Comparison of bioassays by testing whole soil and their water extract from contaminated sites

The harmful effects of contaminants on the ecosystems and humans are characterised by their environmental toxicity. The aim of this study was to assess applicability and reliability of several environmental toxicity tests, comparing the result of the whole soils and their water extracts. In the study real contaminated soils were applied from three different inherited contaminated sites of organic and inorganic pollutants. The measured endpoints were the bioluminescence inhibition of Vibrio fischeri (bacterium), the dehydrogenase activity inhibition of Azomonas agilis (bacterium), the reproduction inhibition of Tetrahymena pyriformis (protozoon), and Panagrellus redivivus (nematode), the mortality of Folsomia candida (springtail), the root and shoot elongation inhibition of Sinapis alba (plant: white mustard) and the nitrification activity inhibition of an uncontaminated garden soil used as "test organism". Besides the standardised or widely used methods some new, direct contact ecotoxicity tests have been developed and introduced, which are useful for characterisation of the risk of contaminated soils due to their interactive nature. Soil no. 1 derived from a site polluted with transformer oil (PCB-free); Soil no. 2 originated from a site contaminated with mazout; Soil no. 3 was contaminated with toxic metals (Zn, Cd, Cu, Pb, As). In most cases, the interactive ecotoxicity tests indicated more harmful effect of the contaminated soil than the tests using soil extracts. The direct contact environmental toxicity tests are able to meet the requirements of environmental toxicology: reliability, sensibility, reproducibility, rapidity and low cost.     

Relative sensitivity of one freshwater and two marine acute toxicity tests as determined by testing 30 offshore E & P chemicals

Acute toxicity of 30 offshore E & P (Exploration and Production) chemicals was measured using the three standard test organisms Daphnia magna (freshwater cladoceran), Acartia tonsa (marine copepod) and Skeletonema costatum (marine diatom alga). Test chemicals included 20 water-soluble and 10 (partially) non-soluble products. For 22 out of the 30 chemicals, the difference in sensitivity between the three tests varied within one order of magnitude. A very good correlation was found between the two marine tests (r = 0.96, P < 0.01, n = 30), and a correlation coefficient of r = 0.78 (P < 0.01, n = 30) was found between D. magna and both A. tonsa and S. costatum, individually. When the comparison of D. magna and A. tonsa sensitivity was based only on the water-soluble chemicals, a significantly higher correlation was obtained (r = 0.84, n = 20), indicating that the sample preparation method used for the (partially) non-soluble chemicals (the water accommodated fraction (WAF) method) induces additional variation between tests performed with different test media. (Partially) non-soluble chemicals are characterised by phase separation or precipitation at the concentrations used for testing. In a WAF-based test, each test concentration/exposure level is prepared separately, and following mixing and separation, only the water phase is used for testing. Toxicity is related to the amount of substance originally added to the mixing vessels. For 25 of the 30 chemicals, D. magna was found to be less sensitive than the marine copepod by a factor >2. The generally higher sensitivity of the marine toxicity tests compared to the Daphnia test emphasise the importance of using marine data for environmental hazard classification as well as for environmental risk assessment purposes.     

A multispecies study to assess the toxic and genotoxic effect of pharmaceuticals: furosemide and its photoproduct

Pharmaceutical products for humans and animals, as well as their related metabolites end up in the aquatic environment after use. Recent investigations show that concentrations of pharmaceuticals are detectable in the order of ng/l-mug/l in municipal wastewater, groundwater and also drinking water. Little is known about the effects, and the hazard of long-term exposure to low concentrations of pharmaceuticals for non-target aquatic organisms. This study was designed to assess the ecotoxicity of furosemide, a potent diuretic agent, and its photoproduct in the aquatic environment. Bioassays were performed on bacteria, algae, rotifers and microcrustaceans to assess acute and chronic toxicity, while the SOS Chromotest and the Ames test were utilized to detect the genotoxic potential of the investigated compounds. A first approach to risk characterization was to calculate the environmental impact of furosemide by measured environmental concentration and predicted no effect concentration ratio (MEC/PNEC). To do so we used occurrence data reported in the literature and our toxicity results. The results showed that acute toxicity was in the order of mg/l for the crustaceans and absent for bacteria and rotifers. Chronic exposure to these compounds caused inhibition of growth population on the consumers, while the algae did not seem to be affected. A mutagenic potential was found for the photoproduct compared to the parental compound suggesting that byproducts ought to be considered in the environmental assessment of drugs. The risk calculated for furosemide suggested its harmlessness on the aquatic compartment.     

