Comparative Analysis of Laboratory-Based and Spectroscopic Methods Used to Estimate the Algal Density of Chlorella vulgaris.

Chlorella vulgaris is of great importance in numerous exploratory or industrial applications (e.g., medicals, food, and feed additives). Rapid quantification of algal biomass is crucial in photobioreactors for the optimization of nutrient management and the estimation of production. The main goal of this study is to provide a simple, rapid, and not-resource-intensive estimation method for determining the algal density of C. vulgaris according to the measured parameters using UV-Vis spectrophotometry. Comparative assessment measurements were conducted with seven different methods (e.g., filtration, evaporation, chlorophyll a extraction, and detection of optical density and fluorescence) to determine algal biomass. By analyzing the entire spectra of diluted algae samples, optimal wavelengths were determined through a stepwise series of linear regression analyses by a novel correlation scanning method, facilitating accurate parameter estimation. Nonlinear formulas for spectrometry-based estimation processes were derived for each parameter. As a result, a general formula for biomass concentration estimation was developed, with recommendations for suitable measuring devices based on algae concentration levels. New values for magnesium content and the average single-cell weight of C. vulgaris were established, in addition to the development of a rapid, semiautomated cell counting method, improving efficiency and accuracy in algae quantification for cultivation and biotechnology applications.

Ecotoxicological Evaluation of Safener and Antimicrobial Additives in Isoxaflutole-Based Herbicide Formulations.

The environmental load by isoxaflutole and its formulated herbicide products has increasingly become apparent because, after the ban of atrazine, isoxaflutole has become its replacement active ingredient (a.i.). Obtaining information regarding the fate of this a.i. in environmental matrices and its ecotoxicological effects on aquatic organisms is essential for the risk assessment of the herbicide. In this study, the effects of Merlin Flexx- and Merlin WG75 formulated isoxaflutole-based herbicide products and two selected additives (cyprosulfamide safener and 1,2-benzisothiazol-3(2H)-one antimicrobial agent) were investigated on Raphidocelis subcapitata in growth inhibition assays. In ecotoxicological tests, two conventional (optical density and chlorophyll-a content) and two induced fluorescence-based (Fv*/Fp: efficiency of the photosystem PSII and Rfd* changes in the observed ratio of fluorescence decrease) endpoints were determined by UV-spectrophotometer and by our FluoroMeter Module, respectively. Furthermore, dissipation of isoxaflutole alone and in its formulated products was examined by an HPLC-UV method. In ecotoxicological assays, the fluorescence-based Rfd* was observed as the most sensitive endpoint. In this study, the effects of the safener cyprosulfamide and the antimicrobial agent 1,2-benzisothiazol-3(2H)-one on R. subcapitata is firstly reported. The results indicated that the isoxaflutole-equivalent toxicity of the mixture of the isoxaflutole-safener-antimicrobial agent triggered lower toxicity (EC50 = 2.81 ± 0.22 mg/L) compared to the individual effect of the a.i. (EC50 = 0.02 ± 0.00 mg/L). The Merlin Flexx formulation (EC50 = 27.04 ± 1.41 mg/L) was found to be approximately 50-fold less toxic than Merlin WG75, which can be explained by the different chemical characteristics and quantity of additives in them. The additives influenced the dissipation of the a.i. in Z8 medium, as the DT50 value decreased by approximately 1.2- and 3.5-fold under light and dark conditions, respectively.

Hormesis, the Individual and Combined Phytotoxicity of the Components of Glyphosate-Based Formulations on Algal Growth and Photosynthetic Activity.

The occurrence of the market-leading glyphosate active ingredient in surface waters is a globally observed phenomenon. Although co-formulants in pesticide formulations were considered inactive components from the aspects of the required main biological effect of the pesticide, several studies have proven the high individual toxicity of formulating agents, as well as the enhanced combined toxicity of the active ingredients and other components. Since the majority of active ingredients are present in the form of chemical mixtures in our environment, the possible combined toxicity between active ingredients and co-formulants is particularly important. To assess the individual and combined phytotoxicity of the components, glyphosate was tested in the form of pure active ingredient (glyphosate isopropylammonium salt) and herbicide formulations (Roundup Classic and Medallon Premium) formulated with a mixture of polyethoxylated tallow amines (POEA) or alkyl polyglucosides (APG), respectively. The order of acute toxicity was as follows for Roundup Classic: glyphosate < herbicide formulation < POEA. However, the following order was demonstrated for Medallon Premium: herbicide formulation < glyphosate < APG. Increased photosynthetic activity was detected after the exposure to the formulation (1.5-5.8 mg glyphosate/L and 0.5-2.2 mg POEA/L) and its components individually (glyphosate: 13-27.2 mg/L, POEA: 0.6-4.8 mg/L), which indicates hormetic effects. However, decreased photosynthetic activity was detected at higher concentrations of POEA (19.2 mg/L) and Roundup Classic (11.6-50.6 mg glyphosate/L). Differences were demonstrated in the sensitivity of the selected algae species and, in addition to the individual and combined toxicity of the components presented in the glyphosate-based herbicides. Both of the observed inhibitory and stimulating effects can adversely affect the aquatic ecosystems and water quality of surface waters.

