Coping with current impacts: The case of Scyliorhinus canicula in the NW Mediterranean Sea and implications for human consumption.

The small-spotted catshark (Scyliorhinus canicula) is a bottom-dwelling elasmobranch that represents the most discarded catch in terms of biomass in the Catalan coast (NW Mediterranean). Potential impacts affecting its population and food safety implications have been assessed in three localities along the Catalan coast. Distinct indicators were integrated, such as biological data, ingested anthropogenic items (plastic and cellulose-like items), parasitological indices, trace metal concentrations and histopathology using liver as target organ. Although high ingestion rates of fibres and levels of some heavy metals, they do not seem negatively affected by any major pathology nor by the current levels of pollutants. Small-scale differences among localities and depths were found and discussed. No zoonotic parasites were found. Encysted larvae of Grillotia adenoplusia and, above all, the levels of Hg found in the musculature, that are well over the European Commission limits, rise concerns regarding human consumption of S. canicula in this region.

Trace metal level variation under strong wind conditions and sediment resuspension in the waters of a coastal lagoon highly impacted by mining activities.

The levels of metals in the waters of the Mar Menor lagoon are higher in the southern than in the northern zone both in the dissolved (As: 1.78 µg L-1 north vs 1.86 µg L-1 south; Cd: 0.020 µg L-1 vs 0.055 µg L-1; Pb: 0. 686 µg L-1 vs 2.714 µg L-1; Zn: 3.06 µg L-1 vs 10.2 µg L-1) as in the particulate fraction (As: 13.6 µg g-1 north vs 27.3 µg g-1 south; Cd: 0.510 µg g-1 vs 2.11 µg g-1; Pb: 146 µg g-1 vs 575 µg g-1; Zn: 266 µg g-1 vs 729 µg g-1). This difference is associated to the influence of historical and recent inputs from the Sierra Minera Cartagena -La Unión located south of the lagoon. Strong winds cause sediment resuspension in this shallow lagoon, increasing metal levels in the dissolved (twofold) and especially in the particulate fraction (threefold) because the resuspended sediments are rich in metals. Distribution among dissolved and particulate fraction is determined by the chemistry of each element and salinity. This increase causes the levels to reach limits very close to those established by the Water Framework Directive, especially in the case of lead, whose annual average level of 1.23 µg L-1 is very close to the 1.3 µg L-1 established in the Directive. Therefore, slight change in environmental variables could make Pb levels to exceed legal limits. Future work should focus on investigating how unique environmental events, enhanced by global change, affect metal cycles in highly anthropised coastal areas.

Effect of dissolved organic matter on copper bioavailability to a coastal dinoflagellate at environmentally relevant concentrations.

The speciation and bioavailability of copper (Cu) in the marine environment are affected by the presence of dissolved organic matter (DOM). Previous studies conducted at dissolved Cu concentrations >100 nM confirmed that Cu bioavailability depends on the concentration of labile Cu, as measured by anodic stripping voltammetry (ASV), which aligns with the expectations of the biotic ligand model (BLM). However, ambient Cu concentrations in coastal waters are generally lower, ranging between 1 and 80 nM, and the effect of DOM on the bioavailability of Cu to marine organisms has not been tested within that range of Cu concentrations. The present study aims to assess the impact of two types of DOM, a commercially available fulvic acid, and marine DOM extracted by ultrafiltration, on Cu bioavailability to phytoplankton using short-term 65Cu internalisation by the marine dinoflagellate Prorocentrum micans. Results showed that Cu internalisation decreases with DOM additions as expected according to the BLM and in agreement with ASV measurements of labile Cu, at the highest tested Cu concentration (100 nM). On the contrary, at a lower Cu concentration (20 nM), organic complexes appear to be partially bioavailable, thereby challenging the general applicability of the BLM model at environmentally relevant concentrations in coastal areas.

Distribution of metals in the queen scallop Aequipecten opercularis during a transplant experiment: Metal rich granules as drivers of Pb bioaccumulation.

The queen scallop Aequipecten opercularis accumulates high concentrations of lead (Pb) in its tissues, what has led to the interruption of this fishery in some extraction areas in Galicia (NW Spain). This study follows the dynamics of bioaccumulation of Pb and other metals in this species, the tissue distribution and the subcellular partitioning in selected organs, in order to understand the mechanisms that provoke the high Pb levels reached in its tissues and to increase our knowledge about metal bioaccumulation dynamics in this species. Scallops originating from a clean area were exposed in cages in two places in the Ría de Vigo (one shipyard and a less impacted location) and 10 individuals were collected every month over a three months period. Metal bioaccumulation and metal distribution in several organs, including gills, digestive gland, kidneys, muscle, gonad and remaining tissues, was studied. The results showed that scallops accumulated similar levels of Cd, Pb and Zn at both sites, while Cu and Ni showed an opposite pattern at the shipyard, with Cu concentrations increasing around 10 times and Ni decreasing during the 3 months of exposure. The preferential organs for metal accumulation were the kidneys for Pb and Zn, the digestive gland for Cd, both organs for Cu and Ni, and the muscle for As. Subcellular partitioning of kidney samples additionally showed an extraordinary ability to accumulate Pb and Zn at very high concentrations in kidney granules, a fraction that accounted for 30 to 60 % of Pb in soft-tissues. It is concluded that Pb bioaccumulation in kidney granules is the mechanism responsible for the high levels of Pb observed in this species.

Use of limpets as alternative to mussels in metal pollution monitoring; application in the Canary Islands.

Metal pollution monitoring programs make use of organisms, such as mussels, as biomonitors to evaluate and compare the metal pollution status of coastal areas worldwide. Despite the widespread distribution of mussels of the genus Mytilus, there are places where these organisms are absent or where their abundance is insufficient for biomonitoring purposes, such as in the Canary Islands (Spain). This study considers the use of limpets of the genus Patella as alternative/complementary species in metal pollution monitoring, and compares the bioaccumulation capacity of mussels and limpets collected simultaneously during several sampling campaigns at 11 sampling sites along the continental Spanish Atlantic coastline. Results show that there are some differences in bioaccumulation capacity between limpets and mussels, with limpets showing in average higher concentrations of Cd, Cr, Ni, As and Cu, and lower concentrations of Zn, while concentrations of Hg and Pb were similar for both types of organisms. Significant correlations between metal concentration in mussels and limpets were found for most metals, with the notable exception of Cd, that showed a very different bioaccumulation pattern in both types of organisms. Limpet to mussel metal concentration ratios were derived that can be used to compare the results of metal biomonitoring using either limpets or mussels, and limpets were successfully used in a monitoring survey in the Canary Islands.

Vitellogenin gene expression in marine mussels exposed to ethinylestradiol: No induction at the transcriptional level.

Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.

A Primer and Guidelines for Shotgun Proteomic Analysis in Non-model Organisms.

During the last decade, we have witnessed outstanding advances in proteomics led mostly by great technological improvements in mass spectrometry field allowing high-throughput production of high-quality data used for massive protein identification and quantification. From a practical viewpoint, these advances have been mainly exploited in research projects involving model organisms with abundant genomic and proteomic information available in public databases. However, there is a growing number of organisms of high interest in different disciplines, such as ecological, biotechnological, and evolutionary research, yet poorly represented in these databases. Important advances in massive parallel sequencing technology and easy accessibility of this technology to many research laboratories have made nowadays possible to produce customized genomic and proteomic databases of any organism. Along this line, the use of proteogenomic approaches by combining in the same analysis the data obtained from different omic levels has emerged as a very useful and powerful strategy to run shotgun proteomic experiments specially focused on non-model organisms. In this chapter, we provide detailed procedures to undertake shotgun quantitative proteomic experiments following either a label-free or an isobaric labeling approach in non-model organisms, emphasizing also a few key aspects related to experimental design and data analysis.

Proteomic analysis and biochemical alterations in marine mussel gills after exposure to the organophosphate flame retardant TDCPP.

Organophosphate flame retardants (OPFRs) are (re-)emergent environmental pollutants increasingly being used because of the restriction of other flame retardants. The chlorinated OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is among those of highest environmental concern, but its potential effects in the marine environment have rarely been investigated. We exposed a widely used sentinel marine mussel species, Mytilus galloprovincialis, to 10 µg L-1 of TDCPP during 28 days and studied: (i) the kinetics of bioaccumulation and elimination of the compound, (ii) the effect on two molecular biomarkers, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities, and (iii) proteomic alterations in the gills, following an isobaric labeling quantitative shotgun proteomic approach, at two exposure times (7 and 28 days). Uptake and elimination of TDCPP by mussels were very fast, and the bioconcentration factor of this compound in mussels was 147 L kgww-1, confirming that this compound is not very bioaccumulative, as predicted by its chemical properties. GST activity was not affected by TDCPP exposure, but AChE activity was inhibited by TDCPP at both 7 and 28 days of exposure. Proteomic analysis revealed subtle effects of TDCPP in mussel gills, since few proteins (less than 2 % of the analysed proteome) were significantly affected by TDCPP, and effect sizes were low. The most relevant effects detected were the up-regulation of epimerase family protein SDR39U1, an enzyme that could be involved in detoxification processes, at both exposure times, and the down-regulation of receptor-type tyrosine-protein phosphatase N2-like (PTPRN2) after 7 days of exposure, which is involved in neurotransmitter secretion and might be related to the neurotoxicity described for this compound. Exposure time rather than TDCPP exposure was the most important driver of protein abundance changes, with 33 % of the proteome being affected by this factor, suggesting that stress caused by laboratory conditions could be an important confounding factor that needs to be controlled in similar ecotoxicology studies. Proteomic data are available via ProteomeXchange with identifier PXD019720.

