DNA Adductomics for the Biological Effect Assessment of Contaminant Exposure in Marine Sediments.

Exposure to chemical pollution can induce genetic and epigenetic alterations, developmental changes, and reproductive disorders, leading to population declines in polluted environments. These effects are triggered by chemical modifications of DNA nucleobases (DNA adducts) and epigenetic dysregulation. However, linking DNA adducts to the pollution load in situ remains challenging, and the lack of evidence-based DNA adductome response to pollution hampers the development and application of DNA adducts as biomarkers for environmental health assessment. Here, we provide the first evidence for pollution effects on the DNA modifications in wild populations of Baltic sentinel species, the amphipod Monoporeia affinis. A workflow based on high-resolution mass spectrometry to screen and characterize genomic DNA modifications was developed, and its applicability was demonstrated by profiling DNA modifications in the amphipods collected in areas with varying pollution loads. Then, the correlations between adducts and the contaminants level (polycyclic aromatic hydrocarbons (PAHs), trace metals, and pollution indices) in the sediments at the collection sites were evaluated. A total of 119 putative adducts were detected, and some (5-me-dC, N6-me-dA, 8-oxo-dG, and dI) were structurally characterized. The DNA adductome profiles, including epigenetic modifications, differed between the animals collected in areas with high and low contaminant levels. Furthermore, the correlations between the adducts and PAHs were similar across the congeners, indicating possible additive effects. Also, high-mass adducts had significantly more positive correlations with PAHs than low-mass adducts. By contrast, correlations between the DNA adducts and trace metals were stronger and more variable than for PAHs, indicating metal-specific effects. These associations between DNA adducts and environmental contaminants provide a new venue for characterizing genome-wide exposure effects in wild populations and apply DNA modifications in the effect-based assessment of chemical pollution.

DNA epigenetic marks are linked to embryo aberrations in amphipods.

Linking exposure to environmental stress factors with diseases is crucial for proposing preventive and regulatory actions. Upon exposure to anthropogenic chemicals, covalent modifications on the genome can drive developmental and reproductive disorders in wild populations, with subsequent effects on the population persistence. Hence, screening of chemical modifications on DNA can be used to provide information on the probability of such disorders in populations of concern. Using a high-resolution mass spectrometry methodology, we identified DNA nucleoside adducts in gravid females of the Baltic amphipods Monoporeia affinis, and linked the adduct profiles to the frequency of embryo malformations in the broods. Twenty-three putative nucleoside adducts were detected in the females and their embryos, and eight modifications were structurally identified using high-resolution accurate mass data. To identify which adducts were significantly associated with embryo malformations, partial least squares regression (PLSR) modelling was applied. The PLSR model yielded three adducts as the key predictors: methylation at two different positions of the DNA (5-methyl-2'-deoxycytidine and N6-methyl-2'-deoxyadenosine) representing epigenetic marks, and a structurally unidentified nucleoside adduct. These adducts predicted the elevated frequency of the malformations with a high classification accuracy (84%). To the best of our knowledge, this is the first application of DNA adductomics for identification of contaminant-induced malformations in field-collected animals. The method can be adapted for a broad range of species and evolve as a new omics tool in environmental health assessment.

Linking consumer physiological status to food-web structure and prey food value in the Baltic Sea.

Declining physiological status in marine top consumers has been observed worldwide. We investigate changes in the physiological status and population/community traits of six consumer species/groups in the Baltic Sea (1993-2014), spanning four trophic levels and using metrics currently operational or proposed as indicators of food-web status. We ask whether the physiological status of consumers can be explained by food-web structure and prey food value. This was tested using partial least square regressions with status metrics for gray seal, cod, herring, sprat and the benthic predatory isopod Saduria as response variables, and abundance and food value of their prey, abundance of competitors and predators as predictors. We find evidence that the physiological status of cod, herring and sprat is influenced by competition, predation, and prey availability; herring and sprat status also by prey size. Our study highlights the need for management approaches that account for species interactions across multiple trophic levels.

Nitrogen fixation by cyanobacteria stimulates production in Baltic food webs.

