Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Identification of fructooligosaccharides in different banana cultivars

Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. This work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and gas chromatography-mass spectrometry (GC-MS). An initial screening of eight cultivars (Ouro, Nanic?o, Prata, Ma??, Mysore, Pacovan, Terra, and Figo) in a full-ripe stage showed that 1-kestose, the first member of the FOS series (amounts between 297 and 1600 microg/g of DM), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level ( approximately 200 mg/g of DM), which seems to trigger the synthesis of 1-kestose (the low amounts of FOS, below the functional recommended dose, indicates that banana cannot be considered a good source of FOS).     

Multivariate approach to the measurement of tomato maturity and gustatory attributes and their rapid assessment by Vis-NIR spectroscopy

Standard methods for determining quality and maturity are time- and labor-consuming and generally measure individual criteria at a specific time, without considering relationships among quality parameters. To propose a rapid and nondestructive analysis method describing multidimensional quality variables, an experiment was undertaken with mature green to overripe tomato fruits found on the North American retail markets. Factor analysis was used to analyze results. Four factors were considered, representing 81% of total variance. The first one, tomato maturity stage (TMS), is related to color, lycopene content, firmness, titratable acidity (TA), pH, and soluble solids (SS). Nondestructive rapid assessment by vis-NIR spectroscopy can predict TMS (r(2)=0.93). Factors 2 and 3 are both related to taste and should be considered simultaneously. Factor 2, called the gustatory index, is linked to electrical conductivity (EC), SS, TA, and pH. Factor 3, defined by SS, can be directly measured by a refractometer. Four categories of taste are proposed; the most desirable one ranks high both in soluble solids (above 4.5 degrees Brix) and in gustatory index (above 0). It was not possible to measure the gustatory index by vis-NIR spectroscopy (r(2)=0.17), but it can be estimated by EC, using a simple formula. The proposed limit between high and low gustatory index then corresponds to an EC of 5.4 mS/cm. Factor 4, variety, mostly discriminates the pink tomato type and field-grown samples from other varieties.     

Nondestructive measurement of fresh tomato lycopene content and other physicochemical characteristics using visible-NIR spectroscopy

Measurement of fresh tomato fruit overall quality, and particularly lycopene content, is challenging in the context of high-volume production. An experiment was conducted to simultaneously measure various quality parameters of tomato in a nondestructive manner using vis-NIR reflectance spectroscopy and chemometrics. The sampling set included different cultivars that are obtainable from both retailers' shelves and two greenhouse producers. Results indicate that lycopene content was accurately predicted [r(2) = 0.98; root mean square error of cross-validation (RMSECV) = 3.15 mg/kg], along with color variables such as Hunter a (r(2) = 0.98), L, and b (r(2) = 0.92). Tomato color index (TCI) was better predicted (r(2) = 0.96) than the a/ b ratio (r(2) = 0.89). Firmness prediction, with an r(2) of 0.75, is comparable to what is reported in the literature for other fruits and may have a practical interest. Prediction of internal quality such as pH, soluble solids, titratable acidity, and electrical conductivity was less accurate, partly due to a low variability of these parameters among samples. Predictions were robust with regard to cultivars, except for pink variety tomato. The 400-1000 nm range gave results almost as accurate as the 400-1500 nm range.     

Classification of Peruvian highland maize races using plant traits

The maize of Latin America, with its enormous diversity, has played an important role in the development of modern maize cultivars of the American continent. Peruvian highland maize shows a high degree of variation stemming from its history of cultivation by Andean farmers. Multivariate statistical methods for classifying accessions have become powerful tools for classifying genetic resources conservation and the formation of core subsets. This study has two objectives: (1) to use a numerical classification strategy for classifying eight Peruvian highland races of maize based on six vegetative traits evaluated in two years and (2) to compare this classification with the existing racial classification. The numerical classification maintained the main structure of the eight races, but reclassified parts of the races into new groups (Gi). The new groups are more separated and well defined with a decreasing accession within group × environment interaction. Most of the accessions from G1 are from Cusco Gigante, all of the accessions from G3 (except one) are from Confite Morocho, and all of the accessions from G7 are from Chullpi. Group G2 has four accessions from Huayleño and four accessions from Paro, whereas G4 has four accessions from Huayleño and five accessions from San Geronimo. Group G5 has accessions from four races, and G6 and G8 formed small groups with two and one accession each, respectively. These groups can be used for forming core subsets for the purpose of germplasm enhancement and assembling gene pools for further breeding.     

