Toxicity assessment of a common laundry detergent using the freshwater flagellate Euglena gracilis

Synthetic detergents are among the commonly used chemicals in everyday life. Detergents, reaching aquatic environments through domestic and municipal wastewater, can cause many different effects in aquatic organisms. The present study was aimed at the toxicity evaluation of a commonly used laundry detergent, Ariel, using the freshwater flagellate Euglena gracilis as a biotest organism. Different parameters of the flagellate like motility, swimming velocity, cell shape, gravitactic orientation, photosynthesis and concentration of light harvesting pigments were used as end points for the toxicity assessment. No Observed Effect Concentration (NOEC) and EC₅₀ values were calculated for the end point parameters at four different incubation times, i.e. 0, 6, 24 and 72 h. After 72 h incubation, swimming velocity of the cells was found to be the most sensitive parameter giving NOEC and EC₅₀ values of 10.8 and 34 mg L⁻¹, respectively. After 72 h exposure to the detergent, chlorophyll a and total carotenoids were significantly decreased in cultures treated with Ariel at concentrations of 50 mg L⁻¹ and above while chlorophyll b significantly decreased at concentrations above 750 mg L⁻¹. The maximum inhibitory effect on the quantum yield of photosystem II was observed after 24 h exposure and thereafter a recovery trend was observed. Motility, gravitaxis and cell shape were strongly impaired immediately upon exposure to the detergent, but with increasing exposure time these parameters showed acclimatization to the stress and thus the NOEC values obtained after 72 h were higher than those immediately after exposure.     

Sandhopper solar orientation as a behavioural biomarker of trace metals contamination

Although many studies have focused on trace metals accumulation, investigations of talitrid amphipods as biomarkers are rare. This study explores the possibility of using the solar orientation capacity of Talitrus saltator as a behavioural marker of exposure to two essential (Cu and Zn) and two non-essential (Cd and Hg) metals. LC₅₀ analyses performed before the solar orientation tests showed that the 72 h LC₅₀ for Hg was 0.02 ppm while the 96 h LC₅₀ values for Cu, Cd and Zn were 13.28 ppm, 27.66 ppm, and 62.74 ppm, respectively. The presence of metals in seawater affects the solar orientation capacity of T. saltator in a concentration-dependent manner and according to the toxicity ranking of the metals (Hg > Cu > Cd > Zn). Therefore, the solar orientation capacity of T. saltator seems to be a promising behavioural marker for exposure to trace metals.     

An evaluation of the toxicity and bioaccumulation of cisplatin in the marine environment using the macroalga, Ulva lactuca

The cytotoxic drug, cisplatin (cis-PtCl₂(NH₃)₂), has been added to cultures of the marine macroalga, Ulva lactuca, under various experimental conditions. Both accumulation and internalisation over a 48 h period was greater when cisplatin was added to coastal sea water (salinity = 33) from a distilled water solution than when added to either sea water or estuarine water (salinity = 16.5) from a saline solution. This effect is attributed to the greater abundance of the more reactive monoaqua complex (cis-PtCl(OH₂)(NH₃)₂⁺) in the distilled water solution and kinetic constraints on its conversion back to cis-PtCl₂(NH₃)₂ in sea water. Despite its mode of action at the cellular level, cisplatin added up to concentrations of 150 nM did not incur a measurable reduction in the efficiency of photochemical energy conversion under any of experimental conditions tested.     

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.     

Below-ground carbon allocation in mature beech and spruce trees following long-term, experimentally enhanced O3 exposure in Southern Germany

Canopies of adult European beech (Fagus sylvatica) and Norway spruce (Picea abies) were labeled with CO₂ depleted in ¹³C to evaluate carbon allocation belowground. One-half the trees were exposed to elevated O₃ for 6 yrs prior to and during the experiment. Soil-gas sampling wells were placed at 8 and 15 cm and soil CO₂ was sampled during labeling in mid-late August, 2006. In beech, δ¹³CO₂ at both depths decreased approximately 50 h after labeling, reflecting rapid translocation of fixed C to roots and release through respiration. In spruce, label was detected in fine-root tissue, but there was no evidence of label in δ¹³CO₂. The results show that C fixed in the canopy rapidly reaches respiratory pools in beech roots, and suggest that spruce may allocate very little of recently-fixed carbon into root respiration during late summer. A change in carbon allocation belowground due to long-term O₃ exposure was not observed.     

