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Article

Effects of Domestic Pollution on European Brook Lamprey Ammocoetes in a Lowland River: Insights from Microbiological Analysis

by
Grzegorz Zięba
1,
Magdalena Moryl
2,
Dominika Drzewiecka
2,
Mirosław Przybylski
1,†,
Kacper Pyrzanowski
1 and
Joanna Grabowska
1,*
1
Department of Ecology and Vertebrate Zoology, Faculty of Biology and Environmental Protection, University of Lodz, 90-136 Lodz, Poland
2
Department of Biology of Bacteria, Faculty of Biology and Environmental Protection, University of Lodz, 90-136 Lodz, Poland
*
Author to whom correspondence should be addressed.
†
Deceased.
Water 2024, 16(16), 2349; https://doi.org/10.3390/w16162349
Submission received: 14 June 2024 / Revised: 14 August 2024 / Accepted: 15 August 2024 / Published: 21 August 2024
(This article belongs to the Special Issue Freshwater Species: Status, Monitoring and Assessment)
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Abstract

:
This study investigates the impact of water faecal contamination on highly threatened European brook lamprey larvae (Lampetra planeri). Water samples and the midgut contents of lampreys collected from a small lowland river upstream (site 1) and downstream from a wastewater treatment plant (WWTP) discharge (site 2) were analysed to check how the faecal microbial load of the habitat is reflected in the intestines of larval lampreys. The counts of viable mesophiles, psychrophiles, Escherichia coli and faecal streptococci as bacterial indicators of microbial (including faecal) water contamination were estimated. Microbial composition and abundance in larval midgut contents depended on the numbers of various microorganisms in the water environment. At site 2, the water was heavily microbiologically contaminated throughout the year by sewage inflow from the WWTP, and the amounts of studied bacteria were also high in the midgut of lampreys inhabiting site 2 regardless of the season. At site 1, water quality was better, and the levels of tested microbial indicators were lower in the intestines of the lampreys living there. The numbers of bacteria dependent on water temperature were growing in warmer seasons both in water and in intestines. Sewage pollution negatively influenced the condition of lampreys in site 2, where they exhibited lower body condition than in site 1.

1. Introduction

Lampreys are primitive, jawless vertebrates that are increasingly rare in freshwater ecosystems, primarily in the northern hemisphere (40 out of 44 species). Apart from species that are anadromous and parasitic after metamorphosis, 26 species are freshwater residents and do not feed at all as adults [1]. Most species spawn in coarse sand or gravel nests in swift-running rivers [2], but their larvae (ammocoetes) prefer to burrow in silt or mud [3]. Such burrowing activity of larvae is known to increase the oxygen availability in interstitial water and improve the physical and chemical properties of benthic sediment, providing therefore important ecosystem services acting as ecosystem engineers [4,5,6]. Larval development is extended typically 5 to 7 years [7], during which lampreys filter feed on detritus, algae and microorganisms [8].
One of the exclusively freshwater residents of the Palearctic is the European brook lamprey (Lampetra planeri), reported as native to 25 countries of Europe, excluding only its southeastern part [9,10]. Due to the small body size, it does not have economic value, but lamprey larval stages are of particular importance in ecosystems, constituting a large portion of biomass and may also ensure a constant source of nutrients to the food web [6]. The European brook lamprey prefers the middle and upper reaches of small streams and larger rivers and, as with most other non-parasitic and non-migratory inland species, has significantly declined throughout Europe [11]. The European brook lamprey is usually classified at a lower threat category, but in many parts of its range distribution, long-term decreases in abundance or even extinction in some locations have raised the level of concern. The European brook lamprey is listed in Annexe II of the Habitats Directive (92/43/EEC) as a species whose conservation requires the designation of Special Areas of Conservation (SACs). The species is also included in Appendix III of the Bern Convention. According to the International Union for Conservation of Nature (IUCN) Red List categories, it is considered critically endangered in Slovakia, Spain and Portugal, endangered in the Czech Republic and Switzerland, and vulnerable in Great Britain, Belgium and Poland [12,13,14,15]. In Poland, a monitoring programme assessing their distribution and abundance is being developed [16].
The main threats to lamprey species, including European brook lamprey, are similar across river basins and countries [1]. Toxins and long-term pollution, habitat destruction and fragmentation due to the construction of dams, weirs and other artificial barriers in rivers, channelisation, riverbank reconstruction, sediment removal and land management practices that lead to an increased siltation of spawning areas have been identified as the main threats [13,14,17]. The pollution of waters with organic waste from agriculture, input of nutrients from diffuse run-off or point sources, such as wastewater treatment plants (WWTPs), and accidental toxic spills are generally of less importance but represent significant threats for individual lamprey populations [17]. However, these threats may be more significant in the case of small watercourses that receive limited controls on the degree of pollution, including faecal contamination, which is a worldwide concern [18].
The detection of faecal contamination in water bodies typically involves an assessment of the water microbiological quality, where the total viable count (TVC) and/or count of indicator microorganisms (IMs) is applied [19]. The cultivation-based methods allow the direct counting of the target microorganisms and an estimation of their numbers. The TVC method covers the analysis of numbers of psychrophilic and mesophilic microorganisms. Their abundances are influenced by water temperature and may be applied as additional indicators of water quality. Under temperate climate conditions, cold-water psychrophiles (with an optimum temperature of 15 °C) may be autochthonous; in running waters, even in summer, the water temperature rarely exceeds 20 °C [20,21], while mesophiles have an optimum growth range from 20 to 45 °C [22]. The detection of IMs such as E. coli (EC) and faecal streptococci (FS) in water is a reliable and effective method to assess faecal water pollution, as their abundance increases with greater levels of pollution [22]. These microorganisms are consistently present in the intestinal tracts of all warm-blooded animals, including humans, in relatively high amounts, and they are believed to be a vital element of the natural faecal microbiota. They are also present in faecal waste, though these microorganisms do not typically occur naturally in the environment and rarely multiply, even in polluted water. EC seems to be the best bacterial indicator of microbiological water quality, as it is present in the intestines of all human beings in higher amounts than pathogenic bacteria. Although present in lower numbers in the intestines, FS are more resistant to harmful environmental factors, so that they may be used as an additional supplementary IM of faecal contamination. Both IMs can survive longer in aquatic environments than enteropathogens, although they are allochthonous in these habitats [19,23,24].
The contamination of aquatic environments by allochthonous microorganisms such as bacteria and viruses poses a significant challenge to aquatic ecosystems. Municipal wastewater, containing various pathogens, is a primary source. As the discharge of wastewater increases, so does the risk of contamination for aquatic organisms [25,26], and high pollution levels potentially pose a threat to rare species. However, sewage effluent also introduces nutrients into food webs, and almost all trophic groups have been shown to have higher mean biomass densities and greater productivity at sewage-exposed sites than at reference sites [27]. Indeed, in several studies, a positive effect of the input of domestic pollution on fish condition was observed [28], but it has yet to be the subject of study on lampreys.
Here, we investigate the condition of European brook lamprey in two adjacent study sites that differ in water quality on the same river. This unusual situation provides an opportunity to study the impact of microbial pollution on body condition, as these filter-feeding animals may absorb microorganisms present in water, e.g., EC as in the case of Pacific lamprey (Entosphenus tridentatus) ammocetes [29]. The aim of our study was, therefore, to (1) confirm the impact of water pollution manifested by the presence of IMs and the increased numbers of psychrophiles and mesophiles on the lamprey’s microbial midgut content and (2) evaluate the effect of the pollution on the condition of lampreys inhabiting stream sections differing in the faecal-based pollutant load. We hypothesise that water microbiological pollution will be reflected in midgut content and, consequently, influence lamprey condition.