Plasma lactate and purine derivatives accumulation after exercise of increasing intensity in standardbred horses

The aim of the experiment was to study the relationship between plasma lactate and allantoin accumulation in horses undergoing five exercises differing in intensity and length. Twenty-five adult trotter horses were used (18 males, two castrated, and five females), housed in three training centers. The horses were assigned to five groups: slow trot, over 2000 m (Group 1); slow trot over 1600 m (Group 2); fast trot over 1600 m (Group 3); fast trot over 2000 m (Group 4); fast trot over 2400 m (Group 5). Plasma was obtained from blood sampled at rest, at the end of the bout of exercise and after 15 and 45 minutes from the end of the bout of exercise and analyzed for glucose, lactate, uric acid, free fatty acids (FFA) and allantoin concentrations. Accumulations of plasma lactate and allantoin (mmol/sec) were calculated as difference between end of exercise and rest and between 45 minutes sample and rest, respectively.Ranking the intensity of exercise using the lactate concentrations at the end of exercise, the level of exertion was highest for Group 3 horses and lowest for Group 5 horses (20.9 and 2.8 mmol/l, respectively). At the end of exercise, glucose concentrations were much higher for horses undertaking the more intensive exercise (Groups 3 and 4 compared to Group 2). FFA concentrations were highest at the end of exercise for Groups 2 and 3 and after 15 minutes for Groups 4 and 5. Plasma uric acid and allantoin concentrations peaked 15 and 45 minutes from the end of exercise, respectively, independently of exercise intensity. The relationship between accumulation of plasma allantoin (y, dependent variables) and lactate (x, independent variable) was non-linear: y=0.15−2.61^*x+68.3^*x² (r²=0.900; se=0.19). This suggests that allantoin accumulation could be used together with plasma lactate to calibrate the workload to muscle conditions to prevent muscle injury.     

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.     

Multiple feather follicle cysts in a Moroseta hen (Gallus gallus)

An 8-month-old white feathered, black skinned Moroseta hen was presented for examination because of numerous 2 mm- to 30 mm-diameter irregularly shaped, hard nodules in the skin of the head, wings, back, and abdomen. The nodules were confined to the skin and did not involve subcutaneous tissues. Nodules consisted of dilated feather follicles packed with a caseous tan-to-pale-yellow material admixed with feather remnants. Histologically, affected feather follicles were markedly dilated and filled with laminated keratin debris. Necrosis of the epidermis and perifollicular lymphocyte infiltration was also present. Bacteriologic investigation of internal organs was negative, while secondary bacteria, Proteus spp. and Bacillus spp., were isolated from skin nodules. A concomitant lice infestation of Menopon spp., as well as leg mange caused by Cnemidocoptes spp., were also present. These bacterial isolates and parasites were not related to the disease condition. The condition observed was differentiated from benign feather follicle tumors, and a diagnosis of multiple feather follicle cysts was made. In addition, a breed predisposition was hypothesized.     

Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumours

Acute phase proteins (APP) and biomarkers of oxidative status change in human and canine mammary tumours, however, they have not been studied in feline mammary tumours. The aims of this study were to investigate the APP and antioxidant responses in feline malignant mammary tumours, to evaluate their relation with tumour features, and to assess their prognostic value. Serum amyloid A (SAA), haptoglobin (Hp), albumin, butyrylcholinesterase (BChE), insulin‐like growth factor1 (IGF1), paraoxonase1 (PON1), total serum thiols (Thiol), glutathione peroxidase (GPox) and total antioxidant capacity determined by different assays, including trolox equivalent antioxidant capacity assessed by two different methodologies (TEAC1/2), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC), were determined in serum of 50 queens with spontaneous mammary carcinomas and of 12 healthy female cats. At diagnosis, diseased queens presented significantly higher SAA and Hp, and lower albumin, BChE, GPox, TEAC1, TEAC2 and CUPRAC than controls. Different tumour features influenced concentrations of APP and antioxidants. Increases in serum Hp, and decreases in albumin, Thiol and FRAP were significantly associated with neoplastic vascular emboli, metastasis in regional lymph nodes and/or in distant organs. Distant metastasis development during the course of the disease was associated with increases in SAA and TEAC1. At diagnosis, decreased albumin was associated with a longer survival, and BChE <1.15 μmoL/mL.minute was associated with a shorter survival time on multivariate analysis. Feline malignant mammary tumours are associated with an APP response and oxidative stress, and different tumour features influence the inflammatory response and the oxidative damage. Furthermore, some of these analytes proved to have prognostic value.     

Comparison of the intravenous and intravaginal route of oxytocin administration for cervical dilation protocol and non‐surgical embryo recovery in oestrous‐induced Santa Inês ewes

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous‐induced Santa Inês ewes (6‐day treatment with progestin‐releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6–7 days after oestrous onset and mating. All ewes received 37.5 μg of d‐cloprostenol via latero‐vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non‐surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17–40 min) and ~96% of flushing fluid could be recovered (range: 85%–100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep.