Assessment of toxic effects of imidacloprid on freshwater zooplankton: An experimental test for 27 species.

The neonicotinoid pesticide imidacloprid has been used worldwide since 1992. As one of the most important chemicals used in pest control, there have been concerns that its run-off into rivers and lakes could adversely affect aquatic ecosystems, where zooplankton play a central role in the energy flow from primary to higher trophic levels. However, studies assessing the effects of pesticides at the species level have relied on a Daphnia-centric approach, and no studies have been conducted using species-level assessments on a broad range of zooplankton taxa. In the present study, we therefore investigated the acute toxicity of imidacloprid on 27 freshwater crustacean zooplankton (18 cladocerans, 3 calanoid copepods and 6 cyclopoid copepods). The experiment showed that a majority of calanoid copepods and cladocerans were not affected at all by imidacloprid, with the exception of one species each of Ceriodaphnia and Diaphasoma, while all six cyclopoid copepods showed high mortality rates, even at concentrations of imidacloprid typically found in nature. In addition, we found a remarkable intra-taxonomic variation in susceptibility to this chemical. As many cyclopoid copepods are omnivorous, they act as predators as well as competitors with other zooplankton. Accordingly, their susceptibility to imidacloprid is likely to cause different responses at the community level through changes in predation pressure as well as changes in competitive interactions. The present results demonstrate the need for species-level assessments of various zooplankton taxa to understand the complex responses of aquatic communities to pesticide disturbance.

Assessment of the relative sensitivity of the copepods Acartia tonsa and Acartia clausi exposed to sediment-derived elutriates from the Bagnoli-Coroglio industrial area.

The sensitivity of the copepods Acartia tonsa, commonly used in standardized tests for environmental risk assessment and A. clausi, the dominant autochthonous congener species in the Mediterranean Sea, was assessed using sediment-derived elutriates from the industrial area of Bagnoli-Coroglio and nickel chloride as referent toxicant. Acute A. clausi naupliar immobilization test showed EC50 for elutriates E25, E56 and E84 of 23.3%, 80.5% and >100%, respectively, compared to 59.5%, 66.6% and >100% in A. tonsa. In the 7 day sublethal test, a reduction in A. clausi egg production rates was observed in all elutriates, but only in E56 for A. tonsa. Elutriate 56, which contained the highest amount of polycyclic aromatic hydrocarbons, also induced 70% mortality in A. clausi females. Although A. clausi was more sensitive than A. tonsa, the two species had convergent responses to the three elutriates, thus opening the venue for a potential use of A. clausi in standardized ecotoxicity tests.

Environmental risk assessment of propranolol in the groundwater bodies of Europe.

A growing concern for contamination due to pharmaceutical compounds in groundwater is expanding globally. The ß-blocker propranolol is a ß-adrenoceptors antagonist commonly detected in European groundwater bodies. The effect of propranolol on stygobiotic species (obligate groundwater dweller species) is compelling in the framework of environmental risk assessment (ERA) of groundwater ecosystems. In fact, in Europe, ERA procedures for pharmaceuticals in groundwater are based on data obtained with surrogate surface water species. The use of surrogates has aroused some concern in the scientific arena since the first ERA guideline for groundwater was issued. We performed an ecotoxicological and a behavioural experiment with the stygobiotic crustacean species Diacyclops belgicus (Copepopda) to estimate a realistic value of the Predicted No Effect Concentration (PNEC) of propranolol for groundwater ecosystems and we compared this value with the PNEC estimated based on EU ERA procedures. The results of this study showed that i) presently, propranolol does not pose a risk to groundwater bodies in Europe at the concentrations shown in this study and ii) the PNEC of propranolol estimated through the EU ERA procedures is very conservative and allows to adequately protect these delicate ecosystems and their dwelling fauna. The methodological approach and the results of this study represent a first contribution to the improvement of ERA of groundwater ecosystems.

Use of scanning and image recognition technology to semi-automate larval development assessment in toxicity tests with a tropical copepod.

