2.1 Study area and species

The study area was the southern coastline of Spain, located to the west of the Mediterranean Sea (Figure 1). The species studied were the Razorbill and the Atlantic puffin, both belonging to the Alcidae family. According to the IUCN (2022), they are classified as ‘least concern’ and ‘vulnerable’, respectively.

2.2 Ethical statement and sample collection

In the winter season, from November 2022 to March 2023, a total of 703 stranded alcids were reported. A subsample of 95 carcasses was selected for further analyses, of which 84 were Razorbills and 11 were Atlantic puffins. Of these, only 86 individuals (76 Razorbills and 10 Atlantic puffins) could be analysed, as individuals that were not fresh were removed in order to avoid any alteration to the results. The main sources of carcasses were (i) stranded seabirds on beaches reported by volunteers and collected by researchers (n = 87) and (ii) seabirds stored in wildlife recovery centres (n = 8) collected in the same season. The individuals were stored in freezers at −20°C until the necropsies could be carried out. The collection of carcasses and tissue analysis has been carried out following authorization and ethical approval from the Ministry for Ecological Transition and the Demographic Challenge (MITECO, Spanish Government, Ref: SGBTM/BDM/AUTSPP/17/2022), and from Junta de Andalucía (Councillor for Sustainability, Environment, and Blue Economy).

2.3 Sample processing

2.4 Literature review of global dataset

We conducted a literature review in the field of stranded Razorbills and Atlantic puffins. The dataset was obtained by searching for scientific articles indexed in the Scopus database up to September 2023. The search string included different terms related to the taxonomic group (‘alcid*’, ‘alca torda’, ‘fratecula artica’, ‘Atlantic Puffin*’, ‘Razorbill*’) and terms related to body condition or physical state (‘body condition’, ‘mortality’, ‘necropsy’). The search was applied to titles, abstracts, and keywords, initially returning a total of 1200 published articles. We then restricted the search based on selection criteria, choosing articles that reported mortality, necropsies, or mass stranded events in one or both of the two species under study. Subsequently, a total of 76 full articles were evaluated, of which 9 were finally selected for inclusion in this study. Articles that did not conduct post-mortem examinations, those that reported instances of mortality caused by oil spread or fishing gear, and those that conducted post-mortem examinations but only evaluated the presence of pollutants were excluded. These 9 articles, published worldwide, were combined with our own data to create a comprehensive database spanning from 1989 to 2023. This dataset included a total of 1695 stranded seabirds of Razorbill (n = 1401) and Atlantic puffin (n = 294) species (Table S2).

In each publication, we extracted data on stranded seabird necropsies in each species, geographic location, study date, number of individuals analysed, sample processing, and identification and analysis technique of body condition (Table S3).

2.5 Statistical analysis

Statistical analysis was conducted using the R Studio package, version 4.4.0 (RStudio Team, 2023). Non-parametric methods were employed for statistical analysis after the data was determined to be abnormally distributed through a Shapiro-Wilk test. Moreover, the Kruskal-Wallis test was performed with a significance level of 0.05 to compare the biometric variables between species. In this test, we included the variables of body mass, wing length, tarsus length, head length, culmen length, and bill length.

Biochemical parameter values from muscle and liver were checked for normality and homogeneity of variance using Kolmogorov-Smirnov and Levene's tests, respectively. Data were analysed by an independent sample T-student test (p ≤ .05). The results are presented as means ± SD.

To propose a standardized global body condition index for stranded seabirds, several analyses were conducted. Initially, a principal component analysis (PCA) was performed to identify patterns and relationships among various variables collected from necropsies. To enhance the robustness of our dataset, we also conducted a Random Forest analysis to assess the correlation of each variable with ‘body mass’, which is a critical component of the body condition index.

3.1 Stranded alcid seabirds in the Western Mediterranean

Razorbills (n = 84) were mostly juveniles (84.5%); the mean weight was 411.89 ± 52.31 g, the wing length measured 188.90 ± 4.58 mm, and the tarsus length was 31.36 ± 1.69 mm. No significant differences were found in the measurements except for wing length. Adults had an average 5.7 mm larger wing length than juveniles. Among the Atlantic puffins, most of the individuals were juveniles (63.6%). In the complete sample (n = 11), the average weight was 257.43 ± 91.51 g, the wing length measured 168.95 ± 30.83 mm, and the tarsus length was 27.30 ± 2.20 mm. Other variables such as head length, culmen length, and bill width and length were also measured (Tables S3 and S4).

