Parasites and Microplastics in the Gastrointestinal Tract of Alosa immaculata from the Black Sea—Implications for Health and Condition

Abstract

Alosa immaculata Bennett, 1835, commonly referred to as the Danube shad, is an anadromous pelagic species of the Clupeidae family, and plays a significant economic role for countries bordering the Black Sea. This study investigates the occurrence of both parasites and microplastics in A. immaculata specimens collected from Sfântu Georghe, with the aim of assessing their potential impact on fish health. The overall physiological condition of the fish was evaluated using Fulton’s condition factor (K) to determine whether the presence of parasites or microplastics had any measurable effect. Five parasitic genera were identified, including one ectoparasitic species from the genus Mazocraes, and four endoparasitic species from the genera Pronoprymna, Lecithaster, Hysterotylacium, and Contracaecum. Microplastic analysis showed a dominance of particles measuring 1–5 mm (62.5%), with fibers and foils being the only morphological forms detected. The most common colors were black (45%), transparent (35%), blue (12.5%), and brown (7.5%). The distribution of microplastics was higher in the stomach than intestines. Our findings offer critical insights into the combined effects of parasitic infection and microplastic pollution on this key Black Sea species. The integrated methodology, combining parasite load, microplastic content, and condition factor analysis, marks a novel approach in fish health assessment.

1. Introduction

Alosa immaculata Bennett, 1835—the Danube shad of the Clupeidae family—is an anadromous and euryhaline pelagic fish with high economic value for all countries bordering the Black Sea [1,2]. The anadromous behavior of the Danube shad, involving migration between marine and freshwater systems during spring and early summer, exposes the species to a broader spectrum of parasitic organisms, which may induce physiological modifications. This ecological plasticity increases susceptibility to parasitic infestation. The parasitofauna of A. immaculata has been extensively investigated throughout the Ponto-Caspian region [3,4,5,6,7,8].

Due to the high quality of its meat, which contains 26% fat, the Danube shad is a highly sought-after fish [1,2]. Alosa immaculata is classified as a vulnerable species on the IUCN Red List and is also protected under the EU Habitats Directive, highlighting the need for conservation efforts to safeguard its declining populations [9].

Marine ecosystems face several significant threats, including increasing environmental contaminants, climate change, and parasitic diseases [10]. Climate change has a multifaceted impact on the world’s oceans, encompassing their physical, chemical and biological systems. In addition, it also influences human exploitation of marine resources. As the Intergovernmental Panel on Climate Change (IPCC) asserts, rising levels of atmospheric carbon dioxide (CO₂) are leading to increased global atmospheric and ocean temperatures. In the absence of significant near-term reductions in CO₂ levels, it is probable that ocean warming will continue [11]. Temperature increases have already had consequences for the survival, growth, reproduction, health and phenology of marine organisms [12]. For instance, periods of thermal stress have been demonstrated to be a contributing factor to the outbreak of disease [13,14].

Plastic pollution is a growing global concern. The Black Sea has been found to be twice as polluted as the Mediterranean [15] due to its semi-enclosed nature and densely populated coasts. A significant source of this pollution is riverine input, with the Danube, the most international river, contributing up to 4.2 tons of plastic per day [16]. Both macroplastics and microplastics have been commonly detected along the coast, in the water, on the seabed, and, more recently, within marine organisms [17,18,19,20,21,22,23,24,25,26].

Recent research has demonstrated that both pelagic and benthic fish ingest microplastics [27,28,29,30], which can result in mortality, reduced feeding behavior, stunted growth and development, endocrine disruption, energy imbalances, impaired immune responses, altered neurotransmission, and potential genotoxic effects [31,32,33]. The detection of microplastics (MPs) in the skin, gills, and gastrointestinal tracts of wild fish underscores the need for further investigation. A deeper understanding of these outcomes could help determine whether MPs increase vulnerability to parasitic infections, as suggested by Collard et al. and Parker et al. [34,35]. The presence of parasites and microplastics significantly impacts the health of vertebrates, as evidenced by a decreased condition index (K), impaired growth, physiological stress, altered reproductive function, and a suppressed immune response [36,37,38,39,40,41].

Consequently, infected fish may exhibit differences from healthy individuals in terms of Fulton’s condition factor and length–weight relationship, reflecting the severity of infection. Alterations in these values may signal potential disruptions in growth rates and reproductive success. A reduced condition factor suggests a compromised physiological state, which can negatively impact both survival and reproduction. The simultaneous presence of microplastics and parasites may intensify these adverse effects, posing broader risks to fish population health and the stability of aquatic ecosystems [42].

