Algal growth inhibition by river water pollutants in the agricultural area around Lake Biwa, Japan

An ecotoxicological study of river water discharged from the agricultural area around Lake Biwa was performed by using algal bioassays to guide chemical analysis. Water samples were collected once a week, at least, for 1 year starting in April 1997 and continuing until April 1998. The toxicities of the dissolved and particulate-adsorbed extracts of water samples were evaluated by the algal growth inhibition test and concentrations of individual pesticides were determined. Most of the river water that was collected during the periods when pesticides were applied to the paddy fields caused algal growth inhibition. Some extracts were found to contain herbicides (molinate, mefenacet, simetryn, or esprocarb) as major compounds. According to chemical assay and bioassay, simetryn was identified as the most toxic compound that caused algal growth inhibition.     

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).     

Effects of cyanobacteria producing microcystins on seed germination and seedling growth of several agricultural plants

The effects of cyanobacteria aqueous extracts containing Microcystin-LR (MC-LR) on the seed germination and growth of Pisum sativum, Lens esculenta, Zea mays and Triticum durum were investigated. Experiments were carried out on a range of doses of the extract (equivalent to 0, 1.6, 2.9, 5.8, 8.7 and 11.6 mu g MC-LR/mL). The results confirm that these plants were sensitive to cell-free extracts of a toxic Microcystis and that germination inhibition was dose dependent. One-way analysis of variance (ANOVA) showed that P. sativum is the most sensitive tested species with a 97% germination rate reduction and L. esculenta was the most resistant. At the 8th day, the exposure to the microcystins (MC) resulted in a significant decrease of plant epicotyls length, roots length and a net inhibition of lateral root formation. It is concluded that MC could affect also terrestrial plants seedling germination and growth. Therefore, the use of water for irrigation contaminated by MC could exert negative biochemical effects on seed and plant metabolism which might influence the agricultural crops.     

Bioavailability of dissolved organic nitrogen in treated effluents.

The research objective was to assess dissolved organic nitrogen (DON) bioavailability in wastewater effluents from a pilot-scale nitrification plant and a laboratory-scale total nitrogen (TN) removal plant. The DON bioavailability was assessed using a 14-day bioassay protocol containing bacterial and algal inocula. Nitrogen species, dissolved organic carbon, chlorophyll a, and biomass (as total suspended solids and culturable cell counts) concentrations were measured to assess DON bioavailability. The results showed an increase in algal chlorophyll a concentration, with a concurrent increase in algal biomass over time; increased bacterial counts and a decrease in DON concentration over time; and increased carbon-to-nitrogen ratio at the end of the 14-day bioassay, indicating effluent DON bioavailability to algae and bacteria. Approximately 18 to 61% of the initial DON in low-total-nitrogen wastewater effluent (TN = 4 to 5 mg/L) sample was bioavailable. The results show that bacteria and algae uptake and release DON during their growth.     

Herbicide impact on Hormosira banksii gametes measured by fluorescence and germination bioassays

The innovative bioassay described here involves chlorophyll a fluorescence measurements of gametes from the macroalgae, Hormosira banksii, where gametes (eggs) were exposed to Diuron, Irgarol and Bromacil. Response was assessed as percent inhibition from control of effective quantum yield (DeltaF/Fm') of photosystem II, herein referred to as % PSII Inhibition. This was measured with the dual-channelled pulse amplitude modulated (PAM) fluorometer, ToxY-PAM. The fluorescence bioassay was run simultaneously with an established H. banksii germination bioassay to compare sensitivity, precision, and time-to-result. The fluorescence bioassay gave highly sensitive results evidenced by EC(50)s (% PSII Inhibition) for Diuron, Irgarol and Bromacil being three, four and three orders of magnitude (respectively) lower than EC50s generated from the germination bioassays. Precision of the fluorescence bioassay was demonstrated with low coefficient of variations (<30%) for all three toxicants. With regard to time, the fluorescence bioassay gave results within 6h, as opposed to more than 50h for the germination bioassay.     

Evaluation and refinement of a continuous seed germination and early seedling growth test for the use in the ecotoxicological assessment of soils.

Four higher plant species (Avena sativa L., oat; Lepidium sativum L., cress; Brassica rapa Metzg., turnip; and Phaseolus vulgaris L., bush bean) and 15 soils including five mineral oil-contaminated soils were tested to assess a soil-based continuous seed germination and early seedling growth bioassay. Seed germination was recorded and the shoot biomass of 14-day seedlings measured. An automatic self-watering system was compared with the daily hand watering. Results obtained from this study show that the self-watering system had minor effects on both seed germination and early seedling growth, and that soil nutrient status rather than soil texture significantly affected both seedling emergence and shoot biomass. Proportional dilution of suspicious samples from primary tests with an appropriate control soil is recommended for secondary tests. Shoot biomass is a more sensitive measurement endpoint than seed germination. Bush bean should not be recommended for use due to its low sensitivity to mineral oil and poor germination in soil.     

Bioherbicidal activity of a germacranolide sesquiterpene dilactone from Ambrosia artemisiifolia L.

Ambrosia artemisiifolia L. (common ragweed) is an invasive plant whose allelopathic properties have been suggested by its field behaviour and demonstrated through phytotoxicity bioassays. However, the nature of the molecules responsible for the allelopathic activity of common ragweed has not been explored. The main objective of this study was to identify the phytotoxic molecules produced by A. artemisiifolia. A preliminary investigation has indicated that a methanol extract of A. artemisiifolia completely inhibited the germination of cress and radish. Semi-preparative fractionation of the methanol extract allowed separating of phytotoxic fraction which contained a single compound. The structure of this compound was elucidated by liquid chromatography–mass spectrometry (LC-MS)/MS, high-resolution mass spectral, nuclear magnetic resonance, and Fourier transform infrared spectra as sesquiterpene lactone isabelin (C₁₅H₁₆O₄). The effect of pure isabelin was tested on four different weed species, confirming the inhibitory activity of molecule. The results indicate directions for the future studies about herbicidal specific activity of isabelin, as pure molecule or in the crude extract, as a potential candidate for biological weed control.     

Phytotoxicity of nanoparticles: inhibition of seed germination and root growth

Plants need to be included to develop a comprehensive toxicity profile for nanoparticles. Effects of five types of nanoparticles (multi-walled carbon nanotube, aluminum, alumina, zinc, and zinc oxide) on seed germination and root growth of six higher plant species (radish, rape, ryegrass, lettuce, corn, and cucumber) were investigated. Seed germination was not affected except for the inhibition of nanoscale zinc (nano-Zn) on ryegrass and zinc oxide (nano-ZnO) on corn at 2000 mg/L. Inhibition on root growth varied greatly among nanoparticles and plants. Suspensions of 2000 mg/L nano-Zn or nano-ZnO practically terminated root elongation of the tested plant species. Fifty percent inhibitory concentrations (IC50) of nano-Zn and nano-ZnO were estimated to be near 50mg/L for radish, and about 20mg/L for rape and ryegrass. The inhibition occurred during the seed incubation process rather than seed soaking stage. These results are significant in terms of use and disposal of engineered nanoparticles.     

Arsenic chemistry in the rhizosphere of Pteris vittata L. and Nephrolepis exaltata L

This greenhouse experiment evaluated the influence of arsenic uptake by arsenic hyperaccumulator Pteris vittata L. and non-arsenic hyperaccumulator Nephrolepis exaltata L. on arsenic chemistry in bulk and rhizosphere soil. The plants were grown for 8 weeks in a rhizopot with a soil containing 105 mg kg(-1) arsenic. The soil arsenic was fractionated into five fractions with decreasing availability: non-specifically bound (N), specifically bound (S), amorphous hydrous-oxide bound (A), crystalline hydrous-oxide bound (C), and residual (R). P. vittata produced larger plant biomass (7.38 vs. 2.32 mg plant(-1)) and removed more arsenic (2.61 vs. 0.09 mg pot(-1) arsenic) than N. exaltata. Plant growth reduced water-soluble arsenic, and increased soil pH (P. vittata only) in the rhizosphere soil. P. vittata was more efficient than N. exaltata to access arsenic from all fractions (39-64% vs. 5-39% reduction). However, most of the arsenic taken up by both plants was from the A fraction (67-77%) in the rhizosphere soil, the most abundant (61.5%) instead of the most available (N fraction).     

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.     

Effects of cyanobacteria producing microcystins on seed germination and seedling growth of several agricultural plants

The effects of cyanobacteria aqueous extracts containing Microcystin-LR (MC-LR) on the seed germination and growth of Pisum sativum, Lens esculenta, Zea mays and Triticum durum were investigated. Experiments were carried out on a range of doses of the extract (equivalent to 0, 1.6, 2.9, 5.8, 8.7 and 11.6 μ g MC-LR/mL). The results confirm that these plants were sensitive to cell-free extracts of a toxic Microcystis and that germination inhibition was dose dependent. One-way analysis of variance (ANOVA) showed that P. sativum is the most sensitive tested species with a 97% germination rate reduction and L. esculenta was the most resistant. At the 8th day, the exposure to the microcystins (MC) resulted in a significant decrease of plant epicotyls length, roots length and a net inhibition of lateral root formation. It is concluded that MC could affect also terrestrial plants seedling germination and growth. Therefore, the use of water for irrigation contaminated by MC could exert negative biochemical effects on seed and plant metabolism which might influence the agricultural crops.     

3种药剂对普通小球藻的抑制作用

针对再生水回用于景观水体生长的主要藻类普通小球藻,进行了药剂抑制实验。通过实验研究发现,在小球藻生长的对数期分别投入不同剂量的2-甲基乙酰乙酸乙酯（EMA）、大麦秸萃取液（BSE）和异噻唑啉酮3种抑藻药剂后,小球藻的生长受到不同程度的抑制。其中,EMA对藻类的抑制效果甚微,最大抑藻率仅为31%,且对小球藻的生长有低促高抑的作用。BSE对小球藻有明显的抑制作用,且药效期长、不复发;当投加浓度为1 mg/L,能起到抑制藻类生长的作用;当投加浓度为10 mg/L,能起到杀灭水体藻类的作用。异噻唑啉酮可有效抑制小球藻生长,5 mg/L的投加浓度就能起到杀灭水体藻类的作用,且药效期长、不复发。     

Effects of copper on spore germination, growth and ultrastructure of Polypodium cambricum L. gametophytes

The effects of different concentrations (10(-5), 5x10(-5) and 10(-4)M) of copper bromide on spore germination, growth and ultrastructure were investigated in Polypodium cambricum L. gametophytes. The inhibitory effect of Cu was observed in spores cultured on medium supplemented with 10(-4)M CuBr(2): germination occurred about 40 days after sowing and was only 25%. Concentrations of 5x10(-5) and 10(-4)M CuBr(2) induced changes in gametophyte development, possibly by re-orientation of growth. Gametophytes treated with 10(-5) and 5x10(-5)M CuBr(2) took up and accumulated a large amount of copper and ultrastructural observations showed that cytoplasmic damage was limited to twisted swollen thylakoids. The ultrastructure of gametophytes treated with 10(-4)M CuBr(2) showed absence of a vacuolar compartment. The present observations suggest that P. cambricum gametophytes could be a suitable material for studying physiological and molecular alterations induced by excess copper.     

Testing of coloured samples for toxicity by the algal-ATP bioassay microplate technique

Algal-ATP bioassay is a relatively simple, inexpensive, reliable and fast testing procedure for toxicity assessment. This algal growth inhibition bioassay technique using Selenastrum capricornutum was used to assess the toxicity levels in pulp and paper mill and thermomechanical mill effluent samples. Pulp and paper mill and thermomechanical mill effluent samples contain a wide range of colours due to certain physico-chemical properties of the test samples. The optical densities of the samples ranged from 0.27 to> 2.0 (a 600 nanometer). The colour variations in the test samples may interfere with ATP measurements. This paper presents data on the nature of interferences due to certain physico-chemical properties of the test sample and the usefulness of developed controls to overcome the problems for a more realistic toxicity assessment of coloured waste samples.     

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.     

Comparison of the ecotoxicological impact of the triazines Irgarol 1051 and atrazine on microalgal cultures and natural microalgal communities in Lake Geneva

The antifouling herbicide Irgarol 1051 has been detected in recent years in numerous estuaries, marinas, harbors and coastal areas, and in some harbors on Lake Geneva, but so far only a few studies have investigated the ecotoxicological effects of this compound on microalgae. The purpose of this study was to assess the ecotoxicological impact of Irgarol 1051 on the algal communities of Lake Geneva, and to compare its phytotoxicity to that of the common triazine herbicide, atrazine. We investigated the response of phytoplanktonic and periphytonic algal communities and single-species isolates collected from the lake, to the PS II inhibitor Irgarol 1051 (growth, proxy of photosynthetic activity and community structure). A short-term bioassay was developed based on in vivo fluorescence, together with nanocosm experiments with natural algal communities, and single-species tests on algal strains isolated from the lake. The toxicity of Irgarol 1051 towards periphyton and phytoplankton was shown to be higher than that of atrazine. Indications of the tolerance induced by this triazine in the algal communities of Lake Geneva, suggests that even at the levels of contamination reported in some parts of the lake, Irgarol 1051 is already exerting selection pressure. Information about sensitivities, selection and tolerance from laboratory experiments are used to explain the observations in natural microalgal communities from the lake.     

Effects of distillery waste on the performance of Cicer arietinum L

The impact of distillery effluent in various concentrations (1, 2.5, 5, 10, 25, 50, 75 and 100%) on the seed germination, Speed of Germination Index, growth behaviour, leaf area, biomass, net primary productivity, pigment content, reproductive capacity, seed output, seed weight, seed density and the seed protein content of Cicer arietinum L. plants was investigated. The percentage and speed of germination of seeds were increasingly retarded with increase in effluent concentration and at 100% concentration there was no germination. The seedlings exhibited maximum shoot length at 5% concentration and maximum root length at 2.5% concentration. The values of root and shoot lengths, leaf area, biomass, net primary productivity, pigment content, reproductive capacity, seed output, seed weight, seed density and seed protein content in pot plants exhibited a gradual increase from the control up to 5% concentration and decreases at higher concentrations. The very high BOD load and the presence of excessive concentrations of soluble salts could be responsible for the toxicity of the effluent. The effluent at up to 5% concentration was, however, beneficial for the overall growth parameters studied and can thus be used as a liquid fertilizer.     

Surface and ground waters characterization in Tuscany (Italy) by using algal bioassay and pesticide determinations: comparative evaluation of the results and hazard assessment of the pesticides impact on primary productivity

Algal bioassays and pesticide analysis were joined in Tuscany regional monitoring of surface and ground waters. Results were compared. Waters affected algal growth in any of the following ways: stimulation, inhibition, stimulation at low concentrations and inhibition at higher concentrations. Most of ground waters inhibited algal growth whereas only a little percentage were contaminated with pesticide. Most of surface waters stimulated algal growth and a higher percentage were contaminated with pesticides. In waters with inhibitory effect toxicological parameters (EC50, EC25, EC10 and NOEC) were measured and compared; EC25 values were very close to NOEC values. Hazard to aquatic primary productivity for each pesticide found during chemical monitoring was evaluated; for this aim the margin of safety (MOS) was calculated by using algal toxicity and the pesticide concentration measured.     

Pollen germination is impeded by tap water

Pollen germination in vitro is totally inhibited in tobacco (Nicotiana tabacum) and other species if tap water is used to prepare the germination medium. This effect is already fully present if tap water accounts for only 25% of the medium. Furthermore, the pollen grains deteriorate rapidly and the culture medium turns yellowish-brown. The water toxicity is not caused by one or several compounds regularly monitored by the water authorities but can be removed by ion exchange purification. Although the factor(s) responsible for the inhibition were not identified, the study clearly shows the presence of such a contaminant in three different Orange County (Southern California) water wells. The fact that a fundamental botanical process like pollen germination is inhibited by a factor in drinking water not included in water quality control causes some general health concern. In addition, crop yield might be largely reduced if overhead spray irrigation with this water is utilized. The experiments also suggest that pollen germination in vitro could serve as a sensitive and simple bioassay for water quality.     

Environmental assessment of ester-based lubricants after application

GOAL, SCOPE AND BACKGROUND: Lubricants based on renewable resources are an environmentally friendly alternative to petrochemical products due to their better ecotoxicological performance and their excellent biodegradability. To improve the technical performance of lubricants, and to reduce friction and wear, the use of additives is nowadays obligatory. The collaborative research center SFB 442 aims at developing environmentally acceptable lubricants that facilitate the avoidance of these additives by transferring their function to suitable coatings. For a complete assessment of the ecological performance of these newly developed lubricants, the whole life cycle including production, application as well as disposal and fate in the environment is studied. In the following study the focus was on the application and its influence on the environmental behavior of the lubricant. The application of lubricants leads, among other things, to the intake of metals due to abrasion from tools, work pieces or mechanical components. Previous examinations indicated a possible influence of metals on the toxicity of eluates prepared from used lubricants (Erlenkaemper et al. 2005). To clarify if the apparent toxicity of used lubricants solely results from the intake of metals, the extractability of these metals from the oil matrix is determined. By combining chemical analyses with bioassays, the bioavailability of metals that are present in the extract is estimated. To further investigate the relevance of metals on toxicity, toxic units (TU) were calculated and related to the results of the bioassays. Interactions between the metals were investigated with aqueous mixtures of metal chlorides and calculations based on the concept of concentration addition and independent action. METHODS: A lubricant mixture was applied to a tribological test bench that simulates real conditions of use and extremely short time load, respectively. Samples were taken at particular times, water soluble fractions (WSF) of these fluids were prepared and dilution series were investigated in several bioassays. Concentration of metals and total organic carbon (TOC) were determined in the eluates. TUs were calculated according to Sprague (1970) and mixture toxicity was calculated according to the concept of concentration addition (Loewe and Muischnek 1926) and independent action (Bliss 1939). RESULTS: Analyses of the metal content of the lubricant and the eluates clearly revealed the availability of the metals in the aqueous extracts. Especially copper, zinc, nickel and chromium were found and their concentrations increased during the time of use. The water extractable fraction, e.g., of copper, rose from 8.8% to 45.3% of the total content in the lubricant after 33.5 hours of use. Tests performed with the algal growth inhibition assay and the luminescence inhibition assay revealed the uptake or absorption by the organisms and, thus, the bioavailability of the metals. The calculation of TUs partly indicated a possible influence of the metals on ecotoxicity of the eluates. Copper reached concentrations equal to or higher than the EC50 value of copper chloride in the growth inhibition assays with algae and Ps. putida as well in the immobilization assay with daphnids. TUs for copper are also larger than 1 for the algal growth inhibition assay. The EL50 values indicated that the luminescence inhibition assay was the most sensitive test system, with values between 4.7% and 9.6%. While the toxicity towards algae and V. fischeri in the growth inhibition assay decreased until both organisms were no longer influenced by the exposure, the EL50 values for the D. magna immobilization assay and the Ps. putida growth inhibition assay decreased with the progressing use of the lubricant. The tested metal salt mixtures showed that Ps. putida, algae and daphnids are the most sensitive organisms with EC50 values below 1 mg/l. DISCUSSION: Although the intake of metals mainly occurred via abrasion of particles, the results revealed the availability of these metals in water. The availability varied for each of the four metals. For both the algal growth inhibition assay and the luminescence inhibition assay, an uptake or absorption of the metals could be demonstrated. The calculated TUs indicated an effect in some bioassays that was not verified in the test itself. The influence of copper on V. fischeri, for example, was not confirmed while the EL50 values for the daphnid bioassay decreased, meaning that the eluates became more toxic with progressing use of the lubricant. The calculations of mixture toxicity based on the concept of concentration addition demonstrated good correlations with the tested metal mixtures, but also a different sensitivity of the organisms. CONCLUSIONS: The results presented here reveal the availability of those metals in water that were taken in during the use of the lubricant in a tribological test bench and, thus, have the possibility of interacting with the organisms. The availability of the metals in the bioassays was proven by chemical analyses. The calculation of TUs and the corresponding EL50 values, however, indicate different availabilities of the metals. The results of the metal salt mixtures show good correlations with calculations of mixture toxicity based on concentration addition. Moreover, the varying sensitivity of the organisms when exposed to eluates or metal mixtures indicates a different bioavailability of the metals and/or the presence of other compounds that exert toxic action. RECOMMENDATIONS AND PERSPECTIVES: For further investigations, the organic oil matrix and its influence on the toxicity have to be taken into account. The toxicity of the eluates may not only be due to metals; additional effects could arise from changes in the lubricant itself.