3.2 Body condition

During the PCA, we identified the variables that constituted the initial two factors, which accounted for over half of the sample (57.69%) (Figure 2). Specifically, Component 1 explained 39.50% of the sample variation and consisted of the measurements of bill, head, and culmen, while Component 2 explained 18.20% of the sample variation and encompassed the measurements of tarsus, body mass, and wing (Table S5 and Table S6).

As shown in Table S7, the variable most highly correlated with weight in the random forest analysis, excluding the variables related to bill measurements, was wing length. Bill measurements were not selected for further analysis because they cannot be standardized across species, as they are also correlated with individual growth. Therefore, we chose two variables from the second factor with the most significance, such as body mass and wing length.

When comparing the body condition index of each species, no significant differences were observed between Razorbills (X = 3.151, df = 1, p = .075) and Atlantic puffins (X = 0.617, df = 1, p = .432) (Figure 3).

3.3 Metabolic condition

Both species exhibited minimal levels of glucose and glycogen in the analysed muscle and liver tissues (Figure 4). Moreover, in both species, muscle tissue showed elevated levels of lactate (Figure 4a,c), while the liver exhibited significant levels of cholesterol and triglycerides, with cholesterol being the main predominant lipid (Figure 4b,d).

Analyses between metabolic condition on muscle and liver of Razorbill and Atlantic puffin denoted the absence of significant differences (T-test; p < .05) between species for the compounds analysed (Figure 5). Similar results are found when metabolic data from tissues are compared, differentiating between juveniles and adults of Razorbill (Figure 6) and Atlantic puffin (Figure 7), since no significant differences were detected (T-test; p < .05).

3.4 Literature review

The reviewed literature indicates a sparse coverage of necropsies conducted on Razorbills and Atlantic puffins globally. The majority of studies were carried out in England and Northern Europe, with additional research conducted along the East Coast of the United States and Southern Europe (Figure 8). Specifically, concerning body condition assessments, only a limited number of studies have provided data for Atlantic puffins. Anker-Nilssen et al. (2003) visually described body condition, finding no evidence of fat deposits and observing that individuals were extremely emaciated. In contrast, Anker-Nilssen et al. (2017) utilized a scale to score muscle, subcutaneous, and intestinal fat deposits, reporting consistently low body condition values along the Norwegian coast. These findings underscore the need for standardized protocols to assess the body condition of Razorbills and Atlantic puffins, as highlighted above.

4.1 Stranded alcid seabirds

Mass bird deaths often occur due to oil spills (Munilla et al., 2011). However, starvation has been seen as a primary factor for massive mortality in alcids globally (Anker-Nilssen et al., 2003, 2017; Diamond et al., 2020; Fullick et al., 2022; Morley et al., 2016). This could also be the case in the Western Mediterranean, because during the season when stranded birds were found, environmental conditions likely could limit their food availability, as previously outlined by Jones et al. (2018) and Newell et al. (2015).

In the winter season of 2022–2023, a total of 703 stranded birds from two alcids species were documented in the western Mediterranean. This number is comparable to the 200 stranded Atlantic puffins found in Norway in 2016 (Anker-Nilssen et al., 2017) and similar to other mass mortality events, including 62 Atlantic puffins, 626 Razorbills, and 474 Guillemots in England in February 1983 (Hope Jones et al., 1984). However, this event is much smaller than the 9148 Cassin's Auklets (Ptychoramphus aleuticus) reported in California in 2014–2015 (Jones et al., 2018) and significantly smaller than the 51,683 dead birds, of which 53.5% were Atlantic puffins, along the European coast in the winter season 2013–2014 (Morley et al., 2016). Most of the areas experiencing these mortalities are situated around the North Atlantic region, where a significant portion of the colonies are located (Guilford et al., 2011; Lavers et al., 2007). However, some events have also been recorded in the Mediterranean in recent years, although they were not as extensive (Belda & Sanchez, 2001; Cooper et al., 2003).