No evidence that vitellogenin protein expression is induced in marine mussels after exposure to an estrogenic chemical.

A wide variety of endocrine disrupting chemicals reach the marine environment and can cause harmful effects in different marine organisms. Vitellogenin (Vtg), the egg-yolk precursor, is a commonly used endocrine disruption biomarker in fish and more recently in marine invertebrates under the assumption of high expected similarities in the endocrine system of vertebrates and invertebrates. However, this assumption has been recently questioned. The results from previous studies focused on bivalve molluscs showed that Vtg induction could be misleading because of the use of either non-robust or indirect techniques to measure Vtg. In this study, mussels (Mytilus galloprovincialis) were exposed to either 10 or 100 ng/L of the synthetic hormone 17α-ethinylestradiol (EE2) at different exposure times (4 and 24 days) and under different feeding regimes (representing different energy balances), and Vtg levels in both male and female mussel gonads were quantified by label free shotgun LC-MS/MS proteomic analysis. Vtg protein was not detected in male gonads. In female gonads, Vtg levels were not significantly affected by EE2 at any exposure time or EE2 concentration tested, whereas a significant correlation was found between the degree of maturation of the gonad and Vtg levels in females. Results obtained in the present study critically question the use of Vtg as a biomarker of endocrine disruption in marine mussels, and show that the degree of maturation of the gonad can be an important confounding factor in the attempts to evaluate estrogenic effects through Vtg measurement in mussel gonads.

In vivo oral bioavailability of Pb sequestered in metal rich granules in bivalves.

The present study was designed to evaluate in vivo the oral bioavailability of lead (Pb) present in the marine bivalve Dosinia exoleta. This infaunal clam, despite inhabiting in clean areas, presents Pb concentrations that are over the 1.5 mg kg-1 wet weight limit for human consumption set by the European Commission. However, Pb is accumulated in this clam in the form of metal rich granules, and it has been shown to be unavailable for trophic transfer to a marine decapod, so it was hypothesised that it might be unavailable for human consumers as well. Twelve Sprague Dawley rats were fed during 14 days with a diet including control mussels (Mytilus galloprovincialis), D. exoleta, or mussels enriched in Pb to the same levels as those found in D. exoleta. Pb accumulation in different rat tissues (blood, bone, kidneys and liver) was analysed. It was observed that Pb assimilation from D. exoleta was about half of Pb assimilation from M. galloprovincialis, and absolute bioavailabilities were around 2% for M. galloprovincialis and 1% for D. exoleta. These results suggest that it might be possible to increase the limit for human consumption for this bivalve to 3 mg kg-1 wet weight without representing an increase in the risk for consumers.

Fifteen years of imposex and tributyltin pollution monitoring along the Portuguese coast.

IMO's Anti-Fouling Systems convention banned the use of organotin-based antifouling systems in 2008 as the ultimate effort to stop tributyltin (TBT) inputs into the marine environment. One of the hazardous effects of TBT is imposex (the superimposition of male sexual characters onto gastropod females), a phenomenon that may cause female sterility and the gastropod populations decline. Despite previous European Union legislation had already been shown effective in reducing the imposex levels along the Portuguese coast, this study intends to confirm these decreasing trends after 2008 and describe the global evolution in the last 15 years. Imposex levels were assessed in two bioindicators - the dog-whelk Nucella lapillus and the netted-whelk Nassarius reticulatus (Gastropoda, Prosobranchia) - in 2011 and 2014, and the results were compared with previous years. Both species showed progressive decreasing trends in imposex levels over the last 15 years; median values of the vas deferens sequence index (VDSI) fell from 3.96 to 0.78 in N. lapillus and from 3.39 to 0.29 in N. reticulatus. The temporal/spatial evolution of imposex suggests an apparent shift of TBT hotspots, being now restricted to fishing ports and marinas in detriment of large commercial harbours where TBT levels fell rapidly. Butyltins were measured in the whole tissues of N. lapillus females collected in 2014: monobutyltin (MBT) varied from < DL (detection limit: 1 ng Sn/g) to 13 ng Sn/g dw, dibutyltin (DBT) from 2.2 to 27 ng Sn/g dw and TBT from 1.5 to 55 ng Sn/g dw. Although TBT body burden has declined over time, the butyltin degradation index ([MBT]+[DBT])/[TBT] exhibited values < 1 in c. a. 90% of the sites assessed, suggesting that recent TBT inputs are still widespread in the Portuguese coast eventually due to illegal use of TBT antifouling systems and TBT desorption from sediments.

