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.

Background: Parrot feather (Myriophyllum aquaticum) is an amphibious macrophyte with an inducible heterophylly. M. aquaticum plants adapted to the emersed state have leaves with a distinct cuticle and water repellent properties. In contrast M. aquaticum plants adapted to the submerged state have leaves typical for submerged hydrophytes with a strongly reduced cuticle. The aim of the study was to evaluate if this heterophylly of M. aquaticum affects the results of macrophyte biotests. Therefore, the two model substances atrazine and 2,4-dichlorophenoxyacetic acid (2,4-D) were tested each with three M. aquaticum biotests, the only modified parameter being the adaptation time to submergence (0, 7, 28 days). Results: Root length was the most sensitive growth endpoint regarding the test substances atrazine and 2,4-D. Biotests with plants adapted to the submerged state show three times more sensitive results (EC50 7d 142.2 μg/L, EC50 28d 154.5 μg/L) than biotests with plants without an adaptation phase (EC50 0d 458.8 μg/L) in case of atrazine and five times more sensitive results (EC50 28d 46.9 μg/L, EC50 0d 246.3 μg/L) in case of 2,4-D. Apart from the differences in sensitivity, the differently adapted M. aquaticum plants show a completely different growth behavior. The growth rates based on shoot length were nearly ten times higher in the biotests with not adapted M. aquaticum plants than in the biotests with plants 28 days adapted to submergence. Additional measurements of the quantum yield of PSII could demonstrate that rapid growth in length is not based on photosynthetic carbon assimilation. Conclusions: The heterophylly of M. aquaticum affects significantly the sensitivity of aquatic macrophyte biotests and should be taken into account in the development of a standardized test design. The 2,4-D results show the importance of an additional macrophyte biotest to the Lemna test, where the no observed effect concentration (NOEC) value is more than 30 times lower (7 μg/L) than the NOEC value of Lemna in the literature (270 μg/L). Furthermore, the growth rate endpoint in macrophyte biotests should not be misinterpreted. Rapid shoot elongation of amphibious macrophytes, which become submerged, is mainly caused by ethylene-triggered endogenous processes that are not connected to photosynthetic carbon assimilation and appear to be part of a stress reaction to avoid adverse environmental conditions.

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Background: In mesocosms, the fate and effects of chemicals can be investigated under more realistic conditions than in laboratory-scale experiments and most mesocosm studies for higher-tier risk assessment are conducted in lentic test systems. However, particularly for the assessment of EPT taxa (Ephemeroptera, Plecoptera, Trichoptera) and gammarids (Amphipoda), which are considered to be ecologically vulnerable macroinvertebrates, lotic systems seem to be more appropriate. Within this pilot study, eight newly constructed stream mesocosms were tested for their suitability in the effect assessment of plant protection products (PPP). By using the insecticide carbaryl as an example test item, five concentrations without replicates were tested under a worst case exposure scenario and three stream mesocosms served as controls. Results: 15 out of the 51 invertebrate taxa found during the study belonged to EPT taxa, while the common lotic amphipod Gammarus sp. (G. pulex and G. roesili) was the most dominant taxa. Estimation of minimum detectable differences based on mean abundances and variance of taxa in the eight mesocosms before application indicate that for five arthropoda taxa and two non-arthropoda taxa, an analysis of effects would have probably would have been possible in a typical test design for a higher-tier study with more test units. Five of these taxa were typical stream taxa, i.e. mayflies, caddisflies and gammarids. With respect to the effects of carbaryl, multivariate analysis (principal response curve) indicates long-term effects on the macroinvertebrates community over several weeks for the highest carbaryl concentration (120 µg a.i./L). Potential effects were observed for the amphipod Gammarus sp., the mayfly Ephemera danica and the caddisfly family Limnephilidae. Conclusions: Through this study it was shown that the stream mesocosms provided suitable habitats for potential sensitive and vulnerable taxa, including those typical for lotic systems. To enable a reliable statistical analysis of the effects within the higher-tier ecological risk assessment, the number of stream mesocosms needs to be enhanced. In addition, colonisation and sampling should be improved to increase the number of taxa with sufficiently high abundances for evaluation.