4.2 Metabolic state of stranded alcids and body condition

Regarding intermediary metabolism, there appears to be a common pattern in both bird species, characterized by probably low carbohydrate values (glucose and glycogen) along with high levels of lactate in muscle tissues. Although it would be important for the correct interpretation of the results to analyse the biochemical parameters evaluated from the tissues of healthy individuals used as control samples, this was not possible due to the nature of the research. However, it is known that carnivorous birds, such as the species studied here, engage in continuous gluconeogenesis primarily in the liver, using amino acids during feeding and fasting periods (Braun & Sweazea, 2008; Pollock, 2002). This process enables them to obtain glucose molecules crucial for producing high-energy biomolecules such as ATP and NADH in the Krebs cycle through predominant aerobic glycolysis (Vock et al., 1996). Complementary, anaerobic glycolysis is fuelled by phosphorylated glucose derived from muscle glycogen, which is converted into lactate that can be accumulated in muscle during the Cory cycle (Meléndez-Morales et al., 2009; Wynkoop et al., 2022). Our results suggested a prevalence of this anaerobic energetic pathway in most of the birds sampled in the present study, possibly indicating prolonged and strenuous exercise, where carbohydrate utilization during exercise closely matches muscle energy demands (Holloszy et al., 1998; Meléndez-Morales et al., 2009). In contrast, fat utilization during exercise is less regulated, as there are no mechanisms for closely matching availability and metabolism to the rate of energy expenditure (Holloszy et al., 1998). Therefore, an optimum level of glucose and tissue glycogen is essential for performing prolonged and strenuous exercise, such as migration (Holloszy et al., 1998; Lupiáñez et al., 1996). The metabolite values found in the tissues of sampled birds are consistent with a state of fatigue that could result from the development of hypoglycaemia (Muneer, 2021) or depletion of muscle glycogen (Braun & Sweazea, 2008). This physiological condition could probably lead to bird mortality, as birds have naturally higher blood glucose levels compared to other vertebrates (Sweazea, 2022), due to their high metabolic rate during migratory flights, which are conducted in a fasting state (Jenni-Eiermann, 2017).

This low body condition could be attributed to exhaustion resulting from possible disorientation and fasting conditions caused by extreme meteorological phenomena, such as the NAO (Durant et al., 2003). During the study period, fluctuations in the NAO index appeared to correlate with changes in bird migration and mortality patterns. In months with positive NAO, such as November 2022 (+0.69), January (+1.25), and February 2023 (+0.92), milder conditions were recorded in northern Europe and drier conditions in the south. These climatic conditions were associated with stronger winds and potential resource scarcity at stopover sites along the flyway. Conversely, months with negative NAO, such as December 2022 (−0.15) and March 2023 (−1.11), were characterized by colder and drier conditions in the breeding and stopover areas, possibly leading to foraging difficulties (National Centers for Environmental Information, 2024). In their study, Yao et al. (2023) identified extreme cold events associated with the NAO in November and December 2022. These findings suggest that NAO fluctuations could significantly have influenced bird migration and mortality.

For interpreting the metabolite values obtained in this assay, it is also important to consider natural post-mortem degradation. The degradation of biological compounds such as carbohydrates and lipids is a complex process influenced by factors including temperature, humidity, microbial population, and aerobic/anaerobic conditions, as well as the specific chemical composition of the compounds (Carter et al., 2007; Forbes & Carter, 2015). This degradation process varies significantly depending on the time elapsed since the animal's death and the environmental condition (Hyde et al., 2013). Thus, although determining the exact post-mortem chronological time of the collected individuals and its effect on the degree of organic matter decomposition was not feasible, it must be highlighted that this factor could impact the concentration of analysed tissue metabolites.

While the results presented here can provide background for future studies, it is crucial for future research to complement our analysis and determine the impact level that biotic and abiotic factors influence the presence/degradation of metabolites in the post-mortem tissues. Considering the low body condition of the studied seabirds and the premature age of the individuals, the decline in forage fish in pre-migration areas could also contribute to their initiation of migratory stages in weakened physical states, as suggested by Breton and Diamond in 2014.

4.3 Conservation implications and recommendations

Although many seabird populations are known to be endangered (Becker & Beissinger, 2006; Gaston & Descamps, 2011), after a thorough examination of the literature, it should be highlighted that no standardized protocols have been used to assess the body condition of stranded alcid seabirds.

Standardizing protocols are crucial for determining the potential causes of death in seabirds, offering a global perspective on the issue, which, in turn, might allow us to implement conservation measures to protect these species or, at the very least, minimize the impacts contributing to their decline. Therefore, we propose a body condition index that considers not only biometric measurements but also information on the physical state of the individual (the pectoral muscle, and subcutaneous and intestinal fat scores). This comprehensive approach would provide more detailed insights into the potential cause of death and facilitate global comparisons.