Filamentous, nitrogen-fixing cyanobacteria form extensive summer blooms in the Baltic Sea. Their ability to fix dissolved N2 allows cyanobacteria to circumvent the general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for the food web. However, the fate of the nitrogen fixed by cyanobacteria remains unresolved, as does its importance for secondary production in the Baltic Sea. Here, we synthesize recent experimental and field studies providing strong empirical evidence that cyanobacterial nitrogen is efficiently assimilated and transferred in Baltic food webs via two major pathways: directly by grazing on fresh or decaying cyanobacteria and indirectly through the uptake by other phytoplankton and microbes of bioavailable nitrogen exuded from cyanobacterial cells. This information is an essential step toward guiding nutrient management to minimize noxious blooms without overly reducing secondary production, and ultimately most probably fish production in the Baltic Sea.

Genetic diversity in Monoporeia affinis at polluted and reference sites of the Baltic Bothnian Bay.

The amphipod Monoporeia affinis plays an important role in the Baltic Sea ecosystem as prey and as detritivore. The species is monitored for contaminant effects, but almost nothing is known about its genetics in this region. A pilot screening for genetic variation at the mitochondrial COI gene was performed in 113 individuals collected at six sites in the northern Baltic. Three coastal sites were polluted by pulp mill effluents, PAHs, and trace metals, and two coastal reference sites were without obvious connection to pollution sources. An off-coastal reference site was also included. Contaminated sites showed lower levels of genetic diversity than the coastal reference ones although the difference was not statistically significant. Divergence patterns measured as ΦST showed no significant differentiation within reference and polluted groups, but there was significant genetic divergence between them. The off-coastal sample differed significantly from all coastal sites and also showed lower genetic variation.

Development of tools for integrated monitoring and assessment of hazardous substances and their biological effects in the Baltic Sea.

The need to develop biological effects monitoring to facilitate a reliable assessment of hazardous substances has been emphasized in the Baltic Sea Action Plan of the Helsinki Commission. An integrated chemical-biological approach is vitally important for the understanding and proper assessment of anthropogenic pressures and their effects on the Baltic Sea. Such an approach is also necessary for prudent management aiming at safeguarding the sustainable use of ecosystem goods and Services. The BEAST project (Biological Effects of Anthropogenic Chemical Stress: Tools for the Assessment of Ecosystem Health) set out to address this topic within the BONUS Programme. BEAST generated a large amount of quality-assured data on several biological effects parameters (biomarkers) in various marine species in different sub-regions of the Baltic Sea. New indicators (biological response measurement methods) and management tools (integrated indices) with regard to the integrated monitoring approach were suggested.

Exposure to contaminants exacerbates oxidative stress in amphipod Monoporeia affinis subjected to fluctuating hypoxia.

Fitness and survival of an organism depend on its ability to mount a successful stress response when challenged by exposure to damaging agents. We hypothesized that co-exposure to contaminants may exacerbate oxidative stress in hypoxia-challenged benthic animals compromising their ability to recover upon reoxygenation. This was tested using the amphipod Monoporeia affinis exposed to hypoxia followed by reoxygenation in sediments collected in polluted and pristine areas. In both sediment types, oxygen radical absorbance capacity (ORAC) and antioxidant enzyme activities [superoxide dismutase (SOD) and catalase (CAT)] increased during hypoxia, suggesting that M. affinis has a strategy of preparation for oxidative stress that facilitates recovery after a hypoxic episode. Exposure to contaminants altered this anticipatory response as indicated by higher baselines of ORAC and SOD during hypoxia and no response upon reoxygenation. This coincided with significantly elevated oxidative damage evidenced by a marked reduction in glutathione redox status (ratio of reduced GSH/oxidized GSSG) and an increase in lipid peroxidation (TBARS levels). Moreover, RNA:DNA ratio, a proxy for protein synthetic activity, decreased in concert with increased TBARS, indicating a linkage between oxidative damage and fitness. Finally, inhibited acetylcholinesterase (AChE) activity in animals exposed to contaminated sediments suggested a neurotoxic impact, whereas significant correlations between AChE and oxidative biomarkers may indicate connections with redox state regulation. The oxidative responses in pristine sediments suggested a typical scenario of ROS production and removal, with no apparent oxidative damage. By contrast, co-exposure to contaminants caused greater increase in antioxidants, lipid peroxidation, and slowed recovery from hypoxia as indicated by CAT, GSH/GSSG, TBARS and AChE responses. These results support the hypothesized potential of xenobiotics to hamper ability of animals to cope with fluctuating hypoxia. They also emphasize the importance of understanding interactions between antioxidant responses to different stressors and physiological mechanisms of oxidative damage.

