Single substance and mixture toxicity of dibutyl-phthalate and sodium dodecyl sulphate to marine zooplankton.

Several contaminants exceed their environmental thresholds in Swedish marine surface waters. We ranked the toxicity-drivers among contaminants detected near heavy industry, based on toxicity to zooplankton, and identified di-butyl phthalate and sodium dodecyl sulphate as contaminants of concern. We tested their acute individual and mixture toxicity by assessing effects on mortality, biodiversity, algal growth, and copepod reproduction in natural mesozooplankton communities. The mixture effects were compared to Independent Action mixture model predictions. Egg production and algae growth were affected at 4 µmol/l DBP, and effects on mortality, algae growth and biodiversity were observed at 12 µmol/l SDS. The mixture (1 µmol/l DBP, 3 µmol/l SDS) affected all endpoints, and the observed effects were underestimated by 21% on average compared to predictions. We found that the successional trajectory in zooplankton communities was compound dependant, and that DBP and SDS are toxic to marine zooplankton, but at levels above measured environmental concentrations.

Short and long-term effects of low-sulphur fuels on marine zooplankton communities.

International shipping is responsible for the release of numerous contaminants to the air and the marine environment. In order to reduce airborne emissions, a global 0.5 % sulphur limit for marine fuels was implemented in January 2020. Recently, a new generation of so-called hybrid fuels that meet these new requirements have appeared on the market. Studies have shown that these fuels have physical properties that make conventional clean-up methods difficult, but few have studied their effects on marine life. We conducted short and long-term microcosm experiments with natural mesozooplankton communities exposed to the water accommodated fractions (WAFs) of the hybrid fuel RMD80 (0.1 % sulphur) and a Marine Gas Oil (MGO). We compared the toxicity of both fuel types in 48h short-term exposures, and studied the effects of the hybrid fuel on community structure over two generations in a 28-day experiment. The F0 generation was exposed for eight days and the F1 generation was raised for 22 days without exposure. GC-MS and GC-FID analysis of the WAFs revealed that the hybrid fuel was dominated by a mixture of volatile organic compounds (VOCs) and poly aromatic hydrocarbons (PAHs), whereas the MGO was mainly composed of VOCs. We observed significant short-term effects on copepod egg production from exposure to 25 % hybrid fuel WAF, but no effects from the MGO WAF at equivalent WAF dilution. In the long-term experiment with RMD80, the feeding rate was initially increased after exposure to 0.5-1.1 % hybrid fuel WAF, but this did not increase the copepod egg production. Significant change in community structure was observed after eight days in the F0 community at 0.5-3.3 % WAF. Indications of further alterations in species abundances was observed in the F1 community. Our results demonstrate that the MGO is a less toxic low-sulphur alternative to the hybrid fuel for marine zooplankton, and that a hybrid fuel spill could result in altered diversity of future generations of copepod communities.

Release of PAHs and heavy metals in coastal environments linked to leisure boats.

Leisure boats are responsible for elevated levels of heavy metals and PAHs in sediments in- and near marinas and natural harbours. As these compounds are released directly into the water column they also pose a threat to organisms in the pelagic environment. Passive samplers were deployed during peak and post tourist season in the water column of natural harbours, leisure boat waterways and small marinas to measure the dissolved fraction of PAHs and metal ions. Differences between seasons indicative of leisure boat activities were found as PAH composition differed between peak and post season for natural harbours and waterways, where heavier PAHs increased during peak season. During peak season, metal samplers were covered by biofouling, which likely affected the uptake. Post season metal concentrations differ between locations, with concentrations exceeding quality standards at near mainland locations where boats are maintained, compared to the sites in the archipelago.

Sediments indicate the continued use of banned antifouling compounds.

Antifouling paints are widely used to avoid organisms settling on boat hulls. The active ingredients in the paints have differed over the years where lead, TBT, irgarol and diuron have been deemed too harmful to non-target organisms and subsequently been banned within the EU. Most of these compounds however are persistent in the environment and can cause problems long after they are deposited. We have examined if present-day and banned substances used in antifouling paints can be found in sediments in a national park on the Swedish west coast. Sampled locations include waterways, natural harbours and small marinas for leisure crafts to investigate if number of visiting boats affect the concentration of antifouling compounds in sediments. Few significant differences were found when comparing the different locations types, suggesting that overall boat presence is more important than specific mooring sites, however, several banned antifouling compounds were found in the surface sediments.

Induced tolerance in situ to chronically PAH exposed ammonium oxidizers.

