New approach in the assessment of impact of arsenic-based chemical warfare agents on marine environment

Recently, abandoned chemical weapons (CWs) containing toxic chemical warfare agents (CWAs) manufactured during the World Wars (WWs) have raised concern not only for environmental but also safety reasons. Until the 1970s, chemical munitions and containers filled with CWAs were disposed of worldwide mainly by sea-dumping. After WWII, huge dumping operations took place in the Baltic Sea area and the Skagerrak Strait, where the loads of dumped CWs were approximately 50,000 and 170,000 tons, respectively. During the last 15 years, the sea-dumped munitions have been addressed in many international, multidisciplinary projects, which have led to growing knowledge on the chemical threat caused by munitions and thus raised public awareness on this issue. Several investigations dealing with the identification, determining of exact location of sites and corrosion stages of dumped warfare objects have been accomplished. Based on the information gained from several sediment sampling campaigns in the Baltic Sea area and the Skagerrak Strait, it has been clearly demonstrated that corroded munitions are releasing toxic CWAs to the surrounding marine environment. To date, very few studies are available regarding the biological effects of sea- dumped CWAs. Negative biological effects have been observed in various fish species like cod, herring and hagfish caught from the different dumping sites in the Baltic Sea and the Skagerrak, as well as in the blue mussels deployed in cages in the dumping area. However, none of these studies have been able to link the observed negative effects to the CWA-related chemicals. Although it has already been proven that the content of the munitions leak releasing toxic CWAs into the marine environment, the impacts of these chemicals in marine biota is still unknown. Currently, the knowledge on environmental toxicity of CWAs is quite scarce, limited to a few studies. Hence, the information on bioaccumulation, biotransformation reactions, as well as toxicity threshold values are crucial for the assessment of the environmental risks possessed by munitions laying on the seabed. The aim of this research was to contribute to negligible knowledge on bioaccumulation and biotransformation of phenylarsenic CWAs in a marine environment. Phenylarsenic chemicals are of great interest due to their existence in marine sediment samples taken from several dumping sites, their potential to bioaccumulate and potential toxicity. Sophisticated liquid chromatographic-mass spectrometric techniques enabled the study of samples collected from the dumping sites and from in vitro metabolism experiments. These techniques together with in vitro toxicity studies led to increased knowledge on the environmental behavior of sea-dumped phenylarsenic CWAs. The results presented in this thesis contribute to enhanced risk assessment of sea-dumped chemical munitions providing new information on the bioaccumulation of CWA-related phenylarsenic chemicals, their metabolism, toxicity, and biotransformation products present in the marine environment.Maailmansotien jälkeen kemiallisia taisteluaineita hävitettiin upottamalla niitä meriin. Itämereen ja Skagerrakin alueelle on upotettu arviolta noin 50 000 ja 170 000 tonnia kemiallisia taisteluaineita sisältäviä ammuksia. Viimeisen 15 vuoden aikana käynnissä olleiden kansainvälisten, poikkitieteellisten tutkimushankkeiden aikana on pystytty tunnistamaan mereen upotettuja ammuksia, paikantamaan niiden tarkkoja sijainteja sekä pystytty määrittämään paikannettujen ammusten korroosioasteita. Sedimenttinäytteiden kemiallisten analyysien perusteella on pystytty todentamaan, että ruostuneet ammukset vuotavat myrkyllisiä kemiallisia yhdisteitä ympäröivään meriympäristöön. On olemassa vain muutama tutkimus mereen upotettujen kemiallisten taisteluaineiden biologisista vaikutuksista. Negatiivisia biologisia vaikutuksia on havaittu eri upotusalueilta pyydetyistä kalalajeista, kuten turskista, silleistä ja viiksiympyräsuisista. Havaittuja negatiivisia vaikutuksia ei ole kuitenkaan pystytty yhdistämään kemiallisiin taisteluaineisiin. Vaikka on pystytty todentamaan, että ammusten sisältö vuotaa ympäröivään meriympäristöön, on vuotavien yhdisteiden vaikutus merieliöstöön vielä tuntematon. Tällä hetkellä tieto yhdisteiden ympäristömyrkyllisyydestä on suppea rajoittuen vain muutamaan tutkimukseen. Tämän takia tietoa yhdisteiden biokertymisestä, biotransformaatioreaktioista sekä myrkyllisyyden raja-arvoista ovat tärkeitä, arvioitaessa merenpohjassa sijaitsevien ammusten ympäristövaikutuksia. Tämän väitöskirjatutkimuksen tavoitteena oli saada tietoa kemiallisina taisteluaineina käytettyjen fenyyliarseeniyhdisteiden biokertymisestä ja biotransformaatiosta meriympäristössä. Fenyyliarseeniyhdisteet valikoituvat tutkimuksen kohteeksi, koska niiden pitoisuudet upotuspaikoita otetuissa sedimenttinäytteissä ovat korkeita sekä niiden on ennustettu olevat biokertyviä. Kehittyneillä nestekromatografia-massaspektrometrisillä menetelmillä pystyttiin tutkimaan upotusaluilta kerättyjä näytteitä sekä in vitro aineenvaihduntaa. Nämä tekniikat yhdistettyinä in vitro myrkyllisyystutkimuksiin johtivat lisääntyneeseen ymmärrykseen tutkittavien yhdisteiden käyttäytymisestä meriympäristössä. Tässä opinnäytetyössä esitetyt tulokset auttavat parantamaan mereen upotettujen kemiallisten ammusten riskinarviointia tarjoamalla uutta tietoa kemiallisina taisteluaineina käytettyjen fenyyliarseeniyhdisteiden biokertymisestä, niiden aineenvaihdunnasta, myrkyllisyydestä ja meriympäristössä esiintyvistä biotransformaatiotuotteista