Herbivorous Juvenile Grass Carp (Ctenopharyngodon idella) Fed with Genetically Modified MON 810 and DAS-59122 Maize Varieties Containing Cry Toxins: Intestinal Histological, Developmental, and Immunological Investigations.

Feeding experiments with juvenile grass carp (Ctenopharyngodon idella) fed with genetically modified maize MON 810 or DAS-59122 dried leaf biomass were carried out with 1-, 3- and 6-month exposures. Dosages of 3-7 µg/fish/day Cry1Ab or 18-55 µg/fish/day Cry34Ab1 toxin did not cause mortality. No difference occurred in body or abdominal sac weights. No differences appeared in levels of inorganic phosphate, calcium, fructosamine, bile acids, triglycerides, cholesterol, and alanine and aspartame aminotransferases. DAS-59122 did not alter blood parameters tested after 3 months of feeding. MON 810 slightly decreased serum albumin levels compared to the control, only in one group. Tapeworm (Bothriocephalus acheilognathi) infection changed the levels of inorganic phosphate and calcium. Cry34Ab1 toxin appeared in blood (12.6 ± 1.9 ng/mL), but not in the muscle. It was detected in B. acheilognathi. Cry1Ab was hardly detectable in certain samples near the limit of detection. Degradation of Cry toxins was extremely quick in the fish gastrointestinal tract. After 6 months of feeding, only mild indications in certain serum parameters were observed: MON 810 slightly increased the level of apoptotic cells in the blood and reduced the number of thrombocytes in one group; DAS-59122 mildly increased the number of granulocytes compared to the near-isogenic line.

Appearance of Thiacloprid in the Guttation Liquid of Coated Maize Seeds.

Thiacloprid (TCL) uptake by maize plants that emerge from coated seeds has been investigated and characterized via measurements of the compound in the guttation liquid. TCL levels were determined in the guttation liquid: (a) under field and semi-field conditions, (b) for different maize varieties, (c) applying different dosages, and (d) as affected by cross-contamination between maize seeds via soil. Cross-contamination was described by uptake interactions between seeds coated with TCL and neighboring seeds not coated or coated with other neonicotinoids, e.g., either thiamethoxam (TMX) or clothianidin (CLO). TCL levels remained under 100 µg/mL in the guttation liquid under field conditions, and were quantifiable even on the 39th day after planting of coated seeds. Higher levels up to 188.6 µg/mL were detected in plants grown under semi-field conditions in pots. Levels in the guttation liquid were also found to be influenced by the applied dosages. The uptake of TCL was found to vary for different maize varieties. Appearance of TCL as a cross-contaminant in the guttation liquid of neighboring plants emerging from non-coated maize seeds indicates translocation of the compound via soil. Peak levels of TCL cross-contamination were found to be lower (43.6 µg/mL) than the corresponding levels in the parent maize plants emerging from coated seeds (107.5 µg/mL), but values converge to each other. Similar trends were observed with neighboring seeds coated with other neonicotinoids (TMX or CLO). The translocation rate of TCL and its uptake by other plants seem to be lower than that of TMX or CLO.

Neonicotinoids: Spreading, Translocation and Aquatic Toxicity.

Various environmental and ecotoxicological aspects related to applications of neonicotinoid insecticides are assessed. Dosages of neonicotinoids applied in seed coating materials were determined and are compared to other applications (spray and granule). Environmental levels in soils and affecting factors in translocation are discussed. Excretion of neonicotinoids via guttation from coated maize seeds up to two months upon emergence, as well as cross-contamination of plants emerged from non-coated seeds or weeds nearby have been demonstrated. Contamination of surface waters is discussed in scope of a worldwide review and the environmental fate of the neonicotinoid active ingredients and the formulating surfactant appeared to be mutually affected by each other. Toxicity of neonicotinoid active ingredients and formulations on Daphnia magna completed with some investigations of activity of the detoxifying glutathione S-transferase enzyme demonstrated the modified toxicity due to the formulating agents. Electrophysiological results on identified central neurons of the terrestrial snail Helixpomatia showed acetylcholine antagonist (inhibitory) effects of neonicotinoid insecticide products, but no agonist (ACh-like) effects were recorded. These data also suggested different molecular targets (nicotinergic acetylcholine receptors and acetylcholine esterase enzyme) of neonicotinoids in the snail central nervous system.

Aquatic toxicity and loss of linear alkylbenzenesulfonates alone and in a neonicotinoid insecticide formulation in surface water.