Sediment was sampled in the vicinity of a long-term source of Polycyclic Aromatic Hydrocarbons (PAHs) to evaluate whether tolerance can be induced in situ. Total PAH concentrations as well as the bioavailable PAHs were measured, and for nine PAHs the pore water concentration could be calculated. An induced tolerance in the ammonium oxidizing community was detected at the site with highest PAH concentration and tolerance was strongest, although not significantly, correlated to bioavailable alkylated PAHs. In addition, the tolerant microbial community showed a significant lower baseline capability for nitrification with an on average 35% reduction compared to the other sites. Meiofaunal community structure differed between all sites, and the difference was significantly correlated to bioavailable alkylated PAHs and PAH31 concentrations. The results suggest that in order to judge magnitude of long-term effects, the bioavailable fraction is to be preferred, and when possible as estimation of the freely dissolved concentration.

PAH effects on meio- and microbial benthic communities strongly depend on bioavailability.

The effects of anthropogenic pollutants in dissimilar habitats can vary depending on differences in bioavailability. The factors determining bioavailability are not yet fully understood. This study was performed to evaluate whether analysis of total PAH concentrations in sediments is a satisfactory measurement to indicate environmental effects or if bioavailability is needed to be taken into account. We have here performed a 60-day experiment, where nominal PAH concentrations of 1,300 µg/kg sediment were added to three different marine sediments. Meiofaunal and microbial communities were analyzed for alterations in community response at 30 and 60 days. Results showed that bioavailability of PAHs varied between the three different sediments. Nonetheless, the petroleum addition gave rise to significant negative effects on all three sediments at both time points. The two direct measurements of toxicity on the microbial community, potential nitrification and denitrification, displayed a lower effect of the PAH addition in the muddy sediment at both time points, compared to the other two sediment types. No effects were seen in the analysis of meiofaunal community structure. Measurements of PAH bioavailability in the three sediment types concurred with the results from the microbial community, revealing a lower bioavailability in the muddy sediment compared to the other two sediment types, 34% compared to sandy and 18% compared to organic at day 0. At day 60 it was 61% lower compared to sandy and 20% lower compared to organic. The negative effects of the PAH addition on the microbial nitrogen cycle were in six out of eight cases best correlated to the amount of alkylated bioavailable PAH in the sediments, and thus microbial nitrogen cycle is a possible good indicator for assessing PAH-induced stress. The results presented here have implications for risk analysis studies of petroleum-contaminated marine sediments; consequently, sediment characteristics and its effects on bioavailability are important to include. In addition, these results add to the understanding that bioavailability measurements of PAHs are a more correct assessment compared to measurements of total PAH concentrations, and need to be included when estimating effects of PAHs in marine benthic communities.

Endocrine-disrupting effects of compounds in Danish streams.

Effluents from municipal wastewater-treatment plants and scattered dwellings, as well as runoff from agricultural fields, are sources of endocrine-disrupting compounds (EDCs) in the aquatic environment. The present study investigated the correlation between the occurrence of EDCs in nine Danish streams using passive samplers (polar organic integrative samplers and silicone membranes) and determined their possible biological effects as assessed by mammal cell cultures and the mussel (Unio tumidus). The passive samplers and mussels were exposed simultaneously at the study sites. The extracts from the passive samplers were used to measure the concentrations of EDCs and the biological effects on the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR)-receptor transactivation. Male mussels were investigated for biomarkers of endocrine effects, such as the levels of vitellogenin-like proteins measured as alkali-labile phosphate (ALP). EDC concentrations, hormone-receptor transactivation (ER, AR, AhR), and level of ALP were greater downstream of wastewater-treatment plants compared with upstream sites and sites supposed to be relatively nonimpacted by wastewater. Furthermore, there was a significant positive correlation between in vitro AhR transactivation and frequency of ALP of male mussels. We conclude that wastewater effluent is an important source of endocrine-disrupting effects in the aquatic environment and that the combination of biological effect measurements and chemical analyses based on passive sampling is useful in the assessment of the ecological state of the aquatic environment.

Abnormalities in eelpout Zoarces viviparus upon chemical exposure.

Elevated frequencies of abnormal embryos in female eelpout Zoarces viviparus have been demonstrated in Danish, Swedish and German monitoring programmes at certain geographic locations with high levels of anthropogenic input. Pollutants present in areas with high malformation frequencies were selected and tested in a controlled laboratory experiment for their potential to induce abnormalities among eelpout embryos upon injection into pregnant eelpout. Tributyltin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, pyrene, nonylphenol, 2,2',4,4'-tetrabromophenylether and heptadecafluorooctanesulfonic acid were tested, either individually or combined. Generally, the chemicals were transferred to eggs and/or embryos. Some of the exposures increased the proportion of broods with more than 10% abnormal or 5% malformed embryos, although the average percentages of abnormal development were not affected. Spinal, cranial and eye deformities were evident, similarly to what is seen in nature. Some of the exposures resulted in increased percentages of females with as well a low reproductive capacity as embryos with a low condition index.

