Use and environmental impact of antifouling paints in the Baltic Sea

Full text

(1)

(2)

(3)

(4) .

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13) ! "#$

(14) #"%"" & '&

(15) '

(16) ()# % . *

(17) +,

(18)

(19)

(20)

(21)

(22) ' %%

(23) -

(24) '

(25)

(26) %

(27)

(28)

(29) +.,

(30) '-

(31) / +0,

(32)

(33)

(34) / %*

(35)

(36)

(37)

(38)

(39) 1

(40)

(41) -

(42) '

(43)

(44)

(45) -

(46)

(47) -

(48) 1%2 '

(49)

(50)

(51) . -

(52)

(53)

(54)

(55) -' -

(56) .

(57)

(58) 3 -

(59)

(60)

(61)

(62)

(63)

(64)

(65) 1

(66)

(67)

(68)

(69) 3

(70) %

(71) 4

(72) ' .-

(73)

(74)

(75) -

(76) '

(77)

(78) " ' -

(79) +, #"5

(80)

(81) '

(82)

(83) + ,

(84)

(85)

(86)

(87). +2*2,'-

(88)

(89)

(90) /

(91)

(92) + #,%6

(93) ' .0-

(94)

(95)

(96)

(97)

(98) '

(99) .

(100)

(101)

(102)

(103)

(104)

(105) %7 -

(106)

(107)

(108)

(109) -

(110) .

(111) '-

(112)

(113) .

(114)

(115) -

(116) -

(117) + ,%2 -

(118)

(119)

(120) 1 1

(121) '

(122)

(123)

(124) . 1

(125) + 8, 9

(126)

(127) '

(128) -

(129)

(130) 9$

(131) '

(132) .

(133)

(134)

(135) +: ;,%! ' -

(136)

(137)

(138)

(139)

(140) +%% . ' '

(141)

(142)

(143)

(144) '

(145) ,-

(146) '

(147)

(148) + ,%

(149)

(150)

(151)

(152)

(153)

(154)

(155) <

(156)

(157)

(158) 1

(159) -

(160)

(161)

(162)

(163) '

(164)

(165)

(166) %2

(167)

(168)

(169)

(170) .

(171)

(172)

(173)

(174)

(175) -

(176)

(177)

(178) .

(179)

(180)

(181)

(182)

(183)

(184)

(185)

(186) -

(187)

(188) /

(189)

(190) %6

(191) '

(192)

(193) 1

(194)

(195) . .

(196)

(197)

(198)

(199)

(200)

(201)

(202)

(203) .

(204) 1

(205)

(206)

(207)

(208) %

(209)

(210)

(211)

(212)

(213)

(214) "#$

(215)

(216) =>> %% > ? @ == = = #8ABA" 6*7A$CA#$9 A9A8 6*7A$CA#$9 A9A8".

(217) !

(218) "

(219)

(220) '#"9A#

(221) .

(222)

(223) Use and environmental impact of antifouling paints in the Baltic Sea. Maria Alexandra Bighiu.

(224)

(225) Use and environmental impact of antifouling paints in the Baltic Sea Maria Alexandra Bighiu.

(226) ©Maria Alexandra Bighiu, Stockholm University 2017 Cover illustration by Anca Cobzaru ISBN print 978-91-7649-692-3 ISBN PDF 978-91-7649-693-0 Printed in Sweden by US-AB, Stockholm 2017 Distributor: Department of Environmental Science and Analytical Chemistry (ACES).

(227) ‘It always seems impossible until it’s done’. Nelson Mandela.

(228)

(229) Contents. Abstract ........................................................................................................... 1 Sammanfattning .............................................................................................. 2 Rezumat .......................................................................................................... 3 List of papers ................................................................................................... 5 Background and Aims ..................................................................................... 6 Methods .......................................................................................................... 7 Model organism ............................................................................................................ 8. Biofouling ...................................................................................................... 10 Biofouling prevention..................................................................................... 12 Antifouling paints ......................................................................................................... 12 Biocide-free paints ...................................................................................................... 14 Non-coating alternatives ............................................................................................. 14. The Baltic Sea ............................................................................................... 15 Environment ................................................................................................................ 15 Boating ........................................................................................................................ 15 Metal concentrations on boat hulls .............................................................................. 16 Metal concentrations in water and sediment ............................................................... 18. Toxicity of antifouling paints .......................................................................... 20 Laboratory studies ...................................................................................................... 20 Metal toxicity .......................................................................................................... 20 Field studies ................................................................................................................ 21. Conclusion and future perspectives .............................................................. 26 References .................................................................................................... 28 Acknowledgements ....................................................................................... 33.

(230) Abbreviations. ANOVA AF BLM COI Cu EU FIAM GC GLM H&E HSD ICP-MS IMO MT N N/A PAH Pb PCA PNEC Psu ROS S SFMS Sn SPC TBT TN TP XRF Zn. Analysis of Variance Antifouling Biotic Ligand Model Cytochrome c Oxidase subunit I Copper European Union Free Ion Activity Model Gas Chromatography Generalized Linear Model Hematoxylin and Eosin Honest Significant Difference Inductively-Coupled Plasma Mass Spectrometry International Maritime Organization Metallothionein North Not Applicable Polycyclic Aromatic Hydrocarbon Lead Principal Component Analysis Predicted No Effect Concentration Practical salinity units Reactive Oxygen Species South Sector Field Mass Spectrometry Tin Self-Polishing Copolymer Tributyltin Total Nitrogen Total Phosphorous X-Ray Fluorescence Zinc.

(231) Abstract. Biocide-based antifouling (AF) paints are the most common method for preventing biofouling, i.e. the growth of algae, barnacles and other organisms on boat hulls. AF paints for leisure boats are predominantly based on copper (Cu) as the main biocide, with zinc (Zn) present as a pigment and stabilizer. Both metals are released from the paint matrix into the water column, leading to contamination of marinas which typically have only limited water exchange. Thus, the aim of this PhD thesis was to describe the use of AF paints in different regions in Sweden, as well as the associated environmental consequences with regard to contamination of the environment and toxicity to non-target aquatic snails. Using a recently developed X-ray fluorescence application, high levels of Cu were detected on boats moored in freshwaters, despite a more than 20-year-old ban, as well as high levels of tin (Sn) on 10 % of the boats, indicating the presence of (old) tributyltin paints (TBT), which might pose an environmental risk and a health hazard for people performing the paint scraping (paper 1). In addition, very high levels of Cu and Zn were measured in the biofouling material collected from the boat hulls, and this is problematic because the biofouling is commonly disposed of on the soil in boatyards at the end of each season. No difference was found in the amount of biofouling on boats coated with Cu or biocide-free paints, which implies that Cu might be currently overused in areas of low salinity and low barnacle density (paper 2). This work also introduces the use of a new species for ecotoxicological field experiments, the snail Theodoxus fluviatilis. Chronic field experiments (paper 3) revealed 6-fold increases in snail mortality, negative growth and up to 67-fold decreased reproduction in marinas, compared to areas not impacted by boating (‘reference areas’). Moreover, a higher prevalence of snails with histopathological alterations (e.g. necrosis of gills, gonads, midgut gland and parasite infestation, among others) was observed in the marinas, compared to the reference areas (paper 4). Statistical modelling indicated that the majority of the toxic effects were best predicted by the metals, most likely originating from AF paints. The results presented in this thesis depict some important aspects of AF paint use in brackish water and highlight the necessity of implementing a suitable management practice for the heavily contaminated biofouling waste in order to minimize the risk to soils. In addition, the evidence of toxicity to snails in marinas can be used as a basis to increase the public understanding of the impact of recreational boating and encourage people to choose less toxic alternatives to AF paints. 1.

