Rapid-behaviour responses as a reliable indicator of estrogenic chemical toxicity in zebrafish juveniles

Whereas biochemical and molecular parameters have been well recognised as important “signposts” of individual disturbance to endocrine disrupting chemical’s (EDCs) exposure, behavioural endpoints are yet greatly overlooked as a routine tool in environmental risk assessment of EDCs. However, life histories are intimately associated with numerous inter- and intra-specific interactions, which invariably depend on the performance of effective behaviours. Within fish species, one of the most important factors influencing energy turnover earlier in the development is locomotor activity. This essential trait reflects the organism’s ability to generate and coordinate the metabolic energy required for both reproductive and non-reproductive behaviours. Inappropriate movement responses due to toxic effects of contaminants may ultimately impact important ecological variables.Therefore, in the present study, the swimming bursts of zebrafish juveniles exposed for 40 d to the synthetic estrogen ethinylestradiol (EE₂), tested at environmentally relevant concentrations (nominal concentrations of 0.5, 1 and 2 ng L⁻¹), were investigated in order to address the potential of rapid-behaviour patterns as an effective response indicator of estrogenic endocrine disrupting chemical’s exposure. This synthetic estrogen was selected due to its high prevalence in aquatic ecosystems, ability to mimic natural estrogens and proven record of causing negative effects in fish reproduction. The behavioural responses were compared with established endpoints used in the screening of EE₂ effects at adulthood. Results indicate that zebrafish juveniles’ swimming activity was significantly decreased upon EE₂ exposure. Since reduced locomotion of zebrafish may impact foraging, predator avoidance, drift and transport, and even interfere with social and reproductive behaviours, a fitness decline of wild fish populations can ultimately be hypothesized. Furthermore, behavioural endpoints were found to display higher sensitivity to EE₂ than either vitellogenin gene induction or reproductive parameters determined at adulthood.Overall, the findings of this work not only demonstrate the power of high-throughput behavioural responses, able to act as sensitive early warning signals of EDC exposure, but also highlight the potential of behavioural endpoints in providing a more comprehensive and non-invasive measure of EDC’s exposure.     

Influence of contaminant burial depth on the bioaccumulation of PCBs and PBDEs by two benthic invertebrates (Monoporeia affinis and Marenzelleria spp.)

The bioaccumulation of buried polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) added to specific depths in sediment (2.0-2.5, 5.0-5.5 and 10.0-10.5 cm) was studied in two infaunal species with similar feeding habits (surface deposit-feeders) but different bioturbation modes. The deep-burrowing polychaetes Marenzelleria spp. (Mz) displayed up to 36 times higher tissue concentrations of buried (spiked) contaminants than the surface-dwelling biodiffusing amphipod Monoporeia affinis. The differences in bioaccumulation were most pronounced for less hydrophobic contaminants due to the bioirrigating activity of Mz. Contaminants buried at shallow depths displayed higher accumulation than more deeply buried contaminants. In contrast, the bioaccumulation of unspiked (native) contaminants with a uniform vertical distribution in the sediment was similar between the species. For Mz, the BSAFs increased with increased K_OW for the uniformly distributed contaminants, but decreased for the buried contaminants, which indicates that the dominant uptake routes of the buried contaminants can differ from the uniformly distributed contaminants. The surface sediment concentration of buried contaminants increased in Mz treatments, showing that Mz bioturbation can remobilize historically buried contaminants to the biologically active surface layer and increase the exposure for surface-dwelling species.     