Low dose TBT exposure decreases amphipod immunocompetence and reproductive fitness.

The antifouling agent tributyltin (TBT) is a highly toxic pollutant present in many aquatic ecosystems. Despite of regulations on the usage of TBT, it remains in high concentrations in sediments both in harbors and in off-shore sites. The toxicity of TBT in mollusks is well documented. However, adverse effects in other aquatic organisms, such as crustaceans, are less well known. This study is an effort to assess the effects of environmentally realistic concentrations of TBT on an ecologically important species in Swedish fresh and brackish water ecosystems, the benthic amphipod Monoporeia affinis. Field collected animals were exposed during gonad maturation to TBT (70 and 170 ng/g sediment d wt) for five weeks in static microcosms with natural sediment. Exposure concentrations were chosen to reflect effects at concentrations found in Swedish coastal sediment, but below expected effects on survival. TBT exposure resulted in a statistically significant adverse effect on oocyte viability and a doubling of the prevalence of microsporidian parasites in females, from 17% in the control to 34% in the 170 ng TBT/g sediment d wt exposure. No effects on survival were observed. Borderline significant effects were observed on male sexual maturation in the 70 ng TBT/g d wt exposure and on ecdysteroid levels in the 170 ng/g sediment d wt exposure. Both reproduction and parasite infection effects are of ecological importance since they have the potential to affect population viability in the field. This study gives further evidence to the connection between low dose contaminant exposure and increases in microsporidian parasite infection.

Single and combined effects of hypoxia and contaminated sediments on the amphipod Monoporeia affinis in laboratory toxicity bioassays based on multiple biomarkers.

In estuaries, hypoxic conditions and pollution are among the major factors responsible for the declines in habitat quality, yet little is known about their combined effects on estuarine organisms. In this study, to investigate single and combined effects of hypoxia and contaminated sediment, the Baltic amphipod Monoporeia affinis was exposed for 5-9 days to four different combinations of oxygen conditions (moderate hypoxia vs. normoxia) and contamination (polluted vs. unpolluted sediments) at environmentally realistic levels. To detect oxidative stress, a suite of biomarkers was used - antioxidant enzymes [superoxide dismutases (SOD), catalase (CAT), and glutathione S-transferases (GST)], acetylcholinesterase (AChE), lipid peroxidation status (TBARS concentration), protein carbonyl content (PCC), and DNA strand breakage (DNA-SB). To assay effects at the organism level, we used RNA:DNA ratio as a proxy for growth and metabolic rate and mortality. There were significant increases in CAT and SOD activities and TBARS levels in response to both moderate hypoxia and contaminated sediment, while GST increased and AChE decreased in response to the contamination only. Significant positive correlations were observed among the antioxidant enzymes and between the enzyme activities and TBARS concentration, suggesting a complex response to the oxidative stress. No significant changes in PCC were recorded in any of the treatments. Furthermore, the negative effect of hypoxia on DNA integrity was significant; with frequency of DNA-SB increasing in animals exposed to hypoxia in contaminated sediment. Despite clear effect at the cellular and biochemical levels, no responses at the organism level were observed. Multivariate analyses of the dataset have allowed us to link exposure factors to individual biomarker responses. Of the potential biomarkers assessed in this study, CAT activity was found to be associated with hypoxia, while SOD, GST and AChE activities appear to predict best the effects of exposure to sediments containing several contaminants (e.g. heavy metals, PCBs and PAHs), and TBARS concentration is particularly indicative of combined effects of hypoxia and contamination. In addition to providing new knowledge on the combined effects of multiple stressors on estuarine organisms, the findings of the present study are also important to understand data from biomonitoring studies in the Baltic Sea and in other regions where multiple stress factors co-occur.