Assessment of heavy metals phytotoxicity using seed germination and root elongation tests: a comparison of two growth substrates

Seed germination and root elongation test is used to evaluate hazardous waste sites and to assess toxicity of organic and inorganic compounds. Paper substrate, especially circular filter paper placed inside a Petri dish has long been used for this test. Same reports indicate that filter paper might interfere with the toxicity of inorganic substances, especially metal cations. This study evaluate toxicity of Cd, Pb, Ni, Cu on lettuce, broccoli, tomato and radish seed using two bed material: agar and filter paper. The results show that percent germination is not affected by substrates; vice versa, as for root elongation, the test in agar showed to be more sensible than that the one on filter paper. The radical growth inhibition depends on the metal, on the tested concentration and on the species; among the tested metals, cadmium was the one determining the highest toxic effects on different species and lettuce was the plant that suffered more. From the comparison, it is clearly evident the greater sensibility of the test in agar; on the other hand, the lower sensibility of the test on the filter paper might be caused by the partial and not homogeneous exposition of the root to metal cations.     

Aquatic toxicity of two Corexit dispersants

The oil spill dispersants, Corexit 9500 and Corexit 9527 have low to moderate toxicity to most aquatic species in laboratory tests. Toxicity estimates are significantly affected by test variables such as species, lifestage, exposure duration, and temperature. Aquatic toxicity data generated from spiked, declining exposures (107 min half-life) are more reflective of actual dispersant use conditions. Decisions to use oil spill response chemicals should not be based solely on aquatic toxicity. Factors to consider include product effectiveness, toxicity of dispersed oil, species/habitats requiring priority protection, and recovery potential of sensitive habitats and populations. An environmental risk assessment approach is recommended where dispersant toxicity data generated under environmentally relevant exposures are compared to estimated environmental concentrations of dispersants.     

Soil ecotoxicity assessment using cadmium sensitive plants

Four crop plant species (sweet corn, Zea may; wheat, Triticum aestivum; cucumber, Cucumis sativus; and sorghum, Sorghum bicolor) were tested to assess an ecotoxicity in cadmium-amended soils. The measurement endpoints used were seed germination and seedling growth (shoot and root). The presence of cadmium decreased the seedling growth. The medium effective concentration values (EC50) for shoot or root growth were calculated by the Trimmed Spearman-Karber method. Due to the greater accumulation of Cd to the roots, root growth was a more sensitive endpoint than shoot growth. Bioavailability and transport of Cd within plant were related to concentration and species. The ratio of bioaccumulation factor (BAF) in the shoots to the roots indicated high immobilization of Cd in the roots. Seed germination was insensitive to Cd toxicity, and is not recommended for a suitable assay. Among the test plants and test endpoints, root growth of sorghum and cucumber appears to be a good protocol to assess ecotoxicity of soils contaminated by Cd.     

