www.kemi.se).

One heavily debated topic in Sweden and in other coun-tries around the Baltic Sea in recent years is the use or non-use of copper and other biocides in anti-fouling paints. TheBaltic Sea is one of the most polluted seas, exposed tomany types of pollutants from all nine surrounding coun-tries. Its young age, low salinity and low biodiversity forma very unique environment. The organisms origin eitherfrom fresh or marine water and are still adapting to theprevailing salinity (Ryden et al., 2003). This makes themexceptionally sensitive to additional stress factors, as theyalready live near their tolerance limit (Kautsky and Teden-gren, 1992). As a consequence the Baltic Sea was classifiedas a Particularly Sensitive Sea Area (PSSA) in 2004 byInternational Maritime Organisation (IMO).

The Swedish authorities banned pleasure boat paintscontaining copper and irgarol 1051 for use on the eastcoast from the 1st of January 2001 (Swedish ChemicalsInspectorate, 1998). Copper and irgarol have shown to bevery toxic to aquatic organisms and is especially a threatagainst the bladderwrack (Fucus vesiculosus), which is akey species in the Baltic Sea (Andersson and Kautsky,1996; Andersson, 1996). The intention of the authoritieswas to direct the protection of hulls to physical meansinstead of using paints based on leakage of toxic chemicals.As a result of the prohibition, a number of new paints haveentered the market. These paints are claimed to be friend-lier to the environment as they do not contain any biocidesand function by physical means. However, a recent studyshows that some of these paints are more or just as toxicas those that have been banned (Karlsson and Eklund,2004).

The main objective of the present study was to use estab-lished test methods with ecologically relevant test organ-isms, i.e., the red macro alga Ceramium tenuicorne andthe copepod Nitocra spinipes, to obtain a practical ranking

system to compare the toxicity of anti-fouling paints. Theaim was in particular to investigate the toxicity of anti-foul-ing paints in low salinity where the organism are morestressed than in marine environment, e.g., the Baltic Sea

and estuaries. A secondary objective was to evaluate thesensitivity of the chosen test methods by exposing C. tenui-

corne and N. spinipes to three common ingredients used inanti-fouling paints, i.e., TBT, irgarol 1051 and diuron.

2. Materials and methods

In this study both leakage waters from different anti-fouling paints as well as single substances commonly usedin anti-fouling paints have been tested.

2.1. Anti-fouling paints (complex mixtures/products)

Six anti-fouling paints available on the Swedish markethave been tested for their toxicity. Two of the tested paintswere polishing paints: Lefant SPF (Lotrec AB) and CruiserEco (International Farg AB). One was based on teflone:Vc17mEco (International Farg AB). Two paints, SafeBoat-Skin (Sailway) and Aurora VS721 (Aurora Marine Indus-tries Inc.) were so-called polymer waxes. According tothe producers, all of the paints function by physical means,do not contain any biocides and are recommended for usein the Baltic Sea. Additionally, the paint EcoMar2000(Thulica AB) containing pepper extract, thus working bychemical means, was included in the study. The SwedishChemicals Inspectorate has approved this paint for useon the east coast of Sweden. Cruiser Superior (Interna-tional Farg AB), which contains copper and irgarol 1051,thus banned in the market, was used as reference paint.

2.2. Anti-fouling agents (single substances)

TBTO (bis(tri-n-butyltin(IV)oxide), diuron (3(3,4-di-chlorophenyl)1-1-dimethylurea) and irgarol 1051 (2-(tert-butylamino)-4-(cyclopropylamino)-6-(methylthio)-s-triazine)were all obtained from Sigma-Aldrich. TBTO is a herbicidethat has been used in anti-fouling paints since the 1960s.The triazine irgarol 1051 is an algaecide, which worksthrough inhibition of photosystem II (Dahl and Blanck,1996). Diuron is a phenylurea that in a similar way inhibitphotosynthesis (Perschbacher and Ludwig, 2004). Apartfrom being an active ingredient in anti-fouling paints, itis also used in weed control on land (Giacomazzi andCochet, 2004).

All three compounds were dissolved in acetone beforeused in the test. The acetone concentration did not exceed0.01% in the test medium.

2.3. The leakage procedure

The paints were applied in 5 cm2 on plastic petri dishesaccording to the instructions from the producers. Thepainted pieces were placed in beakers containing 0.5 L of7& artificial seawater and the leakage proceeded for 14days. This area to volume proportion corresponds to theallowed leakage of copper for the west coast of Sweden(i.e., 200 lg Cu/cm2 in 14 days) (Swedish Chemicals

J. Karlsson et al. / Marine Pollution Bulletin 52 (2006) 1661–1667 1663

Inspectorate, 1998). The 7& artificial sea water corre-sponds to the salinity in the northern Baltic proper of theBaltic Sea.

Before the leaking procedure started, the painted petridishes were put into beakers with 0.5 L of artificial sea-water (Eklund, 2004) for 1 h. This first leakage water wasdiscarded since it may have contained residues of preserva-tives or solvents. The beakers were covered with aluminiumfoil, to prevent growth of photosynthetic organisms duringthe leaking procedure, and placed on a shaking table tosimulate water movement. The leaking was performed at22 ± 2 �C. After the first 14-day period the leakage waterswere frozen for later testing and the painted petri-disheswere transferred to new beakers with 0.5 L of fresh seawa-ter. This procedure was repeated every 14 days for 16weeks. The leakage waters after 2, 8 and 16 weeks weretested for toxicity.

Table 1Results from growth inhibition tests with C. tenuicorne and lethality testswith N. spinipes exposed to leakage waters of six anti-fouling paints

Paint Weeks ofleakage

Ceramium NitocraEC50 (%) (95% CI) LC50 (%)

(95% CI)

Lefant SPF 2 0.56 (0.46–0.69) 49 (31–96)8 0.48 (0.36–0.60) 60 (52–69)16 0.52 (0.45–0.60)

Cruiser Eco 2 0.35 (0.27–0.46) 65 (54–80)8 0.88 (0.75–1.0)16 2.0 (1.3–2.7)

Vc17mEco 2 >100 >80EcoMar2000a 2 >100 >80SafeBoatSkin 2 >100 >80Aurora VS721 2 >100 >80

95% confidence intervals are presented in brackets.a Approved by the Swedish Chemicals Inspectorate.

2.4. Test organisms and procedures

The effect of the leakage water was tested on two organ-isms, which are both common and indigenous species in theBaltic Sea as well as other parts of the world, the red macroalga C. tenuicorne (Eklund, 1998; Bergstrom et al., 2003)and the brackish water harpactacoid copepod N. spinipes

(Lang, 1948).Since, both species has a salinity range between 1& and

32& tests can be performed in relevant salinities for largeareas.

On the alga a growth inhibition test was performedaccording to the procedures described by Bruno and Ekl-und (2003) and Eklund (2004). The tests were performedon the female generation, as it grows faster and more uni-formly than the male generation (Bergstrom et al., 2003;Eklund, 2005). Autoclaved artificial seawater with the addi-tion of nitrogen (3.46 mg/L), phosphorus (0.78 mg/L), iron(0.10 mg/L) and carbon (1.65 mg/L) was used as medium.

The growth inhibition was estimated by measuring thelength of the algae day 0 and again after 7 days. Thegrowth is almost linear during this growth period and thegrowth rate has been calculated as linear regression (Ekl-und, 2005). The Median Effective Concentration (EC50),i.e., the concentrations in which the algae express a 50%growth rate inhibition compared to the control, was cal-culated using REGTOX-EV6.xls (http://eric.vindimian.9online.fr). The method calculates EC50-values for macroalgae with corresponding 95% limits by optimizing thecurve fit with successive iterations.