In their study, Wang et al. [43] reviewed the existing literature on the effects of microplastics and nanoplastics on host–parasite interactions in aquatic environments. Although the findings are varied, the study underscores the complexity of these interactions and the need for further research to fully understand the impact of microplastics on the dynamics of infectious diseases in aquatic ecosystems. Evaluating the potential of microplastics to influence host–parasite interactions is essential, as these contaminants can impair immune function and alter host behaviors—such as antiparasitic avoidance or activity levels—that collectively affect susceptibility to parasitic infections in aquatic ecosystems [44,45,46]. Conversely, microplastics may affect host–parasite interactions by directly impacting parasites with free-living infectious stages—such as trematode cercariae—that rely on limited glycogen reserves for survival and host-seeking behavior in the environment, thereby potentially reducing their infectivity window [47,48].

In the present study, we tested the hypothesis that the presence of parasites and microplastics in A. immaculata specimens caught near the Sfântu Gheorghe area of the Danube River negatively affects their health status, as indicated by a lower Fulton’s condition factor (K).

The aim of this study was to assess the health status of A. immaculata, caught in the Danube River near the Sfântu Gheorghe area, through the detection of parasites and microplastics, with analyses conducted on the same Danube shad specimen. Fulton’s condition factor (K) was considered to assess whether the presence of microplastics or parasites had an influence on the overall condition of the fish.

2. Materials and Methods

2.1. Sample Collection

To assess parasite infestations and the presence of ingested microplastics, 30 Danube shad specimens (A. immaculata) were examined. The fish specimens were collected from the Danube River, Sfântu Gheorghe branch (44.8949° N, 29.58613° E), near its discharge into the northwestern Black Sea, between March and April 2024 (Figure 1), using a seine net. After sampling collection, the specimens were transported to the laboratory and stored frozen at −20 °C until analysis. Furthermore, only adult fish were included in the analysis, resulting in a low length amplitude among the sampled specimens, which corresponds to the size class typically targeted for commercial purposes.

2.2. Sample Preparation

In the laboratory, the fish were thawed 24 h before the analysis.

Each fish was rinsed with ultrapure water, and both the total body length (mm), using an ichthyometer, and weight (g), using an analytical balance, were recorded.

Fulton’s condition factor (K) was calculated for each individual. This was done using the following formula [49]:

$$\mathrm{K}=100\times {\displaystyle \frac{\mathrm{W}}{{\mathrm{L}}^3}}$$

where W = weight (g), L = length (cm).

The condition factor (K) can be divided into five categories according to Morton and Routledge [50]:

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Very Poor (0.8–1.0);

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Poor (1.0–1.2);

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Balanced (1.2–1.4);

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Good (1.4–1.6);

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Very Good (>1.6).

The length–weight relationship was calculated by using Le Cren’s [51] formula to estimate the relationship between the weight (g) of the fish and its total length (cm), as follows:

W = aL^b

The parameters a and b were calculated using the linear regression of the log-transformed equation log(W) = log(a) + b log(L), where a represents the interception and b the slope of the relationship.

When this formula is applied to sampled fish, b may deviate from the “ideal value” of 3, which represents isometric growth [52], due to environmental circumstances or the condition of the fish. If b is less than 3, the fish become slimmer as they grow longer, indicating negative allometric growth. Conversely, when b is greater than 3.0, fish become heavier, showing positive allometric growth and reflecting optimum growth conditions [53].

Also, the relative condition factor (Kn) was calculated based on the formula proposed by Le Cren [51]:

$$\mathrm{K}\mathrm{n}={\displaystyle \frac{\mathrm{W}}{\mathrm{Ŵ}}}$$

where W = actual weight of fish (g), and Ŵ = expected weight of fish (g).

A Kn index of 1.0 or above indicates good growing conditions for the species of fish, whereas an index below 1.0 suggests poorer growing conditions for the species [51].

A longitudinal dissection was performed on the ventral surface of the fish, starting with a cut from the cloaca towards the mouth using sterile dissection scissors. Since examining parasite infestation requires visual inspection of the organs and various processing methods (such as scraping or squashing the material), the gastrointestinal tract (GIT) was divided into the stomach and intestine. Each organ was further divided into equal longitudinal halves, with one half designated for parasite analysis and the other for analysis of ingested microplastics, to prevent airborne contamination of the microplastics samples [54].

2.3. Parasite Infestation Assessment

To identify the parasites and the reactions they provoke in the host, examinations were performed at both the macroscopic and microscopic levels. The parasite species in A. immaculata were identified by examining specific morphological and biological characteristics, such as size, shape, attachment and locomotion organ structure, type of eggs or cysts produced, and their specific localization in the host organism, with identification at the genus level according to the morphological keys of Bruno [55] and Moravec [56]. Microscopic analysis was performed using a Zeiss Axio Imager A1 microscope equipped with a digital camera. Objectives of 3×, 5× and 10× were used to observe larger parasites, while smaller parasites were examined using 20× and 40× objectives in combination with 5× and 10× eyepieces (Figure 2).

