Cyst-based toxicity tests XVII--prefeeding advantages in short-chronic rotifer microbiotests

Attempts were made to overcome too-low reproductive output in the 48-h short-chronic microbiotest with the rotifer Brachionus calyciflorus fed with microalgae (Pseudokirchneriella subcapitata) from algal beads older than 4 months (Chemosphere 50 (2002a) 365). Prefeeding of freshly hatched rotifers prior to the start of the actual growth experiments resulted in a satisfactory reproduction (i.e., an intrinsic growth rate r > 0.65), even with subsequent feeding of the rotifers with microalgae from beads stored up to 1 year. Rotirich is an attractive alternative inert food compared with the mixture of microalgae and Spirulina, originally used to prefeed the rotifers. When the rotifers were prefed for 2 h with 30 microg/mL Rotirich, it was then not necessary to add Spirulina to the microalgal food from beads. The above findings are a substantial simplification of the testing procedure of the new short-chronic "culture/maintenance-free" Rotoxkit microbiotest.     

Complex evaluation of ecotoxicity and genotoxicity of antimicrobials oxytetracycline and flumequine used in aquaculture

Ecotoxicity and genotoxicity of widely used veterinary antimicrobials oxytetracycline and flumequine was studied with six model organisms (Vibrio fischeri, Pseudomonas putida, Pseudokirchneriella subcapitata, Lemna minor, Daphnia magna, Escherichia coli). Overall median effective concentration (EC50) values ranged from 0.22 mg/L to 86 mg/L. Pseudomonas putida was the most sensitive organism (EC50 values for 16-h growth inhibition were 0.22 and 0.82 mg/L for oxytetracycline and flumequine, respectively), followed by duckweed Lemna minor (7-d growth inhibition, EC50 2.1 and 3.0 mg/L) and green alga Pseudokirchneriella subcapitata (4-d growth inhibition, EC50 3.1 and 2.6 mg/L). The least sensitive organism was Daphnia magna (48-h immobilization, lowest-observed-effect concentration [LOEC] of oxytetracycline of 400 mg/L). Oxytetracycline showed limited genotoxicity (SOS-chromotest with Escherichia coli, minimal genotoxic concentration of 500 mg/L), and flumequine was genotoxic at 0.25 mg/L. Based on the reported measured concentrations (MECs) and predicted no-effect concentrations (PNECs), oxytetracycline may be concluded to be of ecotoxicological concern (calculated risk quotient = 8), whereas flumequine seems to represent lower risk.     

Ecotoxicological evaluation of wastewater treatment plant effluent discharges: a case study in Prato (Tuscany, Italy)

Textile wastewaters, which contain numerous chemicals such as dyes, surfactants, solvents, organic and inorganic salts, can cause severe pollution problems for the receiving freshwaters. The ecotoxicity of wastewaters in Prato, where there are about 14,000 textile and related factories, was investigated from 1996-1999 by means of bioassays. 147 samples of reclaimed wastewater were collected at the outlets of 4 centralized wastewater treatment plants. The acute and chronic toxicity of the effluents was measured with bioassays using three different target organisms: green algae (Pseudokirchneriella subcapitata), crustaceans (Daphnia magna) and bioluminescent bacteria (Vibrio fischeri). Toxicity was expressed as Effective Concentration 50 (EC50) and Toxic Units (TU). The results indicated that the effluents did not have significant acute toxicity: only 2.74% (EC50<100%, TU>1) of the 146 samples tested with crustaceans and 6.52% (EC50<50%, TU>2) of the 78 tested with bioluminescent bacteria showed toxic effects. With algae, slight chronic toxicity was found in 49.33% (mean EC50 value=86.56%, mean TU=1.16) of the 140 samples tested. The highest relative response was found with the algal assay using Pseudokirchneriella subcapitata: 49.33% of 140 samples showed chronic toxicity at 96 hours (EC50<100%).     