A lethality test was performed with the crustacean. Thiswas developed over 25 years ago (Bengtsson, 1978) and hassince then been established as Swedish, Danish and Inter-national standards (SIS, 1991; ISO, 1997). Cultivationand tests were performed according to the Swedish stan-dard SS028106 (SIS, 1991). N. spinipes used in the presentstudy were collected from the ITM stock cultures (seeBreitholtz and Bengtsson, 2001).

The organisms were exposed for 96 h after which thenumbers of dead and living animals were counted.

Calculations of 50% lethal concentrations (LC50), with95% confidence intervals were done with Probit Analysis(Finney, 1971).

3. Results

3.1. Anti-fouling paints (complex mixtures/products)

The results from the growth inhibition test and lethalitytest with C. tenuicorne and N. spinipes, respectively, arepresented in Table 1. From four of the leakage waters,i.e., Vc17mEco, EcoMar2000, Aurora VS721 and Safe-BoatSkin, out of the six tested paints no EC/LC50-valuescould be calculated. Thus, these paints were not consideredtoxic to either the alga or the crustacean and were thereforenot further tested. Nonetheless, the paint Vc17mEcoshowed some inhibitory effect on the growth of the algaeand an EC25 was estimated to 75.8% leakage water (95%CI, 55.0–95.6). Conversely, a stimulating effect on thegrowth of the algae exposed to the leakage water from Aur-ora VS721 and SafeBoatSkin was noticed. In 100% leakagewater from Aurora VS721, the algae grew about 41% bettercompared to the control. Similar to Aurora VS721, leakagewater from SafeBoatSkin showed 18% growth stimulationcompared to the control in the highest concentration. Thepaints Lefant SPF and Cruiser Eco were toxic to both testorganisms. Still, the toxicity was low to the crustacean andtests were not repeated after 8 and 16 weeks of leakage forCruiser Eco and 16 weeks for Lefant SPF. However, to thealga these leakage waters showed high toxicity at all threeoccasions. The highest concentration tested in the crusta-cean lethality test was 80% leakage water, thus toxic effectsin 100% leakage water are not known.

In Table 2 test results from previously published results(Karlsson and Eklund, 2004) have been compiled togetherwith results from the current study. With some exceptions,the leakage waters were not so toxic to the crustacean.

Table 2Compilation of previous and new results with C. tenuicorne and N. spinipes

exposed to leakage waters (2 weeks of leakage) of new biocide-freeanti-fouling paint

Paint Ceramium NitocraEC50 (%) (95% CI) LC50 (%) (95% CI)

Lefant H2000a 2.2 (1.2–3.5) 57 (49–68)Mille lighta 2.2 (1.6–3.2) 55 (31–150)Micron Ecoa 0.60 (0.55–0.65) 1.1 (0.91–1.4)SSC-44a 0.19 (0.17–0.22) 9.0 (7.6–11)Lefant SPF 0.56 (0.46–0.69) 49 (31–96)Cruiser Eco 0.35 (0.27–0.46) 65 (54–80)SafeBoatSkin >100 >80Aurora VS721 >100 >80EcoMar2000b >100 >80Vc17mEco >100 >80Cruiser Superiora,c 0.14 (0.12–0.16) 30 (17–300)

95% confidence intervals are presented in brackets.a Karlsson and Eklund (2004).b Approved by the Swedish Chemicals Inspectorate.c Reference paint containing copper and irgarol.