(232) Sammanfattning. Biocidbaserad båtbottenfärg (antifouling, AF) är den vanligaste metoden för att förhindra påväxt av alger, havstulpaner och andra organismer på båtskrov. I Sverige är koppar och zink de vanligaste verksamma ämnena i AF-färger för fritidsbåtar. Färgerna verkar genom att metall frisätts, vilket leder till förorening av marinor och kustnära områden. Syftet med denna avhandling var att bestämma vilken typ av AF-färg som används, uppskatta miljökonsekvenser avseende risk för kontaminering av miljön samt bestämma toxicitet i hamnar hos vattenlevande snäckor. Trots ett mer än 20 år gammalt förbud uppmättes höga kopparhalter på båtar i sötvatten, med hjälp av en nyutvecklad röntgenfluorescensapplikation (XRF). På tio procent av båtarna återfanns dessutom höga halter av tenn, vilket är en indikation på tennorganiska föreningar som exempelvis tributyltenn (TBT). Förutom miljörisken med dessa färger, utgör de även en hälsorisk för människor i samband med underhållsarbete (artikel 1). Höga koncentrationer av koppar och zink uppmättes i påväxt som samlats in från båtskrov. Detta skapar lokala miljöproblem i samband med underhåll eftersom avskrapade färgflagor och påväxt i allmänhet inte samlas in utan hamnar direkt på marken. Studierna indikerar även att kopparbaserad färg inte behövs i områden med låg salthalt och låg förekomst av havstulpaner eftersom ingen skillnad kunde påvisas i mängden påväxt mellan båtar målade med koppar och båtar målade med biocidfri färg (artikel 2). I denna avhandling användes första gången en ny art, snigeln Theodoxus fluviatilis, i ekotoxikologiska experiment. Fältförsök visade kraftiga effekter på snigeln i marinor jämfört med referensområden i form av en sexfaldigt ökad dödlighet, negativ tillväxt och en nära 70-faldigt minskad reproduktion (artikel 3). Dessutom observerades en högre förekomst av sniglar med histopatologiska förändringar (t ex nekros av gälar och könskörtlar samt parasitangrepp) i marinorna, jämfört med referensområden (artikel 4). Statistisk modellering visade att majoriteten av de toxiska effekterna förklarades bäst med metallhalter i vatten och sediment. Eftersom inga andra punktkällor finns i närheten av marinorna härrör metallerna sannolikt från AF-färger. Resultaten som presenteras i denna avhandling visar några viktiga aspekter av båtbottenfärganvändning i bräckt vatten och belyser behovet av att förändra praxis i samband med båtunderhåll för att minimera risken för kontaminering av miljön. Dessutom kan den påvisade toxiciteten hos sniglar användas som grund för att uppmuntra människor att välja mindre giftiga alternativ till AF-färger. 2.

(233) Rezumat. Vopselele antivegetative (antifouling, AF) reprezintă cea mai utilizată metodă de prevenire a biodepunerilor marine (ex. alge, crustacee şi alte organisme) pe carenele bărcilor. Vopselele AF pentru ambarcaţiuni de agrement au la bază Cu (cupru) ca principal biocid şi Zn (zinc) ca stabilizator şi pigment. Ambele metale sunt eliberate din matricea vopselelor în mediul acvatic, ducând la poluarea porturilor de agrement care în general au o capacitate redusă de schimb al apei. Astfel, scopul prezentei teze de doctorat a fost descrierea utilizării vopselelor AF în diferite regiuni din Suedia, precum şi consecinţele privind riscul de contaminare al mediului şi toxicitatea asupra melcilor acvatici. Utilizând o nouă aplicaţie de fluorescenţă de raze X, concentraţii ridicate de Cu au fost detectate pe bărcile ancorate în apă dulce, în ciuda restricţiilor existente de mai bine de 20 de ani. Mai mult, concentraţii ridicate de Sn (staniu) au fost detectate pe 10 % din bărci, ceea ce indică prezenţa straturilor (vechi) de vopsea pe bază de TBT (tributilstaniu), reprezentând atât un risk de mediu, cât şi un potenţial hazard pentru sănătatea persoanelor care desfăşoară activităţi de curăţare a vopselelor de pe carene (articolul 1). În plus, niveluri ridicate de Cu şi Zn au fost măsurate in biodepunerile colectate de pe carenele bărcilor, iar acest lucru este problematic deoarece aceste biodepuneri contaminate sunt depozitate direct pe sol la sfârşitul fiecărui sezon. Nicio diferenţă nu a fost detectată în ceea ce priveşte cantitatea de biodepuneri între carenele acoperite cu vopsele pe bază de Cu şi cele cu vopseluri fără biocid, ceea ce sugerează că gradul curent de utilizare al Cu este mai ridicat decât strictul necesar în condiţii de salinitate scăzută şi densitate scăzută a larvelor de crustacee (articolul 2). Această lucrare introduce, de asemenea, utilizarea unei noi specii pentru experimente de teren în domeniul ecotoxicologiei, şi anume, melcul Theodoxus fluviatilis. Experimentele cronice desfăşurate pe teren (articolul 3) au rezultat în creşterea mortalităţii şi reducerea fecundităţii si creşterii melcilor expuşi în porturi, comparativ cu cei din locaţiile de referenţă. Mai mult, o pondere mai mare de melci cu alterări histologice (ex. necroză a branhiilor, a gonadelor şi a hepatopancreasului, etc) a fost depistată în porturi, comparativ cu locaţiile de referinţă (articolul 4). Modelele statistice au indicat că majoritatea efectelor toxice au fost cel mai bine prezise de către metale (Cu, Zn), cel mai probabil originând din vopselele AF. Rezultatele prezentate în această teză descriu aspecte importante ale utilizării vopselelor AF în apă salmastră şi subliniază necesitatea implementării unei practici de management potrivite pentru 3.

