The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

The auxin herbicide mecoprop-P in new light: Filling the data gap for dicotyledonous macrophytes.

Mecoprop-P (MCPP-P) is an auxin herbicide which has been used against dicotyledonous weed plants since the 1980s. While fate and monitoring data of MCPP-P in the aquatic environment revealing concentrations up to 103 µg/L in freshwaters are well documented, only very few toxicity data and no studies with dicotyledonous macrophytes have been published in open literature so far. To fill up this essential data gap, a microcosm study was conducted in order to test the sensitivity of nine dicotyledonous and one Ceratophyllales macrophyte species. The plant species were exposed to seven MCPP-P concentrations ranging from 8 to 512 µg/L for 21/22 days in one microcosm per concentration, and two further microcosms served as controls. Plant preparation was adapted to each species and endpoints were measured to calculate growth rates. Data were generated to obtain effect concentrations (ECX) which then were used to construct species sensitivity distribution curves (SSD). Eight species proved to be sensitive to MCPP-P in the tested concentration range with EC50 values ranging from 46.9 µg/L for Ranunculus aquatilis to 656.4 µg/L MCPP-P for Ludwigia repens. Taking the EC50 values of this study and published data for autotrophic organisms into account, a hazard concentration (HC5) of 2.7 µg/L was derived from the SSD curve, while an SSD curve without dicotyledonous macrophytes resulted in an about 100 times higher HC5 (360.8 µg/L MCCP-P). This confirms that a re-evaluation for old auxin herbicides by including dicotyledonous test species into the environmental risk assessment may be indicated. Furthermore, the use of MCPP-P in bitumen felts as protection against rooting by plants is not in the focus of any risk regulation so far. This application, however, can lead to high run-off concentrations that can enter surface waters easily, exceeding the new regulatory acceptable concentration values.

Effects of multiple but low pesticide loads on aquatic fungal communities colonizing leaf litter.

In the first tier risk assessment (RA) of pesticides, risk for aquatic communities is estimated by using results from standard laboratory tests with algae, daphnids and fish for single pesticides such as herbicides, fungicides, and insecticides. However, fungi as key organisms for nutrient cycling in ecosystems as well as multiple pesticide applications are not considered in the RA. In this study, the effects of multiple low pesticide pulses using regulatory acceptable concentrations (RACs) on the dynamics of non-target aquatic fungi were investigated in a study using pond mesocosm. For that, fungi colonizing black alder (Alnus glutinosa) leaves were exposed to multiple, low pulses of 11 different pesticides over a period of 60days using a real farmer's pesticide application protocol for apple cropping. Four pond mesocosms served as treatments and 4 as controls. The composition of fungal communities colonizing the litter material was analyzed using a molecular fingerprinting approach based on the terminal Restriction Fragment Length Polymorphism (t-RFLP) of the fungal Internal Transcribed Spacer (ITS) region of the ribonucleic acid (RNA) gene(s). Our data indicated a clear fluctuation of fungal communities based on the degree of leaf litter degradation. However significant effects of the applied spraying sequence were not observed. Consequently also degradation rates of the litter material were not affected by the treatments. Our results indicate that the nutrient rich environment of the leaf litter material gave fungal communities the possibility to express genes that induce tolerance against the applied pesticides. Thus our data may not be transferred to other fresh water habitats with lower nutrient availability.

Structural changes in a macrozoobenthos assemblage after imidacloprid pulses in aquatic field-based microcosms.