The indices used to describe the structure of the identified parasite community included mean abundance, prevalence, mean intensity, and dominance, calculated as follows:

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Abundance—the number of parasites/total number of fish examined (both infected and uninfected) [57].

$$Abundance\left({\displaystyle \frac{parasites}{fish}}\right)={\displaystyle \frac{No.ofparasites}{No.totalfishanalized}}$$

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Prevalence—the percentage of infected fish [57]. The results obtained were interpreted according to

Table 1. Parasite prevalence categories [58].

No.	Prevalence (%)	Category	Remarks
1	100–99	Always	Heavy infection
2	98–90	Nearly always	Bad infection
3	89–70	Usually	Moderate infection
4	69–50	Very often	Very often infection
5	49–30	Common	Common infection
6	29–10	Often	Frequent infection
7	9–1	Sometimes	Occasional infection
8	<1–0.1	Rare	Rare infection
9	<0.1–0.01	Very rare	Very rare infection
10	<0.01	Almost never	Never

[58].

$$Prevalence\left(\%\right)={\displaystyle \frac{No.infectedfish}{No.checkedfish}}\times 100$$

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Parasitic intensity—the average number of parasites/infected host [57]. The results obtained were interpreted according to

Table 2. Parasite intensity categories [58].

No.	Intensity (ind fish−1)	Category
1	<1	Very low
2	1–5	Low
3	6–50	Moderate
4	51–100	Poor
5	>100	Very poor
6	>1000	Overinfected

[58].

$$Intensity({\displaystyle \frac{parasite}{host}})={\displaystyle \frac{No.parasitesfound}{No.infectedfish\left(host\right)}}$$

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Dominance—The dominance of parasites was estimated using the following formula [59]:

$$Dominance\left(\%\right)={\displaystyle \frac{Totalnumberofmembersofthedominantspecies}{Totalnumberofindividualsbelongingtovariousspecies.}}\times 100$$

2.4. Microplastics (MP) Identification

To determine the ingested microplastics, the stomach and intestine halves, collected from each individual, were placed in separate glass containers and chemically digested using a 10% KOH solution, applied in a 1:3 sample to KOH ratio, and incubated at 40 °C for 48–72 h.

Once incubation was complete, the stomach and intestine samples were filtered through 2.7 µm Whatman glass microfiber filters, using a metal vacuum filtration system in a fume hood. The filters were stored in covered Petri dishes to dry at room temperature. Visual examination of the filters was conducted under an Olympus SZX10 microscope (Olympus Co., Hachioji, Tokyo, Japan) with an SC50 camera attached. Plastics found were measured using CellSens Entry software (Version 1.16). To confirm the plastic nature of each item, a hot needle test was performed. If the item melted, curled, shifted, or deformed under the heat of a red-hot needle, it was classified as plastic [60,61].

The length and color of each plastic item were recorded. Microplastics were classified by size into three groups: 1–5 mm (size class 1), 330 µm to 1 mm (size class 2), and 100 µm to 330 µm (size class 3) [62,63]. They were further categorized based on their shape into fibers, filaments, fragments, films, pellets, foam, and granules [62,64]. Since only half of the stomach and the intestine were analyzed, the recorded microplastic values are not absolute and may represent only a fraction of the total ingested by the specimens.

Contamination Control for MP

To maintain proper Quality Assurance and Quality Control (QA/QC) standards and minimize airborne contamination, the laboratory underwent thorough cleaning before analysis, with all surfaces being wiped down using ethanol. Only instruments made of metal, glass, or wood were used, following washing with ultrapure water and covering them with aluminum foil. Researchers wore white cotton lab coats and nitrile gloves, and laboratory access was restricted exclusively to study participants. All solutions were pre-filtered before use and stored in glass containers sealed with aluminum foil.

During the dissection, filtration, and microscopic examination of filters, Petri dishes with moist filters were used as contamination controls. Additionally, a blank sample consisting of only KOH was processed and analyzed alongside each batch of samples. All items found in the blanks were hot needle tested, then measured and characterized by color, type, and number.

The resulting data were corrected by subtracting items of similar size, color, and form identified in the controls and blanks [62,65,66,67].

2.5. Data Analysis

The statistical analysis was conducted using Microsoft Excel, and results were expressed as frequency of occurrence (FO%), mean, and standard deviation (SD).

Shade plots generated in PRIMER (Version 7.0) [68] were used to visualize variation in parasite species abundance and microplastic load. These plots illustrate the density and diversity of parasite taxa, correlations within the dataset, and the distribution of both parasites and microplastics in relation to fish length for each specimen.

For specific statistical analyses, we used R software (Version 4.4.2, 2024-10-31 ucrt) and RStudio (2025.05.0+496). A multiple linear regression model (lm()) was used to estimate the influence of plastic ingestion, parasite load, length, weight, and sex on the condition factor (K). The analysis assessed the effect of each predictor while controlling for potential confounding variables. An independent samples t-test was used to compare the mean condition factor (K) between fish with and without plastic ingestion, as well as between parasitized and non-parasitized individuals. This test assessed whether the observed differences between groups were statistically significant, providing insight into the potential influence of these ecological factors on the physiological condition of the fish. Spearman’s rank correlation was used to assess monotonic relationships between the condition factor (K) and quantitative ecological variables, such as the number of ingested plastic particles and parasite load.