There is a high demand for the development of reliable chronic toxicity tests using tropical marine species for subsequent use in tropical risk assessment. However, many chronic test endpoints can be laborious and time-consuming to assess, particularly if the endpoints require measurements of individuals (e.g. growth, size) or advanced taxonomic expertise (e.g. differentiating between larval development stages). In this study, we used scanning and image recognition (SIR) technology to develop and validate a chronic toxicity test with larvae of the tropical euryhaline copepod, Acartia sinjiensis. Optimisation steps are described, and included egg age, and effect of algal food type and salinity on toxicity. Comparisons were made between traditional endpoints measured using microscopy and those measured using SIR. Traditional endpoints of larval development ratio (LDR) and survival achieved using microscope examination and SIR were almost identical (R2 = 0.96-0.97). Additional endpoints made possible by SIR included larval development index (LDI; based on the number of animals at different stages of development), and a range of size measurements (e.g. surface area, perimeter and length) for individual animals and for total populations (i.e. a proxy for biomass). The SIR-derived endpoints were based on measurements that had concentration-dependant responses to tested toxicants (copper, nickel, ammonia), and were a sub-set of the full range of metrics provided by the software. Toxicity values based on SIR-measurements were similar to or more sensitive than the traditional LDR endpoint. SIR technology provides a major opportunity to improve and modernise larval development tests for a range for species, but comes at a cost of increased data size and complexity. Therefore, as a research tool, SIR has significant advantages over traditional microscope methods, but for routine toxicity testing, SIR incorporation into invertebrate toxicity testing will benefit from further improvements to the associated software and data management systems.

The role of freshwater copepods in the environmental risk assessment of caffeine and propranolol mixtures in the surface water bodies of Spain.

In this study we aimed at assessing: (i) the environmental risk posed by mixtures of caffeine and propranolol to the freshwater ecosystems of Spain; (ii) the sensitivity of freshwater copepod species to the two compounds; (iii) if the toxicity of caffeine and propranolol to freshwater copepods contributes to the environmental risk posed by the two compounds in the freshwater bodies of Spain. The environmental risk was computed as the ratio of MECs (i.e. the measured environmental concentrations) to PNECs (i.e. the respective predicted no-effect concentrations). The effects of caffeine and propranolol on the freshwater cyclopoid Diacyclops crassicaudis crassicaudis were tested both individually and in binary mixtures. Propranolol posed an environmental risk in some but not in all the surface water ecosystems of Spain investigated in this study, while caffeine posed an environmental risk to all the investigated freshwater bodies, both as single compound and in the mixture with propranolol. Propranolol was the most toxic compound to D. crassicaudis crassicaudis, while caffeine was non-toxic to this species. The CA model predicted the toxicity of the propranolol and caffeine mixture for this species. D. crassicaudis crassicaudis was much less sensitive than several other aquatic species to both compounds. The sensitivity of D. crassicaudis crassicaudis does not increase the environmental risk posed by the two compounds in the freshwater bodies of Spain, however, further testing is recommended since the effect of toxicants on freshwater copepods can be more pronounced under multiple stressors and temperature increasing due to climate change.

A multivariable assessment of the spatio-temporal distribution of pyrethroids performance on the sea lice Caligus rogercresseyi in Chile.

Synthetic pyrethroids have been widely used in Chile to control the sea lice Caligus rogercresseyi, a major ectoparasite of farmed salmon. Although resistance of C. rogercresseyi to pyrethroids has been reported in Chile, there is no information regarding the geographic extent of this problem. In this study we explored the spatial and temporal variation of C. rogercresseyi's response to pyrethroids in Chile from 2012 to 2013. We modeled lice abundance one week after treatment with a linear mixed-effects regression, and then we performed spatial and spatio-temporal cluster analyses on farm-level effects and on treatment-level residuals, respectively. Results indicate there were two areas where the post-treatment lice counts were significantly higher than in the rest of the study area. These spatial clusters remained even once we adjusted for environmental and management predictors, suggesting unmeasured factors (e.g. resistance) were causing the clustering. Further investigation should be carried out to confirm this hypothesis.

Sensitivity assessment of sea lice to chemotherapeutants: Current bioassays and best practices.