Identification of phototransformation products of prednisone by sunlight: toxicity of the drug and its derivatives on aquatic organisms

Solar simulator irradiation of an aqueous suspension of prednisone, a widely prescribed drug, produces seven photochemical derivatives. The compounds have been identified on the basis of their physical features. All the chemicals have been tested to evaluate their toxic effects on freshwater organisms from different trophic levels. The rotifer Brachionus calyciflorus and two crustaceans, the cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus, were used to perform acute toxicity tests. Chronic toxicity tests have been performed on the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Ceriodaphnia dubia. The results showed low acute and chronic toxicity of prednisone. Some of the photoproducts had high toxic effects on C. dubia.     

Acute and Chronic Effects of Sodium Tungstate on an Aquatic Invertebrate (Daphnia magna), Green Alga (Pseudokirchneriella subcapitata), and Zebrafish (Danio rerio)

Although aquatic toxicity data exists for tungstate substances, insufficient data of high quality and relevancy are available for conducting an adequate risk assessment. Therefore, a series of acute and chronic toxicity tests with sodium tungstate (Na(2)WO(4)) were conducted on an aquatic invertebrate (Daphnia magna), green alga (Pseudokirchneriella subcapitata), and zebrafish (Danio rerio). Collectively, the data from these studies suggest that sodium tungstate exhibits a relatively low toxicity to these taxa under these test conditions. All studies were conducted in the same laboratory under good laboratory practice standards using Organisation for Economic Co-operation and Development guidelines with the same stock of test material and the same analytical methods. All results are reported as mg W/L. The following toxicity values were based on mean measured concentrations. For D. magna, the 21 day test no-observable effect concentration (NOEC) was 25.9?mg?W/L, and the 48-h median effective concentration (EC(50)) from the acute test was >95.5?mg?W/L (the highest concentration tested). The P. subcapitata test yielded an ErC(50) of 31?mg?W/L. A 38-day test with zebrafish resulted in an NOEC ≥5.74?mg?W/L with no effects at any concentration. The 96-h LC(50) from the acute test with zebrafish was >106?mg?W/L. The results of the current acute study for daphnids and fish are consistent with published literature, whereas the algae results are different from previously reported values. Transformation/dissolution (T/D) studies, which were conducted according to United Nations Globally Harmonized System of Classification and Labelling of Chemicals protocol, confirmed that the WO (4) (-2) anion accounted for most of the tungsten in solution. For classification purposes, the algae ecotoxity reference value was then compared with T/D data and would not classify Na(2)WO(4) as an aquatic toxicant under the European Union Classification, Labelling and Packaging scheme.     

Studies and evaluation of the potential toxicity of decabromodiphenyl ethane to five aquatic and sediment organisms

The potential toxicity of decabromodiphenyl ethane (DBDP-Ethane) was explored in 5 types of organisms residing in the water column and/or sediment, e.g. Oncorhynchus mykiss, Pseudokirchneriella subcapitata, Daphnia magna, Chironmus riparius, and Lumbriculus variegates. Fish, algae or Daphnia were unaffected by acute exposures to water accommodated fractions of 110mg DBDP-Ethane/L. Chronic exposure to DBDP-Ethane at the highest dose tested, 5000mg/kg dry sediment, did not affect midge mean development times, emergence or development rates or oligochaete survival, reproduction or dry weight. The chronic EC50, LOEC and NOEC were ≥5000mg/kg in the two sediment species. Applying an assessment factor of 50, the unbounded predicted no effect concentration (PNEC(sediment)) was 100mg/kg dry sediment. The calculated PNEC indicates DBDPE-Ethane presents little risk to sediment organisms. These results add to DBDP-Ethane's existing database in the terrestrial compartment and mammals.     

