Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.     

Toxicity of chlorophenols to Pseudokirchneriella subcapitata under air-tight test environment

A closed-system algal toxicity test with no headspace was applied to evaluate the toxicity of chlorophenols to Pseudokirchneriella subcapitata. The dissolved oxygen production and the growth rate based on cell density were the response endpoints. Phenol and seven chlorophenols were tested using the above test technique. Median effective concentrations (EC50) range from 0.004 to 25.93 mg/l (based on DO production) and 0.0134 to 20.90 mg/l (based on growth rate). No-observed-effect concentration (NOEC) is within the range of 0.001-8.19 mg/l. In general, growth rate is a more sensitive response endpoint than the oxygen production, except for the case of pentachlorophenol. However, the differences in sensitivity between the two parameters were marginal. Furthermore, quantitative structure-activity relationships (QSAR's) based on the n-octanol/water partition coefficient (log P) and the acid dissociation constant (pK(a)) values were established with R(2) ranged from 0.90 to 0.96. From literature data also based on P. subcapitata, the new test method is 1.65-108 times more sensitive than the conventional algal batch tests. A completely different relative-sensitivity relationship among various aquatic organisms was thus observed. The results of this study indicate that the toxicity data of volatile organic chemicals derived by conventional algal toxicity tests may severely underestimate the impact of these toxicants. Our results show that alga is very sensitive to chlorophenols compared to other aquatic organisms such as the luminescent bacteria (the Microtox test), Daphnia magna, and rainbow trout.     

Effects of planting system design on the toxicological sensitivity of Myriophyllum spicatum and Elodea canadensis to atrazine

The triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-trazine) was selected as a chemical stressor in an investigation of how toxicological responses of individually grown macrophytes reflect those of plants grown in more natural model populations and two-species communities. Phytotoxicity of the compound to Myriophyllumspicatum L. and Elodeacanadensis Michx. was assessed under semi-natural field conditions using 12000l outdoor microcosms. Exposure concentrations of 25, 50, 100, 250mugl(-1) plus controls (n=3) were evaluated, selected to fall within a range of concentrations known to produce a toxic response in the tested macrophytes, and effective concentrations required to cause a decrease in biomass endpoints by 10%, 25%, and 50% were estimated. The sensitivities of aquatic plants to atrazine did not differ substantially between planting systems, and few interactions between the effects of the planting method and atrazine effects on macrophyte biomass were detected using a two-way ANOVA. A lack of significant differences in biomass and relative growth rate measures between plants grown under the various test systems also indicated that interactions between and among species did not influence growth of plants in the model population and communities. Under these test conditions, the use of the "cone-tainer" method provided estimates of toxicity consistent with those from plants grown in assemblages, and potential interactions between plants were not found to modify the response of macrophytes to atrazine.     

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.     

Toxicity of uranium mine receiving waters to early life stage fathead minnows (Pimephales promelas) in the laboratory.

Elevated concentrations of arsenic, nickel, and molybdenum in aquatic systems around northern Saskatchewan uranium mines are an environmental concern. Early life stage fathead minnows were used to assess toxicity from several aquatic systems near the Key Lake and Rabbit Lake uranium operations. Hatching success of fish embryos exposed to waters receiving contaminants associated with uranium ore milling was reduced by 32-61% relative to controls. Mortality differed in two lakes receiving mill effluents because of opposing factors influencing metal toxicity (i.e. low pH and high hardness). In one mill receiving water (Fox Lake), larval mortality was 0%, whereas mortality was 85% in water collected from a downstream location (Unknown Lake). Fish embryos exposed to open-pit dewatering effluent receiving waters, or water from a flooded open pit (i.e. pit waters), hatched 26-39% earlier than those exposed to reference or control water. The combination of low water hardness and elevated nickel concentrations in pit waters contributed to the early hatching. Egg hatchability and hatching time were more sensitive indicators of toxicity than 'standard' endpoints, like larval mortality and growth. Current regulatory emphasis on single contaminants and standard toxicological endpoints should be re-evaluated in light of the complex interaction among confounding variables such as pH, hardness. conductivity, and multi-metal mixtures.     

