Ecotoxicological Risk of World War Relic Munitions in the Sea after Low- and High-Order Blast-in-Place Operations.

Submerged munitions from World War I and II are threatening human activities in the oceans, including fisheries and shipping or the construction of pipelines and offshore facilities. To avoid unforeseen explosions, remotely controlled "blast-in-place" (BiP) operations are a common practice worldwide. However, after underwater BiP detonations, the toxic and carcinogenic energetic compounds (ECs) will not completely combust but rather distribute within the marine ecosphere. To shed light on this question, two comparable World War II mines in Denmark's Sejerø Bay (Baltic Sea) were blown up by either low-order or high-order BiP operations by the Royal Danish Navy. Water and sediment samples were taken before and immediately after the respective BiP operation and analyzed for the presence of ECs with sensitive GC-MS/MS and LC-MS/MS technology. EC concentrations increased after high-order BiP detonations up to 353 ng/L and 175 µg/kg in water and sediment, respectively, while low-order BiP detonations resulted in EC water and sediment concentrations up to 1,000,000 ng/L (1 mg/L) and >10,000,000 µg/kg (>10 g/kg), respectively. Our studies provide unequivocal evidence that BiP operations in general lead to a significant increase of contamination of the marine environment and ecotoxicological risk with toxic ECs. Moreover, as compared to high-order BiP detonations, low-order BiP detonations resulted in a several 1000-fold higher burden on the marine environment.

Warship wrecks and their munition cargos as a threat to the marine environment and humans: The V 1302 "JOHN MAHN" from World War II.

In addition to endangering sea traffic, cable routes, and wind farms, sunken warship wrecks with dangerous cargo, fuel, or munitions on board may emerge as point sources for environmental damage. Energetic compounds such as TNT (which could leak from these munitions) are known for their toxicity, mutagenicity, and carcinogenicity. These compounds may cause potential adverse effects on marine life via contamination of the marine ecosystem, and their entry into the marine and human food chain could directly affect human health. To ascertain the impending danger of an environmental catastrophe posed by sunken warships, the North Sea Wrecks (NSW) project (funded by the Interreg North Sea Region Program) was launched in 2018. Based on historical data (derived from military archives) including the calculated amount of munitions still on board, its known location and accessibility, the German World War II ship "Vorpostenboot 1302" (former civilian name - "JOHN MAHN") was selected as a case study to investigate the leakage and distribution of toxic explosives in the marine environment. The wreck site and surrounding areas were mapped in great detail by scientific divers and a multibeam echosounder. Water and sediment samples were taken in a cross-shaped pattern around the wreck. To assess a possible entry into the marine food chain, caged mussels were exposed at the wreck, and wild fish (pouting), a sedentary species that stays locally at the wreck, were caught. All samples were analyzed for the presence of TNT and derivatives thereof by GC-MS/MS analysis. As a result, we could provide evidence that sunken warship wrecks emerge as a point source of contamination with nitroaromatic energetic compounds leaking from corroding munitions cargo still on board. Not only did we find these explosive substances in bottom water and sediment samples around the wreck, but also in the caged mussels as well as in wild fish living at the wreck. Fortunately so far, the concentrations found in mussel meat and fish filet were only in the one-digit ng per gram range thus indicating no current concern for the human seafood consumer. However, in the future the situation may worsen as the corrosion continues. From our study, it is proposed that wrecks should not only be ranked according to critical infrastructure and human activities at sea, but also to the threats they pose to the environment and the human seafood consumer.

Carbonyl reduction of 4-oxonon-2-enal (4-ONE) by Sniffer from D. magna and D.pulex.

The α, ß-unsaturated aldehydes 4-oxonon-2-enal (4ONE) and 4-hydroxynon-2-enal (4HNE) are products of unsaturated fatty acids and ROS, and can be formed in lipid-rich tissues such as neurons. As strong electrophiles, both compounds react with DNA and proteins, and are capable of inactivating enzymes. However, both the human carbonyl reductase and the carbonyl reductase Drosophila melanogaster Sniffer are known to reduce 4ONE, a major lipid peroxidation product, to a less or non-toxic form. In this study, products formed during carbonyl reduction of 4ONE and 4HNE by recombinant Sniffer proteins from Daphnia magna and Daphnia pulex were investigated. A high-performance liquid chromatography analysis showed that Sniffer from D. magna converted 35.6% of 4ONE to 11.9% HNO and 23.7% 4HNE, while D. pulex converted 34.5% of this substrate to 14.8% HNO and 19.7% 4HNE. Thus, 4HNE is the main product formed from the sniffer-mediated reduction of 4ONE. The kinetic parameters obtained from the reduction of 4ONE were Km = 13.9 ± 2.1 µM, kcat = 1.53 s-1, kcat/km = 0.11 s-1 µM-1 for D. magna Sniffer and Km = 29.2 ± 4.3 µM, kcat = 0.64 s-1, kcat/km = 0.02 s-1 µM-1 for D. pulex Sniffer. These results demonstrate that Sniffer from D. magna and D. pulex are important enzymes involved in the carbonyl reductive biotransformation of 4ONE, a cytotoxic lipid peroxidation product. Noteworthy, the catalytic properties of both Daphnia Sniffer enzymes reflect previous findings with Sniffer from Drosophila melanogaster.