Bioavailability models for predicting acute and chronic toxicity of zinc to algae, daphnids, and fish in natural surface waters

Bioavailability models predicting acute and/or chronic zinc toxicity to a green alga (Pseudokirchneriella subcapitata), a crustacean (Daphnia magna), and a fish (Oncorhynchus mykiss) were evaluated in a series of experiments with spiked natural surface waters. The eight selected freshwater samples had varying levels of bioavailability modifying parameters: pH (5.7-8.4), dissolved organic carbon (DOC, 2.48-22.9 mg/L), Ca (1.5-80 mg/L), Mg (0.79-18 mg/L), and Na (3.8-120 mg/L). In those waters, chronic zinc toxicity (expressed as 10% effective concentrations [EC10]) varied up to 20-fold for the alga (72-h EC10 from 27.3 to 563 microg Zn/L), and approximately sixfold for the crustacean (21-d EC10 from 59.2 to 387 microg Zn/L), and fivefold for the fish (30-d LC10, lethal concentration for 10% of the organisms, from 185 to 902 microg Zn/L). For P. subcapitata a refined bioavailability model was developed by linking an empirical equation, which predicts toxicity expressed as free Zn2+ activity as a function of pH, to the geochemical speciation model WHAM/Model V. This model and previously developed acute and/or chronic biotic ligand models for D. magna and 0. mykiss generally predicted most effect concentrations by an error of less than a factor of two. In waters with pH > 8, however, chronic toxicity to D. magna was underestimated by a factor 3 to 4. Based on the results of this validation exercise and earlier research, we determined applicability ranges for pH (6-8) and Ca (5-160 mg/L) in which all three developed models are valid. Within these ranges, all three models may be considered useful tools for taking into account bioavailability in regulatory assessments of zinc.     

Aquatic toxicity evaluation of para-methylstyrene

The aquatic toxicity of para-methylstyrene was evaluated in acute toxicity studies using fathead minnows (Pimephales promelas), daphnids (Daphnia magna), and freshwater green algae (Selenastrum capricornutum). Static tests were performed in sealed containers with no headspace to minimize loss of this volatile compound to the atmosphere. Concentrations of para-methylstyrene in test solutions were analyzed by gas chromatography equipped with a purge and trap module and flame ionization detection. Test results are based on mean, measured concentrations. para-Methylstyrene was moderately toxic to fathead minnows, daphnids, and green algae. The 96-h LC(50) and NOEC for fathead minnows were 5.2 and 2.6 mg/L, respectively. The 48-h EC(50) and NOEC for daphnids were 1.3 and 0.81 mg/L, respectively. The 72-h EC(50) and NOEC for green algae were 2.3 and 0.53 mg/L, respectively; these effects were algistatic rather than algicidal. para-Methylstyrene's potential impact on aquatic ecosystems is significantly mitigated by its volatility, an important fate process.     

Aquatic ecotoxicity of the selective serotonin reuptake inhibitor sertraline hydrochloride in a battery of freshwater test species

Sertraline hydrochloride is a selective serotonin reuptake inhibitor (SSRI) widely prescribed to patients suffering from psychiatric disorders. Pharmaceutical products such as sertraline have been identified in environmental waters. This study describes the evaluation of sertraline using a battery of freshwater species representing four trophic levels. The species most sensitive to sertraline were Daphnia magna 21 d reproduction test, Pseudokirchneriella subcapitata 72 h growth inhibition, and Oncorhynchus mykiss 96 h mortality, with the Microtox assay being the least sensitive assay. The D. magna 21 d reproduction test was approximately two orders of magnitude more sensitive than the other bioassays. These results show the advantages of having a tiered approach within a test battery. The presented results indicate that sertraline hydrochloride adversely affects aquatic organisms at levels several orders of magnitude higher than that reported in municipal effluent concentrations, however adverse effects may result from lower concentration exposures, further research into chronic toxicity is therefore advocated.     