A field-based microcosm experiment was performed to investigate the effects of repeated pulses of the neonicotinoid insecticide imidacloprid on a lentic benthos assemblage. This specific microcosm method was chosen because it allows for both testing of a wide range of organisms under natural conditions and as well as gaining insight into intraspecific and interspecific interactions. The macrozoobenthos that colonised the microcosms was exposed to three pulses each 1 week apart at nominal concentrations ranging from 0.6 to 40 µg/L. Imidacloprid underwent fast aqueous photolysis due to optimal sunlight conditions during the test phase (half-life = 28 ± 8 h [monitored for 21 days]). Nonetheless, decreased abundance and emergence of Ephemeroptera and decreased survival of chironomid species of the subfamilies Tanypodinae and Orthocladiinae were observed at time-weighted average concentrations of 2.3 µg/L. In contrast, the gastropod Radix sp. became dominant at high imidacloprid concentrations, probably due to decreased competition for food with sensitive species. The results of this study show that repeated short-term contamination of imidacloprid at low concentration levels may affect aquatic ecosystems even under optimal conditions for photodegradation. The microcosm approach, with its simple and field-relevant design, proved to be a useful tool for assessing the effects of imidacloprid contamination.

Effects of repeated insecticide pulses on macroinvertebrate drift in indoor stream mesocosms.

Pesticide contaminations via run-off or spray drift have been reported to result in the mass drift of macroinvertebrates as well as causing structural and functional changes of the corresponding stream sections. However, pesticide pulses in the field are associated with sudden increases in flow velocity, water turbidity, and changes in water temperature, which can also induce drift. Only through replicated community testing under highly controlled conditions can these effects be disentangled. In a stream mesocosm study, 12-h pulses of 12 µg/L imidacloprid were set three times at weekly intervals and are considered a "pulse series". Two pulse series of this neonicotinoid insecticide were run in both spring and summer with 4 treatment and 4 control stream mesocosms used in each pulse series. Prior to the start of the mesocosm experiment, both pulse concentration and duration had been screened for drift responses in larval Baetidae, Chironomidae and adult Gammarus roeseli in laboratory experiments. In the subsequent mesocosm study, each pulse caused a pronounced increase in the drift of insect larvae and gammarids. The drift response was taxon-specific, which was related to preferred habitat and exposure to other stressors like current velocity, in addition to imidacloprid sensitivity. Activity measurements employing a Multispecies Freshwater Biomonitor(®) revealed that in Baetis sp. the diurnal activity pattern became more pronounced even 12h after the pulse though with slightly decreased mean physical activity. Adult G. roeseli showed a drastic pulse by pulse decrease in physical activity which after the 3rd pulse lasted longer than 24h. In conclusion, drift is a sensitive, ecologically relevant endpoint and should be regarded when a specific risk assessment for lotic surface waters is done, e.g. in the context of a spatially explicit risk assessment.

Macroinvertebrate community response to repeated short-term pulses of the insecticide imidacloprid.

Small streams in agricultural landscape can experience short and repeated pulses of fluctuating pesticide concentrations. A single pesticide pulse may not have adverse effects on macrozoobenthos species but repeated pulses may have, especially if the organisms have not yet fully recovered when the second pesticide pulse occurs. Against this background, a comprehensive indoor stream mesocosm study was carried out in order to evaluate the cumulative effects of repeated insecticide pulses on a macrozoobenthos community. Weekly 12h pulses of 12 µg/L of the insecticide imidacloprid were set 3 times in 4 stream mesocosms in 2 series, one in spring and one in summer. Another 4 mesocosms served as controls. Prior to each pulse series, the mesocosms were stocked with macroinvertebrates from an uncontaminated reference stream using straw bags as attraction devices. The straw bag method proved suitable for establishing a functional macroinvertebrate community in the stream mesocosms. The caddisfly species Neureclipsis sp. reacted immediately and most sensitively after a single imidacloprid pulse whilst insect larvae such as ephemerids and dipteran larvae were negatively affected only after repeated imidacloprid pulses. Effects on insect larvae were more pronounced in the summer series most likely due to increased temperature. Abundance was a less sensitive endpoint than sublethal endpoints such as emergence. The results of the study underline that pulse effects are driven by a number of variables like pulse height, pulse duration, number of pulses, time in between pulses and by the species and live stage specific ability of temperature dependent detoxification which all should be taken into account in the risk assessment of pesticides.

AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology.