Acute aquatic toxicity of arsenic-based chemical warfare agents to Daphnia magna

Sea dumping of chemical warfare (CW) took place worldwide during the 20th century. Submerged CW included metal bombs and casings that have been exposed for 50-100 years of corrosion and are now known to be leaking. Therefore, the arsenic-based chemical warfare agents (CWAs), pose a potential threat to the marine ecosystems. The aim of this research was to support a need for real-data measurements for accurate risk assessments and categorization of threats originating from submerged CWAs. This has been achieved by providing a broad insight into arsenic-based CWAs acute toxicity in aquatic ecosystems. Standard tests were performed to provide a solid foundation for acute aquatic toxicity threshold estimations of CWA: Lewisite, Adamsite, Clark I, phenyldichloroarsine (PDCA), CWA-related compounds: TPA, arsenic trichloride and four arsenic-based CWA degradation products. Despite their low solubility, during the 48 h exposure, all CWA caused highly negative effects on Daphnia magna. PDCA was very toxic with 48 h D. magna LC50 at 0.36 mu g x L-1- and Lewisite with EC50 at 3.2 mu g x L-1 . Concentrations at which no immobilization effects were observed were slightly above the analytical Limits of Detection (LOD) and Quantification (LOQ). More water-soluble CWA degradation products showed no effects at concentrations up to 100 mg x L-1.Peer reviewe

Taisteluaineina käytetyt fenyyliarseeniyhdisteet ja niiden glutationimetaboliittien valmistus sekä toksisuus

Fenyyliarseeniyhdisteet luokitellaan myrkyllisiksi yhdisteiksi, joilla on voi olla joko syövyttäviä ja/tai pahoinvointia aiheuttavia vaikutuksia. Niitä on kehitetty kemiallisiksi taisteluaineiksi ensimmäisestä maailmansodasta lähtien. Toisen maailmansodan jälkeen arseeniyhdisteiden hävittäminen on tapahtunut hautaamalla niitä mereen ja maaperään. Ajan kuluessa yhdisteet vuotavat säilytysastioistaan ympäristöön aiheuttaen vaaraa ympäristölle. Yhdisteet hapettuvat, metyloituvat ja hydrolysoituvat joutuessaan ympäristöön joko veden, hapen tai maaperän bakteeritoiminnan johdosta. Tässä tutkielmassa käsitellään kemiallisina taisteluaineina käytettyjä fenyyliarseeniyhdisteitä, niiden hajoamistuotteita sekä arseeniyhdisteiden metaboliaa ja toksisuutta. Koska arseeni(III)yhdisteet reagoivat endogeenisten tioliryhmien kanssa, on niiden todettu erittyvän nisäkkäillä sappeen glutationikonjugaatteina. Glutationi on solunsisäinen tripeptidi, jolla on tärkeä rooli solunsisäisissä puolustusmekanismeissa. Se pystyy myös pelkistämään ja konjugoitumaan elimistöön joutuneen vierasaineen kanssa muodostaen vesiliukoisemman yhdisteen, joka pystytään erittämään sappeen. Muodostuneet konjugaatit ovat epästabiileja ja niiden määrittäminen biologisesta materaalista on haastavaa, mutta ei mahdotonta. Määritettäessä biologisesta materiaalista arseeniyhdisteiden konjugaatteja, tarvitaan yhdisteille puhtaita malliaineita sekä sisäisen standardin menetelmä. Tutkielman kokeellinen osio käsittelee fenyyliarseeniyhdisteiden glutationikompleksien valmistusta, puhdistusta ja tunnistusta. Tunnistusmenetelminä on käytetty pääasiassa LC-MSmenetelmiä sekä 1H NMR-spektroskopiaa. Kokeellinen osio käsittelee myös eri vaihtoehtoja sisäisen standardin valmistukseen. Valmistetut kompleksit osoittautuivat epästabiileiksi vesiliuoksessa ja ne alkavat hajota eientsymaattisen hydrolyysin vaikutuksesta nopeasti; kolmen kuukauden säilytyksen jälkeen kaikki kompleksit olivat hajonneet. Kompleksit hajoavat lisäksi LC-analyysin aikana joko kolonnimateriaalin tai käytetyn eluentin vaikutuksesta

