Claude Amiard-Triquet

Contribution to the ecotoxicological study of cadmium, lead, copper and zinc in the mussel Mytilus edulis

The understanding of natural fluctuations of metal concentrations in mussels used as bio-indicators is indispensable for a good assessment of the disturbances due to pollution. We have studied thoroughly the factors which condition bioaccumulation by using controlled populations of mussels, Mytilus edulis L., sampled monthly over more than two years (March 1982–May 1984) in the Bay of Bourgneuf, France. Seasonal changes in metal levels have been recognized, maximum values being observed in winter and early spring and minimum in later spring and summer. Depending on metal and size group, the ratios between these maximum and minimum values varied between 1.56 and 3.43. The fluctuations in soft-tissue weight appear to be the main explanatory factor of seasonal variations in metal concentrations in mussels. Fluctuations in metal levels related to size of mussels were observed. Except for cadmium in mussels with a mean dry weight of soft tissues >0.2 g, a slight decrease in metal concentrations was observed for growing individuals: the regression coefficient b was -0.10, -0.11, -0.13 and -0.27 for Cd, Cu, Zn and Pb, respectively. The metals examined were rather uniformly distributed among the different groups of organs (visceral mass, gills and palps, “remainder”) and, consequently, several analyses of metals in different organs do not provide much more information than one analysis on the whole soft tissues. It is concluded that fluctuations related to size or season are reflected by only moderate differences in the maximum and minimum concentrations of metals in the mussels, but that they are nevertheless sufficient to conceal low chronic or short-term pollution, except at those sites where the “normal” environmental conditions are well-documented.

Application de la spectrometrie d'absorption atomique zeeman aux dosages de huit elements traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matrices biologiques solides

The use of biological indicators in studies of aquatic pollution (in fresh, estuarine or sea waters), as well as research about the metal transfers in food chains, need a great accuracy of the trace element determination. Therefore, as shown by the results of international intercalibration exercises, the mastery of analytical techniques is far from being perfect in all the laboratories. One of the main sources of error in atomic absorption results from non-specific absorptions due to the presence of important organic and mineral matrixes in biological materials and especially in aquatic and marine organisms. In this case, the correction of unspecific absorption by using deuterium lamp was insufficient and the determination of trace elements had to be preceded by a pre-instrumental stage which allowed the elimination of the organic matter by mineralization and of a large part of the mineral matrix by extraction. The previous separation was long and induced contamination risks. The use of the Zeeman effect background correction allows the transfer of most processes from the pre-instrumental to the instrumental stage. Moreover, the Zeeman effect has three advantages: (1) the background correction is effective up to 2 units of absorbance; (2) the correction is effective from 190 to 900 nm; (3) the method of the double beam is optimalized. The aim of this study was to apply the Zeeman effect to the determination of eight trace elements (Ag, Cd, Cr, Cu, Mn, Ni, Pb, Se) in three different biological solid samples, two originated from the marine environment (lobster hepatopancreas TORT-1, standard reference material from the National Research Council Canada; oyster tissues SRM 1566 from the US National Bureau of Standards) and one from the vegetable kingdom (tomato leaves, SRM 1573 from the US NBS). The experimental procedure is reduced to a minimum since it consists in the digestion of an aliquot of 100 mg of the powdered sample with 1 ml of concentrated nitric acid at 95°C for 1 h. Then the volume is adjusted to 4 ml with deionized water. The metal analysis is carried out using a graphite furnace coated with tantalum carbide. This analysis is achieved according to the method of standard addition. The three added concentrations used for each element are listed in Table 1. The analytical conditions and graphite atomizer program are indicated in Table 2. The temperature program has to be modified according to the type of equipment. The internal quality control of the suggested method related to four criteria: sensitivity, repeatability, accuracy, practicability. The results are shown in Table 3. The threshold of sensitivity (3 times the SD of a series of eight results obtained for a blank of digestion) are low: < 1 μg kg−1 for Ag, Cd and Mn; ∼ 1 μg kg−1 for Cr and Pb; 5 μg kg−1 for Cu and Ni and 15 μg kg−1 for Se. The variation coefficients, calculated for both two series of six determinations each, are generally included between 5 and 10%. The trace element concentrations determined by using this method are in perfect agreement with the certified values of the US NBS and NRC Canada (Table 3.) The quality of the results establishes the possibility of using a very easy and fast method to determine the level of eight trace elements in materials with high mineral and organic matter content.