The toxicity of titanium dioxide nanopowder to early life stages of the Japanese medaka (Oryzias latipes)

In this study, fertilized Japanese medaka (Oryzias latipes) embryos were exposed from fertilization to 5 d post-hatch using static non-renewal assays to aqueous suspensions of titanium dioxide nanoparticles (nTiO₂) ranging in nominal concentrations between 0 and 14 μg mL⁻¹. The average size of the nTiO₂ in the stock solution before addition to the test treatments was 87 nm (±14 nm). TiO₂ materials accumulated in a concentration dependent manner on the chorionic filaments of developing medaka embryos with evidence of pericardial edema occurring during embryo development. However, no significant (p > 0.05) increases in mortality relative to control treatments were observed for the nTiO₂ exposed embryos. A concentration dependent increase in cumulative percent hatch was observed at 11 d, indicating that exposure to increasing concentrations of nTiO₂ resulted in the premature hatch of medaka embryos. Post-hatch, a significant proportion of sac fry from the nTiO₂ exposure groups exhibited moribund swimming behavior and these individuals also experienced greater mortality at 15 d post-hatch. Combined, these results demonstrate that exposure to nTiO₂ can impact the development of early life stages of fish.     

Polybrominated diphenyl ethers (PBDEs) and mercury in fish from lakes of the Tibetan Plateau

High mountains may act as cold traps for globally transported persistent organic pollutants (POPs) and mercury (Hg). In the present study, 60 fish samples were collected from eight alpine lakes across the Tibetan Plateau. Concentrations of polybrominated diphenyl ethers (PBDEs), total mercury (HgT) and methyl mercury (MeHg) were quantified in the fish muscle tissues to improve the understanding of pollution status and factors regulating the transport and fate of these contaminants on the Plateau. The results showed that lake-averaged ∑₁₄PBDEs concentration was between 0.09 ng g⁻¹ dw and 4.32 ng g⁻¹ dw, which was lower than those reported for European mountains. The total mercury concentration in individual fish ranged from 243 to 2384 ng g⁻¹ dw, and that of MeHg from 131 to 1610 ng g⁻¹ dw, which is much higher than those reported in other mountain fish. The spatial variation of PBDEs and mercury in the Plateau is largely controlled by the specific meteorological patterns.     

Effects of the aquatic contaminant human pharmaceuticals and their mixtures on the proliferation and migratory responses of the bioindicator freshwater ciliate Tetrahymena

An increasing attention is paid to the potential harmful effects of aquatic contaminant pharmaceuticals exerted on both biosystems and humans. In the present work the effects of 14 pharmaceuticals including NSAIDs, antibiotics, β-blockers and a frequently used X-ray contrast media on the proliferation and migratory behavior of the freshwater ciliate Tetrahymena pyriformis was investigated. Moreover, the mixture toxicity of four selected pharmaceuticals (diclofenac, ibuprofen, metoprolol and propranolol) was evaluated in binary mixtures using full factorial experimental design. Our results showed that the sensitivity of Tetrahymena to NSAIDs and β-blockers (EC₅₀ ranged from 4.8 mg L⁻¹ to 308.1 mg L⁻¹) was comparable to that of algal or Daphnia bioassays. Based on these elevated EC₅₀ values acute toxic effects of these pharmaceuticals to T. pyriformis are unlikely. Antibiotics and the contrast agent sodium-diatrizoate had no proliferation inhibiting effect. Chemotactic response of Tetrahymena was more sensible than proliferation as significant chemorepellent action was observed in the environmentally realistic concentration range for acetylsalicylic acid, diclofenac, fenoprofen, paracetamol, metoprolol, propranolol, timolol and trimethoprim (Chemotaxis Index ranged from 63% to 88%).Mixture toxicity experiments resulted in a complex, concentration dependent interaction type pattern with antagonism being the predominant interaction type (59%) followed by additivity (37%) and synergism (4%). Hence the concept of concentration addition validated for NSAIDs in other organisms cannot be adopted for this ciliate.In summary authors suggest Tetrahymena as a sensible model of testing aquatic contaminants as well as underline the significance using more specific endpoints to understand the complex mechanisms investigated.     