Sources of variability in plant toxicity testing

Published literature is investigated regarding the response of plants to various substances to determine the sensitivity of agricultural plants versus other species, the similarity of effects seen at different taxonomic levels, sensitivity of plants growing outdoors versus in a greenhouse, and the sensitivity of different measurement endpoints. We find that agricultural species are not consistently more or less sensitive to the herbicides tested than non-crop species. Genus and family taxonomic groupings may show similar responses among species, but this similarity quickly decreases as the comparison progress between orders and classes. Results from field and greenhouse studies are less in agreement between studies than data from the other topics. Shoot length will be affected at concentrations lower than for other vegetative endpoints for most species tested for inorganic substances, but for organic substances root and shoot mass were more sensitive. Overall, there is no one species or endpoint that is consistently the most sensitive for all species or all chemicals in all soils, and differences in bioavailability among compounds may confound comparison of test results. Therefore, species sensitivity distributions, adjusted for bioavailability when possible, should be considered in order to better evaluate effects to non-target terrestrial plants.     

Full life-cycle toxicity assessment using rotifer resting egg production: implications for ecological risk assessment

The majority of standardized toxicity tests incorporate only a fraction of the test organism's life-cycle. However, in natural ecosystems, organisms may be exposed at various times during their life-cycle or throughout their life-cycle. Thus, ecotoxicological data from standardized toxicity tests is of limited ecological relevance. Existing standardized toxicity tests using the freshwater rotifer Brachionus calyciflorus utilize 24-h survival or 48-h asexual reproduction as endpoints, despite evidence that sexual reproduction is more sensitive. A 96-h B. calyciflorus resting egg toxicity test was developed and used to estimate the toxicity of pentachlorophenol (PCP) and copper. Results were compared to a variety of acute and sublethal endpoints for both toxicants. The B. calyciflorus 96-h resting egg production NOEC for PCP of 10 micrograms/l was 20 times lower than the 48-h asexual reproduction no observed effect concentration (NOEC) and 120 times lower than the 24-h acute lethal concentration 50%. The 96-h resting egg production NOEC for copper of 2.8 micrograms/l was 7 times lower than the 48-h asexual reproduction NOEC and nine times lower than the 24-h acute LC50. Resting egg production was a more sensitive indicator of toxicity than several other sublethal endpoints as well. These results indicate that partial life-cycle toxicity tests are not sufficiently sensitive to detect ecologically relevant adverse effects.     

Comparing growth development of <Emphasis Type="Italic">Myriophyllum</Emphasis> spp. in laboratory and field experiments for ecotoxicological testing

BACKGROUND, GOALS AND SCOPE: Risk assessment of herbicides and the evaluation of contaminated sediments based on algae and the macrophyte Lemna sp. alone may underestimate the potential hazard of certain compounds. Therefore, various test systems with Myriophyllum spp. have been developed recently to assess the phytotoxicity in surface waters and natural sediments. In the present study, experiments investigating the growth development of Myriophyllum spp. were performed in the laboratory under defined conditions and in mesocosms under environmentally realistic exposure conditions to evaluate the suitability of these species as potential standard test organisms in ecotoxicological testing. This study provides data on the endpoints biomass, plant length and root development. MATERIALS AND METHODS: Six independent experiments were performed to investigate the plant development of Myriophyllum spp. under control conditions. The main difference in the experiments was the complexity of the test systems ranging from simple laboratory experiments to complex outdoor mesocosm studies. At the start of each experiment, uniform cuttings of Myriophyllum spp. were placed in vessels with or without sediments to reduce variability between replicates. The endpoints considered in this investigation were biomass (fresh weight of the whole plant), length of the main shoot, length of the side shoots, total length of the plant (calculated from the length of the main and side shoots) and root formation. Root to shoot ratios were calculated as a further measure for plant development. Relative growth rates (RGR) based on plant length (RG(L)R) and on biomass (RG(B)R) were calculated. RESULTS: Despite the various experimental conditions, comparable growth was obtained in all test systems and the variability of endpoints, such as total length and biomass of plants, was low. It was observed that the RGR of M. spicatum in the simple laboratory test system with sediment were comparable to growth data obtained for M. verticillatum and M. spicatum grown in indoor and outdoor mesocosms, thus indicating that Myriophyllum growth tends to increase by the addition of sediment. High variability was determined for the endpoints length of the side shoots, total root length and biomass of roots. DISCUSSION: One challenge for a test design to investigate phytotoxicity on aquatic plants is to obtain good growth of the plants. From the results, it can be concluded that the experimental conditions in the various test systems were suitable to study the plant development of Myriophyllum spp. because obtained growth rates were comparable between laboratory and field investigations. Another challenge for developing a plant biotest system is the definition of sensitive endpoints. Low variability is preferred to detect minor effects of chemicals or polluted sediments on plant development. In our studies, the variability of the endpoints biomass and total length of plant was low and, therefore, they have much potential as endpoints for assessing toxicity. CONCLUSIONS: The methodologies presented in this study have applications within the risk assessment for aquatic plants and have the advantage of assessing effects taking into account the relevant exposure pathways via water and/or sediment for compounds under investigation. RECOMMENDATIONS AND PERSPECTIVES: Setting safe quality criteria for surface water and sediments is one of the challenges authorities are facing today. Myriophyllum spp. is recommended as suitable test species to investigate phytotoxicity in surface water and sediments. These results, thus, might serve as a basis for the compilation of a new harmonised guideline for ecotoxicological testing with aquatic macrophytes.     