Perfluorooctane sulfonate accumulation and parasite infestation in a field population of the amphipod Monoporeia affinis after microcosm exposure.

Perfluorooctane sulfonate (PFOS) is the focus of intense toxicity research due to its persistence and widespread occurrence in biota. Studies on benthic invertebrates have shown them to be subjects of high PFOS exposure. However, effects on benthic invertebrates exposed to PFOS in the field are still far from elucidated. To fill a knowledge gap on concentrations and effects in benthic invertebrates, a microcosm study on the benthic amphipod Monoporeia affinis was performed. Field collected M. affinis were analysed for PFOS and showed average background concentrations 39 and 58 ng/g (wet weight) in two different samplings. The field collected animals were exposed to three concentrations of PFOS (50, 200 and 5000 microg PFOS/L water) for 3 weeks during gonad development. Body burdens of PFOS were determined after experiment termination. Results showed negative effects on survival and reproduction effects such as decreased sexual maturation and decreased oocyte viability caused by PFOS exposure. Additionally, a follow-up experiment demonstrated a significant increase in the infection incidence by a microsporidian muscle parasite in animals exposed to PFOS at tissue concentrations in the range of concentrations found in field collected benthic amphipods. This is the first study to demonstrate increased microsporidian infection with pollutant exposure and it suggests that ecologically relevant PFOS concentrations could be sufficient to elicit these effects.

Interactions between climate change and contaminants.

There is now general consensus that climate change is a global threat and a challenge for the 21st century. More and more information is available demonstrating how increased temperature may affect aquatic ecosystems and living resources or how increased water levels may impact coastal zones and their management. Many ecosystems are also affected by human releases of contaminants, for example from land based sources or the atmosphere, which also may cause severe effects. So far these two important stresses on ecosystems have mainly been discussed independently. The present paper is intended to increase awareness among scientists, coastal zone managers and decision makers that climate change will affect contaminant exposure and toxic effects and that both forms of stress will impact aquatic ecosystems and biota. Based on examples from different ecosystems, we discuss risks anticipated from contaminants in a rapidly changing environment and the research required to understand and predict how on-going and future climate change may alter risks from chemical pollution.

Reproductive effects of the endocrine disruptor fenarimol on a Baltic amphipod Monoporeia affinis.

An endocrine disruptor, the fungicide fenarimol, was investigated regarding its effects on reproduction and hormone (ecdysteroid) levels in the deposit-feeding amphipod Monoporeia affinis. In addition, the influence of food shortage, both by itself and in combination with fenarimol, on reproduction was examined. Field-collected amphipods were exposed in flow-through microcosms during the period of sexual maturation and mating in four treatment series: Control with low food, fenarimol with low food, control with high food, and fenarimol with high food. Fenarimol was added at a concentration of 0.3 mg/L in two pulses/ week. Results show that fenarimol has a negative effect on fertilization rate and male mating ability. Results were supported by a tendency toward delayed male sexual development. Food shortage decreased weight in both sexes and retarded female oocyte development. Higher ecdysteroid levels were recorded in males than in females, and food shortage increased male ecdysteroid levels. No effect of fenarimol exposure on ecdysteroid levels was observed. No synergistic effects of fenarimol and food shortage could be distinguished in any variable examined. Thus, M. affinis was vulnerable to reproductive impairment by fenarimol, with effects on the next generation (i.e., a disturbed sexual development and fertilization ability). Food shortage has negative effects on M. affinis, but it does not enhance the effects of fenarimol.

Evaluation of the role of black carbon in attenuating bioaccumulation of polycyclic aromatic hydrocarbons from field-contaminated sediments.

