Macrophytes play an important role in aquatic ecosystems, and thus are often used in ecological risk assessments of potentially deleterious anthropogenic substances. Risk assessments for macrophyte populations or communities are commonly based on inferences drawn from standardized toxicity tests conducted on floating non-rooted Lemna species, or submerged-rooted Myriophyllum species. These tests follow strict guidelines to produce reliable and robust results with legal credibility for environmental regulations. However, results and inferences from these tests may not be transferrable to emergent macrophytes due to their different morphology and physiology. Emergent macrophytes of the genus Typha L. are increasingly used for assessing phytotoxic effects of environmental stressors, although standardized testing protocols have not yet been developed for this genus. In this review we present a synthesis of previous toxicity studies with Typha, based on which we evaluate the potential to develop standard toxicity tests for Typha spp. with seven selection criteria: ecological relevance to the ecosystem; suitability for different exposure pathways; availability of plant material; ease of cultivation; uniform growth; appropriate and easily measurable toxicity endpoints; and sensitivity toward contaminants. Typha meets criteria 1–3 fully, criteria 4 and 5 partly based on current limited data, and we identify knowledge gaps that limit evaluation of the remaining two criteria. We provide suggestions for addressing these gaps, and we summarize the experimental design of ecotoxicology studies that have used Typha. We conclude that Typha spp. can serve as future standard test species for ecological risk assessments of contaminants to emergent macrophytes.

An ecological impact assessment of four herbicides (atrazine, diuron, paraquat and simazine) was assessed using the aquatic floating vascular plants, Lemna gibba, Lemna minor and Lemna paucicostata as test organisms. The sensitivity of several ecologically relevant parameters (increase in frond area, root length after regrowth, maximum and effective quantum yield of PSII and maximum electron transport rate (ETRmax), were compared after a 72 h exposure to herbicides. The present test methods require relatively small sample volume (3 mL), shorter exposure times (72 h), simple and quick analytical procedures as compared with standard Lemna assays. Sensitivity ranking of endpoints, based on EC50 values, differed depending on the herbicide. The most toxic herbicides were diuron and paraquat and the most sensitive endpoints were root length (6.0–12.3 μg L−1) and ETRmax (4.7–10.3 μg L−1) for paraquat and effective quantum yield (6.8–10.4 μg L−1) for diuron. Growth and chlorophyll a fluorescence parameters in all three Lemna species were sensitive enough to detect toxic levels of diuron and paraquat in water samples in excess of allowable concentrations set by international standards. CV values of all EC50s obtained from the Lemna tests were in the range of 2.8–24.33%, indicating a high level of repeatability comparable to the desirable level of <30% for adoption of toxicity test methods as international standards. Our new Lemna methods may provide useful information for the assessment of toxicity risk of residual herbicides in aquatic ecosystems.

Although macrophytes are known to play vital roles in aquatic ecosystems, most quantitative aquatic toxicity data focus on fishes, water fleas, or algae, with limited ecotoxicity data published on macrophytes. Salvinia natans is a fast-growing plant commonly found in freshwater habitats. In this study, we verified a suitable disinfectant for preventing foreign contamination and formulated a culture medium for ensuring high productivity of S. natans. Finally, we established methodology for S. natans to be used in ecotoxicity testing of heavy metals and pesticides. As global regulations are being developed to harmonize guidelines and laboratory test species, S. natans is emerging as a potential candidate. The toxicity data publicly available for S. natans are very limited; hence, this study reports an advantageous culturing technique to optimize healthy growth of this species in the laboratory and presents optimal toxicity results, achieved by modifying the currently available test guidelines for Lemna. Our findings expand the currently limited range of test species for aquatic toxicity assays. We conclude that S. natans could serve as a valuable test species for aquatic toxicity assays.

