Microplastics in Cetaceans Stranded on the Portuguese Coast.

This study characterises microplastics in small cetaceans on the coast of Portugal and assesses the relationship between several biological variables and the amount of detected microplastics. The intestines of 38 stranded dead cetaceans were processed in the laboratory, with digestion methods adapted to the amount of organic matter in each sample. The influence of several biological and health variables (e.g., species, sex, body condition) on the amount of microplastics was tested in all analysed species and particularly in common dolphins, due to the larger number of available samples. Most of the analysed individuals had microplastics in the intestine (92.11%), with harbour porpoises revealing a significantly higher median number of microplastics than common dolphins, probably due to their different diets, use of habitat and feeding strategies. None of the other tested variables significantly influenced the number of microplastics. Moreover, the microplastics found should not be enough to cause physical or chemical sublethal effects, although the correlation between microplastic ingestion and plastic additive bioaccumulation in cetacean tissues requires further investigation. Future monitoring in biota should rely on improved and standardised protocols for microplastic analyses in complex samples to allow for accurate analyses of larger samples and spatio-temporal comparisons.

A straightforward method for microplastic extraction from organic-rich freshwater samples.

The extraction of microplastics from organic-rich freshwater samples is challenging and limited information is available in the literature. This study aims at developing efficient methods for water volume reduction and organic matter removal in freshwater samples, while focusing on the reduction of the economic and environmental costs, maintaining microplastics integrity and avoiding contamination. For the water volume reduction approach, centrifuging freshwater samples (water, sediment, algae, leaves, driftwood, fish tissue) at different speeds (3500, 6000 rpm) and times (5, 10 min) showed that 3500 rpm for 5 min was efficient to settle the mineral and organic material, while preserving the polymers and showing high microplastic recovering rates (93 ± 6%). These recovery rates were significantly higher than the traditional sieving approach (77 ± 22%). The posterior minimal consumption of reagents resulting from the reduction of water volume helped to reduce the economic and environmental costs of the devised methodology, becoming more aligned with green chemistry principles. For biogenic organic matter removal, four digestion solutions were tested on freshwater samples, namely 10% potassium hydroxide, Fenton reagent (30% H2O2 + Fe(II)), 7% and 10% sodium hypochlorite (NaClO), under 3 periods of time (1, 6 and 15 h), at 50 °C. Both 7% and 10% NaClO showed the highest rates of organic matter removal (86 ± 1% and 90 ± 1%, respectively), after 6 h at 50 °C. Exposure of virgin and aged polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, nylon, polyethylene terephthalate) to NaClO showed no weight, visual, surface structure, Fourier transform infrared spectra and carbonyl index changes, except for nylon, although not to an extent that affected its identification. This method resulted in high recovery rates of polymers (92 ± 6%). Thus, 7% NaClO at 50 °C for 6 h (or overnight) may be efficiently used for microplastic analysis in organic-rich freshwater samples.

Preparation of biological samples for microplastic identification by Nile Red.

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 µm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94-100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97-100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

Striped dolphins as trace element biomonitoring tools in oceanic waters: Accounting for health-related variables.

In Europe, monitoring contaminant concentrations and their effects in the marine environment is required under the Marine Strategy Framework Directive (MSFD, 2008/56/EC). The striped dolphin (Stenella coeruleoalba) is the most abundant small cetacean species in Portuguese oceanic waters, representing a potential biomonitoring tool of contaminant levels in offshore waters. Concentrations of nine trace elements were evaluated by ICP-MS in kidney, liver and muscle samples of 31 striped dolphins stranded in the Portuguese continental coast. The mean renal Cd concentration was high (19.3 µg.g-1 wet weight, range 0.1-69.3 µg.g-1 wet weight) comparing to striped dolphins from other locations. Therefore, the present study reports a possibly concerning level of Cd in the oceanic food chain in Portuguese offshore areas. This study also aimed at evaluating potential relationships between trace element concentrations and striped dolphins' biological and health-related variables. Individual length was related with some of the trace element concentrations detected in striped dolphins. Indeed, Cd, Hg and Se bioaccumulated in larger animals, whereas the reverse was observed for Mn and Zn. Striped dolphins with high parasite burdens showed higher levels of Hg, while animals showing gross pathologies presented higher concentrations of Cd and Se. This study reported relationships between trace element concentrations and health-related variables for the first time in striped dolphins and it also provided information on the relative contamination status of Portuguese oceanic waters in comparison to other regions in the world.

Trace elements in loggerhead turtles (Caretta caretta) stranded in mainland Portugal: Bioaccumulation and tissue distribution.