Assessment of the risk posed by the antifouling booster biocides Irgarol 1051 and diuron to freshwater macrophytes

Antifouling paints are used to reduce the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of a biocide. Two 'booster' biocides in common use are the triazine herbicide Irgarol 1051 (N-2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine), and diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea), which are designed to inhibit algal photosynthesis. Previous research has been directed at the effects of these compounds in marine and estuarine environments. In 2001 we sampled the main rivers and shallow freshwater lakes (Broads) of East Anglia UK for Irgarol 1051, its metabolite GS26575 (2-methylamino-4-tert-butylamino-6-amino-s-triazine) and diuron in order to establish the baseline environmental concentrations of these compounds in freshwater systems of eastern UK and to investigate their possible effects on aquatic plants. Irgarol 1051, GS26575 and diuron were found in water samples collected from 21 locations. The highest concentrations were found in the Norfolk and Suffolk Broads in May. The rivers Great Ouse, Wissey, Bure and Yare also contained all three compounds, as did the Great Ouse Cut-off Channel. The toxicity of these biocides to three macrophyte species (Apium nodiflorum, Chara vulgaris, and Myriophyllum spicatum) was investigated. Deleterious effects on relative growth rate, the maximum quantum efficiency (Fv/Fm) of photosystem II and, for Apium, root mass production were found. C. vulgaris was generally most sensitive; growth, especially of roots, was strongly affected in A. nodiflorum; growth rate of M. spicatum was sensitive to diuron. No observed effect concentrations (NOEC) were interpolated using standard toxicological analysis. These were compared with measured environmental concentrations (MEC) to determine the ranges of risk quotients (MEC/NOEC). Both Irgarol 1051 and diuron represented significant risks to A. nodiflorum and C. vulgaris in this area.     

Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes

Toxicity of two azo dyes (Reactive Orange 16 (RO16); Congo Red (CR)) and two anthraquinone dyes (Remazol Brilliant Blue R (RBBR); Disperse Blue 3 (DB3)) were compared using bacterium Vibrio fischeri, microalga Selenastrum capricornutum and ciliate Tetrahymena pyriformis. The following respective endpoints were involved: acute toxicity measured as bacterial luminescence inhibition, algal growth inhibition, and the effects on the protozoa including viability, growth inhibition, grazing effect and morphometric effects. In addition, mutagenicity of the dyes was determined using Ames test with bacterium Salmonella typhimurium His(-). DB3 dye was the most toxic of all dyes in the bacterial, algal and protozoan tests. In contrast to other dyes, DB3 exhibited mutagenic effects after metabolic activation in vitro in all S. typhimurium strains used. Of the methods applied, the algal test was the most sensitive to evaluate toxicity of the dyes tested.     

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).     