Development and application of whole-sediment toxicity test using immobilized freshwater microalgae Pseudokirchneriella subcapitata

A method for a whole-sediment toxicity test using alginate immobilized microalgae Pseudokirchneriella subcapitata was developed using spiked sediments and applied to contaminated field sediment samples. For method development, a growth inhibition test (72 h) with algal beads was conducted for the sediments spiked with Cu or diuron. The method was validated by determining dose-response relationships for Cu and diuron in both fine-grained and coarse-grained sediments. The results of a spiked sediment toxicity test suggested that sediment particle size distribution (clay content) had a significant effect on the growth of P. subcapitata. The developed method using immobilized microalgae P. subcapitata beads was applied successfully in the toxicity test and toxicity identification evaluation (TIE) for the four field sediment samples. After a series of extractions with 0.01 M CaCl(2) solution, acetone, and dichloromethane, the extracted sediment, which was shown to be nontoxic to algae, was used as the control and diluent for the same sediment in the whole-sediment toxicity test. The results showed that all investigated field sediment samples were found to be toxic to the immobilized algae P. subcapitata, with their median effective concentration (EC50) values ranging from 41.4 to 79.0% after 72 h exposure. In the whole sediment TIE, growth of P. subcapitata was improved to varying degrees after adding zeolite, resin, or activated charcoal, suggesting different contributions to toxicity from ammonia, metals, and organic contaminants in the tested sediments.     

Effects of dissolved organic carbon concentration and source, pH, and water hardness on chronic toxicity of copper to Daphnia magna

The effects of pH (5.3-8.7), water hardness (CaCO3 at 25-500 mg/L), dissolved organic carbon (DOC) concentration (1.6-18.4 mg/L), and DOC source on the chronic toxicity of copper to Daphnia magna were investigated by using a multifactorial, central composite test design. Natural dissolved organic matter (DOM) was collected at three sites in Belgium and The Netherlands by using reverse osmosis. For a total number of 35 toxicity tests performed, 21-d no-observed-effect concentrations (NOECs) of copper based on reproduction ranged from 29.4 to 228 microg/L and 21-d concentrations of copper causing 50% reduction of reproduction (EC50s) ranged from 41.5 to 316 microg/L. Statistical analysis revealed that DOC concentration and pH had a significant effect on copper toxicity but hardness (at the levels tested) did not. In general, an increase in pH or DOC resulted in a linear increase of 21-d NOEC and EC50 values. All DOMs (originating from three different sources) reduced copper toxicity to the same extent. Multiple linear regression analysis on the results of all 35 toxicity tests revealed that DOC concentration is the most important factor for chronic toxicity of copper to D. magna, explaining about 60% of the observed variability, whereas pH only explained about 15% of the observed variability. Regression models were developed (with DOC and pH as parameters) that were capable of predicting NOECs and EC50s within a factor of 1.9 from observed NOEC and EC50 values obtained with eight natural surface waters spiked with copper. Until future research further elucidates the mechanisms underpinning the observed bioavailability relations, these empirical regression models can become a first simple tool for regulatory applications.     

Assessment of the toxicity of triasulfuron and its photoproducts using aquatic organisms

The toxicological effects of the sulfonylurea herbicide triasulfuron and its photoproducts were assessed on four aquatic organisms. Toxicity varied with tested organism and with triasulfuron irradiation time. Triasulfuron and its photoproducts had no significant effects on the crustacean (Cladocera) Daphnia magna (causing 50% effective concentration [EC50] [48 h] = 49 +/- 1 mg/L) and the marine bacteria Vibrio fischeri (EC50 [30 min] > 100 mg/L). In contrast, primary producers (the duckweed Lemna minor, the microalgae Pseudokirchneriella subcapitata, and Chlorella vulgaris) were very sensitive to triasulfuron (EC50s < 11 microg/L). For these organisms, triasulfuron photoproducts were less toxic than the parent compound but the residual toxicity observed still represented a potential environmental hazard.     

