Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA, chloridazon and their mixtures

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC?? and EC?? values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC?? and EC?? values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC??CHD + EC??MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC?? after 72 h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.     

Biochemical and standard toxic effects of acetaminophen on the macrophyte species Lemna minor and Lemna gibba

Acetaminophen is globally one of the most prescribed drugs due to its antipyretic and analgesic properties. However, it is highly toxic when the dosage surpasses the detoxification capability of an exposed organism, with involvement of an already described oxidative stress pathway. To address the issue of the ecotoxicity of acetaminophen, we performed acute exposures of two aquatic plant species, Lemna gibba and Lemna minor, to this compound. The selected biomarkers were number of fronds, biomass, chlorophyll content, lipid peroxidation (TBARS assay), and proline content. Our results showed marked differences between the two species. Acetaminophen caused a significant decrease in the number of fronds (EC₅₀?=?446.6 mg/L), and the establishment of a dose-dependent peroxidative damage in L. minor, but not in L. gibba. No effects were reported in both species for the indicative parameters chlorophyll content and total biomass. However, the proline content in L. gibba was substantially reduced. The overall conclusions point to the occurrence of an oxidative stress scenario more prominent for L. minor. However, the mechanisms that allowed L. gibba to cope with acetaminophen exposure were distinct from those reported for L. minor, with the likely involvement of proline as antioxidant.     

Assessing the toxicity of organophosphorous pesticides to indigenous algae with implication for their ecotoxicological impact to aquatic ecosystems

This study aimed to determine the toxicity of three organophosphorous pesticides, chlorpyrifos, terbufos and methamidophos, to three indigenous algal species isolated from local rivers and algal mixtures. The diatom Nitzschia sp. (0.30–1.68 mg L^?1 of EC₅₀ -the estimated concentration related to a 50% growth reduction) and the cyanobacteria Oscillatoria sp. (EC₅₀ of 0.33–7.99 mg L^?1) were sensitive to single pesticide treatment and the chlorophyta Chlorella sp. was the most tolerant (EC₅₀ of 1.29–41.16 mg L^?1). In treatment with the mixture of three pesticides, Chlorella sp. became the most sensitive alga. The antagonistic joint toxic effects on three indigenous algae and algal mixtures were found for most of the two pesticide mixtures. The results suggested that mixture of pesticides might induce the detoxification mechanisms more easily than the single pesticide. The synergistic interactions between terbufos and methamidophos to algal mixtures and between methamidophos and chlorpyrifos to Nitzschia sp. indicated methamidophos might act as a potential synergist. Differential sensitivity of three families of algae to these pesticides might result in changes in the algal community structures after river water has been contaminated with different pesticides, posing great ecological risk on the structure and functioning of the aquatic ecosystem.     

Response of two terrestrial green microalgae (Chlorophyta, Trebouxiophyceae) isolated from Cu-rich and unpolluted soils to copper stress

Some algae inhabit Cu-polluted soils. Intracellular Cu-accumulation and production of non-protein thiols in response to copper stress were compared in Stichococcus minor and Geminella terricola isolated from Cu-polluted and unpolluted soils, respectively. Cu-exposed (0.5 μM) S. minor accumulated lower amounts of copper (0.38 mM) than G. terricola (4.20 mM) and maintained 8.5-fold higher level of glutathione (GSH) than G. terricola. The ratio GSH/0.5 GSSG in the Cu-treated S. minor (7.21) was 7-times higher than in G. terricola. Reduced and oxidized forms of phytochelatins were found in both algae. Under copper stress (5 μM) the ratio -SH_total/Cu_{intracellular} in S. minor ranged from 2.3 to 6.2, while it was lower than 1.0 in G. terricola. Low intracellular Cu-accumulation and maintenance of high GSH level concomitant with PCs production seem to be responsible for a higher Cu-resistance of S. minor than G. terricola.     

Effects of high ammonium level on biomass accumulation of common duckweed Lemna minor L.

Growing common duckweed Lemna minor L. in diluted livestock wastewater is an alternative option for pollutants removal and consequently the accumulated duckweed biomass can be used for bioenergy production. However, the biomass accumulation can be inhibited by high level of ammonium (NH₄ ⁺) in non-diluted livestock wastewater and the mechanism of ammonium inhibition is not fully understood. In this study, the effect of high concentration of NH₄ ⁺ on L. minor biomass accumulation was investigated using NH₄ ⁺ as sole source of nitrogen (N). NH₄ ⁺-induced toxicity symptoms were observed when L. minor was exposed to high concentrations of ammonium nitrogen (NH₄ ⁺-N) after a 7-day cultivation. L. minor exposed to the NH₄ ⁺-N concentration of 840 mg l^?1 exhibited reduced relative growth rate, contents of carbon (C) and photosynthetic pigments, and C/N ratio. Ammonium irons were inhibitory to the synthesis of photosynthetic pigments and caused C/N imbalance in L. minor. These symptoms could further cause premature senescence of the fronds, and restrain their reproduction, growth and biomass accumulation. L. minor could grow at NH₄ ⁺-N concentrations of 7–84 mg l^?1 and the optimal NH₄ ⁺-N concentration was 28 mg l^?1.     

