Ute Feiler

A new plant-based bioassay for aquatic sediments

Background, Goal and ScopeTo date, standardised bioassays for the assessment of the ecotoxicological potential in sediments and dredged material use test organisms like bacteria, algae and crustaceae. This paper presents the development and application of a novel sediment contact test (whole sediment) withMyriophyllum aquaticum, a representative of rooted aquatic macrophytes. The aim of the present study is to demonstrate the value of a sediment contact test with rooted macrophytes as a supplement to existing test batteries in order to improve the assessment of sediment toxicity.MethodsThe newly developed sediment contact test withMyriophylhim aquaticum was applied to natural whole sediments. For performing the test, whorls ofMyriophyllum aquaticum were directly planted in the native sediment and incubated in the light at 24°C (cf. section results and discussion). The end points of the test were the number of the shoots and the fresh weight of the whole plants. The duckweed growth inhibition test withLemna minor according to ISO/DIS 20079 was performed in pore waters from sediment samples. The results of the sediment contact test withMyriophyllum aquaticum were compared with each other and with those of the aquatic duckweed test.Results and DiscussionA test protocol for the new plant-based sediment contact test using the aquatic plantMyriophyllum aquaticum as an indicator was developed. The best control sediment proved to be the OECD sediment (OECD 207). A test period of 10 days appeared to be sufficient for the test. The increase of biomass and the derived growth rate were found to be the most suitable evaluation parameters. The growth behaviour ofMyriophyllum aquaticum differed depending on the origin of sediments. Therefore, plant-affecting contamination, that is bound in sediments, was indicated.ConclusionsThe novel sediment contact test withMyriophyllum aquaticum can indicate phytotoxic effects in sediments. Therefore, it allows a better assessment of the overall-toxicity in whole sediments.Recommendations and OutlookThe sediment contact test withMyriophyllum aquaticum is a valuable tool for the evaluation of the ecotoxicological risk potential of waters and sediments. It should become a complement to a standardised test battery generally used for the assessment of sediment toxicity.

Sediment contact tests as a tool for the assessment of sediment quality in German waters

A sediment contact test (SCT) battery consisting of five ecotoxicological test systems was applied to 21 native freshwater sediments characterized by a broad variety of geochemical properties and anthropogenic contamination. Higher plants (Myriophyllum aquaticum), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), zebrafish embryos (Danio rerio), and bacteria (Arthrobacter globiformis), representing various trophic levels and exposure pathways, were used as test organisms. The test battery detected sediment toxicity caused by anthropogenic pollution, whereas the various tests provided site-specific, nonredundant information to the overall toxicity assessment. Based on the toxicity pattern derived from the test battery, the sediments were classified according to a newly proposed classification system for sediment toxicity assessment. The SCT-derived classification generally agreed well with the application of consensus-based sediment quality guidelines (SQGs), especially with regard to sediments with high toxic potential. For sediments with low to medium toxic potential, the SQGs often underestimated the toxicity that was detected by the SCTs, underpinning the need for toxicity tests in sediment quality assessment.

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

Background, goals and scopeRisk 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 methodsSix 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 (RGLR) and on biomass (RGBR) were calculated.ResultsDespite 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.DiscussionOne 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.ConclusionsThe 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 perspectivesSetting 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.

Inter‐laboratory trial of a standardized sediment contact test with the aquatic plant Myriophyllum aquaticum (ISO 16191)

A whole-sediment toxicity test with Myriophyllum aquaticum has been developed by the German Federal Institute of Hydrology and standardized within the International Organization for Standardization (ISO; ISO 16191). An international ring-test was performed to evaluate the precision of the test method. Four sediments (artificial, natural) were tested. Test duration was 10 d, and test endpoint was inhibition of growth rate (r) based on fresh weight data. Eighteen of 21 laboratories met the validity criterion of r ≥ 0.09 d(-1) in the control. Results from 4 tests that did not conform to test-performance criteria were excluded from statistical evaluation. The inter-laboratory variability of growth rates (20.6%-25.0%) and inhibition (26.6%-39.9%) was comparable with the variability of other standardized bioassays. The mean test-internal variability of the controls was low (7% [control], 9.7% [solvent control]), yielding a high discriminatory power of the given test design (median minimum detectable differences [MDD] 13% to 15%). To ensure these MDDs, an additional validity criterion of CV ≤ 15% of the growth rate in the controls was recommended. As a positive control, 90 mg 3,5-dichlorophenol/kg sediment dry mass was tested. The range of the expected growth inhibition was proposed to be 35 ± 15%. The ring test results demonstrated the reliability of the ISO 16191 toxicity test and its suitability as a tool to assess the toxicity of sediment and dredged material.

