An integrated environmental approach to investigate biomarker fluctuations in the blue mussel Mytilus edulis L. in the Vilaine estuary, France

Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aim of this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in the Vilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.     

Variation patterns in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Platichthys flesus) in the Bay of Biscay

The objective was to describe and model variation patterns in individual fish responses to contaminants among estuaries, season and gender. Two hundred twenty-seven adult European flounders were collected in two seasons (winter and summer) in four estuaries along the Bay of Biscay (South West France), focusing on a pristine system (the Ster), vs. three estuaries displaying contrasted levels of contaminants (the Vilaine, Loire and Gironde). Twenty-three variables were measured by fish, considering the load of contaminants (liver metals, liver and muscle persistent organic pollutants, muscle polycyclic aromatic hydrocarbons); the gene expression (Cyt C oxydase, ATPase, BHMT, Cyt P450 1A1, ferritin); the blood genotoxicity (Comet test); and liver histology (foci of cellular alteration–tumour, steatosis, inflammation, abnormal glycogen storage). Canonical redundancy analysis (RDA) was used to model these variables using gender, season and estuary of origin as explanatory variables. The results underlined the homogeneity of fish responses within the pristine site (Ster) and more important seasonal variability within the three contaminated systems. The complete model RDA was significant and explained 35 % of total variance. Estuary and season respectively explained 30 and 5 % of the total independent variation components, whilst gender was not a significant factor. The first axis of the RDA explains nearly 27 % of the total variance and mostly represents a gradient of contamination. The links between the load of contaminants, the expression of several genes and the biomarkers were analysed considering different levels of chemical stress and a possible multi-stress, particularly in the Vilaine estuary.     

Risk assessment of herbicides and booster biocides along estuarine continuums in the Bay of Vilaine area (Brittany, France)

A 2-year study was implemented to characterize the contamination of estuarine continuums in the Bay of Vilaine area (NW Atlantic Coast, Southern Brittany, France) by 30 pesticide and biocide active substances and metabolites. Among these, 11 triazines (ametryn, atrazine, desethylatrazine, desethylterbuthylazine, desisopropyl atrazine, Irgarol 1051, prometryn, propazine, simazine, terbuthylazine, and terbutryn), 10 phenylureas (chlortoluron, diuron, 1-(3,4-dichlorophenyl)-3-methylurea, fenuron, isoproturon, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl)-urea, linuron, metoxuron, and monuron), and 4 chloroacetanilides (acetochlor, alachlor, metolachlor, and metazachlor) were detected at least once. The objectives were to assess the corresponding risk for aquatic primary producers and to provide exposure information for connected studies on the responses of biological parameters in invertebrate sentinel species. The risk associated with contaminants was assessed using risk quotients based on the comparison of measured concentrations with original species sensitivity distribution-derived hazardous concentration values. For EU Water Framework Directive priority substances, results of monitoring were also compared with regulatory Environmental Quality Standards. The highest residue concentrations and risks for primary producers were recorded for diuron and Irgarol 1051 in Arzal reservoir, close to a marina. Diuron was present during almost the all survey periods, whereas Irgarol 1051 exhibited a clear seasonal pattern, with highest concentrations recorded in June and July. These results suggest that the use of antifouling biocides is responsible for a major part of the contamination of the lower part of the Vilaine River course for Irgarol 1051. For diuron, agricultural sources may also be involved. The presence of isoproturon and chloroacetanilide herbicides on some dates indicated a significant contribution of the use of plant protection products in agriculture to the contamination of Vilaine River. Concentration levels and associated risk were always lower in estuarine sites than in the reservoir, suggesting that Arzal dam reduces downstream transfer of contaminants and favors their degradation in the freshwater part of the estuary. Results of the additional monitoring of two tidal streams located downstream of Arzal dam suggested that, although some compounds may be transferred to the estuary, their impact was probably very low. Dilution by marine water associated with tidal current was also a major factor of concentration reduction. It is concluded that the highest risks associated to herbicides and booster biocides concerned the freshwater part of the estuary and that its brackish/saltwater part was exposed to a moderate risk, although some substances may sometimes exhibit high concentration but mainly at low tide and on an irregular basis.     