Chemical methods have been used for the remediation of arsenic (As)-contaminated water; however, ecological consequences of these methods have not been properly addressed. The present study evaluated the effects of the Fe-oxide-coated sand (IOCS) remediation method on As toxicity to freshwater organisms (Lemna disperma, Chlorella sp. CE-35, and Ceriodaphnia cf. dubia). The As removal efficiency by IOCS decreased substantially with time. The IOCS remediation method was less effective at suppressing the toxicity of Asⱽ than Asᴵᴵᴵ to L. disperma but was highly effective in reducing both the Asᴵᴵᴵ and Asⱽ toxicity to C. cf. dubia. The growth of Chlorella sp. was significantly higher (p < 0.05) in remediated and pre-remediated water than in controls (non-As-contaminated filtered Colo River water) for Asᴵᴵᴵ, while the opposite was observed for Asⱽ, indicating that Asⱽ is more toxic than Asᴵᴵᴵ to this microalga. Although the IOCS can efficiently remove As from contaminated water, residual As and other constituents (e.g. Fe, nitrate) in the remediated water had a significant effect on freshwater organisms.

Lemna minuta Kunth was used to remove Cr(VI) from aqueous solutions, and some of the mechanisms involved in this process were analyzed. In addition, the cellular signaling mediated by phospholipase D activity as well as antioxidant responses was also evaluated during the process. Cr(VI) removal efficiencies were 40% for 0.5 mg/L, after 24 h, and up to 18% at metal concentrations as high as 5 mg/L. Removal mechanisms displayed by these macrophytes include bioadsorption to cell surfaces and, to a greater extent, Cr internalization and bioaccumulation within cells. Inside of them, Cr(VI) was reduced to Cr(III), a less toxic form of this metal. At the first hours of Cr(VI) exposure, plants were able to sense chromium, activating membrane signal transduction pathways mediated by phospholipase D and phosphatidic acid. Moreover, an increase in the activity of antioxidant enzymes such as superoxide dismutases and peroxidases was observed in the same time. These and other components of the antioxidant defense system would help to reduce the stress generated by the metal. The toxicity of the products formed during the removal process was assessed through Lactuca sativa L. and AMPHIAGU test. It was evidenced that Cr(VI) phytoremediation process by L. minuta plants did not generate acute toxicity neither for L. sativa seeds nor for embryos of Rhinella arenarum (Hensel, 1876). Thus, L. minuta plants could be considered as valuable species for the treatment of waters contaminated with Cr(VI).

Toxicity data of substances to higher plants is needed for the purpose of risk assessment, site evaluation, phytoremediation, and plant protection. However, the results from the most common phytotoxicity tests, like the OECD algae and Lemna test, are not necessarily valid for higher terrestrial plants. The willow tree toxicity test uses inhibition of transpiration (aside of growth and water use efficiency) of willow cuttings grown in spiked solutions or soils as end point to quantify toxicity. This overview presents results from 60 studies including 24 new unpublished experiments for 56 different chemicals or substrates. Highest toxicity (EC₅₀ < 1 mg/L) was observed from exposure to heavy metals like copper and cadmium. Also, organotins and free cyanide showed very high toxicity. The toxic effect of chlorophenols on willows was comparable to that on duck weed (Lemna) and green algae, while volatile compounds like chlorinated solvents or benzene, toluene, ethylbenzene, and xylene had less effect on trees than on these aquatic plants, due to volatilization from leaves and test media. In particular low (g/L range) toxicity was observed for tested nanomaterials. Effects of pharmaceuticals (typically weak acids or bases) depended strongly of the solution pH. Like for algae, baseline toxicity was observed for willows, which is related to the water solubility of the compounds, with absolute chemical activity ranging from 0.01 to 0.1, but with several exceptions. We conclude that the willow tree toxicity test is a robust method for relating uptake, accumulation, and metabolism of substances to the toxicity to trees.