3. Results

3.1. Morphometric Data

The morphometric analysis of A. immaculata specimens revealed a mean total length of 31.10 cm ± 1.97 SD and an average body weight of 246.9 g ± 42.86 SD. The b value for A. immaculata was 2.5534, indicating allometric growth [53], with an r² value of 0.8827. This indicates that approximately 88.3% of the weight variation can be explained by length. However, the relationship between length and weight differs within the same species, depending on the condition of each fish. Length–weight relationship values are not constant and can vary significantly due to factors such as food availability and biological, temporal, and sampling factors.

The sex ratio of the analyzed specimens was 73.33% females and 26.67% males.

Table 3. Sex-based differences in Mean Length, Weight, and Condition Factor of A. immaculata (F = female, M = male, SD = standard deviation).

	Sex	Mean	Min	Max
Length (cm)	F	31.35 ± 2.07 SD	27.8	36
	M	30.4 ± 1.6 SD	28.5	32.5
Weight (g)	F	252.29 ± 44.63 SD	184.3	364.6
	M	30.4 ± 36.12 SD	194.3	283.3
Condition Factor (K)	F	0.81 ± 0.06 SD	0.7	0.95
	M	0.82 ± 0.05 SD	0.74	0.91

summarizes the morphometric differences observed between sexes.

Table 4. Individual-level biological metrics and contaminant load: Length, Weight, Sex, Parasite Burden, Microplastic Presence, and Condition Indices (K, Kn) (F = females, M = males).

Sample Code	Length (cm)	Weight (g)	Sex	Parasite	Microplastics	K	Kn
1	36	364.6	F	Yes	Yes	0.78	1.03
2	28.5	184.3	F	Yes	Yes	0.79	0.94
3	32.3	282.3	M	Yes	Yes	0.83	1.05
4	29	186.3	F	Yes	Yes	0.76	0.91
5	28.8	196.2	F	Yes	No	0.82	0.98
6	30.5	236.9	F	Yes	Yes	0.83	1.02
7	30.8	232.8	F	Yes	Yes	0.79	0.97
8	30.5	228.3	F	Yes	No	0.8	0.98
9	33	306	F	Yes	Yes	0.85	1.07
10	30.5	207.1	F	No	No	0.72	0.89
11	32.5	268.7	F	Yes	Yes	0.78	0.98
12	31	253.6	F	Yes	Yes	0.85	1.04
13	33.5	288.9	F	Yes	No	0.76	0.98
14	32	256.9	F	Yes	No	0.78	0.98
15	32.2	289.7	F	Yes	Yes	0.86	1.08
16	31.1	247.6	F	Yes	No	0.82	1.01
17	31.4	258.5	F	No	No	0.83	1.03
18	32.5	283.3	M	Yes	Yes	0.82	1.03
19	29.5	203.2	M	Yes	Yes	0.79	0.95
20	31.8	238.2	M	Yes	No	0.74	0.92
21	28.6	194.3	M	Yes	Yes	0.83	0.98
22	29.8	228.6	F	No	No	0.86	1.04
23	33	275.4	F	Yes	No	0.76	0.97
24	27.8	205.5	F	Yes	Yes	0.95	1.12
25	32	282.3	F	Yes	Yes	0.86	1.07
26	30.4	240.3	F	Yes	Yes	0.85	1.04
27	30	245.9	M	Yes	No	0.91	1.10
28	30	214.5	M	No	Yes	0.79	0.96
29	35.4	311.9	F	Yes	No	0.7	0.92
30	28.5	197.6	M	Yes	No	0.85	1.01

provides the complete dataset recorded for each individual specimen. Fulton’s condition factor (K) ranged from 0.70 to 0.95, with 93.33% of individuals exhibiting values below 0.85. These findings suggest that the majority of the sampled population was in a suboptimal nutritional state, characterized by low fat reserves and overall leanness.

3.2. Parasite Analysis

In the present study, five genera of parasites were identified: one ectoparasite species belonging to the genus Mazocraes, and four endoparasitic species from the genera Pronoprymna, Lecithaster, Hysterotylacium, and Contracaecum (

Table 5. Average intensity, abundance, dominance, and prevalence of parasites in the Danube shad.

Genus Parasites	No. Fish Samples	No. Parasites	Average Intensity (Parasite/Host)	Average Abundance	Dominance (%)	Prevalence (%)
Mazocraes sp.	30	52	3.47	1.73	23.85	50
Pronoprymna sp.	30	11	1.83	0.37	5.05	20
Lecithaster sp.	30	19	1.73	0.63	8.72	36.67
Hysterotylacium sp.	30	103	4.48	3.43	47.25	76.67
Contracecum sp.	30	33	2.36	1.1	15.14	46.67

).