Mixture Toxicity of the Antifouling Compound Irgarol to the Marine Phytoplankton Species Dunaliella tertiolecta

This study examined the toxicity of irgarol, individually and in binary mixtures with three other pesticides (the fungicide chlorothalonil, and the herbicides atrazine and 2,4-D), to the marine phytoplankton species Dunaliella tertiolecta. Standard 96-h static algal bioassays were used to determine pesticide effects on population growth rate. Irgarol significantly inhibited D. tertiolecta growth rate at concentrations ≥ 0.27 μ g/L. Irgarol was significantly more toxic to D. tertiolecta than the other pesticides tested (irgarol 96 h EC₅₀ = 0.7 μ g/L; chlorothalonil 96 h EC₅₀ = 64 μ g/L; atrazine 96 h EC₅₀ = 69 μ g/L; 2,4-D 96 h EC₅₀ = 45,000 μ g/L). Irgarol in mixture with chlorothalonil exhibited synergistic toxicity to D. tertiolecta, with the mixture being approximately 1.5 times more toxic than the individual compounds. Irgarol and atrazine, both triazine herbicides, were additive in mixture. The toxicity threshold of 2,4-D was much greater than typical environmental levels and would not be expected to influence irgarol toxicity. Based on these interactions, overlap of certain pesticide applications in the coastal zone may increase the toxicological risk to resident phytoplankton populations.     

青萍(Lemna minor L.)对氮磷的吸收特征

以西部地区优势浮萍品种青萍为实验对象,研究了青萍对不同浓度硝态氮和磷酸盐的吸收特征。结果表明,硝态氮浓度在1～10 mg/L范围内,青萍对硝态氮有较好的吸收效果,M-M方程可以较好地描述硝态氮浓度与青萍对硝态氮的吸收速率之间的关系,通过M-M方程拟合得到青萍对硝态氮的最大吸收速率为0.1167 mg/(g FW.h),亲和力常数为6.9274。磷酸盐浓度在0.1～1.0 mg/L范围内,青萍对磷酸盐也有较好的吸收效果,二次多项式回归可以较好地描述青萍吸收速率与磷酸盐浓度的关系,回归方程得到青萍对磷酸盐的最大吸收速率为0.0193 mg/(g FW.h),对应磷酸盐浓度为0.6 mg/L。     

Ecotoxicity evaluation of selected sulfonamides

Sulfonamides (SAs) are a group of antibiotic drugs widely used in veterinary medicine. The contamination of the environment by these pharmaceuticals has raised concern in recent years. However, knowledge of their (eco)toxicity is still very basic and is restricted to just a few of these substances. Even though their toxicological analysis has been thoroughly performed and ecotoxicological data are available in the literature, a systematic analysis of their ecotoxicological potential has yet to be carried out. To fill this gap, 12 different SAs were chosen for detailed analysis with the focus on different bacteria as well as non-target organisms (algae and plants). A flexible (eco)toxicological test battery was used, including enzymes (acetylcholinesterase and glutathione reductase), luminescent marine bacteria (Vibrio fischeri), soil bacteria (Arthrobacter globiformis), limnic unicellular green algae (Scenedesmus vacuolatus) and duckweed (Lemna minor), in order to take into account both the aquatic and terrestrial compartments of the environment, as well as different trophic levels. It was found that SAs are not only toxic towards green algae (EC₅₀ = 1.54-32.25 mg L⁻¹) but have even stronger adverse effect on duckweed (EC₅₀ = 0.02-4.89 mg L⁻¹) than atrazine - herbicide (EC₅₀ = 2.59 mg L⁻¹).     