Toxicity of fluoroquinolone antibiotics to aquatic organisms

Toxicity tests were performed with seven fluoroquinolone antibiotics, ciprofloxacin, lomefloxacin, ofloxacin, levofloxacin, clinafloxacin, enrofloxacin, and flumequine, on five aquatic organisms. Overall toxicity values ranged from 7.9 to 23,000 microg/L. The cyanobacterium Microcystis aeruginosa was the most sensitive organism (5-d growth and reproduction, effective concentrations [EC50s] ranging from 7.9 to 1,960 microg/L and a median of 49 microg/L), followed by duckweed (Lemna minor, 7-d reproduction, EC50 values ranged from 53 to 2,470 microg/L with a median of 106 microg/L) and the green alga Pseudokirchneriella subcapitata (3-d growth and reproduction, EC50 values ranged from 1,100 to 22,700 microg/L with a median 7,400 microg/L). Results from tests with the crustacean Daphnia magna (48-h survival) and fathead minnow (Pimephales promelas, 7-d early life stage survival and growth) showed limited toxicity with no-observed-effect concentrations at or near 10 mg/L. Fish dry weights obtained in the ciprofloxacin, levofloxacin, and ofloxacin treatments (10 mg/L) were significantly higher than in control fish. The hazard of adverse effects occurring to the tested organisms in the environment was quantified by using hazard quotients. An estimated environmental concentration of 1 microg/L was chosen based on measured environmental concentrations previously reported in surface water; at this level, only M. aeruginosa may be at risk in surface water. However, the selective toxicity of these compounds may have implications for aquatic community structure.     

Critical evaluation and further development of methods for testing ecotoxicity at multiple pH using Daphnia magna and Pseudokirchneriella subcapitata

To meet the requirements of risk assessment legislature regarding the ecotoxicity of ionizing compounds, the present study attempts to establish easy, robust methods for testing ecotoxicity at various pH levels. An overview is given of the buffering methods found in the literature. This is supplemented by a series of experiments where toxicity and ability to stabilize pH of seven common buffering compounds was tested on Daphnia magna and Pseudokirchneriella subcapitata. We consider a buffer applicable at a given concentration if the pH drift is below 0.2 pH units, and if there are no toxic effects. Twenty-four- and 48-h acute toxicity tests with D. magna were carried on a series of organic buffers with pH monitoring. Based on the experimental results it is possible to give recommendations for buffer concentrations for use in toxicity testing with D. magna at pH levels in the range of pH 6.0-7.8 for 48 h exposure, and pH 6.0-9.5 for 24 h exposure. Forty-eight- and 72-h growth inhibition tests with P. subcapitata were carried out, and recommendations for buffer concentrations at pH 7.5 and 8.0 are made for both 48 and 72 h of exposure.     

Environmental assessment of Norwegian priority pharmaceuticals based on the EMEA guideline

An environmental risk assessment of eleven pharmaceuticals according to the guideline recommended by the European Medicines Evaluation Agency (EMEA) was performed. Cefuroxime, ciprofloxacin, cyclophosphamide, diclofenac, ethinylestradiol, ibuprofen, metoprolol, paracetamol, sulfamethoxazole, tetracycline and trimethoprim were selected for assessment by the Norwegian Pollution Control Authority. Predicted environmental concentrations (PECs) were calculated according to both the EMEA guideline and a conventional model for comparison and ranged from 0.0002 to 45 microg/L. Available acute and chronic toxicity data were collected from the literature, although no data were available for cyclophosphamide. Toxicity tests showed cyclophosphamide to have relatively low acute toxicity with an EC50 for Pseudokirchneriella subcapitata >100 mg/L and a Daphnia magna reproduction NOEC of 56 mg/L. These and the literature data were used to derive predicted no effect concentrations (PNEC). Risk quotients (PEC/PNEC) were then calculated for all 11 pharmaceutical compounds. Risk quotients greater than 1 were obtained for ciprofloxacin, diclofenac, ethinylestradiol, sulfamethoxazole and tetracycline according to the EMEA guideline. Measured environmental concentrations (MECs) confirmed that the release of ciprofloxacin from wastewater treatment works may potentially be of environmental concern in Norway.     

