Are current regulatory log Kow cut-off values fit-for-purpose as a screening tool for bioaccumulation potential in aquatic organisms?

Persistent, Bioaccumulative and Toxic (PBT) and very Persistent and very Bioaccumulative (vPvB) are regulatory hazard categories that have been set to manage the possible risks to humans and the environment from these chemicals. In industrial chemicals regulations, their aquatic Bioaccumulation potential is usually assessed first with a screening based on the octanol/water partition coefficient (Kow). However, current log Kow cut-off values triggering classification, categorisation and/or further fish bioconcentration testing are not harmonised worldwide, and they have never been assessed for their regulatory relevance. In this study, the experimentally determined log Kow and fish bioconcentration factors (BCF) of 532 chemicals were compared. While the analysis underlined the robustness of using log Kow as a screening tool (5/532 were false negatives; log Kow: non-bioaccumulative, but BCF: bioaccumulative), it also demonstrated the conservatism of the cut-offs used worldwide. Indeed, many chemicals were deemed potentially Bioaccumulative based on log Kow when a fish bioaccumulation test showed no concern (false positives), therefore, leading to unnecessary use of vertebrate animals. Our analysis shows that the log Kow cut-off could be increased to 4.5 in all regions for all purposes without leading to a reduced protection of humans and the environment.

Phylogeny and Antagonistic Activities of Culturable Bacteria Associated with the Gut Microbiota of the Sea Urchin (Paracentrotus lividus).

In this study, we have investigated the phylogeny and the antagonistic interactions of culturable bacteria isolated from the sea urchin Paracentrotus lividus collected from Aber and Morgat, both located in Crozon peninsula, France. Bacteria were isolated from the gastrointestinal tracts of ten specimens by using conventional culture-dependent method and then investigated by using phylogenetic analysis based on 16S rRNA gene sequence comparisons. Assays for antagonistic interactions among the bacterial strains were performed; bacteria (including at least one strain representative of each OTU identified) were screened for antimicrobial substance production. So, 367 bacterial strains were isolated on marine-agar. On the basis of morphological characteristics, 180 strains were sequenced and 94 OTUs were classified. The dominant phyla were Proteobacteria, Firmicutes and Actinobacteria, with a high abundance of the strains belonging to the genus Psychrobacter. From the antagonistic interactions assays, it could be determined that 22.7% strains were positive for at least one antagonism interaction, 18.3% of them isolated from the sea urchins collected in Morgat. We hypothesize that the bacteria isolated in this study may represent the transitory microbiota of the gastrointestinal tract of P. lividus, and that this microbiota may be related to the diet of this marine invertebrate. Furthermore, our results suggest that chemical antagonism could play a significant role in shaping the bacterial communities within gastrointestinal tract of the sea urchins. In addition, most isolated bacteria may have promising biotechnology applications.

Ocean Acidification Reduces Spine Mechanical Strength in Euechinoid but Not in Cidaroid Sea Urchins.

Echinoderms are considered particularly sensitive to ocean acidification (OA) as their skeleton is made of high-magnesium calcite, one of the most soluble forms of calcium carbonate. Recent studies have investigated effects of OA on the skeleton of "classical" sea urchins (euechinoids), but the impact of etching on skeleton mechanical properties is almost unknown. Furthermore, the integrity of the skeleton of cidaroids has never been assessed, although their extracellular fluid is under-saturated with respect to their skeleton, and the skeleton of their primary spines is in direct contact with seawater. In this study, we compared the dissolution of test plates and spines as well as the spine mechanical properties (two-points bending tests) in a cidaroid (Eucidaris tribuloides) and a euechinoid (Tripneustes ventricosus) submitted to a 5 week acidification experiment (pHT of 8.1, 7.7, and 7.4). Test plates of both species were not affected by dissolution. The spines of E. tribuloides showed no mechanical effects at pHSW-T 7.4 despite having traces of corrosion on secondary spines. On the contrary, spines of the T. ventricosus were significantly etched at both pHSW-T 7.7 and 7.4 and their fracture force reduced by 16 to 35%, respectively. This increased brittleness is probably of little significance with regards to predation protection but has consequences in terms of energy allocation.

Comparison of three different wastewater sludge and their respective drying processes: Solar, thermal and reed beds - Impact on organic matter characteristics.

Drying process aims at minimising the volume of wastewater sludge (WWS) before disposal, however it can impact sludge characteristics. Due to its high content in organic matter (OM) and lipids, sludge are mainly valorised by land farming but can also be considered as a feedstock for biodiesel production. As sludge composition is a major parameter for the choice of disposal techniques, the objective of this study was to determine the influence of the drying process. To reach this goal, three sludges obtained from solar, reed beds and thermal drying processes were investigated at the global and molecular scales. Before the drying step the sludges presented similar physico-chemical (OM content, elemental analysis, pH, infrared spectra) characteristics and lipid contents. A strong influence of the drying process on lipids and humic-like substances contents was observed through OM fractionation. Thermochemolysis-GCMS of raw sludge and lipids revealed similar molecular content mainly constituted with steroids and fatty acids. Molecular changes were noticeable for thermal drying through differences in branched to linear fatty acids ratio. Finally the thermal drying induced a weakening of OM whereas the solar drying led to a complexification. These findings show that smooth drying processes such as solar or reed-beds are preferable for amendment production whereas thermal process leads to pellets with a high lipid content which could be considered for fuel production.

Could the acid-base status of Antarctic sea urchins indicate a better-than-expected resilience to near-future ocean acidification?

Increasing atmospheric carbon dioxide concentration alters the chemistry of the oceans towards more acidic conditions. Polar oceans are particularly affected due to their low temperature, low carbonate content and mixing patterns, for instance upwellings. Calcifying organisms are expected to be highly impacted by the decrease in the oceans' pH and carbonate ions concentration. In particular, sea urchins, members of the phylum Echinodermata, are hypothesized to be at risk due to their high-magnesium calcite skeleton. However, tolerance to ocean acidification in metazoans is first linked to acid-base regulation capacities of the extracellular fluids. No information on this is available to date for Antarctic echinoderms and inference from temperate and tropical studies needs support. In this study, we investigated the acid-base status of 9 species of sea urchins (3 cidaroids, 2 regular euechinoids and 4 irregular echinoids). It appears that Antarctic regular euechinoids seem equipped with similar acid-base regulation systems as tropical and temperate regular euechinoids but could rely on more passive ion transfer systems, minimizing energy requirements. Cidaroids have an acid-base status similar to that of tropical cidaroids. Therefore Antarctic cidaroids will most probably not be affected by decreasing seawater pH, the pH drop linked to ocean acidification being negligible in comparison of the naturally low pH of the coelomic fluid. Irregular echinoids might not suffer from reduced seawater pH if acidosis of the coelomic fluid pH does not occur but more data on their acid-base regulation are needed. Combining these results with the resilience of Antarctic sea urchin larvae strongly suggests that these organisms might not be the expected victims of ocean acidification. However, data on the impact of other global stressors such as temperature and of the combination of the different stressors needs to be acquired to assess the sensitivity of these organisms to global change.

Warming influences Mg2+ content, while warming and acidification influence calcification and test strength of a sea urchin.

We examined the long-term effects of near-future changes in temperature and acidification on skeletal mineralogy, thickness, and strength in the sea urchin Tripneustes gratilla reared in all combinations of three pH (pH 8.1, 7.8, 7.6) and three temperatures (22 °C, 25 °C, 28 °C) from the early juvenile to adult, over 146 days. As the high-magnesium calcite of the echinoderm skeleton is a biomineral form highly sensitive to acidification, and influenced by temperature, we documented the MgCO3 content of the spines, test plates, and teeth. The percentage of MgCO3 varied systematically, with more Mg2+ in the test and spines. The percentage of MgCO3 in the test and teeth, but not the spines increased with temperature. Acidification did not change the percentage MgCO3. Test thickness increased with warming and decreased at pH 7.6, with no interaction between these factors. In crushing tests live urchins mostly ruptured at sutures between the plates. The force required to crush a live urchin was reduced in animals reared in low pH conditions but increased in those reared in warm conditions, a result driven by differences in urchin size. It appears that the interactive effects of warming and acidification on the Mg2+ content and protective function of the sea urchin skeleton will play out in a complex way as global climatic change unfolds.

Euechinoidea and Cidaroidea respond differently to ocean acidification.

The impact of the chemical changes in the ocean waters due to the increasing atmospheric CO2 depends on the ability of an organism to control extracellular pH. Among sea urchins, this seems specific to the Euechinoidea, sea urchins except Cidaroidea. However, Cidaroidea survived two ocean acidification periods: the Permian-Trias and the Cretaceous-Tertiary crises. We investigated the response of these two sea urchin groups to reduced seawater pH with the tropical cidaroid Eucidaris tribuloides, the sympatric euechinoid Tripneustes ventricosus and the temperate euechinoid Paracentrotus lividus. Both euechinoid showed a compensation of the coelomic fluid pH due to increased buffer capacity. This was linked to an increased concentration of DIC in the coelomic fluid and thus of bicarbonate ions (most probably originating from the surrounding seawater as isotopic signature of the carbon - δ¹³C - was similar). On the other hand, the cidaroid showed no changes within the coelomic fluid. Moreover, the δ¹³C of the coelomic fluid did not match that of the seawater and was not significantly different between the urchins from the different treatments. Feeding rate was not affected in any species. While euechinoids are able to regulate their extracellular acid-base balance, many questions are still unanswered on the costs of this capacity. On the contrary, cidaroids do not seem affected by a reduced seawater pH. Further investigations need to be undertaken to cover more species and physiological and metabolic parameters in order to determine if energy trade-offs occur and how this mechanism of compensation is distributed among sea urchins.

Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

Buffer capacity of the coelomic fluid in echinoderms.

The increase in atmospheric CO2 due to anthropogenic activity results in an acidification of the surface waters of the oceans. The impact of these chemical changes depends on the considered organisms. In particular, it depends on the ability of the organism to control the pH of its inner fluids. Among echinoderms, this ability seems to differ significantly according to species or taxa. In the present paper, we investigated the buffer capacity of the coelomic fluid in different echinoderm taxa as well as factors modifying this capacity. Euechinoidea (sea urchins except Cidaroidea) present a very high buffer capacity of the coelomic fluid (from 0.8 to 1.8mmolkg(-1) SW above that of seawater), while Cidaroidea (other sea urchins), starfish and holothurians have a significantly lower one (from -0.1 to 0.4mmolkg(-1) SW compared to seawater). We hypothesize that this is linked to the more efficient gas exchange structures present in the three last taxa, whereas Euechinoidea evolved specific buffer systems to compensate lower gas exchange abilities. The constituents of the buffer capacity and the factors influencing it were investigated in the sea urchin Paracentrotus lividus and the starfish Asterias rubens. Buffer capacity is primarily due to the bicarbonate buffer system of seawater (representing about 63% for sea urchins and 92% for starfish). It is also partly due to coelomocytes present in the coelomic fluid (around 8% for both) and, in P. lividus only, a compound of an apparent size larger than 3kDa is involved (about 15%). Feeding increased the buffer capacity in P. lividus (to a difference with seawater of about 2.3mmolkg(-1) SW compared to unfed ones who showed a difference of about 0.5mmolkg(-1) SW) but not in A. rubens (difference with seawater of about 0.2 for both conditions). In P. lividus, decreased seawater pH induced an increase of the buffer capacity of individuals maintained at pH7.7 to about twice that of the control individuals and, for those at pH7.4, about three times. This allowed a partial compensation of the coelomic fluid pH for individuals maintained at pH7.7 but not for those at pH7.4.