Ecotoxicological evaluation of the short term effects of fresh and stabilized textile sludges before application in forest soil restoration

The short term (eco)toxicity potential of fresh and stabilized textile sludges, as well as the short term (eco)toxicity of leachates obtained from both fresh and stabilized textile sludges, was evaluated by a battery of toxicity tests carried out with bacteria, algae, daphnids, fish, earthworms, and higher plants. The (eco)toxicological results showed that, after 120 d of stabilization, the experimental loading ratio of 25% sludge:75% soil (v/v) (equivalent to 64.4 ton/ha) did not significantly increase toxicity effects and increased significantly the biomass yield for earthworms and higher plants. The rank of biological sensitivity endpoints was: Algae approximately Plant biomass > Plant germination approximately Daphnids > Bacteria approximately Fish > Annelids. The lack of short term toxicity effects and the stimulant effect observed with higher plants and earthworms are good indications of the fertilizer/conditioner potential of this industrial waste, which after stabilization can be used in the restoration of a non-productive forest soil.     

Assessing the chemical and biological accessibility of the herbicide isoproturon in soil amended with biochar

Decamethylcyclopentasiloxane (D5) is a cyclic volatile methyl siloxane (cVMS) commonly found in commercially available products. D5 is expected to enter the terrestrial environment through the deposit of biosolids from sewage treatment plants onto agricultural fields for nutrient enrichment. Little to no information currently exists as to the risks of D5 to the terrestrial environment. In order to evaluate the potential risk to terrestrial organisms, the toxicity of a D5 contaminated biosolid in an agricultural soil was assessed with a battery of standardized soil toxicity tests.D5 was spiked into a surrogate biosolid and then mixed with a sandy loam soil to create test concentrations ranging from 0 to 4074 mg kg⁻¹. Plant (Hordeum vulgare (barley) and Trifolium pratense (red clover)) and soil invertebrates (Eisenia andrei (earthworm) and Folsomia candida (springtail)) toxicity tests were completed to assess for lethal and sub-lethal effects. Plant testing evaluated the effects on seedling emergence, shoot and root length, and shoot and root dry mass. Invertebrate test endpoints included adult lethality, juvenile production, and individual juvenile dry mass (earthworms only). Soil samples were collected over time to confirm test concentrations and evaluate the loss of chemical over the duration of a test. The toxicity of the D5 was species and endpoint dependent, such that no significant adverse effects were observed for T. pratense or E. andrei test endpoints, however, toxicity was observed for H. vulgare plant growth and F. candida survival and reproduction. Chemical losses of up to 50% were observed throughout the tests, most significantly at high concentrations.     

Review of historical aquatic toxicity and bioconcentration data for the brominated flame retardant tetrabromobisphenol A (TBBPA): effects to fish,invertebrates, algae,and microbial communities

This paper summarizes the historical and recent research on the aquatic toxicology and bioconcentration potential of tetrabromobisphenol A (TBBPA), a major flame retardant in electronics. Historical studies on TBBPA are presented in detail, and are compared with more recent research. The historical studies have not been published to date, though they were pivotal in regulatory assessments by the European Union, Canada, and the USA. These assessments have enabled the use of TBBPA as a flame retardant in electronic applications, to the present. The studies were conducted under a Test Rule by the US Environmental Protection Agency in 1987, and were sponsored by member companies of the North American Flame Retardants Alliance (NAFRA) through the American Chemistry Council. The studies were conducted under Good Laboratory Practice procedures, and include 6 acute toxicity tests of TBBPA with fish, invertebrates, algae, and microbes, eight chronic tests, and three bioconcentration studies with fish and invertebrates. Methods and empirical data for each study are detailed in an electronic supplement. Results of the NAFRA studies are compared with recent findings on TBBPA toxicity. Molluscan shell growth may be uniquely sensitive to TBBPA, more sensitive than chronic fish or crustacean toxicity endpoints. Several of the NAFRA studies and several independent studies have reported toxicities exceeding the empirical water solubility limits of TBBPA (in the range of 2.0 mg/L depending on pH). The validity of these results is discussed.     

用水生生物对重金属污染土壤进行生态毒理评价

应用斜生栅藻（Scenedesmus obliquus）和明亮发光杆菌T3（Photobacterium phosphoreum）急性毒性实验方法对重金属污染土壤的进行生态毒理评价，结果表明，斜生栅藻的生长繁殖率和发光菌的相对发光度与土壤中的重金属含量明显相关，并且随重金属投加量的增加，其生长繁殖率和发光度逐渐降低。对3种测试参数进行比较可知，斜生栅藻细胞数增长率是最敏感的土壤毒性检测指标。土壤的毒性在复合污染的条件下比在单一污染的条件下高很多。本研究的结果可以为污染土壤的优先修复提供理论依据。     