An algal toxicity database of organic toxicants derived by a closed-system technique

The current study presents the toxicity data of 90 organic compounds with various modes of actions to Pseudokirchneriella subcapitata. The assessment was conducted using a closed-system technique, and a biomass-type end point based on the cell density was employed. The above toxicity data were compared with test results from ciliate (Tetrahymena pyriformis), water flea (Daphnia magna), fish (Pimephales promelas), and luminescent bacteria (Photobacterium phosphoreum). Satisfactory correlation relationships between toxicity data from algae and other aquatic organisms were found (r2 = 0.66-0.82). Pseudokirchneriella subcapitata revealed considerably higher sensitivity to organic toxicants compared with other organisms. Benzenes, aldehydes, and alkanes also were highly selective to the test alga. In addition, the results show that conventional algal batch tests tend to underestimate the toxicity of organic compounds, except in the case of 4-chlorophenol. Toxicity observed from the closed-system test is approximately 2- to 380-fold higher than that estimated by conventional batch tests. Such a phenomenon can be found in nearly all organic compounds, regardless of the chemical's Henry's law constant. In the risk assessment of chemicals, following the European Union's practice, approximately 30% (7 of 23) of the cases may result in a more strict classification when the batch test is replaced by the closed-system test. More effort therefore is needed to revise the algal toxicity database using the closed-system test method.     

Development of methods for evaluating toxicity to freshwater ecosystems

This article presents a summary of a collaborative research program involving five European research groups, that was partly funded by the European Commission under its Environmental Research Program. The objective of the program was to develop aquatic toxicity tests that could be used to obtain data for inclusion at Level 2 of the Risk Evaluation Scheme for the Notification of Substances as required by the 7th Amendment to EC Directive 79/831/EEC. Currently only a very limited number of test methods have been described that can be used for this purpose and these are based on an even smaller number of test species. Tests based upon algae (Chlamydomonas reinhardi, Scenedesmus subspicatus, and Euglena gracilis), protozoa (Tetrahymena pyriformis), rotifera (Brachionus calyciflorus), crustacea (Gammarus pulex), and diptera (Chironomus riparius) were developed. The tests encompassed a range of end points and were evaluated against four reference chemicals: lindane, 3, 4-dichloroaniline (DCA), atrazine, and copper. The capacity of the tests to identify concentrations that are chronically toxic in the field was addressed by comparing the effects threshold concentrations determined in the laboratory tests with those determined for similar and/or related species and end points in stream and pond mesocosm studies. The lowest no-observed-effect concentrations (NOEC), EC(x), or LC(x) values obtained for lindane, atrazine, and copper were comparable with the lowest values obtained in the mesocosms. The lowest chronic NOEC determined for DCA using the laboratory tests was approximately 200 times higher than the lowest NOEC in the mesocosms.     

Cross-phylum comparison of a chronic biotic ligand model to predict chronic toxicity of copper to a freshwater rotifer, Brachionus calyciflorus (Pallas)

Short chronic 48-h toxicity tests with the freshwater rotifer Brachionus calyciflorus (Pallas) were conducted to assess the modifying effects of pH and natural dissolved organic carbon (DOC) concentration on reproductive toxicity of Cu. Toxicity tests were carried out in four test waters according to a 2 x 2 design, in which pH (6 and 7.8) and DOC (5 and 15 mg C/L) were the test variables. Concentrations of dissolved Cu with no observed effect at 48 h (NOEC) varied 12-fold between 8.2 and 103 microg/L. Higher DOC and higher pH resulted in a reduction of toxicity, which is in line with the concepts of the biotic ligand model (BLM). A chronic Cu-BLM, originally developed for the cladoceran Daphnia magna, was calibrated to the rotifer dataset and was found to be able to predict all rotifer NOECs with an error factor of less than 1.6. This finding may be of great interest for risk assessment and the establishment of water quality criteria, as it suggests that chronic Cu-BLMs are comparable across phyla (i.e., arthropoda to rotifera).