Converging hazard assessment of gold nanoparticles to aquatic organisms

The gold nanoparticles (Au-NPs) are being increasingly used because of their huge diversity of applications, and consequently, elevated levels in the environment are expected. However, due to their physico-chemical properties and functionalization a high variety of Au-NPs can be found, and complete toxicological information for each type of Au-NPs still lacks, and even, the toxicological information for the same species is sometimes contradictory. Therefore, hazard assessment should be done case by case. Hence, the objective of this study was to obtain ecotoxicological information of the same Au-NPs in aquatic organisms and to find a rationale for Au-NPs toxicity. For such a purpose, bare and hyaluronic acid capped Au-NPs (12.5 nm) along with Au-NPs bulk material were tested on freshwater algae, Daphnia and zebrafish. Results showed that while gold nanoparticles were found to be harmless to the tested organisms, the soluble gold showed to be toxic to algae and Daphnia, with an LC₅₀ between 1 and 2 mg L⁻¹. Comparing our results with those gathered in the literature, it appears that a common hazard assessment of Au-NPs on the studied organisms can be elucidated.     

Acute and chronic effects of nano- and non-nano-scale TiO2 and ZnO particles on mobility and reproduction of the freshwater invertebrate Daphnia magna

Among the emerging literature addressing the biological effects of nanoparticles, very little information exists, particularly on aquatic organisms, that evaluates nanoparticles in comparison to non-nanocounterparts. Therefore, the potential effects of nano-scale and non-nano-scale TiO₂ and ZnO on the water flea, Daphnia magna, were examined in 48-h acute toxicity tests using three different test media, several pigment formulations – including coated nanoparticles – and a variety of preparation steps. In addition, a 21-d chronic Daphnia reproduction study was performed using coated TiO₂ nanoparticles. Analytical ultracentrifugation analyses provided evidence that the nanoparticles were present in a wide range of differently sized aggregates in the tested dispersions. While no pronounced effects on D. magna were observed for nano-scale and non-nano-scale TiO₂ pigments in 19 of 25 acute (48-h) toxicity tests (EC50 > 100 mg L⁻¹), six acute tests with both nano- and non-nano-scale TiO₂ pigments showed slight effects (EC10, 0.5–91.2 mg L⁻¹). For the nano-scale and non-nano-scale ZnO pigments, the acute 48-h EC50 values were close to the 1 mg L⁻¹ level, which is within the reported range of zinc toxicity to Daphnia. In general, the toxicity in the acute tests was independent of particle size (non-nano-scale or nano-scale), coating of particles, aggregation of particles, the type of medium or the applied pre-treatment of the test dispersions. The chronic Daphnia test with coated TiO₂ nanoparticles demonstrated that reproduction was a more sensitive endpoint than adult mortality. After 21 d, the NOEC for adult mortality was 30 mg L⁻¹ and the NOEC for offspring production was 3 mg L⁻¹. The 21-d EC10 and EC50 values for reproductive effects were 5 and 26.6 mg L⁻¹, respectively. This study demonstrates the utility of evaluating nanoparticle effects relative to non-nano-scale counterparts and presents the first report of chronic exposure to TiO₂ nanoparticles in D. magna.     

