Algal Growth Inhibition Assay Research Articles

Toxic Effects of Copper Oxide Nanoparticles on Chlorella vulgaris

Abstract Aim: Exploitation of nano-based materials has increased the disposal of nanoparticles into the environment. Toxic nanomaterials pose their adverse effects and severely impact the human and environmental health. The aim of this study was to explore the toxic effects of chemically-synthesized copper oxide nanoparticles (CuO-NPs) on Chlorella vulgaris using an algal growth inhibition assay. Materials and Methods: Nanoparticles were synthesized using cupric sulfate and sodium hydroxide. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy. C. vulgaris culture was exposed to various concentrations of CuO-NPs at intervals of 3, 6, 12, and 24 h. Growth inhibition assay of algal culture was estimated via the spectroscopic method and cell enumeration was done by Neubauer hemocytometry. Results: The average diameter of CuO-NPs was ~35.85 nm. UV-Vis spectroscopy was used to confirm the configuration of CuO-NPs. Therefore, FTIR results has indicated high purity of synthesized CuO-NPs. Size of the CuO-NPs was determined by XRD. Results showed that growth of C. vulgaris microalgae was notably affected by the exposure of 2 mg/L CuO-NPs concentration exposure for 6 h. Conclusion: The current work showed that CuO-NPs have significant toxic to Chlorella sp. Data analysis showed NPs have broad effects on growth of C. vulgaris and there is a correlation between nanoparticles concentration as well as their toxicity on the microalgae.

Deciphering the toxicity mechanism of haloquinolines on Chlorella pyrenoidosa using QSAR and metabolomics approaches

The hazardous potential of haloquinolines (HQLs) is becoming an issue of great concern due to its wide and long-term usage in many personal care products. We examined the growth inhibition, structure-activity relationship, and toxicity mechanism of 33 HQLs on Chlorella pyrenoidosa using the 72-h algal growth inhibition assay, three-dimensional quantitative structure–activity relationship (3D-QSAR), and metabolomics. We found that the IC50 (half maximal inhibitory concentration) values for 33 compounds ranged from 4.52 to > 150 mg·L−1, most tested compounds were toxic (1 mg·L−1 < IC50 < 10 mg·L−1) or harmful (10 mg·L−1 < IC50 < 100 mg·L−1) for the aquatic ecosystem. Hydrophobic properties of HQLs dominate their toxicity. Halogen atoms with large volume appear at the 2, 3, 4, 5, 6, and 7-positions of the quinoline ring to significantly increase the toxicity. In algal cells, HQLs can block diverse carbohydrates, lipids, and amino acid metabolism pathways, thereby resulting in energy usage, osmotic pressure regulation, membrane integrity, oxidative stress disorder, thus fatally damaging algal cells. Therefore, our results provide insight into the toxicity mechanism and ecological risk of HQLs.

Biodiversity and functional trait effects on copper toxicity in a proof-of-concept multispecies microalgal assay

This study aimed to elucidate biodiversity effects on algal biomass production under toxicant stress and to increase environmental realism in ecotoxicological testing by exposing artificial algal communities to Cu2+. All 15 possible combinations of four functionally distinct microalgal species (green algae Chlamydomonas reinhardtii and Raphidocelis subcapitata, diatom Fistulifera pelliculosa and cyanobacterium Synechocystis sp.) were exposed to 0.01 and 0.02 mg Cu2+ l−1 in a modified algal growth inhibition assay (OECD 201). Compared to monocultures, the replicability of assays in multispecies communities decreased. Copper-induced inhibition of the biomass yield of individual species did not change due to interactions between species in multispecies tests, indicating that Cu toxicity was the driver of competitive outcomes. Biomass yield was increased at higher species richness in controls and Cu-exposed treatments. Selection effect is considered to prevail over trait complementarity in our experiments, because functional diversity, that expresses species trait variability and equality in species' occurrence, had a negative effect on algal biomass yield, while species richness increased the yield. Algal functional traits related to cell size were the best predictors of microalgal biomass production. Species identity was identified as an important determinant of community-level outcomes.