Lead isotopes in marine surface sediments reveal historical use of leaded fuel.

Analyses of lead (Pb) isotopes have been performed in terrestrial and fresh water environments to estimate historical uses of leaded fuel, but so far this method has not been employed in studies of world-wide marine surface sediments. We analyzed Pb and its isotopes in 23 surface sediments from four continents collected during the Galathea 3 expedition in 2006-2007. To enhance the anthropogenic signal, a partial digestion using nitric acid was performed. The concentrations of Pb, Th, U and Al were determined with an ICP-Quadrupole MS, and Pb-isotope ratios with an ICP-multi-collector MS. The samples could be divided into three groups: Harbor areas in larger cities with concentrations of 150 to 265 mg kg(-1) dry weight, smaller towns with concentrations between 20 and 40 mg kg(-1) dry weight, and remotely located sites with concentrations below 15 mg kg(-1) dry weight. Pb-isotope ratios were compared to literature values for gasoline and local or geological background values, and the contribution of leaded-gasoline to total concentrations was calculated for contaminated sites using both a one-dimensional and a novel two-dimensional (vector) method. The North American sites had Pb-isotope ratios corresponding to the US leaded gasoline, with 24-88% of the Pb from leaded gasoline. Samples from Oceania showed Pb-isotope ratios corresponding to Australian gasoline, with 60% attributed to leaded gasoline in Sydney and 21% in Christchurch. Outside Cape Town, 15 to 46% of Pb in sediments was from leaded gasoline.

Local contamination in relation to population genetic diversity and resilience of an arctic marine amphipod.

The objective of this study was to investigate whether populations inhabiting a contaminated environment are affected in terms of decreased genetic diversity due to selection of tolerant genotypes and if such effect carries a cost. Marine arctic amphipod populations (Orchomenella pinguis) were collected from sites within a contaminated fjord, as well as from sites outside the fjord on the west-coast of Greenland over three years (2006-2008). Impacts on genetic diversity, effects on resilience such as development of tolerance and cost were determined. AFLP-analysis was used to explore within and between population genetic diversity, and exposure studies were performed where the populations were subjected to known and unknown stressors to assess resilience. Populations collected at three contaminated sites all had reduced genetic diversity in 2007 compared to populations outside the fjord. This pattern was different in 2008 as all contaminated site populations increased in diversity, whereas a decrease in diversity occurred at the outer sites. However, tolerance, but even more so, cost, was related to contamination exposure in 2008, in spite of the shift in genetic diversity. We suggest that contamination rapidly induces effects that can be captured as tolerance and associated cost, whereas effects on genetic diversity can be difficult to separate from recent migration events that dilute eventual decreases in diversity due to contamination pressure. As long as impacted populations can be influenced by migration events that increase the genetic diversity and add health to an affected population, populations in contaminated areas may have enhanced probability of survival.

Meiofaunal and bacterial community response to diesel additions in a microcosm study.

Effects of low PAH-containing diesel were studied in a 60-day microcosm experiment at PAH concentrations 130, 1300 and 13,000µg/kg sediment. Nutrient fluxes, potential nitrification and meiofaunal community composition were analysed at three time points. Changed ∑NOx-fluxes indicated reduced sediment nitrification in Medium and High with time, in agreement with lowered potential nitrification rates in all treatments. Reduction in silicate and phosphate fluxes over time suggested severe effects on activity of meiofauna. Reduced activity increased the anoxic sediment layer, which could have contributed to the changed ∑NOx-fluxes. There were significant differences in meiofaunal community composition after 30 and 60days in Medium and High. Changes were due to increasing numbers of harpacticoids and the foraminiferan group Rotaliina, as well as decreasing numbers of Nematodes and the foraminiferan group Reophax. In spite of the low PAH-level, small additions of this diesel can still have pronounced effects on meiofaunal and bacterial communities.

Towards a renewed research agenda in ecotoxicology.

New concerns about biodiversity, ecosystem services and human health triggered several new regulations increasing the need for sound ecotoxicological risk assessment. The PEER network aims to share its view on the research issues that this challenges. PEER scientists call for an improved biologically relevant exposure assessment. They promote comprehensive effect assessment at several biological levels. Biological traits should be used for Environmental risk assessment (ERA) as promising tools to better understand relationships between structure and functioning of ecosystems. The use of modern high throughput methods could also enhance the amount of data for a better risk assessment. Improved models coping with multiple stressors or biological levels are necessary to answer for a more scientifically based risk assessment. Those methods must be embedded within life cycle analysis or economical models for efficient regulations. Joint research programmes involving humanities with ecological sciences should be developed for a sound risk management.