Diquat associated with copper sources for algae control: Efficacy and ecotoxicology

The aims of this research were to evaluate the efficacy of copper oxychloride (CuCl₂.3Cu(OH)₂), copper hydroxide (Cu(OH)₂) and diquat (1.1′-ethylene-2.2′-bipyridyldiylium dibromide), isolated and in association with 0.1% of both copper sources, in the control of the unicellular algae Ankistrodesmus gracilis and the filamentous algae Pithophora kewesis, and to determine the acute toxicity of the tested chemicals in Hyphressobrycon eques, Pomacea canaliculata, Lemna minor and Azolla caroliniana. The efficacy was estimated by the methods of chlorophyll a and pheophytin a readings, changed into growth inhibition percentage. Both algae were exposed to the following concentrations: 0.2; 0.4; 0.8; 1.2 mg L^?1 of diquat and its association with the copper sources; and 0.1; 0.3; 0.5; 0.7; 1.0 and 1.5 mg L^?1 in the isolated applications of copper hydroxide and copper oxychloride. An untreated control was kept. The acute toxicity was estimatedby 50% lethal concentration (LC50). The copper sources were effective for A. gracilis control, at rates as high as 0.1 mg L^?1 (>95% efficacy). Isolated diquat and its association with copper hydroxide were both effective at rates as high as 0.4 mg L^?1, with 95 and 88% control efficacy, respectively. The copper oxychloride was effective at 0.2 mg L^?1, with 93% efficacy. None of the tested chemicals and associations was effective on P. kewesis control. The most sensitive non target organism to the tested chemicals was L. minor; the less sensitive was H. eques.     

Duckweed (Lemna gibba) growth inhibition bioassay for evaluating the toxicity of olive mill wastes before and during composting

Two-phase olive mill waste (TPOMW) is considered the main problem confronting the modern oil extraction and processing industry. Composting has been recently proposed as a suitable method to treat TPOMW so that it is suitable for use in agriculture. In the work reported here, the Lemna gibba bioassay was tested to assess the toxicity of TPOMW before and during the composting process. The method was compared with the Lepidium sativum bioassay and with other chemical maturity indices traditionally reported in the literature. The L. gibba test proved to be a simple, sensitive, and accurate method to evaluate toxicity before and during the composting of TPOMW. Plant growth response was measured by two methods: counting the number of fronds (leaves) and measuring total frond area (TFA) with image analysis software. Compared to the counting of fronds (L. gibba) or seeds (L. sativum), the use of area-measuring software permitted a very rapid, unbiased and easy way of analysing the toxicity of TPOMW before and during composting. Although the accuracy of the frond count method was similar to the traditional cress seed test, data analysis showed that the TFA measurement method was statistically more accurate (significantly lower variance) than the frond count approach. Highly significant correlations were found between TFA and some important maturation indices commonly reported in literature indicating that the L. gibba bioassay can be a useful tool to determine the degree of maturity of TPOMW composts.     

Acute and chronic toxicity of benzotriazoles to aquatic organisms

Purpose

Resulting from their intensive use as corrosion inhibitors in aircraft deicing and anti-icing fluids (ADAF) and for silver protection in dishwasher detergents benzotriazoles (BTs) are widespread in European surface waters. The current study aimed on an ecotoxicological characterization of 1H-benzotriazole (1H-BT) and 5-methyl-1H-benzotriazole (5MBT).

Methods

Acute and chronic OECD guideline tests were conducted with primary producers (Desmodesmus subspicatus, Lemna minor) and two daphnia species (Daphnia magna, Daphnia galeata) to characterize the hazard of these chemicals. Additionally, the estrogenic activity of both BTs was analyzed in vitro using a recombinant yeast estrogen screen (YES).

Results

Both BTs revealed significant effects in acute and chronic experiments, but exhibited no estrogenic activity in the YES. The algal growth test displayed an inhibited cell number increase with effect concentration (EC) values of EC₁₀ 1.18 and 2.86?mg?l^-1 for 1H-BT and 5MBT, respectively. In the Lemna test, EC₁₀ values were 3.94?mg?l^-1 (1H-BT) and 2.11?mg?l^-1 (5MBT). D. magna was also affected with EC₅₀ (48?h) values of 107?mg?l^-1 for 1H-BT and 51.6?mg?l^-1 for 5MBT. D. galeata was more sensitive with an EC₅₀ (48?h) of 14.7?mg 1H-BT l^-1 and 8.13?mg 5MBT l^-1. In the 21-day reproduction tests with D. magna, the EC₁₀ for 5MBT was 5.93?mg?l^-1 while 1H-BT showed no adverse effects. D. galeata turned out to be more sensitive in the chronic study with EC₁₀ values of 0.97?mg?l^-1 for 1H-BT and 0.40?mg?l^-1 for 5 MBT.