Effects of black carbon on bioturbation-induced benthic fluxes of polychlorinated biphenyls

It is unknown whether carbonaceous geosorbents, such as black carbon (BC) affect bioturbation by benthic invertebrates, thereby possibly affecting sediment-water exchange of sediment-bound contaminants. Here, we assess the effects of oil soot on polychlorinated biphenyl (PCB) mass transfer from sediment to overlying water, for sediments with and without tubificid oligochaeta as bioturbators. PCB levels were so low that toxicity to the oligochaeta played no role, whereas soot levels and binding affinity of PCBs to soot were so low that pore water PCB concentrations were not significantly affected by binding of PCBs to soot. This setup left direct effects of BC on bioturbation activity as the only explanation for any observed effects on mass transfer. Mass transfer coefficients (K_L) for benthic boundary layer transport were measured by a novel flux method using Empore™ disks as a sink for PCBs in the overlying water. For the PCBs studied (logK_ow 5.2-8.2), K_L values ranged from 0.2 to 2 cm × d⁻¹ in systems without tubificids. Systems with tubificids showed K_L values that were a factor of 10-25 higher. However, in the presence of oil soot, tubificids did not cause an increase in mass transfer coefficients. This suggests that at BC levels as encountered under field conditions, the mechanism for reduction of sediment-water transfer of contaminants may be twofold: (a) reduced mass transfer due to strong binding of the contaminants to BC, and (b) reduced mass transfer of contaminants due to a decrease in bioturbation activity.     

Trophic transfer of pyrene metabolites and nonextractable fraction from Oligochaete (Lumbriculus variegatus) to juvenile brown trout (Salmo trutta)

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC₅₀ values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r² = 0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC₅₀ and the in vivo LC₅₀ data, were highly significant p < 0.001 with r² = 0.977, 0.968 and 0.906 for AB₅₀, NR₅₀, and CB₅₀, respectively.     

Partitioning behaviour of perfluorinated alkyl contaminants between water, sediment and fish in the Orge River (nearby Paris, France)

This paper reports on the partitioning behaviour of 15 perfluorinated compounds (PFCs), including C₄-C₁₀ sulfonates and C₅-C₁₄ carboxylic acids, between water, sediment and fish (European chub, Leuciscus cephalus) in the Orge River (nearby Paris). Total PFC levels were 73.0 ± 3.0 ng L⁻¹ in water and 8.4 ± 0.5 ng g⁻¹ in sediment. They were in the range 43.1-4997.2 ng g⁻¹ in fish, in which PFC tissue distribution followed the order plasma > liver > gills > gonads > muscle. Sediment-water distribution coefficients (log K_d) and bioaccumulation factors (log BAF) were in the range 0.8-4.3 and 0.9-6.7, respectively. Both distribution coefficients positively correlated with perfluoroalkyl chain length. Field-based biota-sediment accumulation factors (BSAFs) are also reported, for the first time for PFCs other than perfluorooctane sulfonate. log BSAF ranged between −1.3 and 1.5 and was negatively correlated with the perfluoroalkyl chain length in the case of carboxylic acids.     

Respiratory uptake kinetics of neutral hydrophobic organic chemicals in a marine benthic fish, Pseudopleuronectes yokohamae

We investigated the respiratory uptake kinetics of polychlorinated biphenyls (PCBs), organohalogen pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), and 2,2′,4,4′-tetrabrominated diphenyl ether (BDE #47) in a marine benthic fish, Pseudopleuronectes yokohamae. The respiratory uptake efficiencies (E_W) of the chemicals, of which there have been no reports for the majority of persistent organic pollutants (POPs), were obtained by measuring the respiratory uptake rate constants (k₁) and the oxygen consumption rates of fish. Fish were exposed to water in which these chemicals were dissolved at environmentally relevant concentrations for 28 d, followed by 168 d of depuration in clean seawater. The k₁ and E_W values for 99 compounds were obtained, and they ranged from 2000 to 42 000 L kg-lipid⁻¹ d⁻¹ and from 0.060 to 1.3, respectively. The E_W values of the chemicals, except for PAHs, tended to increase with increasing values of the log octanol–water partition coefficients (K_OW) of the chemicals up to a log K_OW of 5. For log K_OW in the range 3–5, the E_W values in this study were much lower than those in a published study (about one-third). As a result of analysis by a two-phase resistance model, the resistance of transport rates to the lipid phase in this study was lower than was the case in the published study. These findings indicate that the E_W predicted by the published study for log K_OW in the range 3–5 may differ among fish species and water temperature, and further study is needed.     

