Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus)

The intensive production of farmed fish is at a global all‐time high, and the control of bacteria proliferation in fish farms requires the frequent use of antimicrobials. This practice raises important environmental concerns related to the emergence of antimicrobial‐resistant bacteria strains. Only a few antimicrobial drugs have been approved for use in aquaculture, one of which is florfenicol. This work studies the depletion and withdrawal period of florfenicol and its main metabolite, florfenicol amine, in pacu (Piaractus mesopotamicus), a neotropical characin widely farmed in the southern hemisphere. Juvenile pacu (average weight of 724 g) were stocked in a closed‐loop laboratory system with controlled water temperature (25.8°C), and were fed for 10 consecutive days with a diet containing an intended dose of 10 mg/florfenicol per kg bw. Muscle and skin tissues were collected at 1, 3, 6, 8, 10, 12 and 16 days post‐treatment, and florfenicol and florfenicol amine were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method. The limits of quantitation for florfenicol and florfenicol amine were 10 ng/g in muscle and 50 ng/g in skin. Considering a maximum residue limit of 1000 ng/g for the sum of florfenicol and florfenicol amine in muscle with skin in natural proportions a withdrawal period of 5 days (water temperature 25.8°C) or 129 degree days was calculated on the basis of the upper limit of the one‐sided 95% confidence interval for the 99^th percentile derived from the residue depletion study.     

Acute tolerance and histopathological effects of ammonia on juvenile maroon clownfish Premnas biaculeatus (Block 1790)

This study evaluated the median lethal concentration values (LC₅₀) and the histopathological effects of un‐ionized ammonia (NH₃^‐‐N) on juvenile maroon clownfish Premnas biaculeatus. After 96 h of exposure to different concentrations of ammonia, juveniles were sampled for histopathological evaluation. The 24 and 96 h LC50 values of NH₃^?‐N determined were 1.68 and 0.89 mg L^?1 respectively. Maroon clownfish exposed to different ammonia concentrations displayed histopathological alterations in the gills, kidney, liver and brain. Gill tissue damage included lamellar hyperplasia, lamellar shorting and hyperplasia and hypertrophy of mucous cells. The kidney showed hyperanaemia, enlarged sinusoids within an apparently decreased amount of haematopoietic tissue, oedema on tubular cells and tubular necrosis, and an enlarged Bowman's capsule. The liver presented dilatation of hepatic sinusoids, fatty deposition in hepatocytes and Mallory bodies. Examination of the brain revealed a proliferation of glial cells, and the Virchow‐Robin space indicated a severe perivascular oedema and signs of neuronal suffering with satellitosis. The results of this study indicate that juvenile maroon clownfish are relatively sensitive to ammonia and particular attention must be given to this toxic compound in culture systems.     

Changes in fatty acid and sterol composition during oogenesis in the pearl oyster Pinctada margaritifera

The fatty acid and sterol composition of the oyster Pinctada margaritifera during oogenesis and in eggs was analysed. No major differences were observed during oogenesis, but the egg composition was significantly different from that of gonads. The amount of saturated fatty acids was the highest in eggs and the C16:0 predominant (P<5%); by contrast, the amount of 22:6(n‐3) was significantly lower (P<5%) than in gonads. No major differences were observed for the polar lipid (PL) composition during oogenesis. The main free sterols in gonads and eggs were cholesterol and brassicasterol. Among free sterols, the proportion of cholesterol diminished continuously from the beginning to the end of gonad maturation, and this decrease persisted in eggs after spawning. Cholesterol represented 40% to 55% of the sterol ester encountered in gonad and eggs. This study allowed us to investigate the fatty acid and sterol composition during oogenesis of the pearl oyster P. margaritifera, leading to a clearer understanding of the nutritional requirements of pearl oyster during the reproduction process.     

Effect of the dusk photoperiod change from light to dark on the incubation period of eggs of the spotted rose snapper, Lutjanus guttatus (Steindachner)

Spotted rose snapper, Lutjanus guttatus (Steindachner), eggs were incubated under different photoperiods to examine the effect of photoperiod on incubation. The eggs from two fish were incubated under five artificial photoperiods: constant dark (D), constant light (L) from 06:00 hours and 6, 10 and 14 h of light from 06:00 hours. The eggs from seven other fish were incubated under a natural photoperiod. Different spawning times (21:00 – 01:00 hours) and different photoperiods combined to give the start of the dusk photoperiod change after 11–23 h of incubation. Constant light or applying the dusk photoperiod change after ≥20 h of incubation appeared to extend the hatching period. The mean hatching period for groups of eggs incubated in darkness or that received the dusk photoperiod change after ≤19 h of incubation (n=8 different groups) was 2 h 15±10 min, which was significantly lower (P<0.05) than the mean hatching period of 4 h±37 min for groups that did not receive the dusk photoperiod change or that received the dusk photoperiod change after ≥20 h of incubation (n=9 groups). However, despite these differences, the majority of the eggs hatched during a 2–3 h period from 17 to 20 h of incubation, and a sigmoid regression (r²=0.9) explained the relationship between percentage hatch and hours of incubation for all photoperiod groups.     