Aquatic plant bioassays used in the assessment of water quality in German rivers

A novel aquatic bioassay with Lemna minor (duckweed), a representative of higher plants, was applied to pore waters (interstitial waters) from river sediments. The results have been used for the ecotoxicological assessment of sediments in several German rivers. The aim of the present study is to demonstrate the usefulness of a macrophyte bioassay, as a supplement to the standardised algae bioassays, with regard to the ecotoxicological assessment of aquatic systems. Results relating to the development of a sediment contact test (using the whole sediment) with Lemna minor are also presented.

Der Einsatz von Pflanzentests bei der Sedimentbewertung@@@The application of plant tests for sediment evaluation

ZusammenfassungSedimentuntersuchungen werden bislang hauptsächlich mit aquatischen, standardisierten Biotests mit Organismen der drei Trophieebenen durchgeführt. Algen sind dabei alleinige Vertreter des gesamten Pflanzenreichs. Biotests mit höheren Pflanzen fanden bisher kaum Anwendung bei der Bewertung von Gewässersedimenten. Ziel der hier vorgestellten Untersuchungen war es zu zeigen, dass der Einsatz von höheren Pflanzen in Biotests zur Untersuchung anthropogen belasteter Sedimente wertvolle Ergebnisse liefert, die in einem Konzept zur integrierten Gewässerbewertung verwendet werden können. Die anthropogenen Belastungen durch Umweltchemikalien wurden in der Elbe am Beispiel des Entnahmeortes Fahlberg-List Magdeburg erfasst und mit Untersuchungen in einem Referenzgebiet der Müritz-Elde-Wasserstraße (Eldenburger Kanal) verbunden. Sedimentextrakte und Porenwässer der jeweiligen Sedimente sind mit dem aquatischen Lemnatest untersucht worden. Erste Sedimentkontakttests wurden an Gesamtsedimenten vergleichend erprobt.Im Gesamtextrakt des Sediments aus Fahlberg-List Magdeburg und hauptsächlich in der Fraktion F5 wurden deutliche Pflanzenschädigungen beobachtet. Anscheinend finden sich die pflanzenschädigenden Substanzen aus dem Gesamtextrakt überwiegend in der stark polaren Fraktion F5 wieder. Ein Vergleich mit den chemischen Daten zeigte, dass die Gesamtfraktion F0 und Fraktion F5 stark chemisch belastet sind. In Ansätzen mit Porenwasser dieses Sediments und mit Gesamtsediment traten ebenfalls starke Wachstumsbemmungen auf. Diese Ergebnisse bestätigten die starke Belastung des Sediments aus Magdeburg. Die Untersuchungen des Referenzsediments lieferten Hinweise auf eine nur mäßige toxische Belastung.Die Ergebnisse dieser Arbeit machen deutlich, dass die ausgewählte WasserpflanzeLemna minor Schadstoffbelastungen grundsätzlich anzeigt. Im aquatischen Test der Sedimentextrakte weist sie eine zwar schwache, aber sehr selektive Reaktion auf bestimmte Schadstoffklassen auf. Die Fraktionierung der Probe mit anschließender Stoffanalyse kombiniert mit Toxizitätstests erlaubt die Eingrenzung der toxisch wirksamen Stoffgruppen. Diese toxische Belastung wurde durch die Porenwasser- und Gesamtsedimentuntersuchung bestätigt. Zusammen mit weiteren Toxizitätstests (z.B. standardisierte Biotests) und in Kombination mit benthosbiologischen Untersuchungen ergibt sich eine Gesamtaussage zur integrierten Gewässerbewertung.AbstractUp to now, sediment studies have mainly used standardized aquatic bioassays with organisms of the three trophic levels. Among these, algae are the only representatives of the entire plant kingdom. Bioassays with higher plants have hardly been used for the assessment of sediments. The aim of the present study is to demonstrate that the use of higher plants in biotests for analyses of anthropogenically contaminated sediments yields valuable results, which may be included in a concept for the integrated assessment of waters. The contamination by environmental chemicals was determined in the River Elbe by means of samples taken at the Fahlberg-List industrial site at Magdeburg in combination with studies in a reference area on the Müritz-Elde Waterway (Eldenburger Kanal). Sediment extracts and pore waters were tested with the aquatic lemna test. For comparison, first sediment contact tests withLemna minor were tried out.The whole extract of the sediment from Fahlberg-List Magdeburg and one fraction F5 caused distinct damages of the plants. Obviously, the phytotoxic substances of the whole extract are preferentially concentrated in the highly polar fraction F5. A comparison with chemical analytical data proved that the whole fraction F0 and the fraction F5 were heavily contaminated with chemicals. Assays with pore water from this sediment and with the whole sediment also caused a strong inhibition of growth. These results confirm the high contamination of the sediment from Magdeburg. The tests with the reference sediment suggested only a moderate toxic contamination.The results of this study prove that the selected aquatic plant,Lemna minor, is basically able to indicate contamination. In the aquatic test of the sediment extracts, it showed weak, but very selective, responses to certain classes of contaminants. Fractionating of the sample and subsequent chemical analysis combined with toxicity tests allow to narrow down the groups of substances causing toxic effects. This toxicity was confirmed by analyses of the pore waters and whole sediment samples. Together with other toxicity tests (e.g. standardized bioassays) and combined with biological benthos examinations, an overall judgment can be given for the integrated assessment of waters.