Ectoparasites affect the eyes, gills, skin, nasal cavities, or other parts in direct contact with the external environment.

Mazocraes sp., a gill-specific monogenean, was identified as a parasite associated with fish from the family Clupeidae. In A. immaculata (Danube shad), the average intensity of infestation with Mazocraes sp. was lower, and the dominance value indicated moderate parasite presence (Table 5). Of the 30 A. immaculata specimens examined, 21 exhibited mild irritation of the gill apparatus, suggesting a potential localized impact of the ectoparasite.

In the case of Lecithaster sp., the analysis revealed low values of the parameters under consideration (Table 5). Infected specimens showed no significant pathological alterations in the digestive tract.

Pronoprymna sp. is a trematode commonly found in the intestines of fish species from the Clupeidae family. Pronoprymna sp. was the rarest parasite identified in the A. immaculata specimens examined. It showed a low level of parasitism, with the lowest presence and limited impact on infected hosts (Table 5).

Contracaecum sp. was commonly encountered in the examined hosts, exhibiting a relatively high rate of infection and moderate dominance (Table 5). The parasite was found both freely in the abdominal cavity and attached to the liver in a small number of A. immaculata specimens.

Hysterotylacium sp. was the most dominant parasite species, standing out with the highest prevalence, intensity, abundance, and dominance among all recorded parasites. (Table 5).

3.3. Microplastics

Overall, the specimens analyzed contained a total of 40 microplastic items in the gastrointestinal tract (GIT), with a mean number of 1.6 items/individual, and higher counts observed in the stomach than in the intestines. It is important to note that these values were obtained from only half of the GIT and, therefore, may represent only a fraction of the total ingested microplastics.

Microplastics within class 1 (62.5%) were predominant, followed by size class 2 (30%), and a smaller proportion of size class 3 items (7.5%). A similar pattern, with slight variations in ratios, was observed in both the stomach and intestines. Fibers were the predominant type in the GIT and intestine, accounting for 97.5% and 94.11%, respectively, and were the only type observed in the stomach (100%). Foils were observed in a small proportion in the intestine (5.88%). Only black (45%), transparent (35%), blue (12.5%), and brown (7.5%) microplastics were observed in the GITs, with black and transparent items being predominant in both the stomach and intestines (Figure 3).

3.4. The Impact of Parasites and Microplastics on the Condition Factor (K)

A large proportion of the variability in K (R² = 0.9633, adjusted R² = 0.9556) was statistically significant overall (F (5,24) = 126, p < 2.2 × 10⁻¹⁶). Among the predictors, only length (coefficient = −0.0793, p < 0.001) and weight (coefficient = +0.0033, p < 0.001) had significant effects on K, indicating that body morphology is the primary driver of condition. In contrast, the number of ingested plastic items (coefficient = −0.00066, p = 0.654), number of parasites (coefficient = +0.00021, p = 0.510), and sex (coefficient = +0.00092, p = 0.849) were not statistically significant. These results suggest that, within the analyzed sample, exposure to plastic and parasites did not have a measurable impact on fish physiological condition, while length and weight remained the key factors associated with body condition.

In females, only length and weight had significant effects on K (p < 0.001), while neither plastic ingestion nor parasitic infestation had a statistically significant influence (p > 0.73). In contrast, all predictors were significant in males: plastic ingestion was associated with a significant decrease in K (coefficient = −0.00518, p = 0.001), whereas parasite load was associated with a slight but significant increase in K (coefficient = +0.00031, p = 0.016). These findings suggest a potential sex-specific physiological response to environmental stressors; however, caution is warranted due to the small sample size in the male group (Figure 4 and Figure 5).

A t-test comparing fish with plastic ingestion (mean K = 0.824) to those without plastic (mean K = 0.796) revealed no statistically significant difference (t = −1.399, df = 21.73, p = 0.176), with a 95% confidence interval for the mean difference ranging from −0.069 to 0.014. Similarly, there was no significant difference in K between parasitized fish (mean K = 0.814) and non-parasitized individuals (mean K = 0.800; t = −0.432, df = 3.75, p = 0.689), with a 95% confidence interval of −0.105 to 0.077. These results suggest that, within the analyzed sample, neither plastic ingestion nor parasite load had a statistically detectable effect on fish physiological condition, as measured by the condition factor K.

The results indicated a weak positive correlation between K and plastic ingestion (ρ = 0.271, p = 0.1466), as well as between K and parasite load (ρ = 0.263, p = 0.1607). However, neither correlation was statistically significant, suggesting that the observed variation may reflect natural fluctuations rather than systematic effects. Therefore, within the analyzed sample, there is insufficient evidence to support a significant relationship between fish physiological condition and either parasitic infestation or plastic ingestion, based on Spearman’s correlation analysis.