Organochlorine contaminants in different tissues from Platichthys flesus (Pisces,Pleuronectidea)

Polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB) are organic contaminants that tend to accumulate in fish. Estuaries are exposed to high anthropogenic activities, therefore tending to accumulate more contaminants than the adjacent coastal waters. Platichthys flesus, a fish with high economic value, was analysed for PCBs and HCB along its different age groups, in liver, gills, gonads and muscle. Younger fishes were caught in the estuary, whereas older fishes were caught in the adjacent coastal waters and acquired at Figueira da Foz Regional Office of Docapesca-Portos e Lotas, SA. Both contaminants concentrations follow the pattern: liver > gills > muscle. Hepatosomatic index had the lowest values in younger fishes and the highest values in older fishes. Condition factor values remain stable with age. Younger fishes had higher PCBs concentrations than the older fishes. In opposition HCB was only detected in fish from 3+ to 5+ and its concentration tended to increase with age. Overall P. flesus is considered safe for human intake.     

Risk ranking of multiple-POPs in detritivorous fish from the Río de la Plata

To evaluate the bioaccumulation and the risk associated to consumption of lipid-rich detritivorous fish, a comprehensive set of organic pollutants (n = 213) including polychlorinated biphenyls (PCBs), dioxin like PCBs (dlPCBs), chlorinated pesticides (CHLPs), chlorobenzenes (CBzs), polybrominated diphenyl ethers (PBDEs), polychlorinated dibenzo dioxins and furans (PCDD/F), resolved (ALI) and unresolved aliphatic hydrocarbons (UCM) and linear alkyl benzenes (LABs) were analyzed in Sábalo fish (Prochilodus lineatus) collected in the polluted Metropolitan Buenos Aires coast and in migrating specimens. Fatty fish muscles (lipids: 74 ± 9.3% dry weight) contained homogeneous (24-51% variability) and very high-concentrations of organic pollutants ranging from 60 to 1300 μg g⁻¹ fresh weight (fw) ALI + UCM; 10-40 μg g⁻¹ fw LABs and PCBs; 0.1-1 μg g⁻¹ fw dlPCBs, DDTs, chlordanes, CBzs and PBDEs; 0.01-0.1 μg g⁻¹ fw mirex, endosulfans, aldrin, dieldrin, endrin and 0.07-0.2 ng g⁻¹ PCDD/F. Total toxicity equivalents (TEQs) ranged from 60 to 395 pg g⁻¹ fw (34 ± 17 and 213 ± 124 pg g⁻¹ TEQs for PCDD/F and dlPCBs respectively). These are among the highest concentrations reported for fish and point out the remarkable ability of Sábalo to feed on anthropogenic organic-enriched particles and tolerate a high pollutant load. Contaminant signatures show partial alteration with still abundant lower molecular weight components indicating that fish feeds directly in the outfalls. Consumption limits based on reference doses ranged from 0.1 (PCBs) to >12 000 g d⁻¹ (endosulfan) allowing a comprehensive risk-based ranking of contaminants in this long-range migrating, detritivorous fish.     

Sorption and desorption of carbamazepine, naproxen and triclosan in a soil irrigated with raw wastewater: estimation of the sorption parameters by considering the initial mass of the compounds in the soil

