Bioassays with Vibrio fischeri for the assessment of delayed toxicity

The standardized bioluminescence assay with Vibrio fischeri underestimates the aquatic toxicity of chemicals which interfere with metabolic pathways supporting long term processes like growth and reproduction due to its short incubation time (30 min). Therefore this short term assay was compared with two alternative bioassays with prolonged incubation times using the same test organism: the growth inhibition assay (7 h) and the long term bioluminescence assay (24 h). Two sets of compounds were selected to reflect acute and delayed toxicity. The first group comprised pentachlorophenol, dodecylpyridiniumbromide and 3,4-dichloroaniline and the second nalidixic acid, chloramphenicol and streptomycinsulfate. The effects of compounds with acute toxicity are determined with similar sensitivity in all bioassays. Substances with delayed toxicity show only minor or no toxicities in the standardized short term bioassay but severe effects in both long term bioassays independent of the parameter used. It is concluded that the standardized short term bioluminescence assay exhibits serious limitations for the assessment of aquatic toxicity. The long term bioassays, however, may help to overcome these limitations.     

Ecotoxicological Assessment of Sediment, Suspended Matter and Water Samples in the Upper Danube River. A pilot study in search for the causes for the decline of fish catches (12 pp)

Goals, Scope and Background Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. Methods Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). Results and Discussion The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17β-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that – at least for fish eggs – the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. Conclusions The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. Recommendations and Perspective . In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.     

Sensitivity of the zebrafish (Danio rerio) early life stage test for compounds with different modes of action

The sensitivity of the early life stage (ELS) toxicity test for two compounds with different modes of action was determined, and related to other toxicity tests with the same compounds. The zebrafish. Danio rerio, was used as a test organism, and the two model compounds were 1,2,3-trichlorobenzene (123TCB), a non-polar narcotic, and parathion, an acetylcholinesterase (AChE) inhibitor. Hatching and survival after 28 days were significantly reduced in the highest 123TCB treatment (263 microg/l), but not in any of the parathion treatments. Growth of the larvae was negatively affected at parathion concentrations above 20 microg/l, while AChE was only significantly inhibited at the highest concentration, 93 microg/l. No effects on growth were found in the 123TCB treatments. In comparison with acute and chronic studies with both compounds, the ELS test turned out to be less sensitive than chronic studies and more sensitive than acute studies. The difference in sensitivity between the tests systems seems however, to depend on the mode of action of the compound.     

Inventory of marine biotest methods for the evaluation of dredged material and sediments

An inventory of marine biotest methods for the evaluation of dredged material and sediments was compiled on behalf of the Federal Environmental Agency of Germany. Relevant assays were identified from the literature and experts from several countries contributed to a questionnaire survey on established and developing procedures. The biotest methods are applicable to whole sediment, sediment suspension, sediment elutriate, porewater and/or sediment extract. The endpoints cover acute and long-term toxicity, bioaccumulation, endocrine effects, toxic effects on reproduction, carcinogenicity and mutagenicity. Comparative analyses and evaluation of the biotest methods were conducted with regard to their sensitivity, specificity, applicability (regional specificity, availability and suitability of the test organisms), variability (physicochemical factors, natural factors and factors related to sampling and testing), cost-effectiveness, aspects of animal ethics, standardization (guidelines, intercalibration) and application for monitoring purposes in the areas of the OSPAR and Helsinki Conventions. The available information was integrated to rate the validity of the methods, their relevance for assessing impacts on ecosystems and the suitability of the methods for the evaluation of marine sediments and dredged material. Based on the rating of the individual bioassays, a tiered testing is suggested in a hierarchical approach representing a variety in taxa, biological processes and exposure routes, thereby covering the cellular, species, population and community level with a wide discriminatory and sensitivity range. The toxicological significance and complexity increases with the tiers: (1) screening and detection of impacts, (2) characterization of toxic effects, (3) verification of in situ alterations.     