Metabolism and cytotoxicity of diphenylarsinic acid, a degradation product of sea-dumped chemical warfare agents, in a rainbow trout liver cell line

Recent studies have found primary degradation products of phenylarsenic chemical warfare agents (CWAs) accumulating in fish tissues, while the potential effects of these dumped phenylarsenic CWAs, such as Clark I and II, in the Baltic Sea biota are poorly understood. In this study, the metabolism and cytotoxicity of diphenylarsinic acid (DPA), a primary degradation product of phenylarsenic CWA, was studied by incubating rainbow trout cell line RTL-W1 cells in media with 100 mg/L DPA. Previously undescribed metabolites were identified by ultra-high performance liquid chromatography–high resolution mass spectrometry (UPHLC-HRMS). Moreover, the cytotoxicity of diphenylarsine glutathione conjugate (DPA-SG), the major metabolite of DPA, was studied. Cytotoxicity of the compounds was evaluated using the Neutral Red retention test (NRR), showing an IC50 value of 278 mg/L for DPA and 1.30 mg/L for DPA-SG, indicating that the glutathione (GSH) conjugate of DPA is more than two orders of magnitude toxic than DPA itself, suggesting that toxic properties of DPA are increased after conjugation with intracellular GSH leading enhanced toxicity after uptake. Results gained in this study give more detailed information for elucidating biological effects of dumped chemical munitions in marine environment. Moreover, the results help in assessing the environmental and health risks posed by marine munition continued presence and deterioration in the sea bottom.Peer reviewe

Detection of Chemical Warfare Agent-Related Phenylarsenic Compounds in Marine Biota Samples by LC-HESI/MS/MS

A new method has been developed to determine oxidation products of three chemical warfare agent (CWA) related phenylarsenic compounds from marine biota samples by a liquid chromatography-heated electrospray ionization/tandem mass spectrometry (LC-HESI/MS/MS). The target chemicals were oxidation products of Adamsite (DM­[ox]), Clark I (DPA­[ox]), and triphenylarsine (TPA­[ox]). Method was validated within the concentration range of 1–5, 0.2–5, and 0.2–5 ng/g for DM­[ox], DPA­[ox], and TPA­[ox], respectively. The method was linear, precise and accurate. Limits of quantification (LOQ) were 2.0, 1.3, and 2.1 ng/g for DM­[ox], DPA­[ox], and TPA­[ox], respectively. A total of ten fish samples and one lobster sample collected from near Swedish coast, Måseskär dumpsite were analyzed. Trace concentrations below LOQ values were detected in three samples and the elemental composition of oxidized form of Clark I and/or II was confirmed by LC-HESI/HRMS. To our knowledge, this is the first study that provides the presence of CWA related chemicals in marine biota samples

Biological effects of dumped chemical weapons in the Baltic Sea: A multi-biomarker study using caged mussels at the Bornholm main dumping site