(234) biodepunerile contaminate, cu scopul de a minimiza riscul pentru sol. În plus, dovezile de toxicitate ridicată în rândul melcilor expuşi în porturile de agrement pot fi utilizate ca bază pentru a îmbunătăţi informarea publicului asupra impactului de mediu al bărcilor de agrement şi pentru a încuraja proprietarii de bărci să opteze pentru alternative mai puţin toxice decât vopselele AF.. 4.

(235) List of papers. Paper 1 XRF measurements of tin, copper and zinc in antifouling paints coated on leisure boats. Erik Ytreberg, Maria Alexandra Bighiu, Lennart Lundgren and Britta Eklund. Environmental Pollution 213 (2016) 594-599. Paper 2 Biofouling of leisure boats as a source of metal pollution. Maria Alexandra Bighiu, Ann-Kristin Eriksson-Wiklund and Britta Eklund. Environ Sci Pollut Res DOI 10.1007/s11356-016-7883-7. Paper 3 Metal contamination in harbours impacts life-history traits and metallothionein levels in snails. Maria Alexandra Bighiu, Elena Gorokhova, Bethanie Carney Almroth, Ann-Kristin Eriksson Wiklund. Submitted. Paper 4 Mortality and histopathological effects in harbour-transplanted snails with different exposure histories. Maria Alexandra Bighiu, Burkard Watermann, Xueli Guo, Bethanie Carney Almroth, Ann-Kristin Eriksson Wiklund. Submitted. Author contributions I took the leading role in writing papers 2-4 and contributed to a minor extent to the writing of paper 1. I analysed the XRF spectra for paper 1 and did most of the data analysis for all the papers. I took the lead in designing the experiments from papers 3-4 and planned and carried out the colour experiment from paper 2. I had the main responsibility for the field work in papers 3-4, and I took part in the sampling for paper 2 and in a few of the XRF measurements in paper 1. I performed the laboratory work for papers 2-4 except for the chemical analysis, histopathological evaluation and COI assay (including analysis of the sequences and calculation of diversity indices).. 5.

(236) Background and Aims. Pollution caused by boating activities is a problem that occurs worldwide. In particular, antifouling (AF) paints used on boat hulls are a main source of metal contamination in harbours and may thus represent a risk for biota 1. The main issues regarding biofouling, the need for AF paints and their environmental impact are outlined in the following sections. This PhD thesis aimed at describing the use of antifouling paints on leisure boats in the Baltic Sea in concert with the current salinity-based legislation, as well as the associated environmental consequences, in terms of potential risks of environmental contamination and harm to non-target organisms. Specifically, each paper was focused on the aims presented in Table 1. Table 1 Specific aims of each paper included in this PhD thesis Paper no. 1. Aims Determine the concentrations of Cu, Zn, Sn in AF paints coated on boats from areas with different salinities throughout Sweden.. 2. 1. Quantify the concentrations of metals accumulated in the biofouling matrices at the end of a boating season; 2. Quantify the amount of biofouling on leisure boats in brackish water; 3. Compare the efficiency of different methods for minimizing biofouling (i.e. AF paints vs mechanical hull cleaning);. 3. 4. Investigate the importance of other factors (e.g. hull colour) for biofouling. 1. Assess the chronic toxicity of harbour contaminants on the snail Theodoxus fluviatilis, in terms of physiological effects (growth, reproduction and mortality) and metallothionein (MT) levels;. 4. 2. Evaluate the relationship between the observed toxic effects on snails and several abiotic factors (metals, nutrients, salinity, pH). 1. Assess the histopathological effects on snails exposed in harbours and identify the key biotic and abiotic parameters associated with these effects; 2. Investigate the role of exposure history and genetic diversity for the snails’ tolerance to harbour contaminants.. 6.

(237) Methods. All the four papers included in this PhD thesis are based on field studies in which various techniques were applied. In the first half of the thesis (papers 1-2), boat hulls sampled in dry dock marinas were used for measuring metals in AF paints with XRF and for collecting biofouling material. In the second half, work was focused in the brackish environment in the harbours which were characterised in terms of nutrients, metals and organic AF biocides. These environmental parameters, together with other abiotic factors (salinity, pH, temperature), were used in an attempt to explain toxic effects that I observed and quantified in snails exposed in harbours (papers 3-4). Thus, this thesis comprises multidisciplinary techniques listed in Table 2. For a more detailed description of each method, see the corresponding paper.. Table 2 Overview of the methods used throughout this PhD thesis. A. Analytical methods Paper no.. Analysis. Technique. Method. 2 3-4. Metals in biofouling a Metals in water b. ICP-MS ICP-MS. 3-4. Metals in sediment b. ICP-MS. 3. Organic AF in sediment: TBT/ Irgarol/Diuron Nutrients in water (TN and TP). GC-ICP-SFMS. SS 02 81 50–1 SS EN ISO 172942:2005 SS EN ISO 17294-1 mod DIN ISO 38407-35. 3. SS-EN ISO 119051:1998 and SS-EN ISO 15681-2:2005 3 Metallothionein Spectrophotometry Reference 2 4 Nutrients in water (NO2 Autoanalyzer ISO 13395 and ISO 15681-2 +NO3 and PO4) 4 Mitochondrial COI PCR Reference 3 4 Histopathology Staining H&E and Routine histology microscopic observations a b Cu, Zn and Sn; Cu, Zn (and Pb in paper 4 only). 7. Autoanalyzer.

(238) B. Field methods Paper no. 1. Task. Method description. 2. Metal determination on boat hulls Biofouling sampling from boats Colour experiment. 3-4. In situ exposure. XRF measurement of Cu, Zn, Sn for 30 s; reference 4 Biofouling was collected with a rubber scraper, oven dried (60° C) and weighed. Plastic panels were immersed at 1 m depth for 21 d, after which the abundance of each macrofouling group was determined. Snails kept in plastic cages at 1 m depth for 8 and 16 weeks (paper 3) and 8 weeks (paper 4). 2. C. Statistical methods Paper no. 1 2 3 4. Main tests Steel-Dwass multiple comparisons ANOVA, Kruskal-Wallis, Tukey HSD, Spearman’s ρ Generalized linear models (GLMs), between-group PCA, Log-rank Logistic regressions, GLMs, Linear regression. Model organism In this thesis I chose a nerite gastropod, Theodoxus fluviatilis, as a model organism. This snail species is highly abundant in the Baltic Sea and is not a typical biofouling organism. In this respect, it is a non-target species which is highly representative of the Baltic ecosystem. Therefore it is used as test organism in papers 3 and 4. Since it is a sedentary organism with limited capacity for dispersal (due to the lack of planktonic larvae), T. fluviatilis is a suitable sentinel species for assessing local pollution. However, only basic knowledge is available on the biology of this snail which is not commonly used in environmental monitoring or toxicity testing. This is a challenging aspect of the present thesis because understanding of the natural variation in biological responses (e.g. MT) is lacking for this species. In addition, methodological limitations were encountered in the analysis of genetic diversity, which could not be performed on more sensitive endpoints than COI (paper 4). Nonetheless, this thesis contributes with valuable knowledge on the contaminant effects on both life-history traits and MT responses in T. fluviatilis. Moreover, as this snail is also commonly found in freshwaters throughout Europe 3, our results may be used as a basis for studying toxicity of anthropogenic stressors in other habitats as well. 8.