Effects of light and temperature fluctuations on the growth of Myriophyllum spicatum in toxicity tests—a model-based analysis

Laboratory toxicity tests are a key component of the aquatic risk assessments of chemicals. Toxicity tests with Myriophyllum spicatum are conducted based on working procedures that provide detailed instructions on how to set up the experiment, e.g., which experimental design is necessary to get reproducible and thus comparable results. Approved working procedures are established by analyzing numerous toxicity tests to find a compromise between practical reasons (e.g., acceptable ranges of ambient conditions as they cannot be kept completely constant) and the ability for detecting growth alterations. However, the benefit of each step of a working procedure, e.g., the random repositioning of test beakers, cannot be exactly quantified, although this information might be useful to evaluate working procedures. In this paper, a growth model of M. spicatum was developed and used to assess the impact of temperature and light fluctuations within the standardized setup. It was analyzed how important it is to randomly reassign the location of each plant during laboratory tests to keep differences between the relative growth rates of individual plants low. Moreover, two examples are presented on how modeling can give insight into toxicity testing. Results showed that randomly repositioning of individual plants during an experiment can compensate for fluctuations of light and temperature. A method is presented on how models can be used to improve experimental designs and to quantify their benefits by predicting growth responses.     

Use of freshwater plants for phytotoxicity testing: a review

Phytotoxicity data for aquatic plants have served a relatively minor role in regulatory decisions concerning the environmental hazard of most potential contaminants. A variety of phytotoxicity tests have been conducted with freshwater green algae, duckweed, blue-green algae, diatoms and rooted macrophytes (whole plants and seeds). Several test methods have been standardized for microalgae which are used primarily with chemicals, effluents, contaminated sediment elutriates and hazardous waste leachates. Current scientific understanding concerning the phytotoxic effects of these contaminants is based mostly on results for a few green algae. The greatest limitation of these results is their uncertain environmental relevance due to the large interspecific variation in response of standard algal test species and the unrealistic experimental test conditions. Results of the few field validation toxicity tests conducted to resolve this uncertainty have been chemical-specific and unpredictable. Aquatic vascular plants have been used less frequently than algae as test species. Duckweeds have been used more often than rooted submersed species but the uncertain nature of their sensitivities relative to animal and other plant species has limited their use. Regulatory interest in wetland protection, contaminated sediment evaluations and sediment quality criteria development will result in increased use of whole rooted plants and their seeds as test species. Overall, regardless of the test species, if phytotoxicity data are to be more available and effective in the hazard assessment process, additional information concerning species sensitivity, and environmental relevance of the results will be needed.     

Environmental risk assessment for trisodium [S,S]-ethylene diamine disuccinate, a biodegradable chelator used in detergent applications

Environmental safety data are presented for [S,S]-Ethylene Diamine Disuccinate ([S,S]EDDS), a new, biodegradable, strong transition metal chelator. An environmental risk assessment for its use in detergent applications, which takes into account the chelating properties of [S,S]-EDDS, is proposed.

A property of [S,S]-EDDS that distinguishes it from other strong transition metal chelators is its, “ready” and transparent (no recalcitrant metabolites) biodegradation profile. Because its sorption to activated sludge solids is low ( Kp of 40 1/kg), removal of [S,S]EDDS during sewage treatment, which is greater than 96% as determined by the Continuous Activated Sludge test , is mainly ascribed to biodegradation. At projected use volumes in detergent applications [S,S] - EDDS predicted steady-state concentration in rivers leaving the mixing zone will be below 5 pg/I due to rapid biodegradation. [S,S]-EDDS exhibits low toxicity to fish and Daphnia ( both EC₅₀s> 1000 mg/l). By contrast, due to limitation of the algal test for chelators apparent toxicity was observed (EC₅₀ = 0.290 mg/l, NOEC - No observable Effect Concentration = 0.125 mg/l). Schowanek et al. [1] demonstrated that this is not toxicity sensu stricto but a chelation effect of trace metals in the test medium and of resulting essential nutrients limitation. This requires specific attention when the results of algal toxicity are to be extrapolated to a field situation to perform realistic risk assessment. Metal speciation calculations, using MINEQL+, show that at the predicted environmental concentrations of [S,S] - EDDS (1–5 μg/l), such a chelation effect would be insignificant. These calculations allow to estimate the NOEC for chelation effects in the field to be in the range of 0.250-0.500 mg/l, depending on the background water chemistry. These values are well above the laboratory NOEC.