Conclusion

Because BTs are regularly found in the aquatic environment at lower ??g l^-1 concentrations reflecting their persistence and poor elimination during wastewater treatment processes, a preliminary risk assessment was conducted. There is little indication that BTs pose a risk for aquatic ecosystems at current exposure levels during most of the year. However, it cannot be excluded that in winter with a higher usage of ADAFs environmental concentrations may well exceed the level that is considered safe for aquatic organisms.     

The OH-induced degradation mechanism of 4-chloro-2-methylphenoxyacetic acid (MCPA) with two forms in the water: a DFT comparison

The initial degradation mechanisms of OH and 4-chloro-2-methylphenoxyacetic acid (MCPA) including molecular form and anionic form are studied at the MPWB1K/6-311+G(3df, 2p)//MPWB1K/6-31+G(d, p) level. Possible reaction pathways of H-atom abstraction and OH addition are considered in detail. By result comparison analysis, it is found that the reaction mechanisms for OH and two forms of MCPA are different, and most reactions for anionic MCPA are easier than those for molecular MCPA. For H-atom abstraction reactions, the calculated energies show that OH abstracting H-atom from -CH₃ group of molecular MCPA is the most kinetically favorable process; the potential energy surface for anionic MCPA indicates that H-atom in -CH₂ group is slightly easier to be abstracted than that in -CH₃ group. For OH addition reactions, the addition of OH to the C1 site is the initial step for molecular MCPA and the predominant product is 4-chloro-2-methylphenol (denoted P3), while the C4 site is the most reactive site for anionic MCPA and the primary product results from the hydroxylation of the aromatic ring, which is in good agreement with the experimental observation. In additional, results from PCM calculations show that most reactions in water phase are more kinetically favorable than those in gas phase, though the mechanisms discussed above will not be changed.     

不同氮素浓度与空间条件下青萍生长特征

以西部地区优势浮萍品种青萍为实验对象,研究了它在不同总氮(TN)初始浓度和空间条件下的生长特征。结果表明,在表面积为95 cm2,TN初始浓度为0、1、2、3 mg/L的条件下,青萍的生长能较好地服从经典生长模型,对数生长阶段的相对生长速率(RGR)随TN浓度的增长而线性增长,分别为0.10、0.11、0.13、0.13 d-1。在TN浓度为10 mg/L,表面积为95、130和230 cm2的条件下,青萍的生长能较好地服从Logistic生长模型,其环境最大承载量和对数生长阶段的RGR分别为1 899、2 101和1 962 g/m2以及0.21、0.21和0.20 d-1。     

Identification of a phytotoxic photo-transformation product of diclofenac using effect-directed analysis

The pharmaceutical diclofenac (DCF) is released in considerably high amounts to the aquatic environment. Photo-transformation of DCF was reported as the main degradation pathway in surface waters and was found to produce metabolites with enhanced toxicity to the green algae Scenedesmus vacuolatus. We identified and subsequently confirmed 2-[2-(chlorophenyl)amino]benzaldehyde (CPAB) as a transformation product with enhanced toxicity using effect-directed analysis. The EC₅₀ of CPAB (4.8 mg/L) was a factor of 10 lower than that for DCF (48.1 mg/L), due to the higher hydrophobicity of CPAB (log K_ow = 3.62) compared with DCF (log D_ow = 2.04) at pH 7.0.     

Combined effect of polystyrene plastics and triphenyltin chloride on the green algae Chlorella pyrenoidosa

The combined effect of polystyrene (PS) particles and triphenyltin chloride (TPTCl) to the green algae Chlorella pyrenoidosa was studied. The 96 h IC₅₀ of TPTCl to the green algae C. pyrenoidosa was 30.64 μg/L. The toxicity of PS particles to C. pyrenoidosa was size-dependent, with the 96 h IC₅₀ at 9.10 mg/L for 0.55 μm PS but no toxicity observed for 5.0 μm PS. The exposure to 0.55 μm PS led to damage on structure of algal cells, which could in turn cause inhibition on photosynthesis and population growth of the green algae. TPTCl concentrations in test medium were lowered by 15–19% at presence of 0.55 μm PS particles, indicating a reduced bioavailability of TPTCl. In spite of this reduced bioavailability, the presence of PS increased the toxicity of TPTCl, which might be attributed to facilitated uptake of TPTCl by the green algae after the damage of cell structure. The overall results of the present study provided important information on the effect of PS on the bioavailability and toxicity of TPTCl to phytoplankton species.