The amphipod Hyalella azteca was exposed for 28 d to different combinations of Zn contaminated sediment and food. Sediment exposure (+clean food) resulted in increased Zn body burdens, increased mortality and decreased body mass when the molar concentrations of simultaneously extracted Zn were greater than the molar concentration of Acid Volatile Sulfide (SEM_Zn-AVS > 0), suggesting that dissolved Zn was a dominant route of exposure. No adverse effect was noted in the foodexposure (+clean sediment), suggesting selective feeding or regulation. Combined exposure (sediment + food) significantly increased adverse effects in comparison with sediment exposure, indicating contribution of dietary Zn to toxicity and bioaccumulation. The observed enhanced toxicity also supports the assumption on the presence of an avoidance/selective feeding reaction of the amphipods in the single sediment or food exposures. During 14 d post-exposure in clean medium, the organisms from the same combined exposure history received two feeding regimes, i.e. clean food and Zn spiked food. Elevated Zn bioaccumulation and reduced reproduction were noted in amphipods that were offered Zn spiked food compared to the respective organisms that were fed clean food. This was explained by the failure of avoidance/selective feeding behavior in the absence of an alternative food source (sediment), forcing the amphipods to take up Zn while feeding. Increasing Zn body burdens rejected the assumption that Zn uptake from food was regulated by H. azteca. Our results show that the selective feeding behavior should be accounted for when assessing ecological effects of Zn or other contaminants, especially when contaminated food is a potential exposure route.     

Determination of the dietary biomagnification of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane with the rainbow trout (Oncorhynchus mykiss)

Separate 77-d fish feeding studies were conducted on the cyclic volatile methylsiloxane (cVMS) chemicals octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane with the rainbow trout, Oncorhynchus mykiss, with the determination of biomagnification factor (BMF) and lipid-adjusted BMF (BMF(L)) values as the final experimental metrics. The studies used fish food concentrations of ∼500 μg g⁻¹ for exposure periods of 35 d, followed by a depuration period of 42 d with clean food. The fish tissue concentrations of D4 and D5 achieved empirical steady-state by day 21 in each study. By day 7 of exposure, total ¹⁴C activity of both compounds had moved from the fish gastrointestinal (GI) tract into surrounding tissue. An absence of significant fish growth during the initial depuration phase allowed for measurement of empirical depuration rate constants (k₂) independent of growth dilution for D4 and D5 of 0.035 and 0.040 d⁻¹, respectively, corresponding to elimination half-lives of approximately 20 d. These rate constants indicated that ∼70–75% of steady-state was achieved during exposure in both studies, resulting in empirical steady-state BMF and BMF(L) values of 0.28 and 0.66 for D4, respectively, and 0.32 and 0.85 for D5, respectively. Kinetic modeling using simple first-order uptake and depuration dynamics produced good agreement with experimental data, with D4 and D5 assimilation efficiencies of 40% and 44%, respectively. Growth-corrected depuration rate constants modeled over the entire study data set indicated slower elimination kinetics for D4 (k₂ of 0.007 d⁻¹ or half-life of 100 d) compared to D5 (k₂ of 0.010 d⁻¹ or elimination half-life of 69 d). Kinetic BMF_k values (i.e., k₁/k₂) for D4 and D5 were 1.7 and 1.3, respectively, with lipid-adjusted BMF_k(L) values of 4.0 and 3.4, respectively.     

Bioconcentration and depuration of endosulfan sulfate in mosquito fish (Gambusia affinis)

Endosulfan is an insecticide which has been widely used in agriculture. The technical grade material consists of two isomers (alpha and beta). Under natural environmental conditions, endosulfan is metabolized through oxidation and the main metabolite in the environment is endosulfan sulfate. Most ecotoxicology research has been conducted with technical grade endosulfan to determine effects on non-target aquatic organisms. Little data on the effects of endosulfan sulfate on aquatic organisms are available in the literature. This study characterizes endosulfan sulfate bioconcentration and depuration in mosquito fish (Gambusia affinis). During the study, G. affinis was exposed to an environmentally relevant endosulfan sulfate concentration of 0.25 μg L⁻¹ for 5 weeks (uptake phase) followed by a 3-week period (depuration phase) in clean water. This study found that G. affinis bioconcentrated endosulfan sulfate. During the exposure phase, fish tissue concentrations of endosulfan sulfate increased with time up to 730 μg kg⁻¹ dw or 215 μg kg⁻¹ ww. The bioconcentration data followed Michaelis-Menten kinetics better than the one-compartment first order kinetics (1-CFOK). Using these models, the bioconcentration factors for endosulfan sulfate-exposed G. affinis were from 687 to 888  L kg⁻¹ in wet weight or 2263 to 2936 L kg⁻¹ in dry weight. During the depuration phase, endosulfan sulfate concentrations in tissue significantly decreased and the data followed first order kinetics. The half-life of endosulfan sulfate in G. affinis was about 9 d. There was no significant difference in standard length or weight between control and exposed fish. The growth data followed the von Bertalanffy growth model. However, the condition factor of exposed fish increased with time during the exposure phase.     