Development of a sediment-contact test with rice for the assessment of sediment-bound pollutants

Despite the key role of higher plants in aquatic ecosystems as functional and structural elements, sediment-contact tests with macrophytes are still scarce. Moreover, due to large differences in exposure routes for pollutants as well as in life cycles between the diverse taxa of macrophytes, sensitivities to pollutants vary between taxa. Therefore, the development of new test systems with aquatic macrophytes, in general, is favorable. This study proposes a protocol for a sediment-contact test with Oryza sativa and addresses the main question whether the rice plant is a suitable test organism for sediment toxicity testing with higher plants. As a first evaluation step, the variability and sensitivity of the test was investigated using spiked artificial sediments. Thus, according to the protocol, rice was exposed to arsenic-, cadmium-, chromium-, and nickel-spiked sediments. Additionally, it was investigated which classical endpoints for plant bioassays, such as root and shoot elongation, are suitable for this bioassay. As a second evaluation step, the test system was used for assessment of natural sediments. Thereupon, a sensitivity profile of the presented test protocol was analyzed in comparison to other plant-based test systems. Inhibition of root and shoot elongation turned out to be the most sensitive endpoints for single-substance testing in spiked artificial sediments. However, regarding testing of natural sediments, rice shoots responded more sensitive than rice roots. In conclusion, the rice plant clearly showed pollutant-induced effects on growth in sediments, and thus, it is likely a promising test organism to complement sediment-contact tests with higher plants.

Transcriptional changes measured in rice roots after exposure to arsenite-contaminated sediments

Transcriptional analyses are discussed to provide a deeper understanding of the molecular mechanisms underlying toxic effects. Thus, they can complement classic ecotoxicological test methods and potentially allow the identification of biomarkers associated to the exposure of chemical stressors and or adverse biological effects. This feasibility study intended to identify a set of potential gene expression biomarkers for arsenite-exposure in rice roots that could complement the informative value of an existing sediment-contact test with rice. A sediment-contact test with Oryza sativa with the parameters inhibition of root and shoot elongation as phenotypic endpoints was used as basis. Rice plants were exposed to arsenite-spiked sediments. Transcriptomic changes in response to arsenite were observed by means of cDNA-microarray analysis regarding the whole-transcriptome at two sublethal arsenite concentrations. In order to identify candidate biomarker genes, differentially expressed genes were identified. Arsenite-induced differentially expressed genes were significantly associated with gene ontology (GO)-terms that indicated a general stress response. Of the differentially expressed genes, five genes were selected and their expression was measured at seven arsenite concentrations by means of qPCR in order to obtain their expression profiles. Three candidate biomarker genes showed a dose-dependent upregulation, while two showed no clear dose-dependent expression. The expression of all candidate biomarkers was also assessed in rice plants grown on two arsenic-contaminated natural sediments, but only one biomarker gene showed the expected upregulation.