António Paulo Carvalho

First feeding of common carp larvae on diets with high levels of protein hydrolysates

A 21 day feeding trial was carried out at 24 °C aiming to evaluate the effect of diets containing high levels of protein hydrolysates on growth, survival and body composition of common carp, Cyprinus carpio L., larvae since first feeding. Eight semipurified diets based on a fish protein hydrolysate (CPSP) with or without additional nitrogen sources, such as casein, casein hydrolysates or other hydrolysates from several origins, were tested, using a diet based on yeast as a reference. High survival rates of larvae were observed at the end of the trial for all experimental diets, ranging from 97% for the yeast diet to 71% when CPSP was used as the only nitrogen source. In terms of growth, the best results were achieved with the CPSP-casein diet (1:1) and the yeast-based diet (17.5 mm total length / 82.8 mg wet weight and 16.3 mm total length / 55.4 mg wet weight, respectively). Growth of larvae fed on diets with protein hydrolysates as the only nitrogen source was considerably lower than that of larvae fed a mixture of intact protein and protein hydrolysates.

Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp.

This study compares the effects of pure anatoxin-a and cyanobacterial extracts of an anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.

POPULATION DYNAMICS OF THE RED SWAMP CRAYFISH, PROCAMBARUS CLARKII (GIRARD, 1852) FROM THE AVEIRO REGION, PORTUGAL (DECAPODA, CAMBARIDAE)

The life cycle of the red swamp crayfish, Procambarus clarkii from a shallow lagoon in the Aveiro region was studied for 14 months. Analysis of the size frequency distribution and growth provided evidence for an extended recruitment period with a peak in autumn (October/November). Females clearly outnumbered males and the average sex-ratio (males/females) was 0.36. The values for the Von Bertalanffy growth parameters were L∞ 62.01 mm, K = 0.23, and t0 = 0.11 month. = The high abundance of this species and the extended recruitment period, support the need for a proper management and exploitation of this biological resource. O ciclo de vida do lagostim-vermelho-da-Louisiana Procambarus clarkii da ria de Aveiro foi estudado ao longo de 14 meses. A analise da distribuicao das frequencias de tamanho e de crescimento mostrou um periodo de recrutamento extenso com um maximo no Outono (Outubro/Novembro). As femeas ocorreram com maior abundância que os machos, o que se traduziu numa proporcao de sexos (machos/femeas) de 0,36. Os valores calculados para os parâmetros de crescimento de Von Bertalanffy foram L∞ = 62,01 mm, K = 0,23 e t0 = 0,11 mes. A elevada abundância desta especie e o largo periodo de recrutamento apontam para a necessidade de uma adequada gestao e exploracao deste recurso biologico.

Effect of different microcystin profiles on toxin bioaccumulation in common carp (Cyprinus carpio) larvae via Artemia nauplii

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin (MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B (100 microg L(-1)EqMC-LR) for 2h. The MC concentration in the nauplii was 73.60+/-7.88ngEqMC-LRg(-1)FW (n=4, mean+/-SE) for bloom A and 87.04+/-10.31ngEqMC-LRg(-1)FW for bloom B. These contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval weight and length from day 9 were significantly different between groups A and B, and in both cases lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely correlated with the growth performance, was also significantly different between groups A and B (37.43+/-2.61 and 54.55+/-3.01ngEqMC-LRg(-1) FW, respectively, at the end of the experimental period). These results indicate that MC profile of a bloom may have differential effects on toxin accumulation/transfer and toxicity.

A preliminary study on the molecular weight profile of soluble protein nitrogen in live food organisms for fish larvae

Abstract The solubility of protein nitrogen at pH 8 in the organisms used as live food for fish larvae (rotifers and Artemia) was determined and the molecular weight profile of soluble protein nitrogen was analysed by HPLC-gel filtration chromatography. Soluble nitrogen accounted for 61% of total nitrogen in microalgae-fed rotifers, 47% in starved rotifers and 54% in newly hatched Artemia. A remarkable similarity in the profile of molecular weight distribution of soluble nitrogen was noticed among these three types of live food. The molecular weights of soluble nitrogen was partitioned as follows, respectively, in rotifers and Artemia: 84–88% and 89% >500 Da (corresponding mainly to proteins, poly- and oligopeptides), 8–11% and 4% from 200 to 500 Da (corresponding mainly to di- and tripeptides), 3–4% and 7%

Compensatory Growth Induced in Zebrafish Larvae after Pre-Exposure to a Microcystis aeruginosa Natural Bloom Extract Containing Microcystins

Early life stage tests with zebrafish (Danio rerio) were used to detect toxic effects of compounds from a Microcystis aeruginosa natural bloom extract on their embryolarval development. We carried out the exposure of developing stages of fish to complex cyanobacterial blooms containing hepatotoxic molecules - microcystins. Fish embryo tests performed with the bloom extract containing 3 mg·L−1 Eq microcystin-LR showed that after 24 h of exposure all fish embryos died. The same tests performed with other diluted extracts (containing 0.3, 0.1 and 0.03 mg·L−1 Eq microcystin-LR) were shown to have an influence on zebrafish development and a large number of embryos showed malformation signs (edema, bent and curving tail). After hatching the larvae were transferred to a medium without toxins to follow the larval development under the new conditions. The specific growth of the pre-exposed larvae was significantly more important than that of the control larvae. This may represent a compensatory growth used to reduce the difference in size with the control fish noted after hatching.