Organochlorinated pesticides, PCBs, dioxins, and PBDEs in grey mullets (Liza ramada) and allis shads (Alosa alosa) from the Vilaine estuary (France)

This study aimed to compare the contamination levels of various organohalogenated compounds in two migratory fish species in the Vilaine River in western France. Organochlorinated pesticides, polychlorinated biphenyls (PCBs), dioxins (polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs)), and polybrominated diphenyl ethers (PBDEs) were analyzed in two diadromous species from the Vilaine estuary, the grey mullet (Liza ramada)—an amphihaline species, and the allis shad (Alosa alosa)—an anadromous species. Fish were collected in spring 2004 and spring 2005, upstream and downstream of the Arzal Dam. PCB contamination varied from 27 to 200 ng?g^?1 dry weight (d.w.). PCDDs/Fs, expressed in toxicity equivalent quantity (TEQ) varied from 0.4 to 2.8 pg?g^?1 d.w. Dioxins and dioxin-like PCBs expressed in total TEQ varied from 1.4 to 18.8 pg?g^?1 d.w. PBDE47 was present at around 2–10 ng?g^?1 d.w. and concentrations of the insecticide dichlorodiphenyltrichloroethane breakdown product p,p′-dichlorodiphenylchloroethylene varied from 1 to 14 ng?g^?1 d.w. For both species, specimens collected upstream were more contaminated. The grey mullet specimens were less contaminated than the allis shad when taken downstream of the dam but were more contaminated upstream. The allis shads presented intermediate contaminant concentrations with a less pronounced difference between upstream and downstream specimens. However, it is thought that shads do not feed when they spawn in the upstream parts of rivers, which should modify the contaminant concentrations. However, measurements in upstream shad samples show an unexpected increase of the contamination, which remains unexplained.     

Evidence for selection in response to radiation exposure: Pinus sylvestris in the Chernobyl exclusion zone

Changes of genetic structures due to viability selection are likely to occur in populations exposed to rapidly and extremely changing environmental conditions after catastrophic events. However, very little is known about the extent of selective responses and in particular the proportion of the genome involved in putatively adaptive reactions for non-model plants. We used amplified fragment length polymorphisms (AFLPs) in order to investigate genetic differences between pine (Pinus sylvestris) trees which were partially exposed to extreme environmental conditions. Genetic variation patterns of pines exposed to high radiation in the Chernobyl exclusion zone with or without phenotypic stress symptoms were compared to control trees with a similar origin. Six percent of the investigated loci (15 of 222 loci) were identified as candidates for selective responses. Moderate differentiation was observed between groups of trees showing either weak or strong phenotypic responses to high radiation levels.     

The recovery of oyster (Saccostrea glomerata) populations in Sydney estuary (Australia)

The current work documented a significant and widespread increase in the abundance of the Sydney rock oyster Saccostrea glomerata, in Sydney estuary (Australia) by undertaking surveys of oyster density in the estuary in 1989 and annually from 1994 to 2006. Oyster density at six control sites located in nearby National Parks unaffected by boating and stormwater discharges were compared to 17 study sites widely distributed within Sydney estuary. No oyster populations were evident in Sydney estuary in 1989; however, by 1994 oysters had colonised areas of the lower and central estuary and by 2002 densities were statistically similar to control sites. The timing of estuary-wide increases in oyster abundance suggests that the partial banning of tributyltin in 1989 for vessels under 25 m long may have played a major role in the increase of S. glomerata in this estuary.     

Molecular mechanism for cadmium-induced anthocyanin accumulation in Azolla imbricata

Anthocyanins inducibly synthesized by Cd treatment showed high antioxidant activity and might be involved in internal detoxification mechanisms of Azolla imbricata against Cd toxicity. In order to understand anthocyanin biosynthesis mechanism during Cd stress, the cDNAs encoding chalcone synthase (CHS) and dihydroflavonol reductase (DFR), two key enzymes in the anthocyanin synthesis pathway, were isolated from A. imbricata. Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Expression of AiDFR, and to a lesser extent AiCHS, was significantly induced in Cd treatment plant in comparison with the control. CHS and DFR enzymatic activities showed similar pattern changes with these genes expression during Cd stress. These results strongly indicate that Cd induced anthocyanin accumulation is probably mediated by up-regulation of structural genes including CHS and DFR, which might further increase the activities of enzymes encoded by these structural genes that control the anthocyanin biosynthetic steps.     

Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.     