Ecotoxicological evaluation of in situ bioremediation of soils contaminated by the explosive 2,4,6-trinitrotoluene (TNT)

To evaluate the environmental relevance of in situ bioremediation of contaminated soils, effective and reliable monitoring approaches are of special importance. The presented study was conducted as part of a research project investigating in situ bioremediation of topsoils contaminated by the explosive 2,4,6-trinitrotoluene (TNT). Changes in soil toxicity within different experimental fields at a former ordnance factory were evaluated using a battery of five bioassays (plant growth, Collembola reproduction, soil respiration, luminescent bacteria acute toxicity and mutagenicity test) in combination to chemical contaminant analysis. Resulting data reveal clear differences in sensitivities between methods with the luminescent bacteria assay performed with soil leachates as most sensitive toxicity indicator. Complete test battery results are presented in so-called soil toxicity profiles to visualise and facilitate the interpretation of data. Both biological and chemical monitoring results indicate a reduction of soil toxicity within 17 months of remediation.     

Time-dependent toxicity in the long-term inhibition assay with Vibrio fischeri

The significance of the duration of exposure for the detection of toxicity was evaluated in a 24 h long-term bioluminescence inhibition assay with Vibrio fischeri. Bioluminescence was measured over the time course of 24 h using microplates. The undisturbed luminescence of controls in this assay exhibited characteristic dynamics: a decrease within a period of 12 h with minimal luminescence followed by a continuous increase of luminescence beyond the starting value. To evaluate the toxic influence of compounds chosen to reflect immediate and delayed toxicity to V. fischeri, the bioluminescence was measured for 24 h at 30 min intervals. Luminescence inhibition patterns were recorded for subdinoseb, pentachlorophenol and 2,4,5-trichlorophenol) and for substances causing delayed toxicity (chloramphenicol, nalidixic acid and phosphomycin). The toxic influence of substances with immediate toxicity was observed directly after application, whereas the toxicity patterns of substances with delayed toxicity developed specifically over the time according to the different involved mechanisms of action. It is concluded that knowledge about time to toxicity in bioassays is necessary in order to identify suitable test endpoints as well as to recognize potential hazards related to time-dependent toxicity.     

Reproduction of the estuarine and marine amphipod Corophium volutator (Pallas) in laboratory for toxicity testing

The acute whole sediment bioassay with the estuarine and marine amphipod Corophium volutator (Pallas) is broadly used within Europe. Hitherto, the test is carried out with field-collected animals. In order to provide a more standardized and continuously available test organism reproduction and growth experiments were performed for a period of one year in laboratory under simulated summer conditions (light/dark 16:8 at 15, 19, and 23 degrees C). C. volutator was the first time reproduced successfully in laboratory for several generations and independent from its natural life cycle also in winter. The females produced two to three broods with a mean number of 96 offspring. A mean growth rate of 0.07 mm per day was determined at 15 degrees C. The reproduction and growth experiments provide consequently the essential base for the development of a chronic toxicity test with C. volutator.     

The influence of quinones and phenols on the triple NAD(H)-dependent enzyme systems

Kinetics of the triple bioluminescent enzyme system: alcohol dehydrogenase--NADH:FMN-oxidoreductase--luciferase in the presence of quinones and phenols has been studied. The correspondence between the bioluminescent kinetic parameters, redox potentials and concentrations of the quinones and phenols has been estimated. The substances have been shown to change bioluminescent kinetics through moving off the NAD+/NADH balance in the enzyme processes. This system is proposed to be used as enzymatic biotest in ecological monitoring.     

Investigation of acute toxicity of fenitrothion on peppered corydoras (Corydoras paleatus) (Jenyns, 1842)

Fenitrothion, as an organophosphothionate insecticide, is a contact insecticide and selective acaricide, also used as a vector control agent for malaria in public health programs. A 96 h LC50 value of fenitrothion, a potential toxic pollutant contaminating aquatic ecosystems, was determined on the adult peppered corydoras (Corydoras paleatus). The experiments were repeated three times. The static test method of acute toxicity test was used. Water temperature was regulated at 23 +/- 1 degrees C. In addition, behavioral changes at each fenitrothion concentration were observed for the individual fish. Data obtained from acute toxicity tests were evaluated using the Probit Analysis Statistical Method. The 96 h LC50 value for peppered corydoras was estimated as 3.51 mg/l.     