An environmental risk assessment was performed using the EUSES (1.0) program. EUSES is currently the EU recommended tool for conducting risk assessments (TGD 1995). It was applied to estimate the river water and soil concentrations from production, formulation and private use life stages. The estimated PEC/PNEC ratio in all relevant environmental compartments is smaller than 1, indicating “no immediate concern” at the anticipated usage level.     

Validation of germination rate and root elongation as indicator to assess phytotoxicity with Cucumis sativus

Germination rate and root elongation, as a rapid phytotoxicity test method, possess several advantages, such as sensitivity, simplicity, low cost and suitability for unstable chemicals or samples. These advantages made them suitable for developing a large-scale phytotoxicity database and especially applicable for developing quantitative structure–activity relationship (QSAR) to study mechanisms of phytotoxicity. In this paper, the comparative inhibition of germination rate and root elongation of Cucumis sativus by selected halogen-substituted phenols and anilines were determined. The suitability of germination rate and root elongation as phytotoxicity endpoints was evaluated. Excellent reproducibility and stability of germination rate and root elongation in the control test, relatively greater sensitivity and similar dose–response relations for all tested compounds were observed. These results together with those of a 2-day test were used to demonstrate the suitability of this phytotoxicity test method. A QSAR was developed for the phytotoxicity mode of action of the tested compounds to C. sativus seeds. Models that combined the logarithm of 1-octanol/water partition coefficient (log K_ow) and the energy of the lowest unoccupied molecular orbital (E_lumo) were developed for both germination rate inhibition and root elongation inhibition. The results of these studies indicate that phytotoxicity of substituted phenols and anilines to C. sativus seeds could be explained by a polar narcosis mechanism. This paper will promote the application of germination rate and root elongation method and the development of large-scale phytotoxicity database, which will provide the fundamental data for QSAR and ecological risk assessment of organic pollutants.     

AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology

Introduction and background

Primary producers play critical structural and functional roles in aquatic ecosystems; therefore, it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals. The standard required macrophyte test species is the floating (non-sediment-rooted) duckweed Lemna spp. This macrophyte species might not be representative of all floating, rooted, emergent, and submerged macrophyte species because of differences in the duration and mode of exposure; sensitivity to the specific toxic mode of action of the chemical; and species-specific traits (e.g., duckweed's very short generation time).

Discussion and perspectives

These topics were addressed during the workshop entitled “Aquatic Macrophyte Risk Assessment for Pesticides” (AMRAP) where a risk assessment scheme for aquatic macrophytes was proposed. Four working groups evolved from this workshop and were charged with the task of developing Tier 1 and higher-tier aquatic macrophyte risk assessment procedures. Subsequently, a SETAC Advisory Group, the Macrophyte Ecotoxicology Group (AMEG) was formed as an umbrella organization for various macrophyte working groups. The purpose of AMEG is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field, including prospective as well as retrospective risk assessments for chemicals. As AMEG expands, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services.     

Is an assessment factor of 10 appropriate to account for the variation in chemical toxicity to freshwater ectotherms under different thermal conditions?

Ecotoxicity tests are often conducted following standard methods, and thus carried out at a fixed water temperature under controlled laboratory conditions. Yet, toxicity of a chemical contaminant may vary in a temperature-dependent manner, depending on the physiology of the test organism and physicochemical properties of the chemical. Although an assessment factor of 10 (AF10) is commonly adopted to account for variability in toxicity data related to temperature in the development of water quality guidelines and/or ecological risk assessment, no one has ever rigorously assessed the appropriateness of AF10 to account for potential variation in temperature-dependent chemical toxicity to aquatic organisms. This study, therefore, aims to address this issue through a meta-analysis by comparing median lethal concentration data for nine chemicals (cadmium, copper, nickel, lead, silver, zinc, arsenic, selenium and DDT) on a range of freshwater ectothermic animal species at different temperatures, and to assess whether AF10 is under- or over-protective for tropical and temperate freshwater ecosystems. Our results reveal varying extents of interaction between temperature and different chemicals on organisms and the complexity of these interactions. Applying AF10 sufficiently protects 90 % of the animal species tested over a range of temperatures for cadmium, copper, nickel, silver, zinc and DDT in the tropics, but it is insufficient to adequately encompass a larger temperature variation for most studied chemicals in temperate regions. It is therefore important to set specific AFs for different climatic zones in order to achieve the desired level of ecosystem protection.     