Structure-related distribution of PCDD/Fs, PCBs and HCB in a river-sea system

Water concentrations of PCDD/Fs, HCB, and non-ortho, mono-ortho, and non-dioxin-like PCBs were measured four times during 1 year in a coastal area of the Baltic Sea, to investigate background levels and distribution behaviour. Sampling sites included two rivers, an estuary, and the sea. Particulate and apparently dissolved concentrations were determined using active sampling (filters + PUFs), while freely dissolved concentrations were determined using passive sampling (POM-samplers). The distribution between particulate + colloidal and freely dissolved phases, in the form of TOC-normalized distribution ratios (K_TOC), was found to be near or at equilibrium. The observed K_TOC were not significantly different between sampling sites or seasons. For PCDD/Fs, the concentrations were significantly correlated to suspended particulate matter (SPM), while no correlation to organic carbon (TOC) was observed. In the estuary and the sea, PCB concentrations were correlated to TOC. The sorption of various congeners to SPM and TOC appeared to be related to both hydrophobicity and 3D-structure. The PCDD/F concentration in the sea decreased to one third in May, likely connected to the increased vertical flux of particles during the spring bloom.     

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 μg mL⁻¹. Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.     

PCDD/Fs-induced oxidative damage and antioxidant system responses in tobacco cell suspension cultures

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) are ubiquitous contaminants and can be considerably accumulated by natural plants. In order to elucidate the biochemical and physiological responses of plant to PCDD/Fs, tobacco Bright Yellow-2 (BY-2) cells were selected as model plant and treated with time- and concentration-dependent PCDD/Fs. The toxic effect and oxidative stress caused by PCDD/Fs were evident, which could be indicted by the reduction in fresh mass, the increase in malondialdehyde (MDA) content, and the damage of tobacco cell ultrastructure. PCDD/Fs tolerance was correlated with changes in antioxidant system and hormones of tobacco cells. Superoxide dismutase (SOD) and peroxidase (POD) exhibited peak enzyme activities at the PCDD/Fs concentration of 1000 ng WHO₉₈-TEQ g⁻¹ fresh weight. Glutathione reductase (GR) enzyme activity increased monotonically at high level PCDD/Fs, but the activity of catalase (CAT) was only slightly affected at all treatment. Meanwhile, the exposure to PCDD/Fs resulted in the changes of hormones content. With the increase of exposure concentration of PCDD/Fs, the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) increased, whereas the concentration of jasmonates (JAs) decreased. The above results suggest that tobacco cells had the ability to cope with the oxidative stress induced by low concentration of PCDD/Fs through increasing the activities of antioxidant enzymes and alternating plant hormones levels. However, oxidative stress and toxicity would burst out when plant cells were exposed to the high levels of PCDD/Fs.     

Social–ecological system responses to Hurricane Sandy in the Hudson-Raritan Estuary

The impact of Hurricane Sandy on the Hudson-Raritan estuary (HRE) provided a valuable case study for exploring interactions between long-term environmental degradation, new climatic disturbance stressors, and human behavioral responses. We extend previous research on the ecological effects of major storms to compare water quality and biological parameters three years before and three years after Hurricane Sandy and consider how ecosystem shifts relate to anglers’ perceptions. Results indicate that water clarity and nutrients returned to pre-storm conditions in about one year, while shifts in the biological community, including a significant increase in harmful algal species and declines in zooplankton and Atlantic menhaden, persisted for multiple years, and anglers continued to fish amidst ecosystem decline. Biotic recovery time in the HRE was longer than reports for other shallow estuaries frequently disturbed by hurricanes. Ecological and social responses suggest that the post-storm regime shifts and continued fishing pressure could further environmental degradation.     

Impact of genetic diversity and inbreeding on the life-history of Chironomus midges over consecutive generations

We report results of a multigenerational experiment with Chironomus riparius. Two strains with a high and a low level of genetic variability were exposed to a low, environmentally relevant TBT concentration of 80 μg Sn kg⁻¹ sediment dw nominally (time weighted mean, based on measured concentrations: 4.5 μg Sn kg⁻¹ sediment dw), and various life history traits as well as genetic diversity were monitored for eleven consecutive generations. While TBT effects are hardly visible in the outbred and genetically diverse strain, the inbred and genetically impoverished strain shows a clearly reduced population growth rate compared to the control. Moreover, the impoverished strain shows an increase in fitness over time. Analyses of variation at five microsatellite loci revealed that the level of genetic variation is strongly reduced in the inbred compared to the outbred strain. Moreover, genetic diversity increases over time in the inbred strain. This finding explains the observed increase in fitness in both inbred lineages (control and TBT exposed). The results document that inbreeding and the level of genetic diversity might be of crucial importance in populations under pollution stress. Furthermore, ecotoxicological bioassays have to consider genetic diversity if results between laboratories should be comparable. Our data provides evidence that genetic diversity strongly contributes to the survival of a population exposed to chemical pollution.     

