Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.

Effects of Photosystem II inhibiting herbicides on mangroves - preliminary toxicology trials

Mangroves are sensitive to the root application of Photosystem II inhibiting herbicides and Avicennia marina is more sensitive than other mangroves tested. Seedlings of four mangrove species, including two salt-excreting species (A. marina and Aegiceras corniculatum) and two salt-excluding species (Rhizophora stylosa and Ceriops australis) were treated with a range of concentrations of the herbicides diuron, ametryn and atrazine. Assessment of responses required the separation of seedlings into two groups: those that had only their roots exposed to the herbicides through the water (A. marina and R. stylosa) and those that had both roots and leaves exposed to herbicides through the water (A. corniculatum and C australis). Salt-excreting species in each group were more susceptible to all herbicide treatments than salt-excluding species, indicating that root physiology was a major factor in the uptake of toxic pollutants in mangroves. Submergence of leaves appeared to facilitate herbicide uptake, having serious implications for seedling recruitment in the field. Each herbicide was ranked by its toxicity to mangrove seedlings from most damaging to least effective, with diuron > ametryn > atrazine. The relative sensitivity of A. marina found in these pot trials was consistent with the observed sensitivity of this species in the field, notably where severe dieback had specifically affected A. marina in the Mackay region, north eastern Australia. (c) 2004 Elsevier Ltd. All rights reserved.

Dissipation of acetochlor and its distribution in surface and sub-surface soil fractions during laboratory incubations

Pesticides in soil are subject to a number of processes that result in transformation and biodegradation, sorption to and desorption from soil components, and diffusion and leaching. Pesticides leaching through a soil profile will be exposed to changing environmental conditions as different horizons with distinct physical, chemical and biological properties are encountered. The many ways in which soil properties influence pesticide retention and degradation need to be addressed to allow accurate predictions of environmental fate and the potential for groundwater pollution. Degradation and sorption processes were investigated in a long-term (100 days) study of the chloroacetanilide herbicide, acetochlor. Soil cores were collected from a clay soil profile and samples taken from 0-30cm (surface), 1.0-1.3m (mid) and 2.7-3.0m (deep) and treated with acetochlor (2.5, 1.25, 0.67 mu g acetochlor g(-1) dry wt soil, respectively). In sterile and non-sterile conditions, acetochlor concentration in the aqueous phase declined rapidly from the surface and subsoil layers, predominantly through nonextractable residue (NER) formation on soil surfaces, but also through biodegradation and biotic transformation. Abiotic transformation was also evident in the sterile soils. Several metabolites were produced, including acetochlor-ethane sulphonic acid and acetochlor-oxanilic acid. Transformation was principally microbial in origin, as shown by the differences between non-sterile and sterile soils. NER formation increased rapidly over the first 21 days in all soils and was mainly associated with the macroaggregate (> 2000 mu m diameter) size fractions. It is likely that acetochlor is incorporated into the macroaggregates through oxidative coupling, as humification of particulate organic matter progresses. The dissipation (ie total loss of acetochlor) half-life values were 9.3 (surface), 12.3 (mid) and 12.6 days (deep) in the non-sterile soils, compared with 20.9 [surface], 23.5 [mid], and 24 days [deep] in the sterile soils, demonstrating the importance of microbially driven processes in the rapid dissipation of acetochlor in soil.

Rhizodeposits of Trifolium pratense and Lolium perenne: Their comparative effects on 2,4-D mineralization in two contrasting soils

Rhizosphere enhanced biodegradation of organic pollutants has been reported frequently and a stimulatory role for specific components of rhizodeposits postulated. As rhizodeposit composition is a function of plant species and soil type, we compared the effect of Lolium perenne and Trifolium pratense grown in two different soils (a sandy silt loam: pH 4, 2.8% OC, no previous 2,4-D exposure and a silt loam: pH 6.5, 4.3% OC, previous 2,4-D exposure) on the mineralization of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). We investigated the relationship of mineralization kinetics to dehydrogenase activity, most probable number of 2,4-D degraders (MPN2,4-D) and 2,4-D degrader composition (using sequence analysis of the gene encoding alpha-ketoglutarate/2,4-D dioxygenase (tfdA)). There were significant (P < 0.01) plant-soil interaction effects on MPN2,4-D and 2,4-D mineralization kinetics (e.g. T pratense rhizodeposits enhanced the maximum mineralization rate by 30% in the acid sandy silt loam soil, but not in the neutral silt loam soil). Differences in mineralization kinetics could not be ascribed to 2,4-D degrader composition as both soils had tfdA sequences which clustered with tfdAs representative of two distinct classes of 2,4-D degrader: canonical R. eutropha JMP134-like and oligotrophic alpha-proteobacterial-like. Other explanations for the differential rhizodeposit effect between soils and plants (e.g. nutrient competition effects) are discussed. Our findings stress that complexity of soil-plant-microbe interactions in the rhizosphere make the occurrence and extent of rhizosphere-enhanced xenobiotic degradation difficult to predict.

Application of a novel phytotoxicity assay for the detection of herbicides in Hervey Bay and the Great Sandy Straits

A novel phytotoxicity assay was incorporated into an environmental assessment of Hervey Bay and the Great Sandy Straits, to investigate the role of run-off associated herbicides in the deteriorated health of intertidal seagrass meadows. Dose response curves of common herbicides were performed and their toxicity equivalents elucidated to assist in analysis. The results of the assay were reproducible and corresponded strongly with results of chemical analyses. The incorporation of the assay into the assessment of surface waters added an important aspect to the study by allowing investigation of the toxicity of cumulative herbicide concentrations and yielding biologically relevant data. The highest herbicide concentration detected during the study was equivalent to 0.23 mu g 1(-1) diuron; a concentration known to inhibit photosynthetic efficiency of the assay biomaterial by approximately 3%. (c) 2004 Elsevier Ltd. All rights reserved.

The combined SPE : ToxY-PAM phytotoxicity assay; application and appraisal of a novel biomonitoring tool for the aquatic environment

Mounting concerns regarding the environmental impact of herbicides has meant a growing requirement for accurate, timely information regarding herbicide residue contamination of, in particular, aquatic systems. Conventional methods of detection remain limited in terms of practicality due to high costs of operation and the specialised information that analysis provides. A new phytotoxicity bioassay was trialled for the detection of herbicide residues in filter-purified (Milli-Q) as well as natural waters. The performance of the system, which combines solid-phase extraction (SPE) with the ToxY-PAM dual-channel yield analyser (Heinz Walz GmbH), was tested alongside the traditional method of liquid chromatography-mass spectrometry (LC-MS). The assay methodology was found to be highly sensitive (LOD 0.1 ng L-1 diuron) with good reproducibility. The study showed that the assay protocol is time effective and can be employed for the aquatic screening of herbicide residues in purified as well as natural waters.

Mechanisms of acetohydroxyacid synthases

Acetohydroxyacid synthases are thiamin diphosphate- (ThDP-) dependent biosynthetic enzymes found in all autotrophic organisms. Over the past 4-5 years, their mechanisms have been clarified and illuminated by protein crystallography, engineered mutagenesis and detailed single-step kinetic analysis. Pairs of catalytic subunits form an intimate dimer containing two active sites, each of which lies across a dimer interface and involves both monomers. The ThDP adducts of pyruvate, acetaldehyde and the product acetohydroxyacids can be detected quantitatively after rapid quenching. Determination of the distribution of intermediates by NMR then makes it possible to calculate individual forward unimolecular rate constants. The enzyme is the target of several herbicides and structures of inhibitor-enzyme complexes explain the herbicide-enzyme interaction.

Structure-activity relationships for a new family of sulfonylurea herbicides

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Phytotoxicity of surface waters of the Thames and Brisbane River Estuaries: A combined chemical analysis and bioassay approach for the comparison of two systems

The Thames Estuary, UK, and the Brisbane River, Australia, are comparable in size and catchment area. Both are representative of the large and growing number of the world's estuaries associated with major cities. Principle differences between the two systems relate to climate and human population pressures. In order to assess the potential phytotoxic impact of herbicide residues in the estuaries, surface waters were analysed with a PAM fluorometry-based bioassay that employs the photosynthetic efficiency (photosystem II quantum yield) of laboratory cultured microalgae, as an endpoint measure of phytotoxicity. In addition, surface waters were chemically analysed for a limited number of herbicides. Diuron atrazine and simazine were detected in both systems at comparable concentrations. In contrast, bioassay results revealed that whilst detected herbicides accounted for the observed phytotoxicity of Brisbane River extracts with great accuracy, they consistently explained only around 50% of the phytotoxicity induced by Thames Estuary extracts. Unaccounted for phytotoxicity in Thames surface waters is indicative of unidentified phytotoxins. The greatest phytotoxic response was measured at Charing Cross, Thames Estuary, and corresponded to a diuron equivalent concentration of 180 ng L-1. The study employs relative potencies (REP) of PSII impacting herbicides and demonstrates that chemical analysis alone is prone to omission of valuable information. Results of the study provide support for the incorporation of bioassays into routine monitoring programs where bioassay data may be used to predict and verify chemical contamination data, alert to unidentified compounds and provide the user with information regarding cumulative toxicity of complex mixtures. (c) 2005 Elsevier B.V. All rights reserved.