Ecotoxicity profiling of a library of 24 l-phenylalanine derived surface-active ionic liquids (SAILs)

We evaluated the ecotoxicity of a library of 24 l-phenylalanine derived surface-active ionic liquids (SAILs) with various cationic head groups (pyridinium, Py; imidazolium, Imid and cholinium, Chol) and alkyl ester chains from C2 to C16. For toxicity evaluation we used 72-h algal growth inhibition assay (OECD 201) with Raphidocelis subcapitata and 24-h mortality test with aquatic crustaceans Thamnocephalus platyurus (ISO 14380:2011). The OECD 201 assay was applied to all 24 SAILs while the ISO 14380:2011 test was applied to a subset, specifically all eight pyridinium SAILs and C6 and C8 examples of the imidazolium and cholinium SAILs (total 12 SAILs). For the comparison, 30-min EC50 data (based on inhibition of bioluminescence) previously reported by this group for the 24 SAILs for marine bacteria Vibrio fischeri (ISO 21338:2010) were included and correlated to the algae and aquatic crustaceans data. According to the results of the multritrophic test battery only two studied SAILs - PyC2 and CholC2 – could be considered ‘low toxicity’, (i.e. were ranked not harmful, L(E)C50 > 100 mg/L by the most sensitive test - algal growth inhibition assay). T. platyurus proved about 100-times more tolerant to studied SAILs than algae. An alternative classification scale dependent on the average MW of the compound dataset (based on molar concentrations and not concentrations based on mg/L) was suggested to rank the compounds. When compared to the classification scale independent of the MW of the compound, a more accurate appraisal was achieved for suggesting the greener alternatives for certain commercial SAILs/surfactants.

Chronic exposure of the freshwater alga Pseudokirchneriella subcapitata to five oxide nanoparticles: Hazard assessment and cytotoxicity mechanisms

The increasing use of nanoparticles (NPs) unavoidably enhances their unintended introduction into the aquatic systems, raising concerns about their nanosafety. This work aims to assess the toxicity of five oxide NPs (Al2O3, Mn3O4, In2O3, SiO2 and SnO2) using the freshwater alga Pseudokirchneriella subcapitata as a primary producer of ecological relevance. These NPs, in OECD medium, were poorly soluble and unstable (displayed low zeta potential values and presented the tendency to agglomerate). Using the algal growth inhibition assay and taking into account the respective 72 h-EC50 values, it was possible to categorize the NPs as: toxic (Al2O3 and SnO2); harmful (Mn3O4 and SiO2) and non-toxic/non-classified (In2O3). The toxic effects were mainly due to the NPs, except for SnO2 which toxicity can mainly be attributed to the Sn ions leached from the NPs. A mechanistic study was undertaken using different physiological endpoints (cell membrane integrity, metabolic activity, photosynthetic efficiency and intracellular ROS accumulation). It was observed that Al2O3, Mn3O4 and SiO2 induced an algistatic effect (growth inhibition without loss of membrane integrity) most likely as a consequence of the cumulative effect of adverse outcomes: i) reduction of the photosynthetic efficiency of the photosystem II (ФPSII); ii) intracellular ROS accumulation and iii) loss of metabolic activity. SnO2 NPs also provoked an algistatic effect probably as a consequence of the reduction of ФPSII since no modification of intracellular ROS levels and metabolic activity were observed. Altogether, the results here presented allowed to categorize the toxicity of the five NPs and shed light on the mechanisms behind NPs toxicity in the green alga P. subcapitata.

Application of an algal growth inhibition assay to determine distribution coefficients of benzalkonium ions between kaolinite and water

Benzalkonium compounds are widely used and found in environmental samples. Due to their amphiphilic nature, it is important to know sorption coefficients to account their bioavailability. However, currently available models describing their partitioning were developed using low molecular weight homologues and it cannot be ascertained whether they are applicable to their higher molecular weight homologues. Reasons for the scarcity of data on highly sorptive compounds include the lack of reliable quantification techniques for analyzing these chemicals at environmentally relevant levels. This study, therefore, reports on an algal growth inhibition assay-based method for the determination of kaolinite/water distribution coefficients for benzalkonium compounds at their environmentally relevant concentration range. Sorption to clay was computed using the difference between median effective concentration determined in a culture with kaolinite and that derived from a culture grown in standard medium. A kinetic model was used to account for uptake into algal cells and to calculate free concentrations. Due to the sensitivity of the algal species, Pseudokirchneriella subcapitata, it was possible to determine distribution coefficients below micromole per liter concentrations. The computed distribution coefficients showed a linear increase with number of carbon atoms in the alkyl chain up to 14. The proposed bioassay-based method should be applicable to determine distribution coefficients for highly hydrophobic chemicals and ionic liquids at a concentration range lower than typical analytical limits.