Acute effects of copper and mercury on the estuarine fish Pomatoschistus microps: Linking biomarkers to behaviour

The main objective of the present study was to investigate possible links between biomarkers and swimming performance in the estuarine fish Pomatoschistus microps acutely exposed to metals (copper and mercury). In independent bioassays, P. microps juveniles were individually exposed for 96 h to sub-lethal concentrations of copper or mercury. At the end of the assays, swimming performance of fish was measured using a device specially developed for epibenthic fish (SPEDE). Furthermore, the following biomarkers were measured: lipid peroxidation (LPO) and the activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), glutathione S-transferases (GST), 7-ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx). LC₅₀s of copper and mercury (dissolved throughout metal concentrations) at 96 h were 568 μg L⁻¹ and 62 μg L⁻¹, respectively. Significant and concentration-dependent effects of both metals on swimming resistance and covered distance against water flow were found at concentrations equal or higher than 50 μg L⁻¹ for copper and 3 μg L⁻¹ for mercury (dissolved throughout metal concentrations). These results indicate that SPEDE was efficacious to quantify behavioural alterations in the epibenthic fish P. microps at ecologically relevant concentrations. Significant alterations by both metals on biomarkers were found including: inhibition of AChE and EROD activities, induction of LDH, GST and anti-oxidant enzymes, and increased LPO levels, with LOEC values ranging from 25 to 200 μg L⁻¹ for copper and from 3 to 25 μg L⁻¹ for mercury (dissolved throughout metal concentrations). Furthermore, significant and positive correlations were found between some biomarkers (AChE and EROD) and behavioural parameters, while negative correlations were found for others (LPO, anti-oxidant enzymes and LDH) suggesting that disruption of cholinergic function through AChE inhibition, decreased detoxification capability due to EROD inhibition, additional energetic demands to face chemical stress, and oxidative stress and damage may contribute to decrease the swimming performance of fish. Since a reduced swimming capability of fish may reduce their ability to capture preys, avoid predators, and interfere with social and reproductive behaviour, the exposure of P. microps to copper and/or mercury concentrations similar to those tested here may decrease the fitness of wild populations of this species.     

Uranium uptake and depuration in the aquatic invertebrate Chironomus tentans

Evaluation of aqueous uranium (U) uptake and depuration in larvae of the midge Chironomus tentans were investigated in two separated experiments. First, a static-renewal experiment was performed with 10-d old C. tentans larvae exposed to 300 μg U/L. The animals steadily accumulated U (K_u = 20.3) approaching steady-state conditions (BAF = 56) in approximately 9-11 d. However, accumulated U was readily depurated (K_d = 0.36) with U tissue concentration decreasing rapidly within 3 d of the larvae being placed in clean water (t_1/2 = 1.9 d). Also, the growth of C. tentans larvae appeared to decrease after 6-11 d of U exposure, probably due to the reallocation of resources into U detoxification mechanisms. However, growth significantly increased once C. tentans were transferred to clean water. A separate short-term experiment was performed to evaluate the possible mechanism of U uptake in this invertebrate. Results suggested a passive mechanism of U uptake coupled with an active mechanism of U depuration but no details related to the type of mechanisms or pathway was investigated.     

Development of a combined isotopic and mass-balance approach to determine dissolved organic carbon sources in eutrophic reservoirs

A combined mass-balance and stable isotope approach was set up to identify and quantify dissolved organic carbon (DOC) sources in a DOC-rich (9 mg L⁻¹) eutrophic reservoir located in Western France and used for drinking water supply (so-called Rophemel reservoir). The mass-balance approach consisted in measuring the flux of allochthonous DOC on a daily basis, and in comparing it with the effective (measured) DOC concentration of the reservoir. The isotopic approach consisted, for its part, in measuring the carbon isotope ratios (δ¹³C values) of both allochthonous and autochthonous DOC sources, and comparing these values with the δ¹³C values of the reservoir DOC. Results from both approaches were consistent pointing out for a DOC of 100% allochthonous origin. In particular, the δ¹³C values of the DOC recovered in the reservoir (−28.5 ± 0.2‰; n = 22) during the algal bloom season (May-September) showed no trace of an autochthonous contribution (δ¹³C in algae = −30.1 ± 0.3‰; n = 2) being indistinguishable from the δ¹³C values of allochthonous DOC from inflowing rivers (−28.6 ± 0.1‰; n = 8). These results demonstrate that eutrophication is not responsible for the high DOC concentrations observed in the Rophemel reservoir and that limiting eutrophication of this reservoir will not reduce the potential formation of disinfection by-products during water treatment. The methodology developed in this study based on a complementary isotopic and mass-balance approach provides a powerful tool, suitable to identify and quantify DOC sources in eutrophic, DOC-contaminated reservoirs.     