Pollution is among the most significant threats that endanger sea turtles worldwide. Waters off the Portuguese mainland are acknowledged as important feeding grounds for juvenile loggerheads. However, there is no data on trace element concentrations in marine turtles occurring in these waters. We present the first assessment of trace element concentrations in loggerhead turtles (Caretta caretta) occurring off the coast of mainland Portugal. Also, we compare our results with those from other areas and discuss parameters that may affect element concentrations. Trace element concentrations (As, Cd, Cu, Pb, Mn, Hg, Ni, Se, Zn) were determined in kidney, liver and muscle samples from 38 loggerheads stranded between 2011 and 2013. As was the only element with higher concentrations in muscle (14.78 µg g-1 ww) than in liver or kidney. Considering non-essential elements, Cd presented the highest concentrations in kidney (34.67 µg g-1) and liver (5.03 µg g-1). Only a weak positive link was found between renal Cd and turtle size. Inter-elemental correlations were observed in both liver and kidney tissues. Hepatic Hg values (0.30 ± 0.03 µg g-1) were higher than values reported in loggerheads in the Canary Islands but lower than in Mediterranean loggerheads. Cd concentrations in the present study were only exceeded by values found in turtles from the Pacific. Although many endogenous and exogenous parameters related with complex life cycle changes and wide geographic range may influence trace element accumulation, the concentrations of Cd are probably related to the importance of crustaceans in loggerhead diet in the Portuguese coast.

Bioaccumulation of trace element concentrations in common dolphins (Delphinus delphis) from Portugal.

The common dolphin (Delphinus delphis) is one of the most abundant species in Atlantic Iberia, representing a potentially important tool to assess the bioaccumulation of trace elements in the Iberian marine ecosystem. Nine elements (As, Cd, Cu, Hg, Mn, Ni, Pb, Se and Zn) were evaluated in 36 dolphins stranded in continental Portugal. Dolphins had increasing Hg concentrations (16.72µg·g-1 ww, liver) compared with previous studies in Atlantic Iberia, whereas Cd concentrations (2.26µg·g-1 ww, kidney) fell within reported ranges. The concentrations of some trace elements (including Cd and Hg) presented positive relationships with dolphin length, presence of parasites and gross pathologies. Common dolphins may help biomonitoring more offshore Atlantic Iberian areas in future studies, which would otherwise be difficult to assess.

Biological variables and health status affecting inorganic element concentrations in harbour porpoises (Phocoena phocoena) from Portugal (western Iberian Peninsula).

The coastal preferences of harbour porpoise (Phocoena phocoena) intensify their exposure to human activities. The harbour porpoise Iberian population is presently very small and information about the threats it endures is vital for the conservation efforts that are being implemented to avoid local extinction. The present study explored the possible relation between the accumulation of trace elements by porpoises and their sex, body length, nutritional state, presence of parasites and gross pathologies. The concentrations of arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), manganese (Mn), nickel (Ni), lead (Pb), zinc (Zn) and selenium (Se) were evaluated in 42 porpoises stranded in Portugal between 2005 and 2013. Considering European waters, porpoises stranded in Portugal present the highest Hg concentrations and the lowest Cd concentrations, which may reflect dietary preferences and the geographic availability of these pollutants. While no effect of sex on trace element concentrations was detected, there was a positive relationship between porpoise body length and the concentration of Cd, Hg and Pb. Animals in worse nutritional condition showed higher levels of Zn. Harbour porpoises with high parasite burdens showed lower levels of Zn and As in all analysed tissues and also lower levels of renal Ni, while those showing gross pathologies presented higher Zn and Hg levels. This is the first data on the relationship between trace elements and health-related variables in porpoises from southern European Atlantic waters, providing valuable baseline information about the contamination status of this vulnerable population.

Ecological variables influencing trace element concentrations in bottlenose dolphins (Tursiops truncatus, Montagu 1821) stranded in continental Portugal.

Both the conservation status of bottlenose dolphins (Tursiops truncatus) (Habitats Directive 92/43/CEE, Annex II) and the Marine Strategy Framework Directive demand for data on their ecology and anthropogenic threats. To evaluate the bottlenose dolphin's toxicological status in continental Portugal, several trace elements (As, Cd, Cu, Hg, Mn, Ni, Pb, Se, Zn) were determined in 25 stranded individuals. The potential effect of sex, body length and stranding location on trace element concentrations was analysed. In the present study, bottlenose dolphins presented high mercury levels, only exceeded by animals from the Mediterranean and Adriatic seas. Only essential elements were influenced by dolphin sex, whereas Cd, Hg and Pb bioaccumulated in larger dolphins, and hepatic Hg and Cd concentrations were higher in the northwest coast of continental Portugal. The location effect may relate to variations in bottlenose diet and trace element availability, according to the proximity to anthropogenic sources in the Atlantic Iberian coast.