The influence of natural dissolved organic matter on herbicide toxicity to marine microalgae is species-dependent.

Microalgae, which are the foundation of aquatic food webs, may be the indirect target of herbicides used for agricultural and urban applications. Microalgae also interact with other compounds from their environment, such as natural dissolved organic matter (DOM), which can itself interact with herbicides. This study aimed to evaluate the influence of natural DOM on the toxicity of three herbicides (diuron, irgarol and S-metolachlor), singly and in ternary mixtures, to two marine microalgae, Chaetoceros calcitrans and Tetraselmis suecica, in monospecific, non-axenic cultures. Effects on growth, photosynthetic efficiency (Ф'M) and relative lipid content were evaluated. The chemical environment (herbicide and nutrient concentrations, dissolved organic carbon and DOM optical properties) was also monitored to assess any changes during the experiments. The results show that, without DOM, the highest irgarol concentration (I0.5: 0.5 mg.L-1) and the strongest mixture (M2: irgarol 0.5 µg.L-1 + diuron 0.5 µg.L-1 + S-metolachlor 5.0 µg.L-1) significantly decreased all parameters for both species. Similar impacts were induced by I0.5 and M2 in C. calcitrans (around -56% for growth, -50% for relative lipid content and -28% for Ф'M), but a significantly higher toxicity of M2 was observed in T. suecica (-56% and -62% with I0.5 and M2 for growth, respectively), suggesting a possible interaction between molecules. With DOM added to the culture media, a significant inhibition of these three parameters was also observed with I0.5 and M2 for both species. Furthermore, DOM modulated herbicide toxicity, which was decreased for C. calcitrans (-51% growth at I0.5 and M2) and increased for T. suecica (-64% and -75% growth at I0.5 and M2, respectively). In addition to the direct and/or indirect (via their associated bacteria) use of molecules present in natural DOM, the characterization of the chemical environment showed that the toxic effects observed on microalgae were accompanied by modifications of DOM composition and the quantity of dissolved organic carbon excreted and/or secreted by microorganisms. This toxicity modulation in presence of DOM could be explained by (i) the modification of herbicide bioavailability, (ii) a difference in cell wall composition between the two species, and/or (iii) a higher detoxification capacity of C. calcitrans by the use of molecules contained in DOM. This study therefore demonstrated, for the first time, the major modulating role of natural DOM on the toxicity of herbicides to marine microalgae.

Identification of by-products and toxicity assessment in aqueous-phase hydrodechlorination of diuron with palladium on activated carbon catalysts.

The hydrodechlorination (HDC) of diuron in aqueous phase with hydrogen using two different activated carbon-supported Pd catalysts was studied. A commercial activated carbon and one prepared by chemical activation of grape seeds with phosphoric acid (GS) were evaluated as supports, being the catalysts tested in a wide range of temperature (30-100 °C) and space-time (78-311 kgcat h mol(-1)). Diuron conversion was above 70% under all the conditions tested. The Pd catalyst supported on GS showed the highest activity in terms of diuron conversion within the temperature range studied, allowing nearly complete conversion above 50 °C. However, a gradual loss of activity with time was observed for this catalyst. A complete route of hydrogenation of diuron was elucidated. Two reaction routes one leading to fenuron and another to aniline were identified. As the temperature and space-time were increased, the formation of fenuron (via monuron) was found to be favored. The toxicity of the reaction products was evaluated, being the route to fenuron and monuron, the one giving rise to a significant decrease of ecotoxicity.

Diuron-induced rat bladder epithelial cytotoxicity.

Diuron, a substituted urea herbicide, is carcinogenic to the rat urinary bladder at high dietary levels (2500 ppm). To further elucidate the mode of action, this study aimed to determine the time course and sequence of bladder cytotoxic and proliferative changes induced by diuron treatment of male Wistar rats. Rats were randomized into two groups (control and 2500 ppm diuron) and treated for 28 days. Ten rats from each group were terminated on each of study days 1, 3, 7, or 28. Scanning electron micro scopy (SEM) showed urothelial cell swelling beginning on day 1, and by day 28, showed extensive necrosis, exfoliation and piling up of cells suggestive of hyperplasia. No difference in the bromo deoxyuridine labeling index was detected. In a second experiment, rats were randomized into control and diuron-treated groups and treated for 7 days or 8 weeks. After 7 days, transmission electron microscopy showed cell degenerative changes and distention of the cytoplasm, organelles, and nuclei characteristic of cytolysis. This resulted in protrusion of the superficial cells into the lumen, corresponding to the cell swelling observed previously by SEM. After 8 weeks, bladders in the diuron-treated group showed an increased incidence of simple hyperplasia by light microscopy (6/10, p < 0.05) compared with controls (0/10) and a significantly different SEM classification. In summary, our results support the hypothesis that urothelial cytotoxicity followed by regenerative cell proliferation are the sequential key events that occur with high-dose diuron exposure in rats.