Adaptation of the Daphnia sp. acute toxicity test: miniaturization and prolongation for the testing of nanomaterials

Manufacturing of nanomaterials (NMs) is often complex and expensive, and their environmental risks are poorly understood or even unknown. An economization of testing NMs is therefore desirable, which can be achieved by miniaturizing test systems. However, the downsizing of test vessels and volumes can enlarge the surface/volume ratio (SVR) which in turn can affect the bioavailable concentration of adsorbing substances like NMs. The present study focused on the miniaturization of the acute toxicity test with Daphnia magna. The adaptations were verified with three reference substances, the non-adsorbing potassium dichromate (K₂Cr₂O₇) and as potentially highly-adsorbing substances silver nanoparticles (AgNPs) and silver nitrate (AgNO₃). The miniaturized test was conducted in 24-well microtiter plates (MT) and simultaneously compared to the OECD standard test (ST). Furthermore, the test duration was prolonged from 48 to 96 h since NMs tend to show effects only after extended exposure. The toxicity of K₂Cr₂O₇ and AgNPs continued to increase within the prolonged test span. The test comparisons with K₂Cr₂O₇ did not reveal any significant differences between ST and MT. AgNO₃ toxicity was significantly decreased in MT compared to ST due to the enlarged SVR. The toxicity of AgNPs in MT after 24 h was equal to ST. Contrary to our expectations an exposure longer than 24 h resulted in an increase of AgNP toxicity in MT, possibly due to enhanced dissolution of silver. Microtiter plates are appropriate alternative test vessels for the Daphnia sp. acute toxicity test; thus, its miniaturization is feasible. The enlarged SVR has to be taken into account since it can affect the toxicity of potentially adsorbing substances. Furthermore, the standard test duration of 48 h might underestimate the toxicity of many substances, especially of NMs.     

Evaluation of the Hormosira banksii (Turner) desicaine germination and growth inhibition bioassay for use as a regulatory assay

A germination and growth inhibition bioassay using the marine macroalga, Hormosira banksii, was evaluated for use as a standard method for direct toxicity assessment testing in the Australasian region. H. banksii is the dominant macroalga and an important primary producer in temperate coastal zones of Australasia and is therefore relevant as a test organism. Moreover, it releases gametes readily under laboratory conditions, therefore allowing tests to be conducted year round. The test endpoints' variability and sensitivity was evaluated using ammonia, a common constituent of waste waters. The two test endpoints of germination and growth proved to be both reliable and repeatable bioassay endpoints. The coefficients of variation (CV's) for toxicity to germination and growth by ammonia ranged from 25% to 38% (n=18).     

Heavy metal contamination from mining sites in South Morocco: 2. Assessment of metal accumulation and toxicity in plants

Metalliferous soils cover a relatively large surface area in Morocco, and up to now no hyperaccumulating plants have been identified on these mining or these industrial sites. The aim of this work was to assess the extent of metal accumulation by plants found in three mining areas in southern Morocco with the ultimate goal of finding metal hyperaccumulating species by using the MetPAD biotest. The biotest helps to obtain information on the selective metal toxicity of aqueous extracts from the plants. A strong metal toxicity, as revealed by the biotest is an indication of a hyperaccumulating plant. Toxicity tests were run concurrently with chemicals analyses of metals in plants and their water extracts. The chemical analyses allow the determination of the hyperaccumulated metal(s). Specimens of the plant species mainly growing on and in the vicinity of the three mines were sampled with their corresponding soils. The results show that all plants analyzed had lower heavy metal content and toxicity despite the relatively very high soil concentrations. A comparison of our results with the criterion used to classify the hyperaccumulator plants indicates that plants we collected from mining sites were hypertolerant but not hyperaccumulators. This was confirmed by transfer factors generally lower than 1. Nevertheless, these tolerant plants species can be used as tools for revegetation for erosion control in metals-contaminated sites (phytostabilization).     