Effect of gender on physiological and behavioural responses of Gammarus roeseli (Crustacea Amphipoda) to salinity and temperature

The importance of potentially interacting factors in organisms responses to a stress are often ignored or underestimated in ecotoxicology. In laboratory experiments we investigated how gender, temperature and age influence the behaviour and the physiology of the freshwater amphipod Gammarus roeseli under salinity stress. Our results revealed a significant higher sensitivity of females in survival, ventilation and ionoregulation whereas no inter-age differences were reported. Water temperature also exerted a significant effect in survival and ventilation of G. roeseli. Some of those factors appeared to interact significantly. This study provides evidence that gender can affect organisms responses to a stressor and consequently has to be considered while assessing a stress impact. We discussed the potential relationships between biological and behavioural responses.     

Multi-biomarker approach in wild European bullhead, Cottus sp., exposed to agricultural and urban environmental pressures: practical recommendations for experimental design

In freshwater ecosystems, a large number of chemical substances are able to disturb homeostasis of fish by modulating one or more physiological functions including the immune system. The aim of this study was to assess multi-biomarker responses including immunotoxicity induced by urban and agricultural pressure in European bullheads living in a small French river basin. For this purpose, a set of biochemical, immunological, physiological and histological parameters was measured in wild bullheads from five locations characterized by various environmental pressures. Moreover, to address effects of physiological status and contamination level variation on biomarker responses, fish were sampled during three periods (April, July and October). Results revealed a clear impact of environmental pressure on fish health and particularly on immunological status. An increase of EROD activity was recorded between upstream and downstream sites. Upstream sites were also characterized by neurotoxicological effects. Fish exhibited upstream/downstream variations of immunological status but strong differences were observed according to sampling season. Conversely, regarding biochemical and immunological effects, no significant response of physiological indexes was recorded related to environmental pressures. According to these results, the European bullhead appears as a valuable model fish species to assess adverse effects in wildlife due to urban and agricultural pressures.     

Aquatic environments polluted with antibiotics and heavy metals: a human health hazard

Aquatic environments often receive wastewater containing pollutants such as antibiotics and heavy metals from hospital sewage, as well as contaminants from soil. The presence of these pollutants can increase the rate of exchange of resistant genes between environmental and pathogenic bacteria, which can make the treatment of various types of bacterial infections in humans and animals difficult, in addition to causing environmental problems such as ecological risk. In this study, two tetracycline-resistant Pseudomonas aeruginosa (EW32 and EW33), isolated from aquatic environments close to industries and a hospital in southeastern Brazil, were investigated regarding the possible association between tetracycline and heavy metal resistance. The isolate EW32 presented a conjugative plasmid with coresistance to tetracycline and copper, reinforcing the concern that antibiotic resistance by acquisition of plasmids can be induced by the selective pressure of heavy metals in the environment.     

Mercury accumulation and tissue-specific antioxidant efficiency in the wild European sea bass (Dicentrarchus labrax) with emphasis on seasonality

The main goal of this study was to assess both mercury (Hg) accumulation and organs’ specific oxidative stress responses of gills, liver and kidney of Dicentrarchus labrax with emphasis on seasonality. Fish were collected in cold and warm periods in three stations: reference, moderated and highly contaminated sites. Our results showed that seasonal factors slightly influenced Hg accumulation between year periods (cold and warm) and strongly affected organs’ response basal levels. In contrast, seasonality seemed not to influence oxidative stress responses, since similar response patterns were obtained for both year periods, and moderate degree of antioxidant responses was obtained. Moreover, the oxidative stress profile may be attributed to Hg contamination degree, which showed organ-specific response and accumulation patterns. Hence, gills showed to be able to adapt to Hg contamination, and in opposition, kidney and liver demonstrated some vulnerability to Hg toxicity. The critical Hg concentrations indicated specific threshold limits for each organ. Overall, seasonality should be taken into account in monitoring programmes, helping to characterize the individuals’ reference values of response and thus to discriminate between the effects induced by natural causes or by contamination.     