Traditional bioassays are still necessary to test sensitivity of sea lice species to chemotherapeutants, but the methodology applied by the different scientists has varied over time in respect to that proposed in "Sea lice resistance to chemotherapeutants: A handbook in resistance management" (2006). These divergences motivated the organization of a workshop during the Sea Lice 2016 conference "Standardization of traditional bioassay process by sharing best practices." There was an agreement by the attendants to update the handbook. The objective of this article is to provide a baseline analysis of the methodology for traditional bioassays and to identify procedures that need to be addressed to standardize the protocol. The methodology was divided into the following steps: bioassay design; material and equipment; sea lice collection, transportation and laboratory reception; preparation of dilution; parasite exposure; response evaluation; data analysis; and reporting. Information from the presentations of the workshop, and also from other studies, allowed for the identification of procedures inside a given step that need to be standardized as they were reported to be performed differently by the different working groups. Bioassay design and response evaluation were the targeted steps where more procedures need to be analysed and agreed upon.

The ecotoxicity of binary mixtures of Imazamox and ionized ammonia on freshwater copepods: Implications for environmental risk assessment in groundwater bodies.

Groundwater bodies are impacted by substances such as pesticides and N-fertilizers, which usually occur in the environment as complex mixtures rather than isolated pollutants. The threat that these mixtures pose to groundwater-dwelling organisms is still poorly understood. The aims of the present study were to test the acute effect of a binary mixture of a herbicide (Imazamox) and NH4+ on epigean (Eucyclops serrulatus) and hypogean (Diacyclops belgicus) freshwater copepod species. In addition, to evaluate if the effect of the mixture can be explained by referencing non-interaction models or by more complex interaction models; and the implications for groundwater risk assessment. Compared with the action of the compounds evaluated separately, the effects of Imazamox and NH4+ in the binary mixture were more than additive or synergistic for both species. MixTox models evidenced a dose ratio and dose level deviations from concentration addition and independent action traditional models. The hypogean species was three times more sensitive to NH4+ that the epigean species when assayed as a single chemical. However, D. belgicus was only 1.13 times more sensitive than E. serrulatus when NH4+ was assayed in the mixture. The use of an integrated approach for substances that are known to interact in groundwater, should include copepods species as test organisms.

The occurrence and spatial-temporal distribution of tetrabromobisphenol A in the coastal intertidal zone of Qingdao in China, with a focus on toxicity assessment by biological monitoring.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant that has increasingly been found as contaminant in aquatic environments. The main goal of this study was to evaluate the pollution level of TBBPA at six locations around Qingdao and assess its biotoxicity through a two-generation toxicity study looking at a copepod species. In the chemical monitoring, the concentration of TBBPA in seawater samples ranged from nd to 1.8 µg/L. Next, the biological indicator monitoring used 1.8 µg/L as the middle exposure concentration to perform quantitative evaluations of the influence of TBBPA on the demographic traits of Pseudodiaptomus inopinus. The results showed that copepods became more sensitive to TBBPA exposure even in environmental concentration (1.8 µg/L) as the generations developed. The detrimental effects of TBBPA further increased naupliar mortality and impaired copepodite development to adulthood. This study demonstrated that the water pollution condition of TBBPA was measured at all 6 sampling locations of Qingdao. Therefore, the present results call for a decreased discharge of TBBPA into the marine environment to avoid impairing copepod reproduction and development.

Binary mixture toxicities of triphenyltin with tributyltin or copper to five marine organisms: Implications on environmental risk assessment.

Triphenyltin (TPT) often coexists with tributyltin (TBT) and Cu in coastal waters worldwide. The combined toxic effect of TPT and TBT has always been assumed to be additive without any scientific proof, and the combined effect of Cu and TPT on marine organisms has not been vigorously studied. This study, therefore, investigated the acute toxicity of binary mixture of TPT/Cu and TPT/TBT to five selected marine species including Thalassiosira pseudonana, Skeletonema costatum, Tigriopus japonicus, Brachionus koreanus and Oryzias melastigma. The interaction between TPT and TBT or Cu was modeled antagonistic based on concentration addition (CA) model, while it was synergistic according to response addition (RA) model. Both model well predicted the toxicity of binary mixtures to the five organisms. As for the environmental risk assessment, CA overestimated the toxicity in most cases and thus is a more conservative model than RA model for assessing the toxicity of these chemical mixtures.

Aquatic risk assessment of a novel strobilurin fungicide: A microcosm study compared with the species sensitivity distribution approach.