Bioaccessibility of essential and non-essential metals in commercial shellfish from Western Europe and Asia

Maximum acceptable concentrations of metals in food - based on total concentrations - have been established in many countries. To improve risk assessment, it would be better to take into account bioaccessible concentrations. A total of seven species of molluscs from France, UK and Hong Kong was examined in this study including clams, mussels, oysters, scallops and gastropods. The species which have total metal concentrations higher than the most severe food security limits are mainly oysters (all of the three studied species), the gastropod Buccinum undatum for cadmium and zinc, and scallops for cadmium. The lowest bioaccessibility (in % extractability with gut juices) was observed for silver (median for all of the species: 14%), it was moderate for lead (median: 33%) and higher for cadmium, zinc and copper (medians were respectively 54%, 65%, and 70%). In most cases, bioaccessible concentrations remained higher than the safety limits, except for cadmium in scallops and zinc in B. undatum. The influence of feeding habit (masticated or swallowed, addition of vinegar or lemon) on metal bioaccessibility in oysters is limited. On the contrary, cooking the gastropods decreased the bioaccessibility of metals, except silver.

Behavioral Disturbances: The Missing Link between Sub-Organismal and Supra-Organismal Responses to Stress? Prospects Based on Aquatic Research

ABSTRACT Bridging the gap between early, sensitive responses to stress at the infra-organismal levels and long-term, ecologically relevant responses at the supra-organismal levels is a challenge. Behavioral ecotoxicology provides an approach that clearly links disturbances at the biochemical level (e.g., altered neurotransmitters and thyroid hormones) to effects at the population level. These effects may be direct, such as impairment of the search for a sexual partner, care of juveniles, and avoidance of predators or pollutants. Indirect effects may be alteration of reproduction success due to impairment of feeding and thus, energy metabolism. The sensitivity of behavioral responses can be useful in ecological risk assessment. A major difficulty is extrapolation of observed responses with test species to other species. Thus it is recommended to use behavioral biomarkers, associated with biochemical and physiological markers (neurotoxicity, hormones, energy metabolism) in carefully selected species. These sentinels must be key-species in the structure and functioning of ecosystems because impairments of their responses used as biomarkers will reveal a risk of cascading deleterious effects at the community and ecosystem levels.

A mesocosm study of fate and effects of CuO nanoparticles on endobenthic species (Scrobicularia plana, Hediste diversicolor).

The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 μg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

Biological indices, energy reserves, steroid hormones and sexual maturity in the infaunal bivalve Scrobicularia plana from three sites differing by their level of contamination.

The aim of this work was to investigate in situ biometric (condition index, hepato-somatic index, gonado-somatic index), biochemical (glycogen, lipids, sexual steroids) and histological (sex, sexual maturation stage) variables in the infaunal bivalve Scrobicularia plana. First, the reproductive cycle of S. plana was assessed by collecting bivalves from a reference site (the bay of Bourgneuf, Fr) in April, May, July, September and November 2005 and in January and March 2006. Then, S. plana were collected at three key periods of their sexual cycle (March 2006, beginning of gametogenesis; June 2006, spawning; and September 2006, spent) in three sites differing by their level of contamination (bay of Bourgneuf, reference site; Loire and Seine estuaries; Fr, impacted sites). The reproductive cycle of S. plana was well defined with a clear spawning period between May and July, sexual repose from November to January. Development of the gonad began in January and ended in September. Sex-ratio was determined during spawning and the influence of sex on biochemical variables was examined. Progesterone, 17beta-estradiol and testosterone levels in the gonad of S. plana were close to those reported in other bivalves. This study is the first to demonstrate in situ influence of site, sex and sexual maturity on energy reserves, and sexual steroids in S. plana. Even if interpretation of results is complex due to interferences between natural and anthropogenic factors, S. plana is a suitable species for estuarine studies and a better understanding of its reproduction will permit to assess impacts of environmental pollutants.