4. Discussion

The Black Sea marine ecosystem is subject to a range of anthropogenic activities that exert significant pressure on the aquatic environment. Several studies have highlighted the presence of parasites [69,70] and microplastics in the region, while also emphasizing the key human activities that impact the ecosystem. These activities contribute to changes in water quality and influence the responses of aquatic organisms to factors such as eutrophication, pollution, overfishing, sedimentation, and climate change [71,72,73,74,75,76,77].

The “b” values estimated in this study are lower than those previously reported by Tiganov et al. [78,79] (b = 2.879 and 3.134) and Stroe et al. [80] (b = 2.72), but slightly higher than other values observed by Leonov et al. [81] and Mocanu et al. [82] (b = 2.45 and 2.19) for the Danube shad. The values reported by specialists from the Romanian Black Sea and Danube areas are mostly negative allometric. Also, higher “b” values indicating positive allometric growth have been observed (b = 3.043–3.658; 3.04) [83,84]. The differences in “b” values can be attributed to one or more of the following factors: season and the effects of different regions; differences in water temperature and salinity; sex; food availability; and differences in the number of individuals examined, and in the observed length ranges [83,85,86,87]. In this study, the negative allometric “b” value may be caused by the presence of parasites and microplastics identified in A. immaculata individuals.

The calculated Fulton’s condition factor (K) ranged from 0.70 to 0.95, with an average of 0.812, indicating a moderate to low condition among the analyzed Danube shad specimens, with no significant differences between males and females. These findings are consistent with previous studies conducted along the Romanian Black Sea coast, which reported a condition factor of 0.87 for Pontic shad—attributed primarily to overexploitation and unsustainable fishing practices in the region [79]. On the Samsun coast of the southern Black Sea, Yilmaz & Polat [61] observed K values ranging from 0.550 to 1.064 (mean = 0.794) for females and from 0.557 to 1.047 (mean = 0.761) for males, with peak condition factors recorded in the spring. More recently, in the southeastern region of the basin (Rize coast), [88] reported average K values of 0.763 in females and 0.740 in males, suggesting an overall unfavorable growth environment and ecological pressures during the sampling period. Due to the high consumption of lipid and protein reserves associated with anadromous migration, the Fulton condition factor has been observed to decline—from 1.28 in the Sfântu Gheorghe arm at the beginning of migration, to 1.13 in Chiscani, located 197 km upstream [89]. The lowest K values recorded to date (0.65–0.70) for Pontic shad in the Danube have been observed 861 km upstream [90]. Lower K values are typical during migration, as fish expend significant energy on sustained swimming, resulting in loss of body mass. In the present study, the value of Kn was <1 for 50% of the analyzed Danube shad specimens, indicating poor condition, and >1 for the remaining 50%, indicating good condition. The variation of Kn may be influenced by gonad maturity, feeding activity, and other stress-inducing factors [51,91,92,93]. The specimens analyzed in this study were captured between March and April, corresponding to the early migratory phase, which typically peaks from late April to early May. Therefore, we suggest that the observed low K values are likely attributable to environmental factors associated with the onset of migration.

The study revealed the presence of five species of parasites. Parasite infections can significantly stress fish populations due to their substantial physiological and ecological consequences for hosts [94,95,96]. The monogenean worm Mazocraes sp., a parasite specifically targeting the gills of Clupeidae fish, was identified in 50% of the analyzed specimens. According to Veronika et al., this parasite is frequently considered a primary cause of infections in such hosts [58,97]. In our study, both the mean parasite abundance and intensity were low, ranging from 1.73 parasites per fish to 3.47 parasites per infected host.

These values are consistent with previous research conducted in the Black Sea region. For instance, a study conducted by Plaskina et al. [98] reported different parasitism indices in A. immaculata, with a prevalence of 53%, a mean abundance of 1 parasite/fish, and an average intensity of 1.9. Similarly, Ozer et al. [8] documented a prevalence of 61.3% and an intensity of 3.2 parasites per host, while Popjuk [6] reported a prevalence of 42.1% along with an intensity of 3 parasites per host.

Although previous studies have shown that high loads of blood-feeding monogeneans in the gills can severely affect fish by causing anemia [99,100], our research did not detect a noticeable effect of Mazocraes sp. on the condition factor of fish. In agreement with the findings of Gérard et al. [100], we observed no significant association between infection intensity and the condition of A. immaculata.

The trematode Pronoprymna sp. is usually located in the intestines of various marine fish, mainly from the Clupeidae family, inhabiting the Mediterranean Sea, the Caspian Sea, the Black Sea, and the Sea of Azov [30]. Pronoprymna sp. exhibited the lowest prevalence value (20%), and the average parasitism intensity was 0.37 parasites/host. In A. immaculata, this parasite was previously identified by Ozer (2013) in the Sinop region, with a significantly higher parasitism intensity of 29.7 parasites per host and a prevalence of 35.5% [8]. It was also reported by Popjuk (2011) in the Kerch Strait area, where it showed an intensity of 14 parasites per fish and a prevalence of 33.3% [6].