Aquatic toxicity of PAHs and PAH mixtures at saturation to benthic amphipods: linking toxic effects to chemical activity.

Organisms in marine sediments are usually exposed to mixtures of polycyclic aromatic hydrocarbons (PAHs), whereas risk assessment and management typically focus on the effects of single PAHs. This can lead to an underestimation of risk if the effects of single compounds are additive or synergistic. Because of the virtually infinite number of mixture-combinations, and the many different targeted organisms, it would be advantageous to have a model for the assessment of mixture effects. In this study we tested whether chemical activity, which drives the partitioning of PAHs into organisms, can be used to model the baseline toxicity of mixtures. Experiments were performed with two benthic amphipod species (Orchomonella pinguis and Corophium volutator), using passive dosing to control the external exposure of single PAHs and mixtures of three and four PAHs. The baseline toxicity of individual PAHs at water saturation generally increased with increasing chemical activity of the PAHs. For O. pinguis, the baseline toxicity of PAH mixtures was successfully described by the sum of chemical activities. Some compounds and mixtures showed a delayed expression of toxicity, highlighting the need to adjust the length of the experiment depending on the organism. On the other hand, some of the single compounds had a higher toxicity than expected, possibly due to the toxicity of PAH metabolites. We suggest that chemical activity of mixtures can, and should, be used in addition to toxicity data for single compounds in environmental risk assessment.

Ability of Pseudoalteromonas tunicata to colonize natural biofilms and its effect on microbial community structure.

We investigated the effectiveness of surface colonization by the epiphytic marine bacterium Pseudoalteromonas tunicata firstly on a complex biofilm community on glass slides, and secondly, on the epiphytic community of Ulva australis. The effectiveness of P. tunicata was compared with the performance of Phaeobacter sp. 2.10, also a marine epiphytic isolate in the U. australis colonization experiments. Pseudoalteromonas tunicata cells were able to colonize the glass slide community at densities found naturally in the water column (9.7 x 10(4) cells mL(-1)). However, P. tunicata was a poor invader of the epiphytic community on U. australis at densities of 10(6) cells mL(-1). At densities of 10(8) cells mL(-1), P. tunicata again exerted little impact on the epiphytic community. Phaeobacter sp. 2.10 was also a poor invader at lower densities, but was able to invade and become dominant at densities of 10(8) cells mL(-1). Differences in the ability of P. tunicata and Phaeobacter sp. 2.10 to invade natural communities may be due to differences in the antibacterial compounds produced by the two species. These experiments suggest that epiphytic communities may have protective effects compared with inanimate surfaces.

Pyrene toxicity is affected by the nutrient status of a marine sediment community: implications for risk assessment.

Risk assessment of toxicants often disregards that environmental conditions, like changing nutrient status, may affect ecosystem response to a toxicant even within an ecosystem. We investigated if effects of pyrene on shallow-water sediments depended on nutrient status of the sediment during 58 days of incubation. Natural undisturbed sediment cores were pre-exposed to two concentrations of inorganic nutrients (nitrogen and phosphorous) for 14 days. After terminating nutrient additions, pyrene was applied once to half the Nu(high) and half the Nu(low) cores in a concentration of 2 mg/kg DW, normalized to 1% TOC. Pyrene affected the sediment systems in both Nu(high) and Nu(low), but effects of pyrene differed between nutrient regimes. In the Nu(low) system, effects of pyrene were mainly seen on the sediment community structure, such as meiofauna community structure. On the contrary, effects of pyrene in Nu(high) were mainly seen on community functions, such as changes in inorganic nutrient fluxes. Direct and indirect effects of pyrene were observed in both nutrient regimes, but they operated on different timescales depending on the variable in focus. This study shows the need to include environmental factors such as nutrient status in risk assessment of toxicants.

The amphipod Orchomenella pinguis--a potential bioindicator for contamination in the Arctic.

Indigenous organisms can be used as bioindicators for effects of contaminants, but no such bioindicator has been established for Arctic areas. Orchomenella pinguis is a benthic amphipod, ubiquitous in the Arctic and can be found in high numbers. We collected O. pinguis at sites with different contamination levels. Population characteristics (body length distribution, average dry weight and amphipod organic content) were related to sediment contaminant concentrations, in order to identify suitable endpoints for using this species as a bioindicator. We show that O. pinguis was prevalent in both clean and contaminated areas, easy to sample and that its population characteristics could be linked to both contamination and sediment organic content. We suggest that O. pinguis is a suitable bioindicator for the Arctic, but that endpoints such as reproductive effects and phenotypic and genotypic responses are needed together with population characteristics to assess impacts of contamination.