PO43- dependence of the tolerance of autotrophic and heterotrophic biofilm communities to copper and diuron.

Pollution-induced community tolerance (PICT) concept is based on the assumption that the toxicant exerts selection pressure on the biological communities when exposure reaches a critical level for a sufficient period of time and therefore sensitive species are eliminated. However, induced tolerance of microbial biofilm communities cannot be attributed solely to the presence of toxicants in rivers but also to various environmental factors, such as amount of nutrients. An experimental study was undertaken to highlight the potential impact of a phosphorus gradient on the sensitivity of periphytic microbial community to Cu and diuron. Biofilms were exposed to real-world levels of chronic environmental contamination of toxicants with a phosphorus gradient. Biofilm sensitivity to Cu and diuron was assessed by performing short-term inhibition tests based on photosynthetic efficiency to target photoautotrophs, extracellular enzyme activity (beta-glucosidase and leucine-aminopeptidase) and substrate-induced respiration activity to target heterotrophs. The impact of P-gradient associated to pollution was evaluated by measuring pesticide concentrations in biofilms, biomass parameters (chla, AFDW), bacterial cell density, photosynthetic efficiency and community structure (using 18S and 16S rDNA gene analysis to target eukaryotes and DGGE and HPLC pigment analysis to target bacteria and photoautotrophs). The obtained results show that depending on the studied toxicant and the used structural or functional parameter, the effect of the phosphorus gradient was variable. This highlights the importance of using a range of parameters that target all the biological communities in the biofilm. The PICT method can be regarded as a good tool for assessing anthropogenic environmental contamination, but it is necessary to dissociate the real impact of toxicants from environmental factors.

Spermatogenesis, epididymis morphology and plasma sex steroid secretion in the male lizard Podarcis sicula exposed to diuron.

The present study investigates the effects of diuron, a substituted urea-based herbicide, in the male lizard Podarcis sicula utilizing quantitative and qualitative morphological features of the reproductive system and endocrinological analysis. Besides the control group, lizards were divided into three groups ([a-c]) (n=6/group) and placed for 3 weeks in terraria on polluted soil substrate sprayed with 3.75 L/ha of herbicide Toterbane 50F (50% diuron). Each terrarium was supplemented either with drinking water contaminated by herbicide (i.e. 1.08 microg/mL of diuron; group [a]), or with food contaminated by herbicide (i.e. 5.4 mg of diuron; group [b]), or with drinking water and food contaminated as described above (group [c]). None of the animals exposed to the contaminant showed any signs of general toxicity or death during the course of the experiments. Severe testicular effects are evidenced in all herbicide-treated groups, although, such effects are of a greater magnitude in lizards exposed to contaminated water (groups [a] and [c]). The main degenerative changes observed include: (1) a significant decrease in the mean gonadosomatic index of 55% in group [a] (P<0.001), 21% in group [b] (P<0.01) and 34% in group [c] (P<0.001) compared with control group; (2) a significant shrinking (P<0.001) of seminiferous tubule diameter (more than 60% of the control) in groups [a] and [c], and about 18% in group [b] (P<0.01); (3) a significant decrease in the crude numbers of spermatogonia of 92% in group [a] (P<0.001), 27% in group [b] (P<0.01) and 62% in group [c] (P<0.001) compared with control group. A complete loss of meiotic and mature germ cells in groups [a] and [c], and a reduction of primary spermatocytes, secondary spermatocytes and spermatids (more than 27% of the control) and a decrease of spermatozoa (more than 90% of the control) in group [b]; and (4) an hypertrophy of interstitial connective tissue which contains numerous lymphocytes, neutrophils and monocytes. The decrease and/or loss of germ cells seems to be related to an induction of inflammation (necrosis) rather than to apoptotic processes. Indeed, this hypothesis is supported by a TUNEL-assay, which failed to reveal any apoptotic cells either in the seminiferous epithelium or in the interstitial space in the testis of all exposed groups. Also the epididymis appears affected by diuron exposure. In particular, in experimental groups [a] and [c] it is regressed with abundant connective tissue and low epithelial cells without secretory granules, whereas in group [b] it appears partially regressed, with some secretory granules still present. At the same time, an impairment of the plasma sex-hormone levels is observed in treated lizards, as evidenced by RIA analysis. Testosterone values significantly decreased by 43% in group [a] (P<0.001), 34% in group [b] (P<0.01) and 52% in group [c] compared with control group. Instead, 17beta-estradiol plasma content is undetectable in all diuron-exposed lizards. Taken together, the results presented here indicate that diuron exposure resulted in direct male reproductive toxicity and reveal that this lizard is suitable as a laboratory reptile species for toxicological investigations.