Use of sedimentary metals to predict metal concentrations in black mussel (Mytilus galloprovincialis) tissue and risk to human health (Sydney estuary, Australia)

Filter-feeding bivalves have been used extensively as an indicator of ecosystem condition and in management of estuarine environments. The current study aimed to determine whether sedimentary metals could predict metal concentrations in tissue of filter-feeding mussels (Mytilus galloprovincialis) and to identify areas of the estuary where mussel consumption posed a human health risk. Mussel tissue Cu and Zn concentrations (wet weight) were below guideline values for human consumption in all parts of the waterway, whereas Pb tissue concentrations exceed these guidelines (2.0 μg?g^?1 wet weight) in the upper reaches of some embayments of the estuary. Concentrations of Cu and Pb in the fine fraction (<62.5 μm) of bottom sediment reasonably predicted concentrations (dry weight) of these metals in mussel tissue (r ²?=?0.460 and p?=?0.001 and r ²?=?0.669 and p?<?0.0001, respectively) as these materials are resuspendable and available to filter-feeding estuarine animals, whereas total sediment and mussel tissue were poorly related. Lead concentrations (>350 μg?g^?1) in fine sediments indicated areas of this estuary where human health was at risk due to high tissue concentrations of this metal. These results give encouragement for the use of the metal concentration in fine sediments as an indicator of estuarine condition and risk to human health in this waterway. Mussels were distributed in all parts of the estuary, even in areas where metal concentrations exceeded sediment quality guidelines.     

The negative effects of cadmium on Bermuda grass growth might be offset by submergence

Revegetation in the water-level-fluctuation zone (WLFZ) could stabilize riverbanks, maintain local biodiversity, and improve reservoir water quality in the Three Gorges Reservoir Region (TGRR). However, submergence and cadmium (Cd) may seriously affect the survival of transplantations. Bermuda grass (Cynodon dactylon) is a stoloniferous and rhizomatous prostrate weed displaying high growth rate. A previous study has demonstrated that Bermuda grass can tolerate deep submergence and Cd stress, respectively. In the present study, we further analyzed physiological responses of Bermuda grass induced by Cd-and-submergence stress. The ultimate goal was to explore the possibility of using Bermuda grass for revegetation in the WLFZ of China’s TGRR and other riparian areas. The Cd-and-submergence-treated plants had higher malondialdehyde contents and peroxidase than control, and both increased with the Cd concentration increase. All treated plants catalase activity increased with the experimental duration increases, and their superoxide dismutase also gradually increased with the Cd concentration from 1 day to 15 days. Total biomass of the same Cd-and-submergence plants increased along the experimental duration as well. Plants exposed to Cd-and-submergence stress showed shoot elongation. The heights of all treated plants were taller than those of the control. Leaf chlorophyll contents, maximum leaf length, and soluble sugars contents of all the Cd-and-submergence-treated plants were more than those of the untreated control. Although Cd inhibits plants growth, decreases chlorophyll and biomass content, and with the submergence induced the leaf and shoot elongation, more part of the Cd-and-submergence stress plants appeared in the air, exhibited fast growth with maintenance of leaf color, which guaranteed the plants’ photosynthesis, and ensured the total biomass and carbohydrate sustainability, further promoting Cd-and-submergence tolerance. The results imply that the negative effects of cadmium on Bermuda grass growth might be offset by submergence.     

Reproduction and biochemical responses in Enchytraeus albidus (Oligochaeta) to zinc or cadmium exposures

To better understand chemical modes of action, emphasis has been given to stress responses at lower levels of biological organization. Cholinesterases and antioxidant defenses are among the most used biomarkers due to their crucial role in the neurocholinergic transmission and in cell homeostasis preventing DNA damage, enzymatic inactivation and lipid peroxidation. The main goal of this study was to investigate the effects of zinc and cadmium on survival and reproduction of E. albidus and to assess metals oxidative stress potential and neurotoxic effects at concentrations that affected reproduction. Both metals affected the enchytraeids’ survival and reproduction and induced significant changes in the antioxidant defenses as well as increased lipid peroxidation, indicating oxidative damage. This study demonstrates that determining effects at different levels of biological organization can give better information on the physiological responses of enchytraeids in metal contamination events and further unravel the mechanistic processes dealing with metal stress.