Table 3Presentation of ranking points

Ceramium tenuicorne Nitocra spinipes

EC50 (%) Point LC50 (%) Point

0.1 to 60.2 512 1 to 62 5120.2 to 60.4 256 2 to 64 2560.4 to 60.8 128 4 to 68 1280.8 to 61.6 64 8 to 616 641.6 to 63.2 32 16 to 632 323.2 to 66.4 16 32 to 664 166.4 to 612.8 8 64 to 680 812.8 to 625.6 4 >80 425.6 to 651.2 251.2 to 6100 1>100 0

Table 4Presentation of total points for the anti-fouling paints when points forEC50- and LC50-values are summarised

Paint Function Total point

SafeBoatSkin (sailway) Polymer wax 4Aurora VS721 (Aurora

Marine Industries Inc.)Wax 4

EcoMar2000 (Thulica AB) Capsaicin(peppar extract)

4

Vc17mEco (InternationalFarg AB)

Teflon 4

Lefant H2000 (Lotrec AB) Polishing 48Mille light (Hempel Farg AB) Polishing 48Lefant SPF (Lotrec AB) Polishing 144Cruiser Eco (International

Farg AB)Polishing 264

Cruiser superiora (InternationalFarg AB)

Copper andirgarol

544

SSC-44 (US Gloss Europe AB) Waxy withscale structure

640

Micron Eco (InternationalFarg AB)

Polishing 640

A high total point indicates a high toxicity.a Reference paint.

Table 5Effects of TBTO, diuron and irgarol 1051 on the red macro alga C.

tenuicorne and the crustacean N. spinipes

Compound Ceramium tenuicorne Nitocra spinipes

EC50 (lg/L) (95% CI) LC50 (lg/L) (95% CI)

TBTO 0.49 (0.49-0.50) 13 (10–17)Irgarol 1051 0.96 (0.51–0.98) 4500 (3400–6900)Diuron 3.4 (2.9–3.8) 4000 (3300–5200)

1664 J. Karlsson et al. / Marine Pollution Bulletin 52 (2006) 1661–1667

To make a comparison of the toxicity, the different anti-fouling paints were ranked based on their EC50- and LC50-values. The ranking were conducted as follows (Table 3). Ageometric scale was used, as ecotoxicological responsesnormally follow a geometric pattern. The alga EC50 resultsspanned from 0.1% to 100% leakage water, i.e., from verytoxic to non- toxic. The first interval in the geometric scalewas 0.1–0.2% and then the intervals were increasing by afactor of two up to 100% leakage water. The first interval,0.1–0.2%, was given 512 points and then the points werehalved down to 100%, which was given 0 points.

The LC50 results for the crustacean spanned between1.1% and 80%. Lethality is estimated to a sensitivity aboutten times lower compared to a sublethal endpoint (Envi-ronment Canada, 1999). The first interval, i.e., the mosttoxic, for the lethality test ranged between 1% and 2%and was also given 512 points and then the points werehalved down to 80%, which was given 4 points (Table 3).Since the highest concentration tested in the lethality testwas 80% leakage water and the toxicity in 100% isunknown, the lowest obtainable point was 4. When com-

bining the ranking points from the two tests, the highesttotal point was 1024, which indicates high toxicity.

The total ranking points for the tested anti-foulingpaints are compiled in Table 4. The two most toxic paints,Micron Eco and SSC-44, had a total ranking point of 640,which was ranked 0.2 times more toxic than the referencepaint, Cruiser superior. Four of the paints obtained theranking point 4, which means that they did not produceany leakage water, which exhibited toxic effects to eitherthe alga or the crustacean. These paints were ranked about160 times less toxic than the two most toxic paints. Twopaints, Lefant H2000 and Mille light were ranked 12 timesmore toxic than the ones that did not show a 50% effect ingrowth or lethality.

3.2. Anti-fouling agents (single substances)

TBTO was found to be very toxic to both organisms(Table 5). All three anti-fouling agents presented high tox-icity to the alga with TBTO being the most toxic and diu-ron the least toxic. While TBTO proved to be highly toxicto the crustacean, irgarol and diuron were more than 300times less toxic.

