Additive toxicity of herbicide mixtures and comparative sensitivity of tropical benthic microalgae.

Natural waters often contain complex mixtures of unknown contaminants potentially posing a threat to marine communities through chemical interactions. Here, acute effects of the photosystem II-inhibiting herbicides diuron, tebuthiuron, atrazine, simazine, and hexazinone, herbicide breakdown products (desethyl-atrazine (DEA) and 3,4-dichloroaniline (3,4-DCA)) and binary mixtures, were investigated using three tropical benthic microalgae; Navicula sp. and Cylindrotheca closterium (Ochrophyta) and Nephroselmis pyriformis (Chlorophyta), and one standard test species, Phaeodactylum tricornutum (Ochrophyta), in a high-throughput Maxi-Imaging-PAM bioassay (Maxi-IPAM). The order of toxicity was; diuron > hexazinone > tebuthiuron > atrazine > simazine > DEA > 3,4-DCA for all species. The tropical green alga N. pyriformis was up to 10-fold more sensitive than the diatoms tested here and reported for coral symbionts, and is recommended as a standard tropical test species for future research. All binary mixtures exhibited additive toxicity, and the use of herbicide equivalents (HEq) is therefore recommended in order to incorporate total-maximum-load measures for environmental regulatory purposes.

Rapid exposure assessment of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay.

Recently a new Maxi-Imaging-PAM (Max-I-PAM) instrument for phytotoxicity assessment via chlorophyll fluorescence imaging was introduced. This new instrument allows rapid detection of the effects of PS II inhibiting herbicides which are high use agricultural chemicals frequently detected in surface waters in Australia and elsewhere. Several studies have applied the new instrument for detection of phytotoxicants in water using microalgae suspensions; however, these use preliminary protocols and to date no validated method is available for high throughput testing of environmental samples in 96-well plates. Here we developed and applied a new protocol allowing dose-response assessment of four samples within 2 h (8 dilutions in duplicate). The technique was found to be sensitive, with a detection limit of 2.3 ng l(-1) for the herbicide diuron when testing solid phase extracts (SPE) of 1000 ml water samples, and reproducible both between experiments (coefficient of variation (CV)=0.30) and within the 96-well plate (CV=0.06). Relative potencies were determined for four reference PS II impacting herbicides (diuron>hexazinone>atrazine>simazine). Extracts from 1000 ml environmental samples and diuron spiked ultrapure water as well as passive sampler extracts were evaluated and good agreement was found between diuron equivalent concentrations calculated from bioassay results (DEQ(IPAM)) and DEQ(CHEM) values calculated from LCMS chemical analysis of the four reference compounds in the same samples. Overall, the technique provides a valuable bioanalytical tool for rapid and inexpensive effects-based assessment of PS II impacting herbicides in environmental mixtures.

Toxic equivalent concentrations (TEQs) for baseline toxicity and specific modes of action as a tool to improve interpretation of ecotoxicity testing of environmental samples.

The toxic equivalency concept is a widely applied method to express the toxicity of complex mixtures of compounds that act via receptor-mediated mechanisms such as induction of the arylhydrocarbon or estrogen receptors. Here we propose to extend this concept to baseline toxicity, using the bioluminescence inhibition test with Vibrio fischeri, and an integrative ecotoxicity endpoint, algal growth rate inhibition. Both bioassays were validated by comparison with literature data and quantitative structure-activity relationships (QSARs) for baseline toxicity were developed for all endpoints. The novel combined algae test, with Pseudokirchneriella subcapitata, allows for the simultaneous evaluation of specific inhibition of photosynthesis and growth rate. The contributions of specific inhibition of photosynthesis and non-specific toxicity could be differentiated by comparing the time and endpoint pattern. Photosynthesis efficiency, measured with the saturation pulse method after 2 h of incubation, served as indicator of specific inhibition of photosynthesis by photosystem II inhibitors. Diuron equivalents were defined as toxicity equivalents for this effect. The endpoint of growth rate over 24 h served to derive baseline toxicity equivalent concentrations (baseline-TEQ). By performing binary mixture experiments with reference compounds and complex environmental samples from a sewage treatment plant and a river, the TEQ concept was validated. The proposed method allows for easier interpretation and communication of effect-based water quality monitoring data and provides a basis for comparative analysis with chemical analytical monitoring.

Monitoring of the ecotoxicological hazard potential by polar organic micropollutants in sewage treatment plants and surface waters using a mode-of-action based test battery.

We propose and evaluate a mode-of-action based test battery of low-complexity and in-vitro bioassays that can be used as a routine monitoring tool for sewage treatment efficiency and water quality assessment. The test battery comprises five bioassays covering five different modes of toxic action. The bioluminescence inhibition test with Vibrio fischeri and a growth rate inhibition test with the green algae Pseudokirchneriella subcapitata are measures of non-specific integrative effects. A second endpoint in the algae test, the specific inhibition of the efficiency of photosynthesis, gives an account of the presence of herbicides. An enzymatic assay covers an important aspect of insecticidal activity, the inhibition of the acetylcholine esterase activity. Estrogenic effects are assessed with the yeast estrogen screen (YES) and genotoxicity with the umuC test. Three field studies, each lasting six to seven consecutive days, were undertaken at a sewage treatment plant (STP) in Switzerland. Samples were collected in summer and late autumn, under dry and rainy conditions. None of the bioassays gave positive results with raw water in whole effluent toxicity testing. Therefore, water samples from various sites during wastewater treatment and from surface water were enriched with solid-phase extraction. The focus was on non-volatile compounds of average to moderate hydrophobicity, a range that includes most pesticides, biocides and pharmaceuticals. Various polar solid phases were evaluated for their extraction efficiency, disturbance by matrix components and overall performance. We finally selected a mixture of a polymeric sorbent and a C18-sorbent, Lichrolut EN and RP-18 or, alternatively, Empore SDB-RPS disks. All bioassays gave clear and robust responses with the SPE extracts. With the bioassay data the treatment efficiency of the STP can be assessed with respect to different modes of toxic action and accordingly different groups of micropollutants. Furthermore, the data allowed for a comparison between the effluent and the receiving river. In all bioassays the primary effluent had a strong effect and this effect was reduced after passing the STP. Treatment efficiency was high (typically over 90%) but varied from bioassay to bioassay, which is expected because each bioassay detects different types of micropollutants and therefore we cannot expect a common answer.

Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging.

A new phytotoxicity bioassay based on chlorophyll fluorescence imaging of algae suspensions in multiwell plates is introduced. Phytotoxicity is quantified via inhibition of photosystem II quantum yield, Y(II), assessed with the saturation pulse method. The basics of this approach as well as the factors enhancing and limiting its performance are outlined. Compared to other established techniques the new system allows exceptionally rapid and accurate measurements of phytotoxicity using pulse-amplitude-modulation (PAM) fluorometry. While instrument related errors are negligibly small, optimal performance depends on appropriate choice of algae and illumination conditions. Illustrative examples for the response of Phaeodactylum tricornutum to diuron are presented. The standard deviation involved in the Y(II) determination of a single well amounts to the equivalent of 44 ng/L diuron. A decisive role is played by the light (measuring light, saturation pulses, actinic light) to which samples are exposed during the bioassay: (1) the inhibitor response is enhanced at high measuring light intensity. (2) Saturation pulses may be considered non-invasive only, if applied at low frequency and as long as physiologically healthy algae cultures are used. (3) Continuous actinic light may be problematic, as it induces complex physiological reactions that limit the performance of the approach; it is not required for assessment of diuron-type inhibitors at high measuring light intensity.