River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01 % (standard) up to 0.25 % (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l⁻¹. The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an EᵣC₅₀ (72 h) of 0.00226 g l⁻¹ SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l⁻¹ SEQ. corresponding to 0.01 % DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05 % dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB concentrations analyzed in the sediments. The sediment extracts of Altrip and Veringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9-mix). The findings allow a better ecotoxicological characterization of the sediments extensively analyzed in all subprojects of the DanTox project (e.g., Garcia-Kaeufer et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3894-4 , 2014; Schiwy et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3185-0 , 2014; Hollert and Keiter 2015). In the absence of agreed limit values for sediment extracts in standard tests, further data with unpolluted reference sediments are required for a quantitative risk assessment of the investigated polluted sediments.

Background, aim and scope The applicability of the Whole Effluent Assessment concept for the proof of compliance with the “best available techniques” has been analysed with paper mill wastewater from Germany by considering its persistency (P), potentially bio-accumulative substances (B) and toxicity (T). Materials and methods Twenty wastewater samples from 13 paper mills using different types of cellulose fibres as raw materials have been tested in DIN or ISO standardised bioassays: the algae, daphnia, luminescent bacteria, duckweed (Lemna), fish-egg and umu tests with lowest ineffective dilution (LID) as test result. The potentially bio-accumulative substances (PBS) were determined by solid-phase microextraction and referred to the reference compound 2,3-dimethylnaphthalene. Usually, a primary chemical-physical treatment of the wastewater was followed by a single or multi-stage biological treatment. One indirectly discharged wastewater sample was pre-treated biologically in the Zahn-Wellens test before determining its ecotoxicity. Results No toxicity or genotoxicity at all was detected in the acute daphnia and fish egg as well as the umu assay. In the luminescent bacteria test, moderate toxicity (up to LIDlb = 6) was observed. Wastewater of four paper mills demonstrated elevated or high algae toxicity (up to LIDA = 128), which was in line with the results of the Lemna test, which mostly was less sensitive than the algae test (up to LIDDW = 8). One indirectly discharged wastewater sample was biodegraded in the Zahn-Wellens test by 96% and was not toxic after this treatment. Low levels of PBS have been detected (median 3.27 mmol L⁻¹). The colouration of the wastewater samples in the visible band did not correlate with algae toxicity and thus is not considered as its primary origin. Further analysis with a partial wastewater stream from thermomechanically produced groundwood pulp (TMP) revealed no algae or luminescent bacteria toxicity after pre-treatment of the sample in the Zahn-Wellens test (chemical oxygen demand elimination 85% in 7 days). Thus, the algae toxicity of the respective paper mill cannot be explained with the TMP partial stream; presumably other raw materials such as biocides might be the source of algae toxicity. Discussion Comparative data from wastewater surveillance of authorities confirmed the range of ecotoxicity observed in the study. Wastewater from paper mills generally has no or a moderate ecotoxicity (median LID 1 and 2) while the maximum LID values, especially for the algae and daphnia tests, are considerably elevated (LIDA up to 128, LIDD up to 48). Conclusions Wastewater from paper mills generally is low to moderately ecotoxic to aquatic organisms in acute toxicity tests. Some samples show effects in the chronic algae growth inhibition test which cannot be explained exclusively with colouration of the samples. The origin of elevated algae ecotoxicity could not be determined. In the algae test, often flat dose-response relationships and growth promotion at higher dilution factors have been observed, indicating that several effects are overlapping. Recommendations and perspectives At least one bioassay should be included in routine wastewater control of paper mills because the paper manufacturing industry is among the most water consuming. Although the algae test was the most sensitive test, it might not be the most appropriate test because of the complex relationship of colouration and inhibition and the smooth dose-effect relationship or even promotion of algae growth often observed. The Lemna test would be a suitable method which also detects inhibitors of photosynthesis and is not disturbed by wastewater colouration.

