Chronic toxicity of pharmaceuticals to the benthic green alga Closterium ehrenbergii.

Pharmaceuticals in the environment have emerged to a topic of global concern. Since these substances are designed to be biologically active, hazardous effects on non-target organisms are frequently reported. Here, the effects of five pharmaceuticals, one radiocontrast agent, and one degradation product on the freshwater green alga Closterium ehrenbergii were evaluated after chronic exposure of 168 h. Growth and maximum quantum yield (FV/FM) were used as endpoints and complemented by the assessment of morphology and chlorophyll fluorescence. We found that the tested antibiotics Ciprofloxacin and Ofloxacin impaired chloroplast integrity, resulting in a reduction of FV/FM from 0.1 mg/L. The disintegration of chloroplasts at higher concentrations (c = 0.3 and 0.8 mg/L, respectively) was visualized by brightfield and fluorescence microscopy. In contrast, Sulfamethoxazole interfered with cell division, leading to malformation of cells from 0.8 mg/L. Furthermore, the antibiotics exhibited a latency period of 72 h after which they started to reveal their true effects. Therefore, the importance of long-term toxicity testing is outlined in order to avoid underestimation of toxic effects of pharmaceuticals. Based on the EC10 values obtained, the antibiotics were considered to meet the criteria for classification as toxic to aquatic life with long lasting effects. The other test substances were found to exert no effects on C. ehrenbergii or only at very high concentrations and were classified as nontoxic.

A microplate-based bioassay for toxicity testing using the large benthic algal species Closterium ehrenbergii.

Pollution of water bodies by metals has long been studied but still remains a threat to healthy ecosystems. While most ecotoxicological studies on algae are performed with planktonic standard species such as Raphidocelis subcapitata, benthic algae may depict the majority of the algal flora in rivers and streams. These species encounter different exposure scenarios to pollutants as they are sedentary and not carried away by the current. This particular way of life leads to an integration of toxic effects over time. Therefore, in this study, the effects of six metals on the large unicellular benthic species Closterium ehrenbergii were examined. A miniaturized bioassay with low cell densities of 10-15 cells/mL using microplates was developed. Through chemical analysis, metal complexing properties in the culture medium were demonstrated, that could lead to an underestimation of metal toxicity. Thus, the medium was modified by excluding EDTA and TRIS. The toxicity of the six metals ranked by EC50 values in descending order, was as follows: Cu (5.5 µg/L) > Ag (9.2 µg/L) > Cd (18 µg/L) > Ni (260 µg/L) > Cr (990 µg/L) > Zn (1200 µg/L). In addition, toxic effects on the cell morphology were visualized. Based on a literature review, C. ehrenbergii was shown to be partly more sensitive than R. subcapitata which suggests that it can be a useful addition to ecotoxicological risk assessment.

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.