Size dependent bioaccumulation and ecotoxicity of gold nanoparticles in an endobenthic invertebrate: the Tellinid clam Scrobicularia plana.

Gold nanoparticles (AuNPs) have important technological applications resulting in an increased potential for release to the environment, and a greater possibility of toxicological effects. The marine bivalve Scrobicularia plana was exposed to AuNPs of size 5, 15 and 40 nm during a 16 d laboratory exposure at 100 μg Au L(-1). After exposure to AuNPs forming aggregates (>700 nm), the clams accumulated Au in their soft tissues. Biochemical (biomarkers) and behavioral (burrowing and feeding) responses were investigated. Au NPs were responsible of metallothionein induction (5, 40 nm), increased activities of catalase (15, 40 nm) and superoxide dismutase (40 nm) and of glutathione S-transferase by the three sizes of AuNPs indicating defense against oxidative stress. Exposure to AuNPs impaired burrowing behavior. However, it must be underlined that these effects were observed at a dose much higher than expected in the environment.

Effects of Copper on the Burrowing Behavior of Estuarine and Coastal Invertebrates, the Polychaete Nereis diversicolor and the Bivalve Scrobicularia plana

ABSTRACT Alterations of the burrowing behavior of two benthic invertebrates living in intertidal mudflats, the polychaete Nereis diversicolor and the bivalve Scrobicularia plana, were studied in individuals exposed to soluble copper. The design of the contamination procedure took into account the results assessing the influence of some natural factors potentially able to influence burrowing (artificial vs. natural sediment, lighting, size of the shell for bivalves). Animals were exposed for 4 d to concentrations ranging from 25 to 150 μ g Cu l− 1. At the end of exposure, the burrowing kinetics in clean sediment were determined after 1 and 2 d, then the animals were frozen until acetylcholinesterase (AChE) activity determination. Even at the lowest tested concentrations, copper caused hypoactivity in organisms belonging to both species studied. Behavioral impairments were not related to AChE activity inhibition. Metabolical or physiological disturbances could be the cause of these impairments. Concentrations affecting burrowing behavior were below those responsible for lethality in these species.

METALLOTHIONEINS AND DETOXIFICATION. IS THE USE OF DETOXICATION PROTEIN FOR MTs A LANGUAGE ABUSE

Our own results and a literature review led us to reconsider the detoxifying function of MTs in living organisms. Despite the fact that many authors have observed a synthesis or a level increase of MTs as a response to toxic metal uptake, arguments exist which tend us to give to MTs a strict zinc homeostasic function. Many experiments have been conducted using non-natural routes of exposure and/or concentrations far from those observed even in heavily polluted environments. Zinc is the only metal for which a primary induction has been established. Correlations between zinc and MTs levels are frequently observed, specially at the early stages of life. Our knowledge about metallic cluster structure and our experimental results about inter-metallic competition for binding-sites on the apoprotein, support the idea of substitution processes instead of de novo synthesis in most cases of contamination, leading frequently to acclimation.

A suitable model for the biomonitoring of trace metal bioavailabilities in estuarine sediments: the annelid polychaete Nereis diversicolor

The need to use biomonitors representative of the sedimentary compartment has been recognized, particularly in estuaries. Thus, trace metal contamination has been monitored in an infaunal polychaete worm Nereis diversicolor and sediments in the Seine estuary and comparatively in the relatively clean Authie estuary (French coast of the English Channel) over two years taking samples every three months at both sites. No correlations were shown between total metal (Ag, Cd, Cu, Pb, Zn) concentrations in raw sediments and ragworms. Because these worms are known to be good biomonitors of the bioavailabilities of sedimentary trace metals, it follows that total sediment metal concentrations have a poor predictive ecotoxicological value. Using a correction factor to minimize the influence of weight, it is possible to make a good estimation of the average metal concentrations in a population of worms at a given site, sampling only a limited number of specimens. Metallothioneins are often considered to be good biomarkers of the presence of significant availabilities of trace metals. Metallothionein-like proteins (MTLPs) are present in N. diversicolor, but there is no significant relationship between MTLPs and metal concentrations. This situation might result from the importance of metal-containing granules, both extra- and intracellular, in ragworms.