     

In vitro AHH induction by polychlorinated biphenyl and dibenzofuran mixtures: Additive effects

The activities of several individual polychlorinated biphenyls (PCBs) and dibenzofurans (PCDFs) and several environmentally significant reconstituted mixtures of these compounds as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells were determined. The observed AHH and EROD induction EC₅₀S for the mixtures were compared with the calculated values, which were based on the summation of the relative per cent contributions of the individual components of the reconstituted PCB and PCDF mixtures. The results show that the differences between the observed and calculated EC₅₀s for these mixtures were minimal or not significant and the data supports the use of the rat hepatoma H-4-II E cell system as a bioassay for toxic halogenated aryl hydrocarbons.     

Bioassay for Determining Sensitivity to Sulfosulfuron on Seven Plant Species

This study presents a bioassay procedure, based on the root and shoot growth parameters, for the determination of the herbicide sulfosulfuron (1-(4,6 dimethoxypyrimidin-2-yl)-3-(2-ethylsulfonylimidazo[1,2-a]pyridin-3-ylsulfonil)urea) sensitivity on seven vegetal species. Plant response to sulfosulfuron was calculated with the equations fitted to the root growth data as a function of the logarithm of the herbicide concentration by non-linear regression and was used to calculate the doses for 10, 30 and 50% inhibition of root growth (EC₁₀, EC₃₀ and EC₅₀). The results indicate that the phytotoxic effect of sulfosulfuron in all the species assayed followed the order: flax > maize > onion > vetch > lepidium sativum > tomato > barley. These species showed phytotoxicity at low levels of sulfosulfuron and flax appeared to be the most susceptible species to sulfosulfuron (0.001 mg/L).     

Strategies to evaluate biodegradability: application to chlorinated herbicides

The biodegradability of nitrochlorinated (diuron and atrazine) and chlorophenoxy herbicides (2,4-D and MCPA) has been studied through several bioassays using different testing times and biomass/substrate ratios. A fast biodegradability test using unacclimated activated sludge yielded no biodegradation of the herbicides in 24 h. The inherent biodegradability test gave degradation percentages of around 20–30 % for the nitrochlorinated herbicides and almost complete removal of the chlorophenoxy compounds. Long-term biodegradability assays were performed using sequencing batch reactor (SBR) and sequencing batch membrane bioreactor (SB-MBR). Fixed concentrations of each herbicide below the corresponding EC₅₀ value for activated sludge were used (30 mg L^?1 for diuron and atrazine and 50 mg L^?1 for 2,4-D and MCPA). No signs of herbicide degradation appeared before 35 days in the case of diuron and atrazine and 21 days for 2,4-D, whereas MCPA was partially degraded since the early stages. Around 25–36 % degradation of the nitrochlorinated herbicides and 53–77 % of the chlorophenoxy ones was achieved after 180 and 135 days, respectively, in SBR, whereas complete disappearance of 2,4-D was reached after 80 days in SB-MBR.     

Environmental risk appraisement of disinfection by-products (DBPs) in plant model system: Allium cepa

The organic toxicants formed in chlorinated water cause potential harm to human beings, and it is extensively concentrated all over the world. Various disinfection by-products (DBPs) occur in chlorinated water are genotoxic and carcinogenic. The toxicity is major concern for chlorinated DBPs which has been present more in potable water. The purpose of the work was to evaluate genotoxic properties of DBPs in Allium cepa as a plant model system. The chromosomal aberration and DNA laddering assays were performed to examine the genotoxic effect of trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) in a plant system with distinct concentrations, using ethyl methanesulfonate (EMS) as positive control and tap water as negative control. In Allium cepa root growth inhibition test, the inhibition was concentration dependent, and EC₅₀ values for trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) were 100 mg/L, 160 mg/L, and 120 mg/L respectively. In the chromosome aberration assay, root tip cells were investigated after 120 h exposure. The bridge formation, sticky chromosomes, vagrant chromosomes, fragmented chromosome, c-anaphase, and multipolarity chromosomal aberrations were seen in anaphase–telophase cells. It was noticed that with enhanced concentrations of DBPs, the total chromosomal aberrations were more frequent. The DNA damage was analyzed in roots of Allium cepa exposed with DBPs (TCAA, TCM, TBM) by DNA laddering. The biochemical assays such as lipid peroxidation, H₂O₂ content, ascorbate peroxidase, guaiacol peroxidase, and catalase were concentration dependent. The DNA interaction studies were performed to examine binding mode of TCAA, TCM, and TBM with DNAs. The DNA interaction was evaluated by spectrophotometric and spectrofluorometric studies which revealed that TCAA, TCM, and TBM might interact with Calf thymus DNA (CT- DNA) by non-traditional intercalation manner.