Temperature and water pressure head effects on the degradation of the diketonitrile metabolite of isoxaflutole in a loamy soil under two tillage systems

Laboratory studies were conducted to evaluate the effects of temperature and water pressure head on the degradation of the diketonitrile metabolite (DKN) of isoxaflutole during 84 d in samples collected in a loamy soil under conventional (CT) and conservation (MT) tillage systems. Soil temperature was the major factor controlling DKN degradation in the two tillage systems. The shortest half-lives (T_1/2) were measured in the seedbed samples under MT at 25 °C and −33 cm water pressure head. We found that mouldboard ploughing under CT was responsible for the spatial variability of herbicide degradation properties, whereas under MT herbicide degradation was associated to the vertical distribution of organic matter.     

Evaluation of ABC efflux transporters genes expression in kidney of rainbow trout (Oncorhynchus mykiss) fed with melamine and cyanuric acid diets

Gene expression experiments were targeted in order to monitor the ABC efflux transporters, which is potentially involved in cellular detoxification/defense. Changes in expression levels of different ABC genes in kidney of Oncorhynchus mykiss fed with melamine and melamine + cyanuric acid enriched diets were recorded in both treated groups by mRNA ΔΔ_CT relative quantification method. Expression profiles of eight different ABC genes basically showed low alterations in melamine group and more consistent changes in melamine + cyanuric acid treated fish, compared with own control. In the last group ABCC2 gene over expression was the more evident alteration. These results suggest that ABC efflux system could be involved in mobilization of hydrophilic molecules in the forcing condition of chronic exposure.     

Effects of previous sublethal pulse to ammonium nitrate on mortality and total length on Epidalea calamita larvae

Ammonium nitrate is one of the most widespread contaminants related with the viability of natural amphibian populations. In this study we have evaluated in terms of mortality and total length the effects that a previous sublethal pulse to ammonium nitrate generates in Epidalea calamita. Experiments were divided in two phases. In the first one, tadpoles were exposed to two different treatments, with and without a low ammonium nitrate concentration (22 mg NH₄NO₃ L⁻¹). The second phase consisted in static toxicity experiments from both origin treatments with five different nominal concentrations (0, 22, 45, 90 and 180 mg NH₄NO₃ L⁻¹). Results showed that tadpoles that had experienced a previous sublethal exposure showed a negative effect on survival (57% of reduction in the LC₅₀ value) and total length, throughout an increase in their sensitivity. These results could help us to understand the situation of amphibian populations inhabiting aquatic ecosystems exposed to discontinuous and variable pulses of pollutants.     

Lethal effects of abamectin on the aquatic organisms Daphnia similis, Chironomus xanthus and Danio rerio

Abamectin is used as an acaricide and insecticide for fruits, vegetables and ornamental plants, as well as a parasiticide for animals. One of the major problems of applying pesticides to crops is the likelihood of contaminating aquatic ecosystems by drift or runoff. Therefore, toxicity tests in the laboratory are important tools to predict the effects of chemical substances in aquatic ecosystems. The aim of this study was to assess the potential hazards of abamectin to the freshwater biota and consequently the possible losses of ecological services in contaminated water bodies. For this purpose, we identified the toxicity of abamectin on daphnids, insects and fish. Abamectin was highly toxic, with an EC₅₀ 48 h for Daphnia similis of 5.1 ng L⁻¹, LC₅₀ 96 h for Chironomus xanthus of 2.67 μg L⁻¹ and LC₅₀ 48 h for Danio rerio of 33 μg L⁻¹.