Substance losses of linear alkylbenzene sulfonates (LASs) in a neat surfactant mixture, in an insecticide formulation Mospilan 20 SG, and in solutions with different neonicotinoid active ingredients (AIs) was studied in distilled water and in surface water samples originated from River Danube. Analytical measurements were performed both by HPLC-UV and commercial ELISA methods. Loss rates of LASs were found different in these aqueous matrices, with decomposition rates higher for the neat surfactant mixture than for Mospilan 20 SG (nearly 2- and 9-fold in distilled water and in surface water from River Danube, respectively). Half-lives determined in surface water from River Danube were shown to be affected by the presence of neonicotinoid AIs thiacloprid > imidacloprid > acetamiprid (ACE), while clothianidin and thiamethoxam did not affect LAS decomposition. Aquatic toxicity of Mospilan 20 SG, along with that of its AI ACE and co-formulant LAS, as well as the mixture of ACE and LAS was also investigated in the 48-h acute immobilisation assay on the water flea (Daphnia magna) aquatic indicator organism. LAS appeared to be significantly (8-fold) more toxic in the D. magna test than ACE, and the toxicity of the formulated insecticide was found to be 1.3 and 19.6 times higher than explained by its AI and LAS content, respectively, indicating synergistic toxicity. The strongest synergy between ACE and LASs was observed, when the neat forms of the two substances were applied in combination at concentrations equivalent to those in Mospilan 20 SG.

Radiolysis of sulfonamide antibiotics in aqueous solution: Degradation efficiency and assessment of antibacterial activity, toxicity and biodegradability of products.

Numerous studies have been published on the radiolysis of sulfonamide antibiotic solutions but little effort has been made to monitor the biological properties of degradation products. A complex approach should also clarify the changes in antibacterial activity and biodegradability, besides the usual screening of toxicity. To fill this gap, the ionizing radiation induced degradation of four sulfonamide antibiotics was investigated in dilute aqueous solutions, with emphasis on the biological assessment of decomposition products. Complete removal of sulfonamides was achieved by a low absorbed dose (1.5kGy). 2-2.5kGy dose was needed to transform the persistent initial molecules to substances biodegradable in both river water and activated sludge. The ratio of the biological and chemical oxygen demand increased from <0.21 to at least 0.59, but values as high as 0.80 were also measured. It was demonstrated that antibacterial activity is due to the initial molecules, as it disappeared when the sulfamethoxazole concentration decreased below the minimal inhibitory concentration (30 µM). This means that the products have no antibacterial activity. Toxicity testing performed on test organisms from three different trophic levels and activated sludge evidenced that the toxicity depends both on the test organism and on the sulfonamide used. The degradation of initial molecules is not always enough to eliminate the environmental risk due to the toxic products formed e.g. inhibitory effects to Vibrio fischeri increased by 34% at 2.5kGy. For this reason, complex biological assessment of treated solutions has to play an important role in development and optimization of advanced treatment techniques.

Authorization and Toxicity of Veterinary Drugs and Plant Protection Products: Residues of the Active Ingredients in Food and Feed and Toxicity Problems Related to Adjuvants.

Chemical substances applied in animal husbandry or veterinary medicine and in crop protection represent substantial environmental loads, and their residues occur in food and feed products. Product approval is governed differently in these two sectors in the European Union (EU), and the occurrence of veterinary drug (VD) and pesticide residues indicated by contamination notification cases in the Rapid Alert System for Food and Feed of the EU also show characteristic differences. While the initial high numbers of VD residues reported in 2002 were successfully suppressed to less than 100 cases annually by 2006 and on, the number of notification cases for pesticide residues showed a gradual increase from a low (approximately 50 cases annually) initial level until 2005 to more than 250 cases annually after 2009, with a halt occurring only in 2016. Main notifiers of VD residues include Germany, Belgium, the UK, and Italy (63, 59, 42, and 31 notifications announced, respectively), and main consigning countries of non-compliances are Vietnam, India, China, and Brazil (88, 50, 34, and 23 notifications, respectively). Thus, countries of South and Southeast Asia are considered a vulnerable point with regard to VD residues entering the EU market. Unintended side effects of VDs and plant protection products may be caused not only by the active ingredients but also by various additives in these preparations. Adjuvants (e.g., surfactants) and other co-formulants used in therapeutic agents and feed additives, as well as in pesticide formulations have long been considered as inactive ingredients in the aspects of the required main biological effect of the pharmaceutical or pesticide, and in turn, legal regulations of the approval and marketing of these additives specified significantly less stringent risk assessment requirements, than those specified for the active ingredients. However, numerous studies have shown additive, synergistic, or antagonistic side effects between the active ingredients and their additives in formulated products; moreover, toxicity has been evidenced for various additives. Therefore, toxicological evaluation of surfactants and other additives is essential for proper environmental risk assessment of formulations used in agriculture including animal husbandry and plant protection.