INTRODUCTION AND BACKGROUND: Primary producers play critical structural and functional roles in aquatic ecosystems; therefore, it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals. The standard required macrophyte test species is the floating (non-sediment-rooted) duckweed Lemna spp. This macrophyte species might not be representative of all floating, rooted, emergent, and submerged macrophyte species because of differences in the duration and mode of exposure; sensitivity to the specific toxic mode of action of the chemical; and species-specific traits (e.g., duckweed's very short generation time). DISCUSSION AND PERSPECTIVES: These topics were addressed during the workshop entitled "Aquatic Macrophyte Risk Assessment for Pesticides" (AMRAP) where a risk assessment scheme for aquatic macrophytes was proposed. Four working groups evolved from this workshop and were charged with the task of developing Tier 1 and higher-tier aquatic macrophyte risk assessment procedures. Subsequently, a SETAC Advisory Group, the Macrophyte Ecotoxicology Group (AMEG) was formed as an umbrella organization for various macrophyte working groups. The purpose of AMEG is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field, including prospective as well as retrospective risk assessments for chemicals. As AMEG expands, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services.

Toxic and accumulative potential of the antifouling biocide and TBT successor irgarol on freshwater macrophytes: a pond mesocosm study.

After the ban of tributyltin (TBT) for vessels not longer than 25 m in 1986, Irgarol has become a commonly used antifouling biocide. Irgarol is highly toxic to autotrophic organisms and has the potential to accumulate in organic material. In the literature, environmental concentrations of Irgarol up to 2.4 microg L(-1) were reported forfreshwater. Within a comprehensive freshwater mesocosm study, experiments were conducted to gain more information on the effects of Irgarol on macrophytes. Six indoor pond mesocosms were contaminated once with concentrations between 0.04 and 5 microgl(-1) Irgarol and monitored for 150 days; two mesocosms served as controls. The mesocosm study revealed that all macrophytes were directly affected by this single application. Myriophyllum verticillatum was the most sensitive macrophyte with an EC50 (Day 150) of 0.21 microg L(-1) Irgarol. The duckweed Spirodela polyrhiza was the least sensitive species tested in the mesocosms and number of fronds even increased with increasing Irgarol concentrations. Time-weighted average calculations yielded high BCF values of up to 10,580 L kg(-1) dry weight for M. verticillatum indicating a high potential for accumulation. The results give cause for concern that natural macrophyte communities are impaired at actual environmental concentrations.

Comparing growth development of Myriophyllum spp. in laboratory and field experiments for ecotoxicological testing.

UNLABELLED: BACKGROUND, GOALS AND SCOPE: Risk assessment of herbicides and the evaluation of contaminated sediments based on algae and the macrophyte Lemna sp. alone may underestimate the potential hazard of certain compounds. Therefore, various test systems with Myriophyllum spp. have been developed recently to assess the phytotoxicity in surface waters and natural sediments. In the present study, experiments investigating the growth development of Myriophyllum spp. were performed in the laboratory under defined conditions and in mesocosms under environmentally realistic exposure conditions to evaluate the suitability of these species as potential standard test organisms in ecotoxicological testing. This study provides data on the endpoints biomass, plant length and root development. MATERIALS AND METHODS: Six independent experiments were performed to investigate the plant development of Myriophyllum spp. under control conditions. The main difference in the experiments was the complexity of the test systems ranging from simple laboratory experiments to complex outdoor mesocosm studies. At the start of each experiment, uniform cuttings of Myriophyllum spp. were placed in vessels with or without sediments to reduce variability between replicates. The endpoints considered in this investigation were biomass (fresh weight of the whole plant), length of the main shoot, length of the side shoots, total length of the plant (calculated from the length of the main and side shoots) and root formation. Root to shoot ratios were calculated as a further measure for plant development. Relative growth rates (RGR) based on plant length (RG(L)R) and on biomass (RG(B)R) were calculated. RESULTS: Despite the various experimental conditions, comparable growth was obtained in all test systems and the variability of endpoints, such as total length and biomass of plants, was low. It was observed that the RGR of M. spicatum in the simple laboratory test system with sediment were comparable to growth data obtained for M. verticillatum and M. spicatum grown in indoor and outdoor mesocosms, thus indicating that Myriophyllum growth tends to increase by the addition of sediment. High variability was determined for the endpoints length of the side shoots, total root length and biomass of roots. DISCUSSION: One challenge for a test design to investigate phytotoxicity on aquatic plants is to obtain good growth of the plants. From the results, it can be concluded that the experimental conditions in the various test systems were suitable to study the plant development of Myriophyllum spp. because obtained growth rates were comparable between laboratory and field investigations. Another challenge for developing a plant biotest system is the definition of sensitive endpoints. Low variability is preferred to detect minor effects of chemicals or polluted sediments on plant development. In our studies, the variability of the endpoints biomass and total length of plant was low and, therefore, they have much potential as endpoints for assessing toxicity. CONCLUSIONS: The methodologies presented in this study have applications within the risk assessment for aquatic plants and have the advantage of assessing effects taking into account the relevant exposure pathways via water and/or sediment for compounds under investigation. RECOMMENDATIONS AND PERSPECTIVES: Setting safe quality criteria for surface water and sediments is one of the challenges authorities are facing today. Myriophyllum spp. is recommended as suitable test species to investigate phytotoxicity in surface water and sediments. These results, thus, might serve as a basis for the compilation of a new harmonised guideline for ecotoxicological testing with aquatic macrophytes.