Can seafood from marine sites of dumped World War relicts be eaten?

Since World War I, considerable amounts of warfare materials have been dumped at seas worldwide. After more than 70 years of resting on the seabed, reports suggest that the metal shells of these munitions are corroding, such that explosive chemicals leak out and distribute in the marine environment. Explosives such as TNT (2,4,6-trinitrotoluene) and its derivatives are known for their toxicity and carcinogenicity, thereby posing a threat to the marine environment. Toxicity studies suggest that chemical components of munitions are unlikely to cause acute toxicity to marine organisms. However, there is increasing evidence that they can have sublethal and chronic effects in aquatic biota, especially in organisms that live directly on the sea floor or in subsurface substrates. Moreover, munition-dumping sites could serve as nursery habitats for young biota species, demanding special emphasis on all kinds of developing juvenile marine animals. Unfortunately, these chemicals may also enter the marine food chain and directly affect human health upon consuming contaminated seafood. While uptake and accumulation of toxic munition compounds in marine seafood species such as mussels and fish have already been shown, a reliable risk assessment for the human seafood consumer and the marine ecosphere is lacking and has not been performed until now. In this review, we compile the first data and landmarks for a reliable risk assessment for humans who consume seafood contaminated with munition compounds. We hereby follow the general guidelines for a toxicological risk assessment of food as suggested by authorities.

Exposure to dissolved TNT causes multilevel biological effects in Baltic mussels (Mytilus spp.).

Baltic mussels (Mytilus spp.) were exposed to the explosive trinitrotoluene (TNT) for 96 h (0.31-10.0 mg/L) and 21 d (0.31-2.5 mg/L). Bioaccumulation of TNT and its degradation products (2- and 4-ADNT) as well as biological effects ranging from the gene and cellular levels to behaviour were investigated. Although no mortality occurred in the concentration range tested, uptake and metabolism of TNT and responses in antioxidant enzymes and histochemical biomarkers were observed already at the lowest concentrations. The characteristic shell closure behaviour of bivalves at trigger concentrations led to complex exposure patterns and non-linear responses to the exposure concentrations. Conclusively, exposure to TNT exerts biomarker reponses in mussels already at 0.31 mg/L while effects are recorded also after a prolonged exposure although no mortality occurs. Finally, more attention should be paid on shell closure of bivalves in exposure studies since it plays a marked role in definining toxicity threshold levels.

The explosive trinitrotoluene (TNT) induces gene expression of carbonyl reductase in the blue mussel (Mytilus spp.): a new promising biomarker for sea dumped war relicts?

Millions of tons of all kind of munitions, including mines, bombs and torpedoes have been dumped after World War II in the marine environment and do now pose a new threat to the seas worldwide. Beside the acute risk of unwanted detonation, there is a chronic risk of contamination, because the metal vessels corrode and the toxic and carcinogenic explosives (trinitrotoluene (TNT) and metabolites) leak into the environment. While the mechanism of toxicity and carcinogenicity of TNT and its derivatives occurs through its capability of inducing oxidative stress in the target biota, we had the idea if TNT can induce the gene expression of carbonyl reductase in blue mussels. Carbonyl reductases are members of the short-chain dehydrogenase/reductase (SDR) superfamily. They metabolize xenobiotics bearing carbonyl functions, but also endogenous signal molecules such as steroid hormones, prostaglandins, biogenic amines, as well as sugar and lipid peroxidation derived reactive carbonyls, the latter providing a defence mechanism against oxidative stress and reactive oxygen species (ROS). Here, we identified and cloned the gene coding for carbonyl reductase from the blue mussel Mytilus spp. by a bioinformatics approach. In both laboratory and field studies, we could show that TNT induces a strong and concentration-dependent induction of gene expression of carbonyl reductase in the blue mussel. Carbonyl reductase may thus serve as a biomarker for TNT exposure on a molecular level which is useful to detect TNT contaminations in the environment and to perform a risk assessment both for the ecosphere and the human seafood consumer.

Marine bivalves as bioindicators for environmental pollutants with focus on dumped munitions in the sea: A review.

The seas worldwide are threatened by a "new" source of pollution. Munitions dumped into the seas worldwide will corrode and start to leak. Their impacts on the environment and on human health are now more than ever subject of scientific research. Bivalves are a first choice bioindicator and their importance is demonstrated in numerous worldwide studies as well as their integration in important monitoring programs. In this review, the use of mussels in context with marine pollutants in recent years is pointed out in general but with a special focus on dumped conventional and chemical munitions. Monitoring experiments with mussels are able to generate large data sets, which should be mandatory included in decision support tools to increase their weight of evidence. The usefulness of mussels with regard to dumped munitions has clearly been documented in recent years and the further application of this important biomonitoring system is strongly recommended.