Evaluation of algal photosynthesis inhibition activity for dissolved organic matter with the consideration of inorganic and coloring constituents

The effect of waterborne ingredient on ecosystem has been of great interest. In the present study, the evaluation method using algal photosynthesis inhibition assay with dual-channel pulse amplitude modulation (PAM) system was established for a series of water samples to elucidate the potential effect of the total body of organic compounds including natural organic matter (NOM) on aquatic ecosystems. The more sensitive and less time-consuming monitoring method compared with algal growth inhibition assay was suggested, especially considering inorganic and coloring constituents. Algal photosynthesis inhibition activity was detected with high sensitivity for photosystem II (PSII) inhibitors, whereas the IC10 of the other chemicals was over the environmental standard concentration for Chlamydomonas moewusii (Chlorophyceae) and Pheodactylum tricornutum (Diatomea). The photosynthesis inhibition activity of Lake Biwa dissolved organic matter (LBDOM) and fulvic acid (LBFA) was significantly detected at ≥10 times the concentration and >10 mgC L-1, respectively, whereas prominent activity was confirmed for Suwannee River NOM (SRNOM) on the river original concentration (>30 mgC L-1) for both algae. Significant inhibition activity was detected in both algae at least in twice-concentration for water samples from a wastewater treatment pilot plant. There was no great difference in the activity between sewage secondary effluent and its filtrate with ultrafiltration (UF), and physically washing water for the UF membrane.

Toxic effects of nickel oxide (NiO) nanoparticles on the freshwater alga Pseudokirchneriella subcapitata

Over the last decade, concerns have been raised regarding the potential health and environmental effects associated with the release of metal oxide nanoparticles (NPs) into ecosystems. In the present work, the potential hazards of nickel oxide (NiO) NPs were investigated using the ecologically relevant freshwater alga Pseudokirchneriella subcapitata. NiO NP suspensions in algal OECD medium were characterized with regard to their physicochemical properties: agglomeration, surface charge, stability (dissolution of the NPs) and abiotic reactive oxygen species (ROS) production. NiO NPs formed loose agglomerates and released Ni2+. NiO NPs presented a 72 h-EC50 of 1.6 mg L−1, which was evaluated using the algal growth inhibition assay and allowed this NP to be classified as toxic. NiO NPs caused the loss of esterase activity (metabolic activity), the bleaching of photosynthetic pigments and the intracellular accumulation of reactive oxygen species (ROS) in the absence of the disruption of plasma membrane integrity. NiO NPs also disturbed the photosynthetic process. A reduction in the photosynthetic efficiency (ΦPSII) accompanied by a decrease in the flow rate of electrons through the photosynthetic chain was also observed. The leakage of electrons from the photosynthetic chain may be the origin of the ROS found in the algal cells. The exposure to NiO NPs led to the arrest of the cell cycle prior to the first cell division (primary mitosis), an increase in cell volume and the presence of aberrant morphology in the algal cells. In this work, the use of different approaches allowed new clues related to the toxicity mechanisms of NiO NPs to be obtained. This work also contributes to the characterization of the environmental and toxicological hazards of NiO NPs and provides information on the possible adverse effects of these NPs on aquatic systems.

Potency of (doped) rare earth oxide particles and their constituent metals to inhibit algal growth and induce direct toxic effects

Use of rare earth elements (REEs) has increased rapidly in recent decades due to technological advances. It has been accompanied by recurring rare earth element anomalies in water bodies. In this work we (i) studied the effects of eight novel doped and one non-doped rare earth oxide (REO) particles (aimed to be used in solid oxide fuel cells and gas separation membranes) on algae, (ii) quantified the individual adverse effects of the elements that constitute the (doped) REO particles and (iii) attempted to find a discernible pattern to relate REO particle physicochemical characteristics to algal growth inhibitory properties. Green algae Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) were used as a test species in two different formats: a standard OECD201 algal growth inhibition assay and the algal viability assay (a ‘spot test’) that avoids nutrient removal effects. In the 24h ‘spot’ test that demonstrated direct toxicity, algae were not viable at REE concentrations above 1mgmetal/L. 72-hour algal growth inhibition EC50 values for four REE salts (Ce, Gd, La, Pr) were between 1.2 and 1.4mg/L, whereas the EC50 for REO particles ranged from 1 to 98mg/L. The growth inhibition of REEs was presumably the result of nutrient sequestration from the algal growth medium. The adverse effects of REO particles were at least in part due to the entrapment of algae within particle agglomerates. Adverse effects due to the dissolution of constituent elements from (doped) REO particles and the size or specific surface area of particles were excluded, except for La2NiO4. However, the structure of the particles and/or the varying effects of oxide composition might have played a role in the observed effects. As the production rates of these REO particles are negligible compared to other forms of REEs, there is presumably no acute risk for aquatic unicellular algae.