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.     

The efficiency of tobacco Bel-W3 and native species for ozone biomonitoring in subtropical climate, as revealed by histo-cytochemical techniques

We aimed to verify whether hydrogen peroxide (H₂O₂) accumulation and cell death are detected early in three bioindicators of ozone (O₃), Nicotiana tabacum ‘Bel-W3’, Ipomoea nil ‘Scarlet O’Hara’ and Psidium guajava ‘Paluma’, and whether environmental factors also affect those microscopic markers. The three species were exposed to chronic levels of O₃ in a subtropical area and a histo-cytochemical technique that combines 3,3′-diaminobenzidine (DAB) with Evans blue staining was used in the assessments. The three species accumulated H₂O₂, but a positive correlation with O₃ concentration was only observed in N. tabacum. A positive correlation between O₃ and cellular death was also observed in N. tabacum. In I. nil and P. guajava, environmental factors were responsible for symptoms at the microscopic level, especially in P. guajava. We conclude that the most appropriate and least appropriate bioindicator plant for O₃ monitoring in the subtropics are N. tabacum ‘Bel-W3’ and P. guajava ‘Paluma’, respectively.     

Toxicity of tire wear particle leachate to the marine macroalga, Ulva lactuca

Tire wear particles filed from the treads of end-of-life vehicle tires have been added to sea water to examine the release of Zn and the toxicity of the resulting leachate and dilutions thereof to the marine macroalga, Ulva lactuca. Zinc release appeared to be diffusion-controlled, with a conditional rate constant of 5.4 μg[L(h)^1/2]⁻¹, and about 1.6% of total Zn was released after 120 h incubation. Exposure to increasing concentrations of leachate resulted in a non-linear reduction in the efficiency of photochemical energy conversion of U. lactuca and, with the exception of the undiluted leachate, increasing accumulation of Zn. Phototoxicity was significantly lower on exposure to equivalent concentrations of Zn added as Zn(NO₃)₂, suggesting that organic components of leachate are largely responsible for the overall toxicity to the alga. Given the ubiquity and abundance of TWP in urban coastal sediments, the generation, biogeochemistry and toxicity of tire leachate in the marine setting merit further attention.     

Toxicity of mixtures of perfluorooctane sulphonic acid with chlorinated chemicals and lipid regulators

The toxicological interaction of perfluorooctane sulphonic acid (PFOS) with the chlorinated pollutants triclosan and 2,4,6-trichlorophenol and the lipid regulators gemfibrozil and bezafibrate was evaluated using the combination index-isobologram equation. The endpoint for bioassays was the growth rate inhibition of the green alga Pseudokirchneriella subcapitata. The results showed that most of the binary combinations assayed exhibited antagonism at all effect levels. The addition of a third component induced a less antagonistic or even synergistic behaviour. This was particularly marked for the ternary mixture of triclosan and 2,4,6-trichlorophenol with PFOS, for which synergism was very strong at all effect levels, with a combination index as low as 0.034 ± 0.002 at EC₅₀ for the mixture. The results obtained indicate that the evaluation of mixture toxicity from single component data using the concentration addition approach could severely underestimate combined toxicity.     

In situ studies on growth,oxidative stress responses,and gene expression of juvenile bighead carp (Hypophthalmichthys nobilis) to eutrophic lake water dominated by cyanobacterial blooms

Cyanobacterial blooms have received increasing attention as a public biohazard for human and animal health. To assess the effect of cyanobacteria-dominant lake water on juvenile fish, we measured the responses of specific growth rate, condition factor, body weight and body length, oxidative stress, and related gene expression of juvenile bighead carp Hypophthalmichthys nobilis exposed to in situ eutrophic lake (Chl a was around 7.0 μg L⁻¹). Results showed in situ cyanobacteria-dominant lake water had no effect on the growth performance, but significantly elevated the contents of malondialdehyde, the expression of heat shock protein 70, and the activity of superoxide dismutase, indicating that oxidative stress occurred. Meanwhile in situ lake water significantly decreased the expression of catalase and glutathione S-transferase genes. We conclude that in situ cyanobacteria-dominated lake water was harmful to juvenile bighead carp based on the oxidative stress and changes in the related gene expression levels.     