The ecotoxicological effects of pyraoxystrobin, a novel strobilurin fungicide, were studied using outdoor freshwater microcosms and the species sensitivity distribution approach. The microcosms were treated with pyraoxystrobin at concentrations of 0, 1.0, 3.0, 10, 30 and 100µg/L. Species sensitivity distribution (SSD) curves were constructed by means of acute toxicity data using the BurrliOZ model for fourteen representatives of sensitive invertebrates, algae and fish and eleven taxa of invertebrates and algae, respectively. The responses of zooplankton, phytoplankton and physical and chemical endpoints in microcosms were studied. Zooplankton, especially Sinodiaptomus sarsi was the most sensitive to pyraoxystrobin exposure in the microcosms. Short-term toxic effects (<8 weeks) on zooplankton occurred in 1µg/L treatment group. The duration of toxic effects on S. sarsi could not be evaluated within the initial 56 days. Significant long-term toxic effects were observed at 10, 30 and 100µg/L (>281 days) for S. sarsi and the zooplankton community. Based on the results obtained from the organisms in the microcosm system, 1µg/L was recommended as the NOEAEC (no observed ecologically adverse effect concentration). Also, 0.33µg/L was derived as the Regulatory Acceptable Concentration based on the ecological recovery option (ERO-RAC) of pyraoxystrobin. For all fourteen tested species, the median HC5 (hazardous concentration affecting 5% of species) was 0.86µg/L, and the lower limit HC5 (LL-HC5) was 0.39µg/L. For the eleven taxa of invertebrates and algae tested, the median HC5 was 1.1µg/L, and the LL-HC5 was 0.26µg/L. The present study positively contributes to the suggestion of adequately using acute L(E)C50-based HC5/ LL-HC5 for deriving protective concentrations for strobilurin fungicides, and it should be valuable for full comprehension of the potential toxicity of pyraoxystrobin in aquatic ecosystems.

Determining the Advantages, Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST).

The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.

Chronic aquatic effect assessment for the fungicide azoxystrobin.

The present study examined the ecological effects of a range of chronic exposure concentrations of the fungicide azoxystrobin in freshwater experimental systems (1270-L outdoor microcosms). Intended and environmentally relevant test concentrations of azoxystrobin were 0 µg active ingredient (a.i.)/L, 0.33 µg a.i./L, 1 µg a.i./L, 3.3 µg a.i./L, 10 µg a.i./L, and 33 µg a.i./L, kept at constant values. Responses of freshwater populations and community parameters were studied. During the 42-d experimental period, the time-weighted average concentrations of azoxystrobin ranged from 93.5% to 99.3% of intended values. Zooplankton, especially copepods and the Daphnia longispina group, were the most sensitive groups. At the population level, a consistent no-observed-effect concentration (NOEC) of 1 µg a.i./L was calculated for Copepoda. The NOEC at the zooplankton community level was 10 µg azoxystrobin/L. The principle of the European Union pesticide directive is that lower-tier regulatory acceptable concentrations (RACs) are protective of higher-tier RACs. This was tested for chronic risks from azoxystrobin. With the exception of the microcosm community chronic RAC (highest tier), all other chronic RAC values were similar to each other (0.5-1 µg a.i./L). The new and stricter first-tier species requirements of the European Union pesticide regulation (1107/2009/EC) are not protective for the most sensitive populations in the microcosm study, when based on the higher tier population RAC. In comparison, the Water Framework Directive generates environmental quality standards that are 5 to 10 times lower than the derived chronic RACs.

Toxicological assessment of the anti-salmon lice drug diflubenzuron on Atlantic cod Gadus morhua.

Increasing use of the chitin synthesis inhibitor diflubenzuron against the ectoparasitic salmon louse Lepeophtheirus salmonis in marine aquaculture has raised concerns over its environmental impacts. This study evaluated how diflubenzuron affects Atlantic cod Gadus morhua, a fish species often found near Atlantic salmon Salmo salar farms, focusing on uptake kinetics and hepatic transcriptional responses. Two experiments were conducted, one time-series trial in which the fish were given a daily dose (3 mg kg-1 fish) of diflubenzuron for 14 d followed by a 3 wk depuration period, and one dose-response trial with increasing concentrations (3, 10 and 50 mg kg-1 fish). The highest diflubenzuron concentrations were found in the liver at Day 15. No detectable levels of diflubenzuron were found in liver or muscle 3 wk after the end of the treatment. At the molecular level, small effects of diflubenzuron treatment on gene transcription were observed. In the time-series experiment, the strongest effects were seen at Day 8, with 2 transcripts being upregulated (bclx2 and cpt1a) and 8 transcripts being downregulated (gstp1, gstm1, gstt1, ugt1a, nat2, cat, p53 and slc16a9a). Five transcripts (cyp3a, cpt1a, ptgs2, elovl5 and mapk1) responded significantly to diflubenzuron exposure in the dose-response experiment. This study shows that diflubenzuron can be taken up by Atlantic cod, that it is rapidly cleared from the body and that when present this pharmaceutical causes only small effects on the expression of genes involved in detoxification pathways. Taken together, our data suggest that accumulated diflubenzuron at the levels studied would have a relatively small effect on wild Atlantic cod.