Effects of density on growth and survival of juvenile Red Swamp Crayfish, Procambarus clarkii (Girard), reared under laboratory conditions

The aim of this study was to quantify the importance of population density on the growth and survival of juvenile red swamp crayfish, Procambarus clarkii (Girard), and to understand the effects of density on both the period between moults and the increment per moult at high densities without food limitation. A laboratory experiment was performed to determine growth and survival of juvenile crayfish at five densities (20, 40, 60, 80 and 100 crayfish m^?2). Density clearly affected crayfish growth. The impact of density on final weight was higher than on total length increase, growth rate and final length (FL) (by decreasing order of effect). When comparing minimum and maximum densities, we observed that FL suffered a 34% reduction. Number of moults, mean intermoult period (IP) and survival were not significantly affected by density. Our results indicated that the IP and the percentage of increment per moult are affected by the size of crayfish before moult and we provide equations for these relations. Our findings are relevant both for crayfish aquaculture management and for the management of wild or harvested rice‐field crayfish populations.     

Nutrients removed by Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in integrated cultivation with fishes in re-circulating water

The potential of the red alga Kappaphycus alvarezii to remove nutrients was tested to treat effluents of Trachinotus carolinus fish cultivation, and the production of carrageenan in this condition was analyzed. Experiments were conducted in four tanks of 8000 L with approximately 1200 fishes of 30 g each integrated with three tanks of 100 L with 700 g of K. alvarezii, as initial biomass per tank. Seawater was re-circulated between tanks with seaweed and with fish. As a control, three tanks with seawater circulating in an open system were utilized. Seawater samples were collected daily for 10 days and concentrations of nitrate, nitrite, ammonium and phosphate were determined in the inflow and outflow water of the tanks. Significant differences between both collecting points were considered as nutrient removal by the seaweed. Growth rates and carrageenan yields were also analyzed in seaweed cultivated in seawater and in effluents. Growth rates of seaweed cultivated in tanks were lower than those obtained in open sea and in laboratory cultivation. Effluents had concentrations of nitrate and nitrite ca. 100 times higher than in the control. Maximum values of nutrient removal on effluents were: nitrate = 18.2%; nitrite = 50.8%; ammonium = 70.5% and phosphate = 26.8%. All plants survived throughout the experimental period, but some developed “ice–ice”, a disease associated with physiological stress. After the experimental period, some plants selected and cultivated in open sea presented higher growth rates in 40 days, indicating nutrient storage. No significant differences between carrageenan yields of K. alvarezii cultivated in seawater and in the effluents were observed. Our results show that K. alvarezii can be utilized as a biofilter for fish cultivation effluents, reducing the eutrophication process and can also be processed for carrageenan production, which provides an additional benefit to the fisheries.     

Perkinsus sp. infestation in carpet-shell clams, Ruditapes decussatus (L), along the Portuguese coast. Results from a 2-year survey

This study characterises Perkinsus atlanticus disease prevalence and intensity within the economically important beds of the clam Ruditapes decussatus along the Iberian Atlantic coast. Samples were collected from five different sites along the Portuguese coast (Ria Formosa, Ria do Alvor, V.N. Milfontes, Aveiro, Lagoa de Obidos) and in Galicia (Ria de Arosa) between winter 2000/2001 and 2002/2003. The infection level was evaluated by the Ray Fluid Thioglycollate medium (RFTM) method using the body burden assay. In addition, the measure of the condition index (CI) (percentage between the edible part and the total weight of the clams) was conducted to investigate a possible correlation between this parameter and the intensity of the infection. However, no clear relationship between infection intensity and condition index (CI) could be determined although a significant decline in CI was found for heavily infected clams. Results of a 2-year survey demonstrate the presence of Perkinsus sp. in all sampling sites albeit with different intensities. Sites where human interference was minimal showed the lowest levels of infection. There were no significant differences in Perkinsus sp. infection intensity between samples collected in winter and summer, in contrast with several studies describing higher intensities at the end of the summer. Nevertheless, major differences were observed from year to year and site to site, indicating that factors other than those responsible for seasonal climatic variations might affect the prevalence and the intensity of Perkinsus sp. infection.     