Endpoint 'floating leaves' of potamogeton natans: a new method to evaluate the development of macrophytes in pond mesocosms.

GOAL, SCOPE AND BACKGROUND: One of the advantages of long-term mesocosm experiments as compared to short-term standard toxicity tests in the laboratory is the potential for detecting secondary effects due to the interaction of species and recovery with biomass of macrophytes being an important endpoint. However, generating biomass data by harvesting is often laborious, time-consuming, costly and restricted to the end of the experiment. Moreover, valuable information may get lost, in particular in single application studies, since maximal primary effects and secondary effects or recovery occur per se at different times. Potamogeton natans was used as an example in order to test whether number and area of floating leaves can be reliably measured and be used as intermediate and final end-points in mesocosm effect studies. METHODS: Digital photos, which were taken of the water surface in the course of an indoor pond mesocosm study on herbicide effects, were subjected to image analysis. The results were compared to wet weight and ash-free dry weight of Potamogeton at the end of the herbicide study. RESULTS AND DISCUSSION: Both number and area of floating leaves indicated the same herbicide effects as wet weight and ash-free dry weight of Potamogeton. Error introduced by the different work steps is small and can be further minimised by a number of method improvements. RECOMMENDATIONS AND PERSPECTIVES: In indoor mesocosm studies, errors due to the perspective adjustment may be circumvented by taking the photos perpendicular to the water surface. Correction for lens aberration, identical light conditions and the use of fluorescence images are considered promising. Field applications are proposed.

Enhanced experimental flexibility and control in ecotoxicological mesocosm experiments--a new outdoor and indoor pond and stream system.

SCOPE: The German Federal Environmental Agency has put into operation a new modular mesocosm system consisting of eight outdoor and eight indoor ponds and streams in order to investigate fate and effects of chemicals and municipal wastewater in aquatic ecosystems. General design and special characteristics are given to demonstrate the wide range of possibilities for experimental research. GENERAL DESIGN: Each of the 16 streams with riffle and pool sections can be varied in length up to 106 m. The streams can be run as circular or flow-through systems at a flow velocity of 0.02 to 0.6 m/s. Physico-chemical standard parameters are measured on-line. The 16 ponds, which can be connected to the stream systems, are equipped with drainage and pore water-sampling devices for simulating processes in the littoral zone including influent and effluent ground water flow. PERSPECTIVES: Since the highly flexible and controllable construction also allows treating a wide range of hydrological and ecological experiments external institutions are invited to submit proposals.