(239) Figure 1 Theodoxus fluviatilis, brackish water specimen.. 9.

(240)

(241) colour on the settlement of barnacles (Balanus improvisus), mussels (Cerastoderma glaucum) and ostracods in brackish water. Both the barnacles and the mussels were present at significantly higher densities (i.e. 48 and 33 % higher, respectively) on the black panels compared to the white ones. Thus, by choosing white over black for the hull colour (assuming that the active ingredients in the paints are the same), one can minimize barnacle growth. Although it is a natural and ubiquitous phenomenon, biofouling can have detrimental ecological and economic consequences. When it comes to vessel hulls, biofouling increases the drag, thereby leading to a higher fuel consumption, which translates into higher costs and higher CO2 emissions 5. In addition, the manoeuvrability of the ships can be affected and the risk of corrosion can increase. From an ecological perspective, biofouling of vessels represents a risk of transporting invasive species to new habitats 5. All these negative effects of biofouling are of great importance because 90 % of the world’s trade is done via shipping 11. Thus, several types of antifouling methods are currently in use and will be described in the following section.. 11.

(242) Biofouling prevention. Historically, various methods have been used for preventing biofouling on ship hulls, ranging from copper and lead sheets, to tar or lime, to metallic paints 1. In addition to paints, other non-coating methods for biofouling prevention or removal have been developed in the recent decades, and these are presented below.. Antifouling paints Tributyl tin (TBT) paints were introduced on the market in the 1960s and proved to be very efficient, having a life time of up to 5 years 1. However, the detrimental environmental impact of TBT paints was discovered in the 1980s when malformations of oyster shells and imposex in the dogwhelk were observed 1. The link between these toxic effects and the use of TBT has been well established, leading to the European Union (EU) ban of TBT paints in 1989 for boats <25 m (Directive 89/677/EC). The International Maritime Organization (IMO) extended the ban to include all ships in 2008, through the International Convention on the Control of Harmful Anti-fouling Systems on Ships (AFS), at present signed by 74 countries 12. As a consequence of these regulations, modern antifouling paints are mainly based on copper (Cu2O, CuSCN or metallic copper) as the active ingredient 1 and zinc as pigment or stabilizer. Organic ‘booster’ biocides (e.g. SeaNine, irgarol, diuron, dichlofluanid, zineb) have also been used in the last decades, mainly as additions to copper paints in order to prevent the growth of copper-resistant algae 1. However, these organic biocides have gradually been restricted in several countries throughout Europe 13, including Sweden 14. Emerging AF paints contain pharmaceuticals such as medetomidine that prevent the settlement of barnacle larvae 15. Copper-based antifouling paints can be formulated in different matrices, depending on the release method needed for different types of boats. In Sweden, there are two main categories of copper paints, namely ‘contact leaching’ (hard paints) and ‘self-polishing copolymers’ (SPC). Contact leaching paints release copper through diffusion until the biocide is entirely 12.

(243)

(244) Biocide-free paints The so-called ‘foul-release coatings’ are alternatives to biocidal paints, based on silicone or fluoropolymers. These act by creating a smooth layer on the boat hull (low surface energy), to which the fouling organisms cannot attach strongly and are thus removed by hydrodynamic forces when the boat is moving. However, these paints are mechanically fragile and less suitable for boats which typically don’t reach a speed of at least 15 knots 5. Other ‘biocide-free’ paints contain zinc as the main ingredient and claim to have a physical, rather than a chemical mode of action 21,22. Research on new technologies based on ‘superhydrophobic’ surfaces is ongoing, with hopes of developing materials that mimic the ‘Lotus leaf effect’ which can reduce the adhesive strength of biofoulers 5. However, such micro- and nanotopographic coatings have yet to appear on the market and prove their efficiency to the consumers.. Non-coating alternatives In Sweden, the main alternatives to hull painting are boat lifts, hull protectors, in-water brushing of the hulls (manually or on boat washers) and, to a smaller extent, ultrasound systems. Boat washers work by in-water brushing of the boat hulls and collection of biofouling material and potential paint residues for treatment as hazardous waste. In paper 2 we showed that, at the end of the boating season, the boats which used these devices had 50 % less biofouling mass compared to the boats not brushed (and not coated with AF). This mechanical cleaning technique seems to have potential in keeping the boats hulls clean and should therefore be tested on a larger scale in order to produce a clear assessment of costs and benefits, compared to AF paints.. 14.

(245) The Baltic Sea. Environment The Baltic Sea is the second largest brackish sea in the world, with strong vertical and horizontal salinity gradients (e.g. surface salinity ranges from 4 to 33 psu from N to S, i.e. zones A-C shown in Fig. 4) 23. The relatively young ‘ecological’ age of the Baltic Sea (ca 8000 years) is signified in the rather low biodiversity of the area 24. In addition to the presence of stressors such as hypoxia and eutrophication, the salinity gradient results in an environment in which most organisms live in constant osmotic stress. Moreover, stress caused by contaminants (e.g. heavy metals, dioxins, PAHs) increases the vulnerability of the Baltic ecosystem, which was therefore classified as a Particularly Sensitive Sea Area by the International Maritime Organisation 25 . Coastal hard-bottom communities are the most species-rich habitats in the Baltic Sea, with keystone species such as the bladderwrack (Fucus vesiculosus) and the blue mussel (Mytilus edulis) 26. Besides these keystone species, other groups of organisms also play an important role in the functioning of the ecosystem. For example, the model species used in this thesis, T. fluviatilis, is the second most widespread gastropod in the Baltic Sea 27. It is also an important grazer of benthic algae and diatoms and serves as a food source to several other species including the grayling or the whitefish 28. The reproductive period of this snail peaks during the boating season in the Baltic Sea, which means that the snail may be exposed to high metal concentrations during one of its most sensitive life stages.. Boating The Baltic Sea hosts approximately 2 million leisure boats (i.e. boats < 25 m), and half of them are located in Sweden 29. Unlike larger ships, leisure boats sail in shallow coastal areas where many species reproduce and where the water exchange is limited. This is problematic because many leisure boats (e.g. 40 % in paper 2) are coated with copper-based AF paints and are stationary 90 % of the time, meaning that the leaching of copper from AF. 15.