Effects of Tributyltin and Other Retinoid Receptor Agonists in Reproductive-Related Endpoints in the Zebrafish (Danio rerio).

Both field and experimental data examined the influence of exposure to environmental contaminant tributyltin (TBT) on marine organisms. Although most attention focused on the imposex phenomenon in gastropods, adverse effects were also observed in other taxonomic groups. It has been shown that imposex induction involves modulation of retinoid signaling in gastropods. Whether TBT influences similar pathways in fish is yet to be addressed. In this study, larvae of the model teleost Danio rerio were exposed to natural retinoids, all-trans-retinoic acid, 9-cis-retinoic acid, and all-trans-retinol, as well as to the RXR synthetic pan-agonist methoprene acid (MA) and to TBT. Larvae were exposed to TBT from 5 days post fertilization (dpf) to adulthood, and reproductive capacity was assessed and correlated with mode of action. TBT significantly decreased fecundity at environmentally relevant levels at 1 μg TBT Sn/g in diet. Interestingly, in contrast to previous reports, TBT altered zebrafish sex ratio toward females, whereas MA exposure biased sex toward males. Since fecundity was significantly altered in the TBT-exposed group with up to 62% decrease, the potentially affected pathways were investigated. Significant downregulation was observed in brain mRNA levels of aromatase b (CYP19a1b) in females and peroxisome proliferator activated receptor gamma (PPARg) in both males and females, suggesting an involvement of these pathways in reproductive impairment associated with TBT.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Effects of storage, processing and proteolytic digestion on microcystin-LR concentration in edible clams.

Accumulation of microcystin-LR (MC-LR) in edible aquatic organisms, particularly in bivalves, is widely documented. In this study, the effects of food storage and processing conditions on the free MC-LR concentration in clams (Corbicula fluminea) fed MC-LR-producing Microcystisaeruginosa (1×10(5) cell/mL) for four days, and the bioaccessibility of MC-LR after in vitro proteolytic digestion were investigated. The concentration of free MC-LR in clams decreased sequentially over the time with unrefrigerated and refrigerated storage and increased with freezing storage. Overall, cooking for short periods of time resulted in a significantly higher concentration (P<0.05) of free MC-LR in clams, specifically microwave (MW) radiation treatment for 0.5 (57.5%) and 1 min (59%) and boiling treatment for 5 (163.4%) and 15 min (213.4%). The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of MC-LR degradation by pancreatic enzymes. Our results suggest that risk assessment based on direct comparison between MC-LR concentrations determined in raw food products and the tolerable daily intake (TDI) value set for the MC-LR might not be representative of true human exposure.

Water Quality Concerns Due to Forest Fires: Polycyclic Aromatic Hydrocarbons (PAH) Contamination of Groundwater From Mountain Areas

Water quality alterations due to forest fires may considerably affect aquatic organisms and water resources. These impacts are cumulative as a result of pollutants mobilized from fires, chemicals used to fight fire, and postfire responses. Few studies have examined postfire transport into water resources of trace elements, including the polycyclic aromatic hydrocarbons (PAH), which are organic pollutants produced during combustion and are considered carcinogenic and harmful to humans. PAH are also known to adversely affect survival, growth, and reproduction of many aquatic species. This study assessed the effects of forest wildfires on groundwater from two mountain regions located in protected areas from north and central Portugal. Two campaigns to collect water samples were performed in order to measure PAH levels. Fifteen of 16 studied PAH were found in groundwater samples collected at burned areas, most of them at concentrations significantly higher than those found in control regions, indicating aquifer contamination. The total sum of PAH in burned areas ranged from 23.1to 95.1 ng/L with a median of 62.9 ng/L, which is one- to sixfold higher than the average level measured in controls (16.2 ng/L). In addition, in control samples, the levels of light PAH with two to four rings were at higher levels than heavy PAH with five or six rings, thus showing a different profile between control and burned sites. The contribution of wildfires to groundwater contamination by PAH was demonstrated, enabling a reliable assessment of the impacts on water quality and preparation of scientifically based decision criteria for postfire forest management practices.