A new protocol for ecotoxicological assessment of seawater using nauplii of Tisbe biminiensis (Copepoda:Harpacticoida).

Copepods are largely used in toxicity tests. The nauplii of these organisms are more sensitive to contaminants than the adult stage. The aim of the present study was to test a protocol for the use of nauplii of the copepod Tisbe biminiensis in the ecotoxicological assessment of seawater. The sensitivity of these organisms to zinc sulphate (ZnSO4·7H2O) was also determined. The following conditions were established for the protocol based on the best development of nauplii to copepodites: 72-h duration, the microalga Chaetocerus gracilis at 2.5×10(5)cellsmL(-1) as feed and incubation temperature of 28°C. In the zinc sulphate sensitivity tests, EC50-72 h and LC50/72 h were 3.25±0.59 mg L(-1) and 3.46±0.72 mg L(-1), respectively, as estimated by the final number of copepodites and total number of live animals in relation to the mean number of inoculated nauplii. The estimated NOEC was 2.0 mg L(-1). The test developed is fast and not labour intensive. T. biminiensis nauplii exhibit sensitivity to zinc sulphate similar to that of other species of copepods employed in water toxicity tests, demonstrating the usefulness of these organisms in ecotoxicological studies involving samples of environmental seawater.

Sediment quality assessment in a tropical estuary: the case of Ceará River, Northeastern Brazil.

The present study aimed to assess the sediment quality in a tropical estuary located in the northeast of Brazil under semi-arid conditions and multiple sources of contamination, using both toxicity bioassays and metal distribution. The metal distribution followed a concentration gradient decreasing one order of magnitude from the inner station toward the outer estuary, with amounts in the following order: Fe > Al > Zn > Cr > Pb > Cu. The index of geoaccumulation indicated a metal enrichment in the Ceará river sediment, mainly at inner sites, considered from moderately to strongly contaminated by Al, Cu, Cr and Zn. Sediment samples were considered toxic by means of whole sediment tests with copepods (reproduction) and amphipods (survival), and also elutriate fraction and sediment-water interface with sea urchin embryos (development). Acute and chronic toxicity did not exhibit a significant correlation with metals, emphasizing the influence of other contaminants mainly related to the pollution sources installed in the mid-estuary.

A short life-cycle test with the epibenthic copepod Nitocra spinipes for sediment toxicity assessment.

A new short life-cycle test methodology is presented to evaluate sublethal effects of contaminated sediments to the harpacticoid copepod species Nitocra spinipes. The method combines a 4-d survival-gravidity test with a 7-d development test. For water-only Cu exposures, the sensitivity of the development test endpoints were compared with tests using nonexposed gravid females to initiate the 7-d development test phase, and also with a multiple generation test comprising three successive phases: 9-d development; 21-d survival-gravidity; second-generation 9-d development. The results indicated that the development endpoints were the most sensitive, with median effective concentration (EC50) values of 95 µg Cu/L for nauplii/gravid female and 101 µg Cu/L copepodites/gravid female endpoints, followed by the gravidity (144 µg Cu/L) and survival (347 µg Cu/L). The sensitivity of the short life-cycle test endpoints was similar to the multiple-generation test endpoints, and the shorter test had less variability in controls. The multiple-generation test showed a large amount of stimulation of reproduction and development at Cu concentrations of 50 to 100 µg/L. The suitability of the short life-cycle test for assessing sediment toxicity was demonstrated using Cu-spiked and naturally contaminated whole sediments. Although the small nauplii were more difficult to isolate from sediments, the small amounts of sediments used for the tests and the large effects of the contaminated sediments on nauplii and copepodite numbers resulted in significant differences to controls. For sediment exposures, the sensitivity of the endpoints was in the order development > gravidity > survival. The short life-cycle test was demonstrated to detect, within 11 d of exposure, similar levels of effects on reproduction and development to those detected using a 39-d multiple-generation exposure.