(246) paints occurs primarily in the marinas, thus causing increased levels of pollution in such enclosed locations (Fig. 5). In paper 2, we measured the amount of biofouling growing on boat hulls during a boating season, which typically lasts from late May to early October in Sweden. We found, on average, 7.63 g/m2 of biofouling (dry weight; max 49.3 g/m2, corresponding to ca 74 g/m2 wet weight), represented mainly by soft biofouling (e.g. bacteria, algae). This amount of biofouling is considerably lower than what was reported in warmer climates and at higher salinities, such as the Gulf of Oman, i.e. 2500-5000 g/m2 (wet weight) 30. In a survey by the Swedish Transport Agency 31, 45 % of the boaters from zone B (see map in Fig. 4) stated that they do not have biofouling on their boats, which is another indication that biofouling pressure is generally not high on the east coast of Sweden. The biofouling matrix is rich in organic matter and thus has a high capacity to bind metals 32. Paper 2 is, to our knowledge, the first study to investigate accumulation of metals in biofouling. We showed that the metal concentrations accumulated in the biofouling material can be as high as 28 g Cu/kg dw and 171 g Zn/kg dw. These levels exceed the Swedish guidelines for least sensitive land use by a factor of 140 for copper and 340 for zinc 33. Biofouling material can be problematic since it, together with metals bound to the organic matter, is commonly disposed of on the soil in boatyards. Therefore, we emphasize the need for improving the current practices in boatyards by proper collection and disposal of the heavily contaminated biofouling waste.. Metal concentrations on boat hulls Due to the special conditions in the Baltic Sea (in terms of tides and salinity), the leaching rate and concentration of copper in AF paints are regulated differently for the east and west coast of Sweden. Different PNEC (predicted no effect concentration) values are used for the two salinity regimes, since copper toxicity usually increases with decreasing salinity 34,35. This led to a ban against copper in AF paints north of Örskär (Fig. 4, zone A), while maximum 8.5 % copper was allowed for the remaining part of the east coast (Fig. 4, zone B), and up to 35 % copper for the west coast (Fig. 4, zone C) in 2012 when the survey from paper 2 was conducted. At present, the maximum copper concentration allowed in AF paints is 18.7 % for zone B 36 and 39.8 % for zone C 37. However, not all boaters followed the regulation, as shown in paper 2 where 14 % of the boaters admitted to having used paints which were not allowed in the studied area. This issue has also been illustrated in a recent survey carried out on a larger scale (i.e. ca 750 000 boats) by the Swedish Transport Agency 31, which showed that 5 % of the boaters used paints that were forbidden in the respective areas. Thus, it appears that 16.

(247)

(248) tons characteristic for each element 40. Only the concentration of the elements can be measured with XRF, but not the chemical speciation. The XRF technique makes possible in situ measurements of Cu, Zn and Sn directly on boat hulls. Sn is measured as a proxy for organotin compounds (e.g. TBT), since a strong relationship was demonstrated between inorganic and organic Sn in AF paints 41. In paper 1, this XRF technique was applied on more than 600 boats from areas of different salinities, in order to see if the metal content varies as expected under the current regulation. Indeed, the highest copper concentrations were measured on boats from the west coast, where the salinity is the highest. This area also corresponded to the highest Sn levels, up to 6 and 4-fold higher than on east coast and inland (freshwater), respectively. Surprisingly, the boats moored in freshwater had very high levels of copper in their coatings, despite a more than 20-year-old ban. It is possible that some boat owners were not aware of the type of AF paint used on their boats if they purchased the boats on the second-hand market or did not apply the paint themselves. A similar situation was detected in paper 2, when 21 % of the boaters did not know what type of AF paint their boats were coated with. Thus, our study underlines the problem of both lack of knowledge and potentially illegal use of copper paints in freshwater, an issue which has not been addressed before. Moreover, our results indicated the presence of high levels of Sn on 10 % of the investigated boats, most probably due to old TBT paint layers which might pose the above-mentioned risks. In addition, paper 1 depicts the overuse of copper paints in the Baltic Sea by the build-up of unnecessary paint layers and this finding complements that from paper 2, where we also imply that copper paints might not be needed to the high extent at which they are currently used, as they have not proven to be significantly more efficient than other paints (e.g. biocidefree paints). Therefore, it seems that the benefit of using Cu paints does not outweigh the environmental risk.. Metal concentrations in water and sediment The levels of dissolved metals were elevated in all the studied marinas (papers 3-4), compared to areas not impacted by boating (Fig. 5). Several PNEC values have been proposed for Cu in the marine environment, such as 5.6 µg/L 42 which is in the range found in our marinas, or 0.64 µg/L 43, which is considerably lower. In addition to Cu and Zn which are associated with AF paints, Pb was also considered in paper 4. Although Pb has been phased out from boat paints in the last 2 decades and it is only associated with boating to a small extent through leaching from lead keels 44, elevated Pb concentrations are used in paper 4 as another measure of anthropogenic pollu18.

(249)

(250) Toxicity of antifouling paints. Laboratory studies Several laboratory studies have shown that leachates from antifouling paints, including those marketed as ‘biocide-free’, are toxic to aquatic organisms. Some examples include growth inhibition of macroalgae, as well as mortality and inhibition of larval development in crustaceans 21,47. These studies have concluded that the risk caused by AF paints is underestimated in toxicity testing based solely on the active substance (instead of the whole paint leachate). We used a different approach for assessing the toxicity of AF paints, while improving the environmental relevance, namely by conducting field studies (papers 3-4). For this, some basic understanding of the mechanisms of copper and zinc toxicity are needed, in order to compare the effects observed in the field with the expected toxic outcome demonstrated in laboratory studies.. Metal toxicity Trace metals represent an interesting group of pollutants because they are naturally found in the environment and are essential for life but become toxic at elevated concentrations. Copper, in particular, has a very narrow window between essential and toxic concentrations 48. Both copper and zinc are needed for normal metabolism, and are part of several enzymatic or nonenzymatic proteins, Cu in hemocyanin and cytochromes, Zn in RNA and DNA polymerases and carbonic anhydrase, Cu and Zn in the antioxidant superoxide dismutase, among others 49. Metals may induce toxicity through several mechanisms: 1) binding to proteins and impairing their functionality 50 , 2) outcompeting other ions (Na+, K+, Ca2+) at the binding sites, thus causing an imbalance in the mineral concentration in the organism 51 or 3) increased production of reactive oxygen species (ROS) in the mitochondria or inhibition of antioxidant defences, leading to oxidative stress 52. Bioavailability of metals is a key prerequisite for triggering toxicity. Several factors affect the bioavailability of metals, such as the levels of dissolved organic matter, water hardness, pH and redox potential 53. Therefore, in order to predict acute metal toxicity, several models have been developed to 20.