Sorption of silver nanoparticles to laboratory plastic during (eco)toxicological testing

Here, we evaluate the extent of sorption of silver nanoparticles (AgNPs) with different primary sizes (30 and 70 nm) and surface properties (branched polyethylene imine, “bPEI” and citrate coating) to laboratory plastic during (eco)toxicological testing. Under conditions of algal growth inhibition assay, up to 97% of the added AgNPs were sorbed onto the test vessels whereas under conditions of in vitro toxicological assay with mammalian cells, the maximum loss of AgNPs was 15%. We propose that the high concentration of proteins and biomolecules in the in vitro toxicological assay originating from serum-containing cell culture medium prevented NP sorption due to steric stabilisation. The sorption of AgNPs to test vessels was clearly concentration dependent. In the conditions of algal growth inhibition assay at 10 ng AgNPs/mL, up to 97% of AgNPs were lost from the test while at higher concentrations (1000 ng AgNPs/mL), the loss of AgNPs was remarkably smaller, up to 64%. Sorption of positively charged bPEI-coated AgNPs was more extensive than the sorption of negatively charged citrate-coated AgNPs and, when calculated on a mass basis, more 70 nm-sized Ag than 30 nm Ag sorbed to plastic surfaces. In summary, this study demonstrates that the loss of AgNPs during (eco)toxicological tests due to sorption on test vessel surfaces is significant, especially in diluted media (e.g. in algal growth medium) and at low NP concentrations. Thus, to ensure the accurate interpretation of (eco)toxicological results, the loss of AgNPs due to adsorption to test vessels should not be overlooked and considered for each specific case.

Toxicity of 58 substituted anilines and phenols to algae Pseudokirchneriella subcapitata and bacteria Vibrio fischeri: Comparison with published data and QSARs

A congeneric set of 58 substituted anilines and phenols was tested using the 72-h algal growth inhibition assay with Pseudokirchneriella subcapitata and 15-min Vibrio fischeri luminescence inhibition assay. The set contained molecules substituted with one, two or three groups chosen from -chloro, -methyl or -ethyl. For 48 compounds there was no REACH-compatible algal toxicity data available before. The experimentally obtained EC50 values (mg L −1) for algae ranged from 1.43 (3,4,5-trichloroaniline) to 197 (phenol) and for V. fischeri from 0.37 (2,3,5-trichlorophenol) to 491 (aniline). Only five of the tested 58 chemicals showed inhibitory effect to algae at concentrations >100 mg L −1, i.e. could be classified as “not harmful”, 32 chemicals as “harmful” (10–100 mg L −1) and 21 as “toxic” (1–10 mg L −1). The occupied para-position tended to increase toxicity whereas most of the ortho-substituted congeners were the least toxic. As a rule, the higher the number of substituents the higher the hydrophobicity and toxicity. However, in case of both assays, the compounds of similar hydrophobicity showed up to 30-fold different toxicities. There were also assay/organism dependent tendencies: phenols were more toxic than anilines in the V. fischeri assay but not in the algal test. The comparison of the experimental toxicity data to the data available from the literature as well as to QSAR predictions showed that toxicity of phenols to algae can be modeled based on hydrophobicity, whereas the toxicity of anilines to algae as well as toxicity of both anilines and phenols to V. fischeri depended on other characteristics in addition to log K ow.

Molecular Characterization of Microbial Population Dynamics during Sildenafil Citrate Degradation

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.

Performance of a miniaturized algal bioassay in phytotoxicity screening

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC(50) results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r (2) = 0.975, p < 0.0017) or environmental samples toxicity (r (2) = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r (2) = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.