An assessment of three harpacticoid copepod species for use in ecotoxicological testing.

The relatively short life cycles of harpacticoid copepods makes them appropriate animals for use in tests that rapidly assess the acute, sublethal, or chronic effects of sediment contaminants. In this study, four harpacticoid copepod species (Nitocra spinipes, Tisbe tenuimana, Robertgurneya hopkinsi, and Halectinosoma sp.) were isolated from clean marine sediments, and procedures for laboratory culturing were developed. Halectinosoma sp. was abandoned due to handling difficulties. For the remaining species, the influence of food type and quantity on life-cycle progression was assessed. A mixed diet, comprising two species of algae (Tetraselmis sp. and Isochrysis sp.) and fish food (Sera Micron) was found to maintain healthy cultures and was fed during laboratory tests. Water-only exposure to dissolved copper (Cu) showed that the times (range) required to cause 50% lethality (LT(50)) were 24 (22-27) h at 50 µg Cu/l for T. tenuimana; 114 (100-131) and 36 (32-40) h for 200 and 400 µg Cu/l, respectively, for N. spinipes; and 119 (71-201) and 25 (18-33) h for 200 and 400 µg Cu/l, respectively, for R. hopkinsi. 96-h LC(50) (concentration causing 50% lethality) were also determined for adult N. spinipes exposed to cadmium, copper, zinc, ammonia, and phenol. A ranking system was generated based on the ease handling and culturing, rate of maturity, food selectivity and sensitivity to Cu. From this ranking, N. spinipes was determined to be the most suitable species for use in developing sediment-toxicity tests. The measurement of total reproductive output of N. spinipes during 10-day exposure to whole sediment was found to provide a useful end point for assessing the effects of sediment contamination.

Sediment integrative assessment of the Bay of Cádiz (Spain): an ecotoxicological and chemical approach.

This study consisted of the sediment toxicity assessment of the Bay of Cádiz based on two endpoints: growth inhibition for Cylindrotheca closterium (benthic microalgae) and fecundity inhibition for Tisbe battagliai (harpacticoid copepod). A new methodology to eliminate (but not as storage technique) the autochthonous biota present in the sediment samples by immersing them in liquid nitrogen (-196 degrees C) was also assessed. Sediment toxicity data showed different toxicity levels for both organisms. In general, T. battagliai was more sensitive; however a good correlation (r=0.75; p<0.05) between sediment toxicity results for both species was found. Data in pore water (pH, redox potential, and toxicity for microalgae and copepod) and sediment (pH, redox potential, organic carbon, and metal concentrations) demonstrated that ultra-freezing did not alter sample characteristics; thus, this technique can be adopted as a pre-treatment in whole-sediment toxicity tests in order to avoid misleading results due to presence of autochthonous biota. Multivariate statistical analysis such as cluster and principal component analysis using chemical and ecotoxicological data were employed. Silt and organic matter percentage and lead concentration were found to be the factors that explain about 77% of sediment toxicity in the Bay of Cádiz. Assay methodology determined in this study for both assayed species is considered adequate to be used in sediment toxicity monitoring programs. Results obtained using both species show that the Bay of Cádiz can be considered a moderately polluted zone.

Acclimation effect and fitness cost of copper resistance in the marine copepod Tigriopus japonicus.

Copper (Cu) contamination is common and widespread in coastal marine environments. This study used the marine copepod Tigriopus japonicus to test whether Cu resistance can be developed through multigeneration acclimation to elevated Cu levels and whether the resistance has a fitness cost. T. japonicus (F0) were acclimated to three Cu concentrations (0, 10, and 100 microg l(-1)) and offspring (F1 and F2) of each treatment were subsequently acclimated at these three concentrations, respectively. Our results evidently indicated that Cu resistance of the copepod was increased even after one generation of acclimation to 100 microg Cu l(-1). The acquired Cu resistance had a fitness cost, as the intrinsic population growth rate of this Cu resistant lineage was significantly lower than the control. The Cu resistance of the offspring from Cu resistant copepods, when raised under control conditions, returned to a level comparable to the control implying a plastic physiological adaptation.