"Don't Blast": blast-in-place (BiP) operations of dumped World War munitions in the oceans significantly increase hazards to the environment and the human seafood consumer.

The seas worldwide are threatened by a "new" source of pollution: millions of tons of all kind of warfare material have been dumped intentionally after World War I and II, in addition to mine barriers, failed detonations as well as shot down military planes and sunken ship wrecks carrying munitions. For example, in the German parts of the North and Baltic Sea approximately 1.6 million metric tons of toxic conventional explosives (TNT and others) and more than 5000 metric tons of chemical weapons are present. Such unexploded ordnance (UXO) constitutes a direct risk of detonation with increased human access (fisheries, water sports, cable constructions, wind farms and pipelines). Moreover, after more than 70 years of resting on the seabed, the metal shells of these munitions items corrode, such that chemicals leak out and distribute in the marine environment. Explosive chemicals such as TNT and its derivatives are known for their toxicity and carcinogenicity. In order not to endanger today's shipping traffic or the installation of pipelines and offshore plants by uncontrolled explosions, controlled blast-in-place (BiP) operations of these dangerous relics is a common practice worldwide. However, blast-in-place methods of in situ munitions disposal often result in incomplete (low-order) detonation, leaving substantial quantities of the explosive material in the environment. In the present free field investigation, we placed mussels (Mytilus spp.) as a biomonitoring system in an area of the Baltic Sea where BiP operations took place and where, by visual inspections by scientific divers, smaller and larger pieces of munitions-related materials were scattered on the seafloor. After recovery, the mussels were transferred to our laboratory and analyzed for TNT and its derivatives via gas chromatography and mass spectroscopy. Our data unequivocally demonstrate that low-order BiP operations of dumped munitions in the sea lead to multiple increases in the concentration of TNT and its metabolites in the mussels when compared to similar studies at corroding but still encased mines. For this reason, we explicitly criticize BiP operations because of the resulting environmental hazards, which can ultimately even endanger human seafood consumers.

Carbonyl reductase sniffer from the model organism daphnia: Cloning, substrate determination and inhibitory sensitivity.

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (kcat/Km = 2.6 s-1 x µM-1) and isatin (kcat/Km = 1.5 s-1 x µM-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with kcat/Km = 0.23 s-1 µM-1 and kcat/Km = 0.35 s-1 µM-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 µM for sniffer from D. magna, Ki = 12.0 µM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.

Bioaccumulation of 2,4,6-trinitrotoluene (TNT) and its metabolites leaking from corroded munition in transplanted blue mussels (M. edulis).

Bioaccumulation of 2,4,6-trinitrotoluene (TNT) and its main metabolites 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) leaking from corroded munitions at a munitions dumping site (Kolberger Heide, Germany) was evaluated in transplanted blue mussels (Mytilus edulis). Six moorings with mussel bags were placed east and west at varying positions near the mine mound. In order to monitor any differences resulting from changing seasons, three exposure times were chosen. First exposure period: April-July 2016 (106 days); second exposure period: July-December 2016 (146 days); third exposure period: December 2016-March 2017 (92 days). We found amounts of 4-ADNT in mussel tissue ranging from 2.40 ±â€¯2.13 to 7.76 ±â€¯1.97 ng/(g mussel wet weight). Neither TNT nor 2-ADNT could be detected. Considering seasonal differences, orientation and distances of the moorings to the mine mound no correlation between levels in mussel tissue was evident.

Biomonitoring of 2,4,6-trinitrotoluene and degradation products in the marine environment with transplanted blue mussels (M. edulis).

Since World War I considerable amounts of warfare material have been dumped at sea worldwide, but little is known about the fate of the explosive components in the marine environment. Sea dumped munitions are able to contaminate the surroundings because of the release of explosive chemicals due to corrosion and breaching or by detonation after blast-operations. This implies the risk of accumulation of toxic compounds in human and wildlife food chains. With the help of divers, we performed an active biomonitoring study with transplanted blue mussels (M. edulis) in a burdened area (Kolberger Heide, Germany) with explosive compounds near blast craters over an exposure time of 93days. With this biomonitoring system, we could show that blue mussels accumulate 2,4,6-trinitrotoluene (TNT) and its metabolites 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) in their tissues. In all mussels deployed at the ground, we found a body burden with 2-ADNT of 103.75±12.77ng/g wet weight and with 4-ADNT of 131.31±9.53ng/g wet weight. TNT itself has been found in six mussels with an average concentration of 31.04±3.26ng/g mussel wet weight. In the mussels positioned at one meter above the ground no TNT nor 2-ADNT could be detected, but 4-ADNT was found in all samples with an average concentration of 8.71±2.88ng/g mussel wet weight. To the best of our knowledge, this is the first study using blue mussels M. edulis as an active biomonitoring system for TNT and its metabolites 2-ADNT and 4-ADNT in a free field experiment in a burdened area. Moreover, with this system, we unequivocally proved that these toxic explosives accumulate in the marine biota resp. in the marine food chain, thereby posing a possible risk to the marine ecosphere and human health.