Environmental assessment of ester-based lubricants after application

Lubricants based on renewable resources are an environmentally friendly alternative to petrochemical products due to their better ecotoxicological performance and their excellent biodegradability. To improve the technical performance of lubricants, and to reduce friction and wear, the use of additives is nowadays obligatory. The collaborative research center SFB 442 aims at developing environmentally acceptable lubricants that facilitate the avoidance of these additives by transferring their function to suitable coatings. For a complete assessment of the ecological performance of these newly developed lubricants, the whole life cycle including production, application as well as disposal and fate in the environment is studied. In the following study the focus was on the application and its influence on the environmental behavior of the lubricant. The application of lubricants leads, among other things, to the intake of metals due to abrasion from tools, work pieces or mechanical components. Previous examinations indicated a possible influence of metals on the toxicity of eluates prepared from used lubricants (Erlenkaemper et al. 2005). To clarify if the apparent toxicity of used lubricants solely results from the intake of metals, the extractability of these metals from the oil matrix is determined. By combining chemical analyses with bioassays, the bioavailability of metals that are present in the extract is estimated. To further investigate the relevance of metals on toxicity, toxic units (TU) were calculated and related to the results of the bioassays. Interactions between the metals were investigated with aqueous mixtures of metal chlorides and calculations based on the concept of concentration addition and independent action. A lubricant mixture was applied to a tribological test bench that simulates real conditions of use and extremely short time load, respectively. Samples were taken at particular times, water soluble fractions (WSF) of these fluids were prepared and dilution series were investigated in several bioassays. Concentration of metals and total organic carbon (TOC) were determined in the eluates. TUs were calculated according to Sprague (1970) and mixture toxicity was calculated according to the concept of concentration addition (Loewe and Muischnek 1926) and independent action (Bliss 1939). Analyses of the metal content of the lubricant and the eluates clearly revealed the availability of the metals in the aqueous extracts. Especially copper, zinc, nickel and chromium were found and their concentrations increased during the time of use. The water extractable fraction, e.g., of copper, rose from 8.8% to 45.3% of the total content in the lubricant after 33.5 hours of use. Tests performed with the algal growth inhibition assay and the luminescence inhibition assay revealed the uptake or absorption by the organisms and, thus, the bioavailability of the metals. The calculation of TUs partly indicated a possible influence of the metals on ecotoxicity of the eluates. Copper reached concentrations equal to or higher than the EC50 value of copper chloride in the growth inhibition assays with algae and Ps. putida as well in the immobilization assay with daphnids. TUs for copper are also larger than 1 for the algal growth inhibition assay. The EL50 values indicated that the luminescence inhibition assay was the most sensitive test system, with values between 4.7% and 9.6%. While the toxicity towards algae and V. fischeri in the growth inhibition assay decreased until both organisms were no longer influenced by the exposure, the EL50 values for the D. magna immobilization assay and the Ps. putida growth inhibition assay decreased with the progressing use of the lubricant. The tested metal salt mixtures showed that Ps. putida, algae and daphnids are the most sensitive organisms with EC50 values below 1 mg/l. Although the intake of metals mainly occurred via abrasion of particles, the results revealed the availability of these metals in water. The availability varied for each of the four metals. For both the algal growth inhibition assay and the luminescence inhibition assay, an uptake or absorption of the metals could be demonstrated. The calculated TUs indicated an effect in some bioassays that was not verified in the test itself. The influence of copper on V. fischeri, for example, was not confirmed while the EL50 values for the daphnid bioassay decreased, meaning that the eluates became more toxic with progressing use of the lubricant. The calculations of mixture toxicity based on the concept of concentration addition demonstrated good correlations with the tested metal mixtures, but also a different sensitivity of the organisms. The results presented here reveal the availability of those metals in water that were taken in during the use of the lubricant in a tribological test bench and, thus, have the possibility of interacting with the organisms. The availability of the metals in the bioassays was proven by chemical analyses. The calculation of TUs and the corresponding EL50 values, however, indicate different availabilities of the metals. The results of the metal salt mixtures show good correlations with calculations of mixture toxicity based on concentration addition. Moreover, the varying sensitivity of the organisms when exposed to eluates or metal mixtures indicates a different bioavailability of the metals and/or the presence of other compounds that exert toxic action. For further investigations, the organic oil matrix and its influence on the toxicity have to be taken into account. The toxicity of the eluates may not only be due to metals; additional effects could arise from changes in the lubricant itself.