(251) take into account the reactivity of the metal (FIAM – Free Ion Activity Model) and the water chemistry (BLM - Biotic Ligand Model). Although we did not investigate the speciation of metals in our study, we addressed the issue of bioavailability to some extent by measuring the dissolved metals instead of the total metal concentrations. A study by 17 showed that both the dissolved Cu and the labile fraction were correlated with the number of recreational boats in a marina. Effects of chronic laboratory exposure of gastropods to copper are welldocumented. Some of the main toxic effects described include reductions in feeding rates 54, declined growth 55,56, reduced fecundity 54,55, loss of chemoception and reduced egg hatching 54, as well as decreased survival 4,56. In contrast to Cu, the chronic toxicity of Zn on gastropods (and molluscs in general) is by far less studied. Münzinger and Guarducci 57 showed reductions in fecundity, growth and egg-hatching in the snail Biomphalaria glabrata exposed to low Zn concentrations. Chronic Pb exposure caused inhibition of Ca2+ uptake and subsequent growth reductions in the snail Lymnaea stagnalis 58. Metal uptake in aquatic snails occurs mainly via the digestive tract (by ingesting contaminated food) and via the gills. In Baltic Sea specimens of T. fluviatilis, metal uptake via the gills is expected to be lower than for freshwater populations, due to the lower activity of the Ca2+ pumps, which also facilitate the entry of other metal ions 59 into the gill tissues. Hence, in our field experiment we consider dietary uptake to be the main route of exposure for the snails. Metal coergisms are very diverse; mixtures of Cu and Zn can interact both synergistically and antagonistically, depending on the test concentrations and model organism 60,61. Moreover, in the environment, metals may interact with other biotic (e.g. parasites) and abiotic factors (e.g. nutrients, temperature), thus increasing the difficulty of extrapolating toxic effects from the laboratory into the field.. Field studies Field experiments allow for testing of research questions under natural conditions and are thus considered to have a higher external validity (i.e. environmental relevance) compared to laboratory studies. Field studies are at the base of all the papers presented in this PhD thesis. We have shown that high concentrations of metals are present in the paint of the boat hulls and that these metals are transferred into the biofouling matrix. This may have a negative impact both on animals feeding on contaminated biofouling, as well as 21.

(252) on the quality of the soil on which the biofouling is discarded. Furthermore, we evaluated the chronic toxicity of AF paints on populations of snails by combining the natural variability in the environment with some degree of experimental manipulation in order to control specific parameters (e.g. we controlled exposure duration as well as the presence of food and protection from predators). In both papers 3 and 4, we transplanted caged snails from their original habitat to test sites. In this way, we could control not only the exposure time, but also ensure that the individuals used in the experiments had similar background exposures and similar genetic diversity at the start of the experiments. Exposure history is important because contaminants exert a selective pressure that can lead to adaptation or acclimatisation of organisms, in which case their responses to contaminants would be different than that of organisms lacking exposure history. Moreover, high genetic diversity is a trait that may increase the populations’ resilience to stressors, by selection of favourable genes. This lack of consideration of prior exposure burdens and possible adaptations are a weakness in current risk assessment strategies of organisms in the field. We therefore address the role of both exposure history and genetic diversity of the snails with respect to toxic responses (paper 4). In addition, the presence of multiple stressors (both biotic and abiotic) and mixture toxicity (focused on metals) are taken into consideration in papers 3 and 4, in an attempt to provide a relevant assessment of the toxic effects observed in harbour-exposed snails. In paper 3 we investigated the effects of harbour contaminants on the main life-history traits of snails, namely growth, reproduction and mortality. Growth and reproduction decreased considerably and mortality increased in harbour-exposed snails, compared to snails from the reference sites. The same pattern of mortality was observed in paper 4 as well (Fig. 6). In addition to the effects on life-history traits, we investigated MT levels in the snails (paper 3). MTs are non-enzymatic proteins that are involved in the normal metabolism of essential metals and which are upregulated by exposure to elevated concentrations of both essential and non-essential metals 62. In our study, the MT levels were the highest in snails caged at the most contaminated site, most likely indicating the snails’ efforts to detoxify the metals. However, on average, the MT levels were not significantly lower at the reference sites, compared to the harbours, indicating that other environmental parameters, rather than contaminants, are affecting the MT expression. Reproduction, for instance, is a process known to indirectly increase the MT levels in some invertebrates, by causing changes in body weight and oestrogen levels 63. Thus, due to the natural variability in biomarker responses, it is important to investigate multiple types of endpoints simultaneously (e.g. molecular and physiological) in order to correctly interpret observations and obtain more conclusive results on the toxicity of contaminants.. 22.

(253)

(254)

(255)

(256) Conclusion and future perspectives. This thesis illustrates several concerning aspects regarding the use of AF paints in the Baltic Sea, ranging from the extent of their use on boat hulls, to metal contamination of the biofouling and of the environment and toxicity to non-target species. We have shown that high levels of metals are present in AF paints on leisure boats, sometimes contrary to the regulations, and that these metals are transferred to the biofouling mass which often is discarded on the boatyard soil at the end of the boating season. Moreover, this contaminated biofouling may represent a risk for organisms feeding upon it. Furthermore, we have shown that the use of AF paints is associated with chronic toxic effects on the snail Theodoxus fluviatilis (e.g. reduced reproduction and growth, increased frequency of tissue alterations, increased mortality). We believe that consideration of mixture effects and multiple stressors (e.g. parasites, nutrient levels) in the natural environment is crucial for managing existing and emerging AF contaminants. In order to further improve the environmental assessment of AF paint use, several studies can be employed to complement our work. For example, the toxic responses seen in the field should, ideally, be confirmed in controlled laboratory experiments that would reduce the signal to noise ratio. Interactions between parasites and metals require more study. These will be best understood by designing experiments in which each of these stressors is carefully controlled so that a cause-effect relationship can be established. Knowledge about the infection process, immune responses in snails and the sensitivity of parasites to metals is needed. Well-defined Cu:Zn ratios could be used in mixture exposure scenarios to investigate MT induction in snails. Moreover, information on the background levels of MT is lacking from our field studies (i.e. time zero) and thus would be an important aspect to look into. There are currently no published papers on any molecular biomarkers measured in T. fluviatilis so little is known about variations with abiotic factors, inducibility, sensitivity or fluctuations with biological changes in, for example, nutritional or reproductive status. In a future field experiment, MT should also be measured at different time points in order to see how this biomarker is affected by shortterm versus long term exposure and find the optimal response time. 26.

(257) Further investigations may also include studying the F1 generation of experimental snails in order to assess whether the chronically-exposed snails produce viable offspring. Observations of the snails’ behaviour might also be valuable for identifying the presence of avoidance mechanisms of e.g. contaminated food in T. fluviatilis. Different behaviours might explain the difference in mortality between the two populations studied in paper 4.. 27.