J. Karlsson et al. / Marine Pollution Bulletin 52 (2006) 1661–1667 1665

4. Discussion

The intention of the responsible Swedish environmentalauthorities’ restrictions on anti-fouling paints was toreduce the use and discharge of toxic substances to theaquatic environment. In favour of the traditional paintsthat leak toxic substances, other mechanical/physical alter-natives, e.g., boat washers or use of paints based on phys-ical (e.g., polishing) fouling prevention, were consideredsufficient in low salinity areas where the problem with foul-ing is not as severe. However, most of the paints claimed towork by physical means were toxic to either one or both ofthe tested non-target organisms and some were even moretoxic than the reference paint in the current study. Appar-ently, they do leak something that negatively affects the twotested organisms. A possible reason for this drawbackcould be found in how the regulation is used and inter-preted. That is, only anti-fouling paints based on biocidalsubstances needs to receive approval from the SwedishChemicals Inspectorate (KemI) before they can be releasedon the market for sale (KIFS, 2004). A biocide is defined asa substance intended to have a negative effect on livingorganisms (Directive 98/8/EC, 1998). In a mixture or aproduct it is up to the producer to determine whether theirproduct contains a substance with the purpose to nega-tively affect living organisms or not. If the producers donot consider that a biocide has been added to the productit only needs to be notified to the authorities (KIFS, 2004).Along with the notification, all ingredients known to causea risk to the environment or human health must be speci-fied. However, if an ingredient is added in concentrationsbelow 1% by weight and is not intended to have an activeeffect it does not need to be specified (KIFS, 2004). Thismeans that very potent substances may be added in smallamounts and thereby it is possible to avoid the approvalprocedure.

The toxicity of the tested paints varied in the sense thatthey showed toxic effects to either one, both or none of thetest organisms. Different methods measure different end-points and as a consequence EC50- and LC50-values willvary. This makes it difficult to estimate the relative toxicitybetween the paints. The ranking procedure presented in thecurrent study however allows comparison of the toxicitybetween the different paints according to their EC50- andLC50-values. When the ranking points of both tests wereadded, a comparison of total toxicity was received. Theranking results show a variation of 160 times between themost toxic paints and the least toxic ones. Thus, there isa wide span between the new anti-fouling paints in termsof toxicity. One of the paints (Vc17mEco) ranked non-toxicshowed some growth inhibiting effect towards the alga.Even so, it ended up in the group with non-toxic paintssince it did not show a 50% growth inhibition in the highestconcentration. In addition, two of the paints ranked leasttoxic had some growth stimulating effect on the alga. Thisis another reason why products and not only single chem-icals should be evaluated.

The polishing paints, i.e., paints that function by gradu-ally peeling off, were most toxic whereas paints functioningin other ways, e.g., teflon or wax, seem to be non-toxic.Four of the products were not toxic to the two test organ-isms according to our ranking system. This means that sev-eral non-toxic products do exist on the market, which mayprovide an alternative to the toxic leaking paints.

Only one of the tested paints (EcoMar2000) in the pres-ent study is approved for use on the east coast of Sweden.All the others have not been evaluated, as the producersconsider that they have not added anything in their paintacting as a biocide. In anti-fouling paints as well as in manyother complex products various toxic chemicals are used.This aspect is never considered in the current legislation,and possible synergistic effects, which may play an impor-tant role, will be missed as only the toxicity of individualchemical substances exerting biocidal effects are investi-gated. In our study, we have tested the effect of the totalproduct, i.e., the leakage waters from anti-fouling paints.This is the way the product will be used and distributedin the environment and the test set-up mimic a realisticway of exposure. Our results show that several of the paintsregistered as containing no biocides were still toxic to twocommon organisms in the marine and brackish water envi-ronment. The reason for this toxicity could be either syner-gistic effects or that some very potent substances is added inamounts below 1% by weight. With the existing regulationthese toxic products will not be discovered. This means thatthe legislation for products needs to be improved. We pro-pose that the principle within the new European chemicallegislation REACH, i.e., that chemicals should be testedbefore they can be sold on the market should also includemixtures such as anti-fouling paints.