Lecithaster sp. is a widely distributed digenetic trematode found in marine fish. Its second intermediate host is typically marine copepods, which explains the frequent infection of Clupeidae, as copepods constitute their primary food source [101,102]. In the present study, Lecithaster sp. exhibited a prevalence of 36.67%, indicating a common infection [58], and a dominance of 8.72%. The parasite was previously reported in A. immaculata from the Sinop area of the Black Sea, with a prevalence of 29% and a parasitism intensity of 8.2 parasites per fish [8].

Parasites from the Contracaecum and Hysterothylacium genera are among the most widespread nematodes in the marine environment. Koie [103] suggested that these nematodes require at least one intermediate host—typically a crustacean—for transmission to fish, which become infected through the ingestion of parasitized copepods. Hysterothylacium sp. was found in the intestines of most specimens examined in this study, with a mean of 4.48 parasites per host and a prevalence of 76.6%. This parasite has been previously identified in A. immaculata populations from the Black Sea, including the Anatolian coast (Sinop), Sevastopol, and the Kerch Strait [5,6,8,97].

The pathogenicity of Hysterothylacium sp. larvae and adults is particularly evident in heavy infestations, where they can cause obstruction of the digestive tract and damage to parasitized organs. In extreme cases, worms may emerge from the gills and surface of the body if the fish is kept out of water for extended periods. In mild infestations, fish may tolerate parasitism with minimal physiological impact; in the current study, the effect was limited to a reduction in growth rate. However, severe infestations can result in fish mortality.

The analysis of microplastics in the gastrointestinal tract of A. immaculata indicated that fibers (97.5%) and black-colored particles (45%) were the predominant type and color observed. An assessment of various biota groups revealed that fish predominantly prefer black, blue, and transparent microplastics [104], with darker plastics more commonly ingested by underwater feeders compared to surface feeders that favor lighter-colored plastics, [105], and black microplastics likely originating from tire wear or wastewater sources [106,107]. Similar outcomes have been reported in the Black Sea region, particularly along the Sinop coast, where microplastics were identified in the brain, gills, muscle, and gastrointestinal tract of the Pontic shad [108]. Moreover, the same study found that microplastics ranging from 50–200 µm were the most common across all tissue types, with particles measuring 1–5 mm found at very low levels in the gills and gastrointestinal tract.

In contrast, our study found that microplastics between 1 and 5 mm (size class 1) were the most frequently observed (62%) in both the stomach and intestines. These results support previous findings suggesting that larger fish are more likely to ingest larger microplastic particles [109]. This pattern is also influenced by mouth aperture, as the size of the mouth opening plays a critical role in determining the maximum and optimal size of particles—including microplastics—that a fish can ingest [110].

Considering that only half of the gastrointestinal tract was analyzed to minimize environmental contamination, the observed mean of 1.6 microplastic particles per individual is likely an underestimation. According to a review study on aquatic biota, the average number of plastic particles found per fish was approximately 2.6 [104].

The feeding behavior of adult A. immaculata—which includes the consumption of large zooplankton, crustaceans, and small fish—may represent an indirect pathway for microplastic ingestion. This is supported by the presence of microplastics in several Black Sea species, including anchovy (Engraulis encrasicolus), sprat (Sprattus sprattus), and various zooplankton taxa, highlighting the widespread contamination of the food web [111,112,113,114].

However, we believe that the main uptake route for microplastics for A. immaculata within this study is through environmental contamination. Owing to its transboundary nature and extensive drainage basin spanning 19 countries, the Danube is considered the dominant contributor of plastic debris to the Black Sea [16,115]. This role is further supported by research showing significantly higher microplastic concentrations near the Danube Delta compared to other areas along the Romanian and Bulgarian coasts [17,115,116]. In the Danube–Black Sea system, microplastic composition differs between beach and marine sediments, likely due to variations in source and local hydrodynamics. Upstream of Iron Gates I, concentrations were low (<87 particles kg⁻¹), increased sharply downstream, then declined below Iron Gates II. Moderate levels (~165 particles kg⁻¹) were observed near the Sulina and Sfântu Gheorghe bifurcation and at coastal sites, reflecting both riverine and marine influences [116]. Notable differences were also observed in water microplastic concentrations between the Danube Delta front and adjacent Romanian harbor areas, with values ranging from an average of 0.63 MPs/m³ to a peak of 1.37 MPs/m³ near Sfântu Gheorghe, suggesting localized inputs and hydrodynamic influences, with fragments and fibers predominating across all sites [115]. Procop et al. [117] estimate that suspended sediments in the Romanian lower Danube, between Moldova Veche and Isaccea, carry annually between 46 and 51 tonnes of microplastics and 93–100 tonnes of total plastic debris, spanning all size classes from micro- to macroplastics.

It was observed that fish without any detected parasites also lacked microplastics, suggesting a possible connection between the two factors (Figure 6). Nonetheless, research by Alves et al. [118] and Parker et al. [35] indicates that the relationship between microplastic burden and parasitic infection remains inconclusive. This association has been explored in more detail only by Hernandez-Milian et al. [119], specifically in grey seals (Halichoerus grypus).

Although no statistically significant correlation was found within our study between parasites, microplastics ingestion, and the condition factor, previous findings suggest that microplastics may accumulate in areas with higher parasite densities. For instance, a study examining the stomach contents of European sardines (Sardina pilchardus) and anchovies (Engraulis encrasicolus) in the northwestern Mediterranean Sea reported a positive association between the presence of parasites—specifically trematode and nematode larvae—and microplastic ingestion. These observations imply that regions with greater microplastic pollution may also exhibit increased parasitic prevalence in certain fish species, highlighting a potential link that warrants further investigation [120]. Parker et al. [121] also found that dietary microplastic exposure did not affect parasite abundance or most condition indices, while parasitism significantly reduced feeding rates and had stronger individual effects than microplastics, the impacts of which appear relatively minor and lack clear interactive effects.

The ingestion of microplastics can cause various health and toxicological effects. Studies on zebrafish (Danio rerio) have shown that exposure to microplastics disrupts gut microbiota balance and induces intestinal inflammation, particularly with an increased presence of Proteobacteria. Among the microplastic types used, fibers were found to cause the most severe intestinal toxicity [122]. Similarly, Gaulke et al. [123] identified multiple associations between taxon abundance, microbial burden, and pathological changes in the gut of zebra fish, suggesting that the extent of microbiome disruption correlates with Pseudocapillaria tomentosa infection severity. Additionally, through a random forest classifier that successfully predicted parasite exposure to fish based on gut phylotype abundance, they highlighted the diagnostic potential of gut microbiome profiles for intestinal parasite infections.

In addition, additives or environmental organic pollutants that become adsorbed onto plastic surfaces can leach out, potentially causing disruptions in endocrine and reproductive functions or altering hormone levels [37]. Evidence suggests that microplastics (MPs) can lead to premature maturation and spawning in fish, accompanied by declines in fecundity, gonadosomatic index, fertilization rates, and gamete quality [37,124].

Parasites can have a significant impact on the reproduction of fish by impairing their physiological functions and altering their behavior. Parasitic infections can result in reduced gamete quality, hormonal imbalances, and damage to the reproductive organs. For instance, certain parasites infect gonadal tissues, thereby decreasing egg production [125,126]. For instance, infected fish may exhibit reduced courtship behavior, diminished mobility, or diminished attractiveness to potential mates. Furthermore, parasites often trigger energy trade-offs, diverting resources from reproduction to immune responses and resulting in fewer or lower-quality offspring [95,127,128,129].

Stressors affecting the ecosystem can significantly impact its overall health and that of its components, including humans [130]. In contaminated seawater, protozoan pathogens—including Toxoplasma gondii, Cryptosporidium parvum, and Giardia enterica—tend to associate with microplastics, showing a higher tendency to bind to fibers rather than microbeads [131].

The results of the study represent a first step towards filling a gap within the Black Sea region regarding the presence of microplastics and parasites in A. immaculata. Therefore, we acknowledge that our study’s primary limitation is that, due to its baseline nature, sampling was restricted to a single station, from which only 30 individuals were collected, which, however, is in accordance with the recommended number of individuals within Mattidi et al.’s [62] protocol for monitoring microplastics ingested by fish.

5. Conclusions

The morphometric analysis of A. immaculata revealed a predominance of females and generally low body condition, with most individuals (93.33%) showing Fulton’s condition factor (K) values below 0.85. The lower “b” values observed in this study suggest negative allometric growth in A. immaculata, likely influenced by regional environmental conditions and stressors such as parasites and microplastic contamination. These findings align with similar trends reported in the Danube and Black Sea regions. Five parasitic genera were identified, with Hysterotylacium sp. and Contracaecum sp. posing the greatest health impact due to their high prevalence and intensity. Mazocraes sp. caused mild gill irritation, while Lecithaster sp. and Pronoprymna sp. had limited pathological effects. Microplastic analysis showed a dominance of 1–5 mm fibers, mainly black and transparent, present throughout the gastrointestinal tract. These findings highlight the combined burden of parasitic infection and microplastic ingestion on A. immaculata, suggesting potential health risks and emphasizing the need for further research into their synergistic effects.

Currently, there are few studies that simultaneously assess fish health using Fulton’s condition factor alongside microplastic and parasite loads in the same specimens, underscoring the novelty and importance of this integrated approach.