Phototoxicity of pyrene affects benthic algae and bacteria from the Arctic.

Phototoxicity of polycyclic aromatic hydrocarbons (PAHs) in the Arctic is important to study since the future PAH load is likely to increase. In combination with the increased UV-light penetration due to ozone layer thinning, phototoxicity may be a potential problem for arctic areas. The aim of this study was to evaluate effects of pyrene and phototoxicity of pyrene on natural algae and bacteria from arctic sediments. Sediments from a shallow-water marine baywere spiked with different pyrene concentrations. Microcosms containing the sediment were incubated under three light regimes, natural sunlight with UV-light, natural sunlight without UV-light, and dark. Significant effects were evident at low pyrene concentrations, particularly in presence of UV-light, indicating phototoxicity. The microalgae were especially sensitive to the phototoxicity of pyrene. Already atthe lowest pyrene concentration (Cfree: 4 nM) algal 14C-incorporation and chlorophyll a content were reduced. The toxic effects of pyrene on the microalgae probably led to the release of organic matter. In agreement with this, bacterial activity increased at high pyrene concentrations indicated by increased oxygen consumption and increased release of inorganic N and P from the sediment. This study indicates that phototoxicity of PAHs may be relevant for sediment communities from shallow marine arctic areas at environmentally relevant pyrene concentrations.

Effects of pyrene on grazing and reproduction of Calanus finmarchicus and Calanus glacialis from Disko Bay, West Greenland.

The effects of pyrene on grazing and egg production of the ecologically important arctic copepods Calanus finmarchicus and Calanus glacialis were studied in the Disko Bay, Western Greenland. The effects of both passive uptake of pyrene by diffusion through membranes, and active uptake through ingestion of contaminated food were investigated for both species. Furthermore, the hatching success for eggs from exposed females and for eggs directly exposed to pyrene was also studied. There was no reduction in egg production in starved Calanus spp., which indicates that the uptake of pyrene through passive diffusion was limited. A significant reduction was observed in grazing and egg production for feeding C. finmarchicus exposed to the highest concentration of 100 nM pyrene within the experimental period. Furthermore, the time-dependent development in grazing and egg production was reduced for both species exposed to 100 nM, and we suggest that the effect of pyrene on these endpoints would increase with time. The observed difference in response time between the two species can be attributed to the differences in the amount of storage lipid and in their reproductive strategies. There was a lower hatching percentage for eggs from C. finmarchicus females exposed to 10nM pyrene, whereas the egg hatching percentage was not reduced for eggs exposed directly.

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples.

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.

Direct and indirect effects of the herbicides Glyphosate, Bentazone and MCPA on eelgrass (Zostera marina).

Eelgrass beds are important habitats for many organisms, but there has been a decline in the area covered by eelgrass during the last decades due to increased eutrophication resulting in increased shading from phytoplankton. The use of herbicides in terrestrial agriculture has also increased over the last century, and while the effects of herbicides on non-target organisms have been well studied in freshwater they are overlooked in coastal waters. It is not known if herbicides have any effect on the distribution of eelgrass (Zostera marina), or how natural phytoplankton communities respond to the same herbicides. Direct and indirect effects of the herbicides Glyphosate, Bentazone and MCPA both as single toxicants and as mixtures, on the eelgrass plants were investigated in this study. The direct effects on eelgrass were examined by measuring the four different endpoints; the relatively growth rate as length and weight, the chlorophyll a and b ratio, as well as the RNA-DNA ratio, at the end of a 3 days exposure period. The indirect effect was investigated by measuring the effect on a natural phytoplankton production from Roskilde Fjord six times during 1 year. The results showed that the chlorophyll a-b and RNA-DNA ratios were the most sensitive endpoints in single herbicide experiments. The effects of herbicide mixtures on eelgrass were much larger compared to the single substances. Nearly a halving was found for both the relatively growth rate in length and weight, and the RNA-DNA as well as the chlorophyll a-b ratios were also significant reduced. This indicates a possible synergistic effect, and calculations based on the concentration addition model indicate that the low concentrations mixture has a synergistic effect, whereas the high concentration mixture has an antagonistic effect on eelgrass (Z. marina). The low concentrations mixture is the one with the highest relevance for coastal areas. The effect on phytoplankton showed some variation over the year but mainly in a stimulation in primary production. This means that herbicides affect eelgrass both directly and indirectly.