After World War II, thousands of tons of highly toxic chemical warfare agents (CWA) were deposited in the Baltic Sea, the main dumping site locating in the Bornholm Basin. In the present study, Baltic mussels (Mytilus trossulus) were transplanted in the area in cages at two hotspot sites and a reference site at the depths of 35 and 65 m for 2.5 months to study bioaccumulation and biological effects of CWA possibly leaking from the corroding warfare materials. No traces of degradation products of the measured phenylarsenic CWA could be detected in the tissues of mussels. Nevertheless, several biochemical and histochemical biomarkers, geno- and cytotoxicity indicators, and bioenergetic parameters showed significant responses. The Integrated Biomarker Index calculated from the single biomarkers also showed a higher total response at the two hotspot areas compared to the reference site. Although no direct evidence could be obtained confirming the responses being caused specifically by exposure to CWA, the field exposure experiment showed unambiguously that organisms in this sea area are confronting environmental stress affecting negatively their health and this is likely related to chemical contamination, which is possibly connected to the sea-dumped CWA

Chemical warfare agents and their risk assessment in <i>Daphnia magna</i> and fish in the Baltic Sea - 15 years of measurements

The Baltic Sea is one of the world's largest brackish waters, it is drained through the Danish Straits into the Kattegat, and almost enclosed by nine countries. The Baltic Sea is one of the world's most polluted water bodies thus facing a wide range of environmental threats towards its water resources, such as fish stocks, and coastal environments and economics. Chemical warfare agents (CWAs) that were dumped following the Second World War are known to occur in intact or degraded states in sediments and are documented to affect benthic fauna and fish as well as having injured fishermen having accidentally caught lumps of CWAs in their nets. However, a thorough mapping of remaining CWAs and degradation products and an understanding of the environmental risks does not exist although more than 75 years have passed. This study compiles and analyzes published/peer-reviewed data, generated since 2005 in five selected comprehensive studies, on sediment measurements of known dumped CWAs and degradation products in the Baltic Sea. As a worst-case approach, sediment con-centrations are transformed to concentrations in near-bottom water, which represents Predicted Environmental Concentrations (PECs) to marine biota. To investigate the accuracy and representativeness of toxicological values, which are fundamental in risk assessments, two cases are considered: Case 1 (specificity) uses toxico-logical data (EC50 or NOEC) for Daphnia magna and fish with applied assessment factors (AF) to derive Predicted No-Effect Concentrations (PNECs) for organoarsenical and non-arsenical CWAs; Case 2 (robustness) uses partly Danish Environmental Quality Standards (DK EQS) for arsenicals, and partly a Water Quality Criterion (WQC) for arsenicals, representing the marine environment. From 872 data points risk quotients (RQs=PEC/PNEC) are calculated. In Case 1 exceedances of risk for the sum of chemicals (sumRQ>1) occur 24 and 1 times for Daphnia magna and fish, respectively, without applying AFs. 263 and 120 exceedances are found for Daphnia magna and fish, respectively, when applying AFs. Case 2 shows 0 (WQC) and 25 (DK EQS) exceedances for arsenicals when using more robust toxicological values, however, at the expense of specificity of chemicals and target species. The results underline the importance of obtaining more representative and accurate toxicological data (lowering AFs) in order to increase the accuracy of the risk estimates. This quantitative state of risk towards representative marine species indicates that there are indeed potential risks, and it qualifies the understanding and debate on the challenges and future actions regarding dumped chemical munitions in the Baltic Sea.Peer reviewe

Detection of chemical warfare agents-related phenylarsenic compounds and multi-biomarker responses in cod (Gadus morhua) from chemical munition dumpsites in the Baltic Sea

Recently, sea-dumped chemical weapons (CWs) containing toxic chemical warfare agents (CWAs) have raised international attention. It is well known that CWAs are leaking from corroded munitions causing a risk to the surrounding marine environment, while the impact on marine biota is still unknown. In this study, cod (Gadus morhua) was used as a model species to study the possible bioaccumulation of phenylarsenic CWAs and their negative effects at multiple levels of biological organization on fish living in the vicinity of a major CWs dumpsite in the Bornholm Basin in the Baltic Sea. In total, 14% of the cod muscle samples collected close to the main dumpsite contained trace levels of phenylarsenic CWAs. However, most of the biomarkers measured did not show clear differences between this area compared with a lesser contaminated reference area. On the other hand, significant changes in some biomarkers were observed in individuals containing trace levels of CWA-related chemicals. The results gained in this study have significant importance for environmental risk assessment and for evaluating the risk of CWA contamination for human seafood consumers