Background, aim, and scope Many pollutants have received significant attention due to their potential estrogenic effect and are classified as endocrine disrupting compounds (EDCs). Because of possible ecological effects and increased attention for water reuse schemes, it is important to increase our understanding of the EDC removal capacities of various wastewater treatment systems. However, there has so far been little research on the fate and behavior of EDCs in stabilization pond systems for wastewater treatment, which represent an important class of wastewater treatment systems in developing countries because of their cost-effectiveness. The aim of this work is to study the fate and behavior of EDCs in algae and duckweed ponds. Because the synthetic hormone 17α-ethinylestradiol (EE2) and the natural hormones estrone (E1), as well as 17β-estradiol (E2), have been detected in effluents of sewage treatment plants and been suggested as the major compounds responsible for endocrine disruption in domestic sewage; E1, E2, and EE2 were therefore chosen as target chemicals in this current work. Materials and methods Both batch tests and continuous-flow tests were carried out to investigate the sorption and biodegradation of estrogens in algae and duckweed pond systems. The applied duckweed was a Lemna species. The applied algae was a mixture of pure cultures of six different algae genera, i.e., Anabaena cylindrica, Chlorococcus, Spirulina platensis, Chlorella, Scenedesmus quadricauda, and Anaebena var. Synthetic wastewater were used in all tests. The concentrations of estrogens were measured with three different enzyme-linked immunosorbent assay kits specific for E1, E2, or EE2. When the concentrations of estrogens in water samples were below the lowest quantitative analysis range (0.05 µg/l), preconcentration of the water samples were performed by means of solid phase extraction (SPE) with C18 cartridges. Results The 6-day batch tests show that the presence of algae or duckweed accelerated the removal of the three estrogens from the synthetic wastewater. More estrogens were removed in the tests with duckweed than in tests with algae or with wastewater. In the sorption tests, a swift sorption of the three estrogens was observed when the estrogens were contacted with duckweed or algae, while the estrogen concentrations in tap water kept unchanged during the 3-h sorption tests. The mass balances indicated that only about 5% of the estrogens were bound to the algae sediment or duckweed at the end of the 6-day tests. Results of the continuous-flow tests revealed that the algae and duckweed ponds effectively removed E1, E2, and EE2 even at nanograms per liter level. Interconversion of E1 and E2 occurred both in batch and continuous-flow tests. E2 could be readily transformed to E1, especially in the tests with algae. Discussion Different processes like sorption, biodegradation and photolytic degradation might play an important role in the removal of estrogens from the aquatic phase. The 3-h sorption tests support the importance of sorption for estrogen removal, in which a rapid initial sorption was observed over the first 2 min for E1/E2/EE2 to both duckweed and algae. In the 6-day batch tests, estrogens were sorbed by algae or duckweed during the early stage when algae and duckweed were contacted with the synthetic wastewater and the sorbed estrogens were further biodegraded by the microorganisms developed in the wastewater. The persistent estrogen concentrations in tap water, however, implied that no sorption, biodegradation, or photolytic degradation occurred in tap water under the specific experimental conditions. Under aerobic or anoxic conditions, E2 could be first oxidized to E1, which is further oxidized to unknown metabolites and finally to CO₂ and water. Under anaerobic conditions, E1 can also be reduced to E2. However, the interconversion might be much more complex especially in the tests with algae because both aerobic and anaerobic conditions occurred in these tests due to the variation of the dissolved oxygen concentration induced by the light regime. Conclusions This study shows that estrogens, E1, E2, and EE2, can be effectively removed from the continuous-flow algae and duckweed ponds even when their concentrations are at nanograms per liter level. The presence of algae and duckweed accelerate the removal of estrogens from the synthetic wastewater because estrogens can be quickly sorbed on duckweed or algae. The sorbed estrogens are subsequently degraded by microorganisms, algae, or duckweed in the wastewater treatment system. E1 and E2 are interconvertible in both duckweed and algae pond systems. E2 can be readily transformed to E1, especially in the tests with algae. Recommendation and perspectives Based on the tests performed so far, one can conclude that both sorption and biodegradation are important to the estrogens removal from stabilization pond systems for wastewater treatment. Further research using, e.g., radioimmunoassay is needed to investigate the biodegradation pathway of estrogens in algae and duckweed ponds.