The sensitivity of the test organisms was also tested byexposing them to common anti-fouling compounds. Theresults showed EC50- and LC50-values in line with earlierstudies on similar organisms (Fargasova, 1998; Okamuraet al., 2000a; Ohji et al., 2002; Fernandez-Alba et al.,2002). The EC50-values of TBT (0.49 lg/L) and irgarol(0.96 lg/L) for C. tenuicorne are in agreement with earlierEC50-values reported for other alga species (Fargasova,1998; Okamura et al., 2000a,b). However, C. tenuicorne

seems to be more sensitive to diuron (EC50 3.4 lg/L) com-pared to an earlier study that showed EC50-values of 45 lg/L for S. capricornotum (Fernandez-Alba et al., 2002).

Compared to the copepod Tigriopus japonicus (LC50

0.149 lg/L), N. spinipes was less sensitive to TBT (LC50

of 13 lg/L) (Kwok and Leung, 2005). Still, the LC50 forN. spinipes is in accordance with other crustacean species(Ohji et al., 2002). N. spinipes showed to be about twiceas sensitive to irgarol in comparison with Daphnia magna

(Okamura et al., 2000a). An earlier toxicity test with diuronhas shown an EC50-value (motility) of 8.6 mg/L for D.

magna (Fernandez-Alba et al., 2002), which is about twicethe LC50-value in the present study. Even though thisalgaecide shows low toxicity to crustaceans the previousstudy proved that a degradation product to diuron resulted

1666 J. Karlsson et al. / Marine Pollution Bulletin 52 (2006) 1661–1667

in an EC50-value that was 215 times more toxic to D.

magna than the original product.When it comes to the relative toxicity of the three tested

biocides, TBT were most toxic to both organisms. Com-pared to irgarol, TBT was about twice as toxic to the algawhereas TBT was almost 7 times as toxic as diuron. Irgaroland diuron exhibited about the same toxicity to the crusta-cean. Compared to TBT, irgarol and diuron were over 300times less toxic to the crustacean, which could be expected,as they are both algaecides.

Concentrations in the range of the EC50-values pre-sented in the present study as well as previous studies(Andersson, 1996; Okamura et al., 2000a), have been mea-sured in coastal waters all over the world (Cleary and Steb-bing, 1985; Dahl and Blanck, 1996; Haglund et al., 2001;Hernando et al., 2001; Okamura et al., 2003; Konstantinouand Albanis, 2004). Consequently, these substances havethe potential to cause harm to ecosystems.

The ranking system presented in this study permitsranking and comparison of total toxicity of mixtures andproducts. A central objective of the present study was touse ecological relevant organisms from different trophiclevels and to use tests that are fast, cost-effective and sensi-tive. Whereas, the alga was very sensitive for both anti-fouling paints as well as the single anti-fouling compounds,most of the leakage waters showed low toxicity towards thecrustacean. However, the lethality endpoint used for N.

spinipes is a more rough variable compared to a sublethalendpoint like growth inhibition, which was used for thealga. The sensitivity of the crustacean may be elevated byusing sublethal endpoints like larval development, repro-duction or life cycle tests (e.g., Breitholtz and Wollenber-ger, 2003). These tests are more sensitive but also morelabour demanding and consequently more costly.

The use of toxic leaking anti-fouling paints may causemost harm to ecosystems in areas of low salinity since mostorganisms are stressed in lower salinities (Kautsky andTedengren, 1992). For this reason, the tests in the presentstudy were performed in 7&, which is relevant for the Bal-tic Sea as well as many estuaries. Both test organisms arenaturally found in areas between 1& and 32&, whichmean that the proposed test strategy and ranking systemmay be relevant to use also for the marine environment.

In conclusion, both tests and organisms showed to besensitive to the leakage waters from the anti-fouling paints.This makes the presented ranking system suitable for use infuture ecotoxicological evaluations of potentially harmfulproducts, such as anti-fouling paints and can thereby bea powerful screening tool to identify the most toxic prod-ucts before they reach the market.

Acknowledgement

We would like to thank the Swedish Maritime Adminis-tration for financial support of this work.

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