Aquatic Toxicity of Leachates Generated from Electronic Devices

Heavy metal leaching of electronic waste has been documented in recent literature. Heavy metal aquatic toxicity in the toxicity characteristic (TC) leachates produced from 56 electronic devices were tested using the aquatic toxicity assays such as Ceriodaphnia dubia 48-hr acute toxicity assay, the Selenatastrum capricornutum chronic algal growth inhibition assay (test used only for circuit-board leachates), and the MetPLATE acute heavy metal toxicity tests. The electronic devices tested, include 9 circuit boards (printed wire boards), 2 videocassette recorders, 4 remote controls, 1 cathode ray tube, 15 cellular phones, 1 calculator, 5 smoke detectors and their PC board components, 3 printers, 4 laptop computers, and 7 personal computer central processing units (CPUs). The toxicity tests showed toxicity in 51 of the 56 Toxicity Characteristic Leaching Procedure leachates of electronic devices assayed.

Hazard characterization and identification of a former ammunition site using microarrays, bioassays, and chemical analysis

More than 100,000 tons of 2,4,6-trinitrotoluene were produced at the former ammunition site Werk Tanne in Clausthal-Zellerfeld, Germany. The production of explosives and consequent detonation in approximately 1944 by the Allies caused great pollution in this area. Four soil samples and three water samples were taken from this site and characterized by applying chemical-analytical methods and several bioassays. Ecotoxicological test systems, such as the algal growth inhibition assay with Desmodesmus subspicatus, and genotoxicity tests, such as the umu and NM2009 tests, were performed. Also applied were the Ames test, according to International Organization for Standardization 16240, and an Ames fluctuation test. The toxic mode of action was examined using bacterial gene profiling assays with a battery of Escherichia coli strains and with the human liver cell line hepG2 using the PIQOR Toxicology cDNA microarray. Additionally, the molecular mechanism of 2,4,6-trinitrotoluene in hepG2 cells was analyzed. The present assessment indicates a danger of pollutant leaching for the soil-groundwater path. A possible impact for human health is discussed, because the groundwater in this area serves as drinking water.

Development of automated high-throughput ecotoxicity and genotoxicity test systems and fields of application

Bioassays like growth inhibition and genotoxicity assays are frequently used for the characterization of chemicals and contaminated environmental samples. In this work two standardized bioassays are automated completely using newly developed liquid handling stations and robotics. A high-throughput algal growth inhibition assay prototype is set up and optimized in cooperation with Hoelle & Huettner AG (Tuebingen, Germany) and Polygen GmbH (Langen, Germany). A software package for both the control of the test system and for data evaluation has been developed (Biolane Supervisor, BioLane Manager HTT). The applicability of the prototype is demonstrated by testing reference compounds with the automated assay in parallel with the Erlenmeyer flask assay. It is shown that EC50-values of chemicals do not differ significantly when incubation parameters like homogeneity of light intensity, temperature and evaporation during 72 hours of incubation are optimized. The umu-genotoxicity test is automated completely using a "RoboSeq 4204 SE" pipetting station (MWG AG, Ebersberg, Germany) equipped with two shakers, microplate photometer, four pipettes, stacker for ten microplates and incubation cabin with temperature regulation. As a result of miniaturization and automation large numbers of toxicants and far more treatments and parallels can be tested and, additionally, only low sample volumes are needed.

Assessment of the influence of use on ecotoxicological characteristics of synthetic ester lubricants.

Synthetic ester lubricants need optimisation about their technical and their ecotoxicological characteristics. To determine the ecotoxicological potential the required examinations can be based on the procedure for a risk assessment of chemicals. At present risk classification of lubricant oils is carried out with new oil fluids that are normally prepared before application in aqueous bioassays. In order to improve the ecotoxicological characteristics of some lubricant oils, the quality of the preparation method has been optimised. The resulting preparation protocol leads to aqueous extracts of the oil fluids that can be tested using biological assays. The extent of the changes of the chemical composition caused by the use as well as the ecotoxicological effects caused by additives have to be taken into consideration. For this reason various used lubricants are tested in addition to new oil fluids. In this work various lubricant samples were examined with standardised bacterial growth assays with Vibrio fischeri and Pseudomonas putida, luminescence inhibition assay with V. fischeri, survival assay with Daphnia magna and algal growth inhibition assay with Scenedesmus subspicatus. The chemical characterisation of the aqueous extracts included the determination of pH, conductivity, heavy metals, the content of dissolved organic carbon, inorganic anions and the content of phosphorus. The results emphasize the thesis that environmentally acceptable lubricants can undergo a change of their ecotoxicological potential during the use. Some of the substances that are normally added to base fluids in order to enhance the applicability of the oils may possess a high toxicological potential.