(258) References. 1. Dafforn KA, Lewis JA, Johnston EL. Antifouling strategies: History and regulation, ecological impacts and mitigation. Marine Pollution Bulletin. 2011;62(3):453– 465. 2. Viarengo A, Ponzano E, Dondero F, Fabbri R. A Simple Spectrophotometric Method for Metallothionein Evaluation in Marine Organisms: an Application to Mediterranean and Antarctic Molluscs. 1997;44(1):69–84. 3. Gergs R, Koester M, Grabow K, Schöll F, Thielsch A, Martens A. Theodoxus fluviatilis’ re-establishment in the River Rhine: a native relict or a cryptic invader? Conservation Genetics. 2015;16(1):247–251. 4. Ytreberg E, Lundgren L, Bighiu MA, Eklund B. New analytical application for metal determination in antifouling paints. Talanta. 2015;143:121–126. 5. Dürr S, Thomason J, editors. Biofouling. 1st ed. Chichester, West Sussex [England] ; Ames, Iowa: Wiley-Blackwell; 2010. 429 p. 6. Chambers LD, Stokes KR, Walsh FC, Wood RJK. Modern approaches to marine antifouling coatings. Surface and Coatings Technology. 2006;201(6):3642–3652. 7. Lehaitre M, Delauney L, Compère C. Biofouling and underwater measurements. Real-time observation systems for ecosystem dynamics and harmful algal blooms: Theory, instrumentation and modelling. Oceanographic Methodology Series. UNESCO, Paris. 2008:463–493. 8. Woods Hole Oceanographic Institution. Marine fouling and its prevention. Annapolis, Maryland: United States Naval Institute; 1952. 9. Dobretsov S, Abed RMM, Voolstra CR. The effect of surface colour on the formation of marine micro and macrofouling communities. Biofouling. 2013;29(6):617–627. 10. Satheesh S, Wesley SG. Influence of substratum colour on the recruitment of macrofouling communities. Journal of the Marine Biological Association of the United Kingdom. 2010;90(05):941–946. 11. IMO, International Maritime Organization. International Shipping Facts and Figures – Information Resources on Trade, Safety, Security, Environment. 2012. 12. IMO, International Maritime Organization. Status of multilateral Conventions and instruments in respect of which the International Maritime Organization or its Secretary-General performs depositary or other functions. 2016. 13. European Environment Agency. Late lessons from early warnings: science, precaution, innovation. 2013. Report No.: 1. 28.

(259) 14. Swedish Chemicals Agency. Antifouling products: pleasure boats, commercial vessels, nets, fish cages and other underwater equipment. 1993. Report No.: 2/93. 15. Guardiola FA, Cuesta A, Meseguer J, Esteban MA. Risks of Using Antifouling Biocides in Aquaculture. International Journal of Molecular Sciences. 2012;13(12):1541–1560. 16. Schiff K, Diehl D, Valkirs A. Copper emissions from antifouling paint on recreational vessels. Marine Pollution Bulletin. 2004;48(3–4):371–377. 17. Warnken J, Dunn RJK, Teasdale PR. Investigation of recreational boats as a source of copper at anchorage sites using time-integrated diffusive gradients in thin film and sediment measurements. Marine Pollution Bulletin. 2004;49(9–10):833– 843. 18. Swedish Chemicals Agency. Kemiska ämnen i båtbottenfärger – en undersökning av koppar, zink och Irgarol 1051 runt Bullandö marina 2004. 2006. Report No.: 2/06 (in Swedish). 19. Schiff K, Brown J, Diehl D, Greenstein D. Extent and magnitude of copper contamination in marinas of the San Diego region, California, USA. Marine Pollution Bulletin. 2007;54(3):322–328. 20. Ejhed H, Palm Cousins A, Karlsson M, Köhler SJ, Huser B, Westerberg I. Feasibility study of net load of metals: Particulate fraction and retention of metals in lakes and rivers. 2011. 21. Karlsson J, Eklund B. New biocide-free anti-fouling paints are toxic. Marine Pollution Bulletin. 2004;49(5–6):456–464. 22. Watermann BT, Daehne B, Sievers S, Dannenberg R, Overbeke JC, Klijnstra JW, Heemken O. Bioassays and selected chemical analysis of biocide-free antifouling coatings. Chemosphere. 2005;60(11):1530–1541. 23. Jaspers C, Møller LF, Kiørboe T. Salinity Gradient of the Baltic Sea Limits the Reproduction and Population Expansion of the Newly Invaded Comb Jelly Mnemiopsis leidyi Browman H, editor. PLoS ONE. 2011;6(8):e24065. 24. Ojaveer H, Jaanus A, MacKenzie BR, Martin G, Olenin S, Radziejewska T, Telesh I, Zettler ML, Zaiko A. Status of Biodiversity in the Baltic Sea. PLoS ONE. 2010;5(9):e12467. 25. IMO, International Maritime Organization. Particularly Sensitive Sea Areas. 2005 http://www.imo.org/en/OurWork/Environment/PSSAs/Pages/Default.aspx Accessed 2017.01.19 26. Nekoro M. State of the Baltic Sea: Background paper. 2013. 27. HELCOM. Checklist of Baltic Sea Macro-species. 2012. Report No.: Baltic Sea Environment Proceedings No. 130. 28. Skoog G. Aspects on the biology and ecology of Theodoxus fluviatilis (L) and Lymnea peregra (O.F Müller) (Gastropoda) in the Northern Baltic. Sweden: Stockholm University; 1978.. 29.

(260) 29. Eklund B. Disposal of plastic end-of-life-boats. Nordic Council of Ministers; 2013. 30. Dobretsov S. Biofouling on artificial substrata in Muscat waters. Journal of Agricultural and Marine Sciences Vol. 2011;19(1):24–29. 31. Swedish Transport Agency. Båtlivsundersökningen 2015 - En undersökning om svenska fritidsbåtar och hur de används. 2016. Report No.: TSG 2016-534 (in Swedish). 32. Barranguet C, Charantoni E, Plans M, Admiraal W. Short-term response of monospecific and natural algal biofilms to copper exposure. European Journal of Phycology. 2000;35(4):397–406. 33. Swedish Environmental Protection Agency. Riktvärden för förorenad mark. 2009. Report No.: 5976. 34. Coglianese M. The Effects of Salinity on Copper and Silver Toxicity to Embryos of the Pacific Oyster. 1982;11:297–303. 35. Grosell M, Blanchard J, Brix KV, Gerdes R. Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates. Aquatic Toxicology. 2007;84(2):162–172. 36. Swedish Chemicals Agency. 2017. http://www.kemi.se/global/bekampningsmed el/biocidprodukter/batbottenfarger/batbottenfarger-for-ostkusten.pdf 37. Swedish Chemicals Agency. 2017. http://www.kemi.se/global/bekampningsmed el/biocidprodukter/batbottenfarger/batbottenfarger-for-vastkusten.pdf 38. Eklund B, Elfström M, Borg H. Tributyltin originates from pleasure boats in Sweden in spite of firm restrictions. Open environmental sciences. 2008;2:124–132. 39. Eklund B, Eklund D. Pleasure Boatyard Soils are Often Highly Contaminated. Environmental Management. 2014;53(5):930–946. 40. Gauglitz G, Vo-Dinh T, editors. Handbook of spectroscopy. Weinheim ; [Cambridge]: Wiley-VCH; 2003. 2 p. 41. Lagerström M, Strand J, Eklund B, Ytreberg E. Total tin and organotin speciation in historic layers of antifouling paint on leisure boat hulls. Environmental Pollution. 2017;220:1333–1341. 42. Hall L, Anderson R. A Deterministic Ecological Risk Assessment for Copper in European Saltwater Environments. 1999;38:207–218. 43. Norwegian Institute for Water Research. PNEC for metals in the marine environment derived from species sensitivity distributions. 2007. Report No.: 27053. 44. Swedish Chemicals Agency. Lead in articles – a government assignment reported by the Swedish Chemicals Agency and the Swedish Environmental Protection Agency. 2007. Report No.: 5/07. 45. Neira C, Levin LA, Mendoza G, Zirino A. Alteration of benthic communities associated with copper contamination linked to boat moorings. Marine Ecology. 2014;35(1):46–66.. 30.