Persistence and Mobility (defined as organic­carbon partitioning) do not correlate to the detection of substances found in surface and groundwater: Criticism of the regulatory concept of Persistent and mobile substances.

The Chemical Strategy for Sustainability (CSS) includes actions to ensure the protection of drinking water resources from chemical pollution. To proactively identify potential pollutants, the German Environment Agency (UBA) proposed the Persistent and Mobile (PM) concept according to which Persistence (criteria of REACH Annex XIII) and Mobility (log Koc < 4) would be proxies for a substance's degradation potential and transport velocity, two processes believed to drive the potential for contamination of surface and groundwater as drinking water sources. Two studies identified hundreds of PM substances while three subsequent studies have selected some of these substances for monitoring in surface, ground- and/or drinking water to support the concept. In the present work, the Persistence of the aforementioned substances was reassessed based on all experimental data publicly available. Depending on the exact study examined, it was found that 15 % to 40 % of the substances were erroneously concluded as P. The reinterpretation of the data indicates that a PM substance does not have a higher likelihood to be detected in surface or groundwater than a non-PM substance. In addition, the PM properties do not have any influence on the level of contamination. Twenty-six to 75 % of the substances selected because they were identified as PM were not found in surface or ground water despite being selected for their high emission pattern. Regulations based primarily on the PM concept, like the CLP and possibly REACH and UN-GHS, are unlikely to appropriately identify substances of concern for drinking water sources. It is more likely that chemical presence in surface and groundwater is driven by emission patterns or local factors. The development of specific exposure models would better contribute to the protection of drinking water resources and consumers.

Bottom-up effects on biomechanical properties of the skeletal plates of the sea urchin Paracentrotus lividus (Lamarck, 1816) in an acidified ocean scenario.

Sea urchins, ecologically important herbivores of shallow subtidal temperate reefs, are considered particularly threatened in a future ocean acidification scenario, since their carbonate structures (skeleton and grazing apparatus) are made up of the very soluble high-magnesium calcite, particularly sensitive to a decrease in pH. The biomechanical properties of their skeletal structures are of great importance for their individual fitness, because the skeleton provides the means for locomotion, grazing and protection from predators. Sea urchin skeleton is composed of discrete calcite plates attached to each other at sutures by organic ligaments. The present study addressed the fate of the sea urchin Paracentrotus lividus (Lamarck, 1816) skeleton in acidified oceans, taking into account the combined effect of reduced pH and macroalgal diet, with potential cascading consequences at the ecosystem level. A breaking test on individual plates of juvenile specimens fed different macroalgal diets has been performed, teasing apart plate strength and stiffness from general robustness. Results showed no direct short-term effect of a decrease in seawater pH nor of the macroalgal diet on single plate mechanical properties. Nevertheless, results from apical plates, the ones presumably formed during the experimental period, provided an indication of a possible diet-mediated response, with sea urchins fed the more calcified macroalga sustaining higher forces before breakage than the one fed the non-calcified algae. This, on the long term, may produce bottom-up effects on sea urchins, leading to potential shifts in the ecosystem equilibrium under an ocean acidified scenario.

Parameter Estimations of Dynamic Energy Budget (DEB) Model over the Life History of a Key Antarctic Species: The Antarctic Sea Star Odontaster validus Koehler, 1906.