Fine-earth translocation by tillage in stony soils in the Guadalentin, south-east Spain: an investigation using caesium-134

Tillage erosion is increasingly recognised as an important soil erosion process on agricultural land. In view of its potential significance, there is a clear need to broaden the experimental database for the magnitude of tillage erosion to include a range of tillage implements and agricultural environments. The study discussed in this paper sought to address the need for such data by examining tillage erosion by a duckfoot chisel plough in stony soils on steep slopes in a semi-arid environment. Results of the investigation of coarse fraction (rock fragment) translocation by tillage in this environment have been presented elsewhere and the paper focuses on tillage translocation and erosion of the fine earth. Tillage translocation was measured at 10 sites, representing both upslope and downslope tillage by a duckfoot chisel plough on five different slopes, with tangents ranging from 0.02 to 0.41. A fine-earth tracer, comprising fine earth labelled with ¹³⁴Cs, was introduced into the plough layer before tillage. After a single pass of the plough, incremental samples of plough soil were excavated and sieved to separate the fine earth from the rock fragments. Translocation of the fine-earth tracer was established by analysing the ¹³⁴Cs content of the samples of fine earth. These data were used to establish translocation distances for each combination of slope and tillage direction. Translocation distances of the fine earth were not significantly different from translocation distances of the coarse fraction. For all sites, except uphill on the 0.41 slope, translocation distances were found to be linearly related to slope tangent. The soil flux due to tillage for each site was calculated using the translocation distance and the mass per unit area of the plough layer. For slopes with tangents <0.25, the relationship between soil flux and tangent was linear and the soil flux coefficient derived was 520–660 kg m⁻¹ per pass. This is much larger than the coefficients found in other studies and this high magnitude is attributed to the non-cohesive nature and high rock fragment content of the soil in this investigation. A second contrast with previous studies was found in non-linearity in the relationship between soil flux and tangent when steeper slopes were included. This was a product of variation in plough depth between the steepest slopes and the remainder of the study area. On the basis of the study it is suggested that an improved understanding of tillage erosion may be obtained by considering the dual processes of tillage detachment (mass per unit area of soil subject to tillage) and tillage displacement (equivalent to translocation distance per pass) in assessing, comparing and modelling tillage translocation. An improved model is proposed that recognises the complexity of soil redistribution by tillage, provides a framework for process-based investigation of the controls on tillage fluxes, and allows identification of potential self-limiting conditions for tillage erosion.     

Effects of substrate sterilization,fungicide treatment,and mycorrhization helper bacteria on ectomycorrhizal formation of pedunculate oak (Quercus robur) inoculated with Laccaria laccata in two peat bare-root nurseries

Summary Pedunculate oak seedlings (Quercus robur) inoculated with the ectomycorrhizal fungus Laccaria lacata were grown for 1 year on fertilized sphagnum peat in two nurseries. Three factors affecting microbial populations in the substrate were studied, fungicide treatment of the seeds, peat disinfection before sowing (methyl bromide or steam pasteurization), and inoculation with mycorrhization helper bacteria. Treatment of acorns with Iprodione had no depressive effect on mycorrhiza formation. Both disinfection techniques were equivalent, stimulating or depressing mycorrhiza formation depending on the initial microflora in the peat. The introduction of two previously selected mycorrhization helper bacteria (one Pseudomonas fluorescens and one unidentified fluorescent pseudomonad), isolated from L. laccata sporocarps associated with Douglas fir—L. laccata ectomycorrhizas in other nurseries, significantly increased the mycorrhizal rate from 30 to 53% of the short roots. The implications of these results for the controlled mycorrhization of planting stocks and the specificity of mycorrhization helper bacteria are discussed.     

Recovery of pigments from Origanum majorana L. by extraction with supercritical carbon dioxide

Extraction of pigments (chlorophylls and carotenoids) from marjoram (Origanum majorana L.) with supercritical carbon dioxide was investigated. The aim of this study was to map the effects of extraction pressure and temperature on the yield of coloring materials by applying a 3(2) full factorial design with three repeated tests in the center of the design. For comparison, laboratory and pilot plant Soxhlet extractions were carried out using ethanol and n-hexane solvents. The compositions of pigments in marjoram extracts were determined by HPLC. Similar amounts of carotenoids, in addition to 40% of chlorophylls and their derivatives, were recovered from the supercritical fluid extraction, in comparison to the ethanol Soxhlet extraction.