(261) 46. Sim VXY, Dafforn KA, Simpson SL, Kelaher BP, Johnston EL. Sediment Contaminants and Infauna Associated with Recreational Boating Structures in a MultiUse Marine Park McKindsey CW, editor. PLOS ONE. 2015;10(6):e0130537. 47. Karlsson J, Ytreberg E, Eklund B. Toxicity of anti-fouling paints for use on ships and leisure boats to non-target organisms representing three trophic levels. Environmental Pollution. 2010;158(3):681–687. 48. Wang W-X, Rainbow PS. Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates. Ecotoxicology and Environmental Safety. 2005;61(2):145–159. 49. White SL, Rainbow PS. On the metabolic requirements for copper and zinc in molluscs and crustaceans. Marine Environmental Research. 1985;16(3):215–229. 50. Viarengo A. Biochemical effects of trace metals. Marine Pollution Bulletin. 1985;16(4):153–158. 51. Santore RC, Di Toro DM, Paquin PR, Allen HE, Meyer JS. Biotic ligand model of the acute toxicity of metals. 2. Application to acute copper toxicity in freshwater fish and Daphnia. Environmental Toxicology and Chemistry. 2001;20(10):2397– 2402. 52. Sokolova I, Lannig G. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change. Climate Research. 2008;37(2–3):181–201. 53. Flemming CA, Trevors JT. Copper toxicity and chemistry in the environment: a review. Water, Air, and Soil Pollution. 1989;44(1–2):143–158. 54. Das S, Khangarot BS. Bioaccumulation of copper and toxic effects on feeding, growth, fecundity and development of pond snail Lymnaea luteola L. Journal of Hazardous Materials. 2011;185(1):295–305. 55. Rogevich EC, Hoang TC, Rand GM. Effects of Sublethal Chronic Copper Exposure on the Growth and Reproductive Success of the Florida Apple Snail (Pomacea paludosa). Archives of Environmental Contamination and Toxicology. 2009;56(3):450–458. 56. Besser JM, Dorman RA, Hardesty DL, Ingersoll CG. Survival and Growth of Freshwater Pulmonate and Nonpulmonate Snails in 28-Day Exposures to Copper, Ammonia, and Pentachlorophenol. Archives of Environmental Contamination and Toxicology. 2016;70(2):321–331. 57. Münzinger A, Guarducci M-L. The effect of low zinc concentrations on some demographic parameters of Biomphalaria glabrata (Say), Mollusca: Gastropoda. Aquatic toxicology. 1988;12(1):51–61. 58. Grosell M, Brix KV. High net calcium uptake explains the hypersensitivity of the freshwater pulmonate snail, Lymnaea stagnalis, to chronic lead exposure. Aquatic Toxicology. 2009;91(4):302–311. 59. Marigómez I, Soto M, Cajaraville MP, Angulo E, Giamberini L. Cellular and subcellular distribution of metals in molluscs: Distribution of Metals in Molluscs. Microscopy Research and Technique. 2002;56(5):358–392.. 31.

(262) 60. Daka ER, Hawkins SJ. Interactive Effects of Copper, Cadmium and Lead on Zinc Accumulation in the Gastropod Mollusc Littorina Saxatilis. Water, Air, & Soil Pollution. 2006;171(1–4):19–28. 61. Obiakor M. and Ezeonyejiaku C. Copper–zinc coergisms and metal toxicity at predefined ratio concentrations: Predictions based on synergistic ratio model. Ecotoxicology and Environmental Safety. 2015;117:149–154. 62. Amiard J, Amiardtriquet C, Barka S, Pellerin J, Rainbow P. Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers. Aquatic Toxicology. 2006;76(2):160–202. 63. Machreki-Ajmi M, Rebai T, Hamza-Chaffai A. Variation of metallothionein-like protein and metal concentrations during the reproductive cycle of the cockle Cerastoderma glaucum from an uncontaminated site: A 1-year study in the Gulf of Gabès area (Tunisia). Marine Biology Research. 2011;7(3):261–271.. 32.

(263) Acknowledgements. There are many people who had an impact on my life as a PhD student and to whom I am really thankful. First of all, my parents, without whom I wouldn’t have been here. I know it has not been easy, but I do appreciate all your efforts supporting me throughout my studies. I am also grateful for the help and patience shown by all of my supervisors. Thank you, Ann-Kristin, for always being there for me and for all your efforts! Thank you, Britta, for your enthusiasm that made everything sound so exciting and easy (though the easy part was a trap!). Thank you, Bethanie, for bringing in some fresh ideas towards the last bit of my thesis and for the positive thoughts. Elena, thank you for the huge amount of work you’ve put in our manuscript. I’ve learnt a lot from you, though it was quite a painful process. Anna Sobek, thank you for listening and providing sensible advice! Matt Bennett, you’ve saved me so many times from total despair caused by centrifuge issues and for that I am really grateful! Karin Nyström, thanks for always being so helpful and not getting (too) mad when I’m late with my bills! All the lovely TA ladies from ACESx, thank you for your time answering my questions and for always being so kind to ask me how it is going -. All the ‘ITM chicks’ who have made my breaks, weekends, trips and birthdays more enjoyable, thank you! You are awesome! ♥ Time is one of the things I value the most. So, I am grateful to all my other colleagues and co-authors who were kind enough to share their time with me, either for scientific discussions or just for fun. I’m also happy for the human interactions outside the walls of ACES, so thanks, Cristina T., for all the fun times and Brian H. for the ‘wise’ life and chemistry advice. My friends in Romania, thanks for the good times we have whenever we meet or just chat! Damien, there are a gazillion things to thank you for, but you know I like stuff to fit on one page. Thanks for everything! You’re the nicest human! 33.

(264)

(265)

No results found