Shotgun Proteomics Analysis Discards Alkali Labile Phosphate as a Reliable Method To Assess Vitellogenin Levels in Mytilus galloprovincialis.

Vitellogenin, the egg yolk precursor, is a well-known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless of sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels, and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Subcellular distribution and trophic transfer of Pb from bivalves to the common prawn Palaemon serratus.

The edible clam Dosinia exoleta has been reported to accumulate high contents of lead (Pb) in soft tissues disregarding the levels of Pb in the environment. This is due to the retention of Pb in the form of metal rich granules (MRG) in their kidneys throughout the mollusc lifespan. The potential for trophic transfer of Pb in this form to predators is expected to be low, since metals in the form of MRG are generally supposed to be trophically unavailable, but this assumption is based on studies with other metals (Ag, Cd, Cu or Zn) and has not been demonstrated with Pb until now. This study was designed to test if the Pb present in D. exoleta in the form of MRG is available to a decapod consumer, the common prawn Palaemon serratus, in comparison with a mussel diet showing a different subcellular distribution of Pb. As hypothesised, despite the high Pb concentrations (15µgg-1ww) offered to the prawns as D. exoleta tissues, Pb was almost completely unavailable for trophic transfer, and the prawns fed with this diet during 28 days showed the same Pb accumulation as prawns fed with a control diet with a much lower Pb concentration. On the contrary, individuals fed with mussel tissues containing the same Pb concentrations as the diet based on D. exoleta tissues showed 10 times higher Pb bioaccumulation, corresponding to a trophic transfer factor of 1.1%. Subcellular fractionation experiments revealed that the fraction of Pb in the form of MRG was much lower for the mussel, confirming, as observed for other metals, that MRG-associated Pb is not available for trophic transfer to decapod crustaceans.

Dynamic modeling of copper bioaccumulation by Mytilus edulis in the presence of humic acid aggregates.

Copper (Cu) complexation by humic acids (HA) is expected to decrease Cu bioavailability for aquatic organisms as predicted by metal bioavailability models, such as the biotic ligand model (BLM). This has been confirmed for non-feeding organisms such as marine invertebrate embryos or microalgae, but for filter-feeding organisms such as the mussel Mytilus edulis, Cu bioaccumulation was higher in the presence of HA, suggesting that part of the Cu-HA complexes were available for uptake. This study shows the dynamic modeling of Cu accumulation kinetics in the gills and rest of the soft-body of M. edulis in the absence and presence of HA. Assuming that truly dissolved Cu is taken in the body via the gills following BLM premises, and including uptake of Cu-HA aggregates via the gut into the rest compartment, this two-compartmental model could successfully explain the observed bioaccumulation data. This modeling approach gives strong evidence to the hypothesis that Cu-HA aggregates can be ingested by mussels leading to Cu absorption in the digestive system.

Triphenyltin induces imposex in Nucella lapillus through an aphallic route.

Triphenyltin (TPT) was used until recently as a biocide in antifouling systems and nowadays is still applied as an agriculture pesticide in some countries. This compound is known to cause imposex (the imposition of male characters in females of gastropod molluscs) in a very limited number of species, when compared with tributyltin (TBT), the universally recognized imposex-causing agent. In this study, we tested if TPT could induce imposex in females of the dog-whelk Nucella lapillus. Experimental groups of 40 females were injected with a volume of 2µL/g of soft tissue wet weight (ww) of one of the following treatments, using DMSO as a solvent carrier: DMSO (solvent control); 1µg/g ww of TBT (positive control); 0.2, 1 and 5µg/g ww of TPT and a non-injected group (negative control). Concentrations were confirmed in the organism tissues by means of chemical analyses of a pool of 10 specimens at T0 and then after the imposex analysis at T56days. After 8-week trial, results pointed out statistically significant differences between treatments, with both TPT and TBT positively inducing imposex. However, imposex development in TPT-injected females differed from that of TBT, since females that developed imposex presented an aphallic condition (no penis development) while the TBT-treated females developed standard imposex (with penis formation). These results suggest that TPT and TBT act differently in the sequential process of female masculinization, casting new insights about the hypothetical pathways underlying imposex development.