Heavy metal contamination from mining sites in South Morocco: 1. Use of a biotest to assess metal toxicity of tailings and soils

Our work was conducted to investigate the heavy metal toxicity of tailings and soils collected from five metal mines located in the south of Morocco. We used the MetPAD biotest Kit which detects the toxicity specifically due to the heavy metals in environmental samples. This biotest initially developed to assess the toxicity of aquatic samples was adapted to the heterogeneous physico-chemical conditions of anthropogenic soils. Contrasted industrial soils were collected from four abandoned mines (A, B, C and E) and one mine (D) still active. The toxicity test was run concurrently with chemical analyses on the aqueous extracts of tailings materials and soils in order to assess the potential availability of heavy metals. Soil pH was variable, ranging from very acidic (pH 2.6) to alkaline values (pH 8.0-8.8). The tailings from polymetallic mines (B and D) contained very high concentrations of Zn (38,000-108,000 mg kg(-1)), Pb (20,412-30,100 mg kg(-1)), Cu (2,019-8,635 mg kg(-1)) and Cd (148-228 mg kg(-1)). Water-extractable metal concentrations (i.e., soil extracts) were much lower but were highly toxic as shown by the MetPAD test, except for soils from mines A, E and site C3 from mine C. The soil extracts from mine D were the most toxic amongst all the soils tested. On this site, the toxicity of soil water extracts was mainly due to high concentrations of Zn (785-1,753 mg l(-1)), Cu (1.8-82 mg l(-1)) and Cd (2.0-2.7 mg l(-1)). The general trend observed was an increase in metal toxicity measured by the biotest with increasing available metal contents in tailings materials and soils. Therefore, the MetPAD test can be used as a rapid and sensitive predictive tool to assess the heavy metal availability in soils highly contaminated by mining activities.     

Toxicity of lead on Ceriodaphnia dubia in the presence of nano-CeO(2) and nano-TiO(2)

Although engineered nanoparticles (NPs) could negatively impact environmental organisms, the synergistic effect of NPs and other toxic substances, which could be more significant than that of NP alone, have seldom been examined. The effect of two common NPs, nano-CeO₂ and nano-TiO₂, on the toxicity of Pb was evaluated using Ceriodaphnia dubia (C. dubia) as the model organism. Standard EPA procedures were followed in the toxicity evaluation. The toxicity of bare NPs (without Pb) was first evaluated and safe doses (levels without causing lethal effect) of NPs were used in the synergistic studies. It was found that the overall toxicity of Pb in the system containing NPs was greater than that of Pb alone, as indicted by the reduced median lethal concentration (LC₅₀) of soluble Pb. The sorption of Pb onto the NP, and the uptake of NPs in the gastrointestinal tract of C. dubia were validated. Therefore, the uptake of Pb-loaded NPs increased the exposure of C. dubia to Pb, resulting in the enhanced toxicity. Reducing the solution pH could shift Pb speciation and enhance the overall toxicity of Pb, with or without the presence of NPs.     

Bioassay-directed chemical analysis of River Elbe surface water including large volume extractions and high performance fractionation

A bioassay-directed fractionation and identification (toxicity identification evaluation procedure) was performed on extracts of 10 1 River Elbe water samples. The experimental method included a SDB-1 solid phase extraction followed by RP-HPLC fractionation and subfractionation. Chemical analysis by GC-MS as well as acute toxicity testing using a luminescent bacteria assay were conducted in the respective fractions. Many substances were identified, among which were pesticides and pharmaceuticals, but many compounds remained unknown.     

Toxicity characterisation of sediment porewaters collected from UK estuaries using a Tisbe battagliai bioassay

The lethal toxicity of sediment porewater isolated from samples collected from six United Kingdom estuaries was tested using a Tisbe battagliai bioassay. A selection of Phase 1 toxicity identification evaluation (TIE) procedures was then used to characterise the substances responsible for the measured toxicity. In samples collected in 2000, cationic metals, ammonia, organic compounds were identified as the cause of toxicity in the most toxic sample collected. Some of the toxicity remained uncharacterised. It was shown that the toxicity in samples collected from the same locations in 2001 was due to ammonia. The successful application of the T. battagliai bioassay demonstrates that it is a suitable assay for marine sediment porewater TIE and that combined with simple characterisation procedures allow an initial assessment to be made on the type of substances responsible for the measured toxicity.     

Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (log K_AW from −6.53 to −2.13) and lipophilicity (log K_OW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC₅₀ values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC₅₀ values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a log K_OW higher than 3 or a Henry coefficient log K_AW higher than −4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay’s one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.     

Toxicity testing with luminescent bacteria – Characterization of an automated method for the combined assessment of acute and chronic effects

The luminescent bacteria test according to EN ISO 11348 is frequently applied in (eco) toxicity testing and is applicable for a huge variety of environmental and industrial samples. A big disadvantage of this method is the very short exposure time, which is expressed in a low sensitivity in regard to substances with a delayed effect. Chronic effects, i.e. interference with cell growth, cannot be assessed with this conventional standard method. The goal of this research was to develop an automated testing system for long term toxicity towards the luminescent bacteria Vibrio fischeri by implementing microtitration-based instrumentation. The optimized method, hereinafter referred to as “kinetic luminescent bacteria test”, can be described as a miniaturized combination of the conventional short-term luminescence inhibition test according to EN ISO 11348 and the Photobacterium phosphoreum growth inhibition test (DIN 38412-37). The validation procedure included the evaluation of six reference compounds (3,4-Dichloroaniline, 3,5-Dichlorophenol, Chloramphenicol, Streptomycin sulfate, Potassium dichromate, Zinc sulfate heptahydrate) and three different endpoints that are acute luminescence inhibition (acute LI) after 30 min, chronic luminescence inhibition (chronic LI) after 24 h and growth inhibition (GI) after 14 h. The optimized method allows the assessment of acute and chronic effects within one test, by what a misinterpretation of the toxicity of substances with delayed bacterial toxicity can be prevented, without abandoning most of the advantages of the conventional short-term test. Therefore, the kinetic luminescent bacteria test is exceptional as an initial screening test for environmental samples or substances with unknown (eco) toxicological characteristics.     

The SeKT joint research project: definition of reference conditions, control sediments and toxicity thresholds for limnic sediment contact tests

- In current biotest approaches, intact organisms or in vitro systems are exposed to sediments using different exposure scenarios. The most important issue in sediment toxicity testing protocols is the question which test phase (solid or liquid) should be used. Whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions in the laboratory. However, until now there is no agreement in how to acquire and to evaluate the data of the various available sediment contact assays. The SeKT joint research project was initiated with the aim to compare recently developed sediment contact assays by addressing reference conditions, control sediments and toxicity thresholds for limnic sediment contact tests.     

Evaluation of surface water quality using an ecotoxicological approach: a case study of the Alqueva Reservoir (Portugal)

Background, aim, and scope  

Freshwater reservoirs can be impacted by several hazardous substances through inputs from agricultural activity, sewage discharges, and groundwater leaching and runoff. The water quality assessment is very important for implementation of the monitoring and remediation programs to minimize the risk promoted by hazardous substances in aquatic ecosystems. Evaluation of the degree of contamination of aquatic environments must not take in account only its chemical characterization but it must be complemented with biological assays, which determine potential toxic effects and allows an integrated evaluation of its effects in populations and aquatic ecosystem communities. The application of this type of strategy has clear advantages allowing a general evaluation of the effects from all the water components, including those due to unknown substances and synergic, antagonistic, or additive effects. There are only a few studies that reported ecotoxicological acute end points, for the assessment of surface water quality, and the relationship among toxicity results and the anthropogenic pollution sources and the seasonal period. The aim of this study was to assess the ecotoxicological characterization of the surface water from Alqueva reservoir (South of Portugal) and to evaluate the influence of anthropogenic sources of pollution and their seasonal variation in its toxicity. The construction of Alqueva reservoir was recently finished (2002) and, to our knowledge, an ecotoxicological assessment of its surface water has not been performed. Because of that, no information is available on the possible impact of pollutants on the biota. The surface water toxicity was assessed using acute and chronic bioassays. The results are to be used for developing a monitoring program, including biological methods.