The significance of black carbon (BC) for the bioavailability of polycyclic aromatic hydrocarbons (PAHs) was examined by using historically contaminated intact sediment cores in laboratory exposure experiments with the deposit-feeding amphipod Monoporeia affinis. Log values of amphipod biota-sediment accumulation factors (BSAFs) were significantly related to log BC, whereas log BSAFs were related to log octanol-water partition coefficients only in background sediments containing less BC. In the background sediments, the BSAF for polycyclic aromatic hydrocarbons (PAHs) was 1 to 2 for phenanthrene, with lower values for more hydrophobic PAHs, indicating an increase in nonequilibrium conditions with increasing PAH molecular size. For the near-equilibrated phenanthrene and fluoranthene, higher BSAFs were measured during exposure to background sediments, with BSAF decreasing to <0.1 in contaminated sediments in the Stockholm waterways. In situ caged mussels (Dreissena polymorpha) exhibited field BSAF values (relative to sediment-trap-collected suspended matter) for polychlorinated biphenyls (PCBs) of 0.1 to 0.4, but for PAHs of similar hydrophobicity and molecular size, the field BSAFs were much lower and in the range 0.002 to 0.05. This PAH-PCB dichotomy is consistent with recently reported much stronger binding to diesel soot (a form of BC) for PAHs than for PCBs of equal hydrophobicities. Lower BSAFs for the near-equilibrated PAHs (phenanthrene and fluoranthene) in the urban sediments relative to the background sediments were consistent with the larger presence of BC in the urban sediments. This study provides the first linked BSAF-BC field data that supports a causal relationship between strong soot sorption and reduced bioavailability for PAHs.

Dynamics of lipids and polychlorinated biphenyls in a Baltic amphipod (Monoporeia affinis): a field study.

Processes such as accumulation and elimination, which control tissue concentration of polychlorinated biphenyls (PCBs), were examined over time in an in situ study of the amphipod Monoporeia affinis. These processes were studied with respect to individual PCB congeners, percentage lipid and composition, and body weight. A secondary objective was to examine the impact of seasonal variability in percentage lipids and lipid composition on PCB concentration in two coexisting Baltic amphipods, M. affinis and Pontoporeia femorata. Polycholorinated biphenyl concentrations tended to be higher in P. femorata than in M. affinis, possibly because of P. femorata's lower respiration rate and larger size. The net accumulation of PCBs was congener dependent and negatively correlated to lipid concentration. The relation between the net concentration change rate of 16 PCB congeners over time and log Kow was not significant during the spring and summer months, a time when lipid accumulation and strong growth occur. In contrast, the net concentration change rate of the corresponding PCB congeners over time during autumn and early winter, that is, from the period before gonad maturation to the period after mating and early embryogenesis, showed a significant relation to tog Kow (r2 = 0.62, p < 0.001, n = 16). During the latter period, amphipod lipid weight was reduced while the PCB body burden increased. Results strongly indicate that elimination rather than accumulation is the main process controlling amphipod body burden. This pattern results in a transfer of PCBs from the female to the developing embryos, which is reflected in high PCB body burden in newly released offspring.

Bioavailability of metals to the amphipod Monoporeia affinis: interactions with authigenic sulfides in urban brackish-water and freshwater sediments.

Could reduced eutrophication be a potential environmental threat because of increased mobility and bioavailability of trace metals? This question was addressed by oxygenating intact sediment cores, varying in redox potential and salinity, in a test system containing the amphipod Monoporeia affinis. Results show a low mobility of metals during oxygenation, and despite high concentrations of metals in sediments, only Pb showed a notable degree of bioaccumulation. Cadmium was bioaccumulated particularly in freshwater sediment, and body burden of Cd was related to salinity, porewater, and sediment concentrations. Despite high sediment and porewater concentrations of Cu and Zn, no relationship was recorded to body burden. For three of four tested metals, Cd, Pb, and Zn, metals in sediment were more important for body burdens in amphipods as compared to metals in porewater. Food, rather than interstitial water, therefore seems to be the main route of metal contaminants to these amphipods. Furthermore, this observed low release of metals from sediments and low body burden significance of porewater metals indicate that ameliorated oxygen conditions in contaminated sediments may be regarded as a minor environmental threat for one of the most important Baltic benthic organisms.