An evaluation of a short-term chronic effluent toxicity test using sheepshead minnow (Cyprinodon variegatus) larvae

In response to recent changes in National Pollutant Discharge Elimination System (NPDES) permit regulations, rapid (7-day) static renewal toxicity tests have been developed to detect chronic (sublethal and lethal) effect concentrations of municipal and industrial effluents on freshwater and marine/estuarine organisms. This paper evaluates results from short-term (7-day) chronic effluent tests measuring effects on the growth and survival of the larvae of estuarine sheepshead minnow (Cyprinodon variegatus). Growth was the more sensitive endpoint in 52% of the effluent tests; survival was most sensitive in 7% of the tests. Growth and survival were equally sensitive endpoints in 26% of the tests, while there was no effect on either endpoint at the highest test concentration in 15% of the tests. Four short-term chronic tests were conducted concurrently with early life stage (ELS) tests using a common effluent. Effect concentrations were generally (two of three) within one dilution (a factor of three). Comparison of short-term chronic and ELS tests were also conducted with reference single chemicals and the results were slightly better than the effluent comparisons. A series of ten intralaboratory tests and seven interlaboratory tests were conducted to assess the variability among tests results. Toxicity in these tests varied by no more than one test concentration interval. We concluded that this sheepshead minnow test is suitable to estimate chronic values for complex effluents discharged to marine/estuarine environments within the NPDES permit regulations.     

Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes

A mixture of urban and hospital effluents (50 % v/v) was evaluated for ecotoxicity with an advanced bioassay battery. Mixed effluents were tested before any treatment, after biological treatment alone, and after biological treatment followed by a tertiary ozonation (15 mg O₃/L). Laying a high value on the continuance of organisms’ fitness, essential to preserve a healthy receiving ecosystem, the main objective of this study was to combine normalized bioassays with newly developed in vivo and in vitro tests in order to assess alteration of embryo development, growth and reproduction, as well as genotoxic effects in aquatic organisms exposed to complex wastewater effluents. Comparison of the bioassays sensitivity was considered. Contrary to the lack of toxicity observed with normalized ecotoxicity tests, endpoints measured on zebrafish embryos such as developmental abnormalities and genotoxicity demonstrated a residual toxicity in wastewater both after a biological treatment followed or not by a tertiary O₃ treatment. However, the ozonation step allowed to alleviate the residual endocrine disrupting potential measure in the biologically treated effluent. This study shows that normalized bioassays are not sensitive enough for the ecotoxicological evaluation of wastewaters and that there is a great need for the development of suitable sensitive bioassays in order to characterize properly the possible residual toxicity of treated effluents.     

Toxicological evaluation of constructed wetland habitat sediments utilizing 10-day sediment toxicity test and bacterial bioluminescence

A toxicological evaluation was conducted on wetland habitats created as a result of run-off from agricultural areas. These temporary wetlands were created by using drop pipes as a means of reducing erosional cutting in agricultural fields. Toxicity bioassays utilizing bacterial bioluminescence and Hyalella azteca were used to assess sediment pore water and whole sediment, respectively. Inhibition of bacterial bioluminescence was initially used to determine relative toxicities of pore water from ten wetland sites. Constructed wetland sites were compared to the University of Mississippi Biological Field Station, a relatively pristine reference site. The H. azteca ten day sediment toxicity test was utilized to assess sediment from four selected sites using survival and growth as toxicological endpoints. Results from the toxicological evaluation, along with extensive ecological evaluations, were used to assess the best approach for implementation of temporary wetland habitats with existing agricultural practices.     

用水生生物对重金属污染土壤进行生态毒理评价

应用斜生栅藻(Scenedesmusobliquus)和明亮发光杆菌T3(Photobacteriumphosphoreum)急性毒性实验方法对重金属污染土壤的进行生态毒理评价,结果表明,斜生栅藻的生长繁殖率和发光菌的相对发光度与土壤中的重金属含量明显相关,并且随重金属投加量的增加,其生长繁殖率和发光度逐渐降低。对3种测试参数进行比较可知,斜生栅藻细胞数增长率是最敏感的土壤毒性检测指标。土壤的毒性在复合污染的条件下比在单一污染的条件下高很多。本研究的结果可以为污染土壤的优先修复提供理论依据。