Lead (Pb) and copper (Cu) share a common uptake transporter in the unicellular alga Chlamydomonas reinhardtii.

The unicellular alga Chlamydomonas reinhardtii has a very high rate of lead (Pb) internalization and is known to be highly sensitive to dissolved Pb. However, the transport pathway that this metal uses to cross cellular membranes in microalgae is still unknown. To identify the Pb(2+) transport pathway in C. reinhartdii, we performed several competition experiments with environmentally relevant concentrations of Pb(2+) (~10 nM) and a variety of divalent cations. Among the essential trace metals tested, cobalt, manganese, nickel and zinc had no effect on Pb internalization. A greater than tenfold increase in the concentrations of the major ions calcium and magnesium led to a slight decrease (~34 %) in short-term Pb internalization by the algae. Copper (Cu) was even more effective: at a Cu concentration 50 times higher than that of Pb, Pb internalization by the algae decreased by 87 %. Pre-exposure of the algae to Cu showed that the effect was not due to a physiological effect of Cu on the algae, but rather to competition for the same transporter. A reciprocal effect of Pb on Cu internalization was also observed. These results suggest that Cu and Pb share a common transport pathway in C. reinhardtii at environmentally relevant metal concentrations.

Copper uptake by the marine mussel Mytilus edulis in the presence of fulvic acids.

Copper uptake and accumulation by the marine mussel Mytilus edulis were studied at different Cu concentrations in chemically defined artificial seawater in the presence and absence of fulvic acids. Both short-term uptake of Cu by excised mussel gills and Cu accumulation in whole mussels after 24 h of exposure decreased in the presence of fulvic acids compared with their absence at similar dissolved Cu concentrations. Calculations of Cu speciation based on previous measurements of labile Cu by anodic stripping voltammetry demonstrated that Cu uptake and accumulation depended on the concentration of labile Cu, in agreement with the free ion activity model. No evidence of a significant uptake of Cu-fulvic acid complexes was observed.

Dependence of Cu, Pb and Zn remobilization on physicochemical properties of marine sediments.

The resuspension of 65 marine sediments was simulated in the laboratory with elutriates from 30 different sites from the north coast of Spain. The partitioning of Cu, Pb and Zn between sediment and elutriate was studied as a function of different physicochemical characteristics of the sediment: organic matter (OM), fine fraction (FF), redox potential and acid volatile sulfides (AVS). Mean remobilization factors (RF) -calculated as metal concentration in the elutriate (µg/L) divided by metal concentration in the sediment (µg/g dry weight)- were 0.072 for Cu, 0.012 for Pb and 0.071 for Zn. Remobilization of Pb was significantly lower than that of Cu and Zn. Although AVS, OM and FF presented a strong intercorrelation, OM explained great part of the variability on Cu and Pb remobilization while AVS did it for Zn. A multiple regression model considering both OM and AVS explained slightly better the remobilization of Pb and Cu, but not that of Zn.

Quantification of the increase in Pb bioavailability to marine organisms caused by different types of DOM from terrestrial and river origin.

Dissolved organic matter (DOM) has metal binding properties and decreases the concentration of free metal ions in the aquatic environments. However, humic acids (HA) have been shown to increase Pb bioavailability for marine invertebrates, including Pb toxicity to sea urchin embryos. The low solubility and other properties of commercially available HA, which may not be the most representative fraction of DOM in natural waters, limit the environmental relevance of these findings. The present study tested the effect of more soluble DOM, fulvic acids (FA) and DOM extracted from the Suwannee River (SRDOM), on the acute toxicity of Pb to Paracentrotus lividus embryos. It was confirmed that FA and SRDOM, despite their Pb binding properties, also increased Pb toxicity for sea urchin embryos, even though this increase was not as high as that previously observed for HA. The enhancing effect of DOM on Pb bioavailavility was quantified using multiple regression equations for the present data, as well as for previously published data on Pb uptake by the gills of the marine mussel Mytilus sp. and Pb internalization by the marine microalgae Isochrysis galbana and Thalassiosira weissflogii. These results confirm that dissolved Pb bioavailability in seawater in the presence of DOM is higher than predicted using current bioavailability models based on chemical equilibrium chemistry. The experimental evidence suggests that the mechanisms by which DOM enhances Pb uptake and toxicity implies direct contact of the organic compounds with the plasma membrane.