Statistical uncertainty in hazardous terrestrial concentrations estimated with aquatic ecotoxicity data

Since chemicals’ ecotoxic effects depend for most soil species on the dissolved concentration in pore water, the equilibrium partitioning (EP) method is generally used to estimate hazardous concentrations (HC50) in the soil from aquatic toxicity tests. The present study analyzes the statistical uncertainty in terrestrial HC50s derived by the EP-method. For 47 organic chemicals, we compared freshwater HC50s derived from standard aquatic ecotoxicity tests with porewater HC50s derived from terrestrial ecotoxicity tests. Statistical uncertainty in the HC50s due to limited species sample size and in organic carbon–water partitioning coefficients due to predictive error was treated with probability distributions propagated by Monte Carlo simulations. Particularly for specifically acting chemicals, it is very important to base the HC50 on a representative sample of species, composed of both target and non-target species. For most chemical groups, porewater HC50 values were approximately a factor of 3 higher than freshwater HC50 values. The ratio of the porewater HC50/freshwater HC50 was typically 3.0 for narcotic chemicals (2.8 for nonpolar and 3.4 for polar narcotics), 0.8 for reactive chemicals, 2.9 for neurotoxic chemicals (4.3 for AChE agents and 0.1 for the cyclodiene type), and 2.5 for herbicides–fungicides. However, the statistical uncertainty associated with this ratio was large (typically 2.3 orders of magnitude). For 81% of the organic chemicals studied, there was no statistical difference between the hazardous concentration of aquatic and terrestrial species. We conclude that possible systematic deviations between the HC50s of aquatic and terrestrial species appear to be less prominent than the overall statistical uncertainty.     

Effects of ethylene glycol ethers on the reproduction of Ceriodaphnia dubia

Seven-day static renewal tests with Ceriodaphnia dubia were used to document the chronic toxicity of ethylene glycol ethers and acetates to this invertebrate. The 7-d EC10 (effective concentrations inducing an inhibition of 10% of the reproduction of the tested organisms) values ranged from 0.06 to 1025 mg/l. While a survey of the literature showed that the acute toxicity of these chemicals appeared negligible, our results clearly revealed the potential chronic effects of some of them to this organism occupying an important trophic level in the aquatic ecosystems. The usefulness of this kind of test to better estimate the adverse effects of glycol ethers was stressed.     

Evaluation of the Hormosira banksii (Turner) desicaine germination and growth inhibition bioassay for use as a regulatory assay

A germination and growth inhibition bioassay using the marine macroalga, Hormosira banksii, was evaluated for use as a standard method for direct toxicity assessment testing in the Australasian region. H. banksii is the dominant macroalga and an important primary producer in temperate coastal zones of Australasia and is therefore relevant as a test organism. Moreover, it releases gametes readily under laboratory conditions, therefore allowing tests to be conducted year round. The test endpoints' variability and sensitivity was evaluated using ammonia, a common constituent of waste waters. The two test endpoints of germination and growth proved to be both reliable and repeatable bioassay endpoints. The coefficients of variation (CV's) for toxicity to germination and growth by ammonia ranged from 25% to 38% (n=18).     