Influence of environmental factors on the response of a natural population of Daphnia magna (Crustacea: Cladocera) to spinosad and Bacillus thuringiensis israelensis in Mediterranean coastal wetlands

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 μg L⁻¹) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 μL L⁻¹) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.     

A protocol for the evaluation of genotoxicity in bile of carp (Cyprinus carpio) exposed to lake water treated with different disinfectants

A sensitive and rapid method to evaluate toxic and genotoxic properties of drinking water supplied from Lake Trasimeno (Umbria, Central Italy) was worked out analysing bile in Cyprinus carpio exposed for 20 d to lake water treated with 3 different disinfectants, sodium hypochlorite (NaClO), chlorine dioxide (ClO₂) and peracetic acid (PAA). Fish were sacrificed at 0, 10 and 20 d in order to investigate the time course of these endpoints. An aliquot of bile samples was fractionated by adsorption on C₁₈ silica cartridges and the genotoxic potential of whole bile and of bile fractions was evaluated by the single-cell microgel-electrophoresis (comet) assay on human colonic adenocarcinoma cells (Caco-2). Bile (both whole and fractionated) from specimens exposed to the three disinfectants always showed a genotoxic activity as compared to the control group. The results of this study provide evidence that all three disinfectants cause an increase in bile genotoxicity of chronically exposed fish.     

Influence of flooding and metal immobilising soil amendments on availability of metals for willows and earthworms in calcareous dredged sediment-derived soils

Soil amendments previously shown to be effective in reducing metal bioavailability and/or mobility in calcareous metal-polluted soils were tested on a calcareous dredged sediment-derived soil with 26 mg Cd/kg dry soil, 2200 mg Cr/kg dry soil, 220 mg Pb/kg dry soil, and 3000 mg Zn/kg dry soil. The amendments were 5% modified aluminosilicate (AS), 10% w/w lignin, 1% w/w diammonium phosphate (DAP, (NH₄)₂HPO₄), 1% w/w MnO, and 5% w/w CaSO₄. In an additional treatment, the contaminated soil was submerged. Endpoints were metal uptake in Salix cinerea and Lumbricus terrestris, and effect on oxidation-reduction potential (ORP) in submerged soils. Results illustrated that the selected soil amendments were not effective in reducing ecological risk to vegetation or soil inhabiting invertebrates, as metal uptake in willows and earthworms did not significantly decrease following their application. Flooding the polluted soil resulted in metal uptake in S. cinerea comparable with concentrations for an uncontaminated soil.     

Effects of ammonia from livestock farming on lichen photosynthesis

This study investigated if atmospheric ammonia (NH₃) pollution around a sheep farm influences the photosynthetic performance of the lichens Evernia prunastri and Pseudevernia furfuracea. Thalli of both species were transplanted for up to 30 days in a semi-arid region (Crete, Greece), at sites with concentrations of atmospheric ammonia of ca. 60 μg/m³ (at a sheep farm), ca. 15 μg/m³ (60 m from the sheep farm) and ca. 2 μg/m³ (a remote area 5 km away). Lichen photosynthesis was analysed by the chlorophyll a fluorescence emission to identify targets of ammonia pollution. The results indicated that the photosystem II of the two lichens exposed to NH₃ is susceptible to this pollutant in the gas-phase. The parameter PI_ABS, a global index of photosynthetic performance that combines in a single expression the three functional steps of the photosynthetic activity (light absorption, excitation energy trapping, and conversion of excitation energy to electron transport) was much more sensitive to NH₃ than the F_V/F_M ratio, one of the most commonly used stress indicators.