Marine organisms in Antarctica are adapted to an extreme ecosystem including extremely stable temperatures and strong seasonality due to changes in day length. It is now largely accepted that Southern Ocean organisms are particularly vulnerable to global warming with some regions already being challenged by a rapid increase of temperature. Climate change affects both the physical and biotic components of marine ecosystems and will have an impact on the distribution and population dynamics of Antarctic marine organisms. To predict and assess the effect of climate change on marine ecosystems a more comprehensive knowledge of the life history and physiology of key species is urgently needed. In this study we estimate the Dynamic Energy Budget (DEB) model parameters for key benthic Antarctic species the sea star Odontaster validus using available information from literature and experiments. The DEB theory is unique in capturing the metabolic processes of an organism through its entire life cycle as a function of temperature and food availability. The DEB model allows for the inclusion of the different life history stages, and thus, becomes a tool that can be used to model lifetime feeding, growth, reproduction, and their responses to changes in biotic and abiotic conditions. The DEB model presented here includes the estimation of reproduction handling rules for the development of simultaneous oocyte cohorts within the gonad. Additionally it links the DEB model reserves to the pyloric caeca an organ whose function has long been ascribed to energy storage. Model parameters described a slowed down metabolism of long living animals that mature slowly. O. validus has a large reserve that-matching low maintenance costs- allow withstanding long periods of starvation. Gonad development is continuous and individual cohorts developed within the gonads grow in biomass following a power function of the age of the cohort. The DEB model developed here for O. validus allowed us to increase our knowledge on the ecophysiology of this species, providing new insights on the role of food availability and temperature on its life cycle and reproduction strategy.

Long-term mesocosms study of the effects of ocean acidification on growth and physiology of the sea urchin Echinometra mathaei.

Recent research on the impact of ocean acidification (OA) has highlighted that it is important to conduct long-term experiments including ecosystem interactions in order to better predict the possible effects of elevated pCO2. The goal of the present study was to assess the long-term impact of OA on a suite of physiological parameters of the sea urchin Echinometra mathaei in more realistic food conditions. A long-term experiment was conducted in mesocosms provided with an artificial reef in which the urchins principally fed on algae attached to the reef calcareous substrate. Contrasted pH conditions (pH 7.7 vs control) were established gradually over six months and then maintained for seven more months. Acid-base parameters of the coelomic fluid, growth and respiration rate were monitored throughout the experiment. Results indicate that E. mathaei should be able to regulate its extracellular pH at long-term, through bicarbonate compensation. We suggest that, within sea urchins species, the ability to accumulate bicarbonates is related to their phylogeny but also on the quantity and quality of available food. Growth, respiration rate and mechanical properties of the test were not affected. This ability to resist OA levels expected for 2100 at long-term could determine the future of coral reefs, particularly reefs where E. mathaei is the major bioeroder.

Acid-base physiology response to ocean acidification of two ecologically and economically important holothuroids from contrasting habitats, Holothuria scabra and Holothuria parva.

Sea cucumbers are dominant invertebrates in several ecosystems such as coral reefs, seagrass meadows and mangroves. As bioturbators, they have an important ecological role in making available calcium carbonate and nutrients to the rest of the community. However, due to their commercial value, they face overexploitation in the natural environment. On top of that, occurring ocean acidification could impact these organisms, considered sensitive as echinoderms are osmoconformers, high-magnesium calcite producers and have a low metabolism. As a first investigation of the impact of ocean acidification on sea cucumbers, we tested the impact of short-term (6 to 12 days) exposure to ocean acidification (seawater pH 7.7 and 7.4) on two sea cucumbers collected in SW Madagascar, Holothuria scabra, a high commercial value species living in the seagrass meadows, and H. parva, inhabiting the mangroves. The former lives in a habitat with moderate fluctuations of seawater chemistry (driven by day-night differences) while the second lives in a highly variable intertidal environment. In both species, pH of the coelomic fluid was significantly negatively affected by reduced seawater pH, with a pronounced extracellular acidosis in individuals maintained at pH 7.7 and 7.4. This acidosis was due to an increased dissolved inorganic carbon content and pCO2 of the coelomic fluid, indicating a limited diffusion of the CO2 towards the external medium. However, respiration and ammonium excretion rates were not affected. No evidence of accumulation of bicarbonate was observed to buffer the coelomic fluid pH. If this acidosis stays uncompensated for when facing long-term exposure, other processes could be affected in both species, eventually leading to impacts on their ecological role.