Growth rate effects, responses of antioxidant enzymes and metabolic fate of the herbicide Propanil in the aquatic plant Lemna minor

Propanil (3,4-dichloropropionanilide) is a selective contact pesticide, recommended for post-emergence use in rice. This herbicide may end up in surface waters and present potential risk for aquatic vascular plants. Therefore, its toxicity was evaluated on Lemna minor L., an aquatic plant regularly used for toxicological studies, during time- and concentration-dependent exposure. Toxicity assessments were based on inhibition of growth of L. minor cultures after 24 days. The obtained results showed that the growth of Lemna was affected by the herbicide. The responses of the guaiacol peroxidase (G-POD) and glutathione S-transferase (GST) involved in the xenobiotic metabolism and antioxidative system were also investigated following Propanil exposure. Our results showed that Propanil has not induced enzymatic antioxidative defenses of L. minor. Both 3,4-dichloroaniline (3,4-DCA) and 3,4-dichloroacetanilide are the major metabolites in this plant. On the contrary, only 3,4-DCA was found in culture media after 4 days. Probably, the enzymatic hydrolysis by acyl acylamidase and the acetylation by acetyl-CoA are the major pathways for these transformation products, respectively. The results of this study showed that the selected aquatic plant has the potential to accumulate and metabolize rice herbicide, like Propanil. Based on these toxicity data this herbicide should impair the establishment of non-target aquatic plants.     

Toxicological evaluation for the hazard assessment of tire crumb for use in public playgrounds

Disposal of used tires has been a major problem in solid waste management. New uses will have to be found to consume recycled tire products. One such proposed use is as ground cover in playgrounds. However, concern has been expressed regarding exposure of children to hazardous chemicals and the environmental impact of such chemicals. We designed a comprehensive hazard assessment to evaluate and address potential human health and environmental concerns associated with the use of tire crumb in playgrounds. Human health concerns were addressed using conventional hazard analyses, mutagenicity assays, and aquatic toxicity tests of extracted tire crumb. Hazard to children appears to be minimal. Toxicity to all aquatic organisms (bacteria, invertebrates, fish, and green algae) was observed; however, this activity disappeared with aging of the tire crumb for three months in place in the playground. We conclude that the use of tire crumb in playgrounds results in minimal hazard to children and the receiving environment.     

Germination of Medicago sativa is inhibited by soluble compounds in cement dust

Deposition of cement dust on soils and plant surfaces is known to affect plant growth and the species composition of plant communities, but little is known about its effects (and those of its pH and constituents) on germination. Therefore, the aim of this study was to assess the toxicity of an aqueous cement extract, constituents of the extract and pH on the germination of seeds of a selected species, Medicago sativa. First, the effects of the extract were tested in assays with concentrations and exposure durations ranging from 0 to 1.0 g/mL and 4 to 96 h, respectively. At 0.8 g/mL, the extract strongly inhibited germination; a 4-h exposure reduced the germination rate, from 77?±?1.8 to 50?±?2.6 % (mean ± SE), while 8-h exposure completely inhibited it. Further, treatment at this concentration killed the non-germinating seeds, thus the inhibition was due to toxic effects. Neither the pH of the extract nor the concentration of its main soluble elements separately (K, Ca, S, Na, or Cr) caused the toxicity since germination rates were not significantly reduced when these variables were tested individually. However, a mixture of the elements in solution reduced germination rates, suggesting that they have adverse synergistic effects.     

The impact of pesticides toward parrotfeather when applied at the predicted environmental concentration

Pesticides pose a serious risk for aquatic macrophytes in the environment. They are also detrimental to the rooted macrophytes used in bioassays for assessment. Currently, no data is available for impact of pesticides toward parrotfeather when present at the predicted environmental concentration. The calculated expected environmental concentration was applied to the plants and the effect was compared. Eight of the 18 pesticides showed significantly different impact. All of the other tested pesticides induced a significant change in pigment content of parrotfeather. The RQ values for risk quotient had a value higher than 0.5, so need regulatory action for environment. This study may be the first to evaluate the predicted environmental concentrations reported by pesticide registration in Europe. Additional studies are required to test all pesticides within one group since the compounds tested may depict a wide toxicity level. Furthermore, the tests should include more than one macrophyte, e.g. one rooted and one non-rooted species, in order to provide a better understanding on pesticide toxicity.