2. Materials and Methods

2.1. Study Site

The River Gać is an 18.6 km 3rd-order stream in central Poland. Poland is located in a moderate climate zone and is dominated by four distinct seasons, autumn from September to November; winter from December to February; spring from March to May; and summer from June to August. An upstream, relatively unpolluted stretch (site 1) (51.560749 N, 20.138290 E) is separated from the WWTP discharge by two dams, preventing the migration of fish and lampreys. Downstream, a polluted section (site 2) (51.538980 N, 20.139011 E) is highly influenced by the outflow from a communal mechanical-biological sewage treatment plant in Spała village (capacity of 202.8 m3/24 h). The amount of sewage discharge depends seasonally on the number of residents in the village from around 410 (local residents only, as of the census dated 2021) to around 1700 (local residents and visitors) at the peak of the tourist season (June–September). The upper stretch, including site 1, aggregates relatively unpolluted water from a deciduous forest and farmland catchment. Both sample sites were 3–4 m wide and 0.3–0.7 m deep, mostly sandy along riffles, with thick layers of organic deposits in pools, with limited submerged vegetation comprising Elodea canadensis and Potamogeton sp. due to a dense riparian tree canopy.

2.2. Microbiological Study of Lamprey Gut Content and Water

2.2.1. Water and Larval Lamprey Sampling

Water samples and lamprey ammocoetes were collected in summer (June 2020), autumn (September 2020), winter (January 2021) and spring (April 2021) at each sampling location described below (Table 1). Water samples (0.5 L) were collected 0.25 m above the substrate and stored in sterile vessels in a chilled cabinet. Microbial analyses were performed directly after transfer to the laboratory. A total of 24 European brook lamprey (3 individuals in each season inhabiting each of the two sample sites; mean ± SD total length (TL) [mm]: 148.2 ± 12.1; min 110; max 170) were collected using pulsed backpack electrofishing equipment (EFGI 650, BSE Bretschneider Spezialelektronik, Chemnitz, Germany). As the European brook lamprey is a protected species in Poland, lamprey capture was conducted with permission from the Regional Directorate of Environmental Protection (WPN.6401.282.2020.JHi/BWo). Immediately after capture, lampreys were placed individually in plastic containers filled with water from the respective sample site and transferred to the laboratory. Lampreys were euthanised by decapitation and brain pithing (regulated procedures for fish recommended by the American Veterinary Medical Association [AVMA] Panel on Euthanasia), as the use of anaesthetics was incompatible with the study design and could impact the survival of microorganisms. According to the Polish Animal Welfare Authorities, this euthanasia procedure on fish and lampreys is in accordance with AVMA guidelines and does not require approval of the Local Institutional Animal Care and Use Committee (IACUC) of the Medical University in Lodz, Poland. No experiments on live vertebrates were conducted. All methods and procedures were carried out under relevant EU regulations and according to the relevant Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. In the laboratory, under aseptic conditions, lampreys were measured for total length (TL) to the nearest 1 mm and weighed (W), and the alimentary tract of each specimen was removed. While conducting all procedures, surfaces and tools were cleaned and disinfected in advance and between dissections using 70% ethanol (POCH S.A., Gliwice, Poland), and sterile gloves were exchanged after each tissue and gut content sampling. The time between euthanasia and sample collection was less than 5 min. Microbiological analysis started immediately after isolation of the midgut content even though the survival of IMs from fish intestines is reported to remain stable or increase slightly over a 48 h incubation period [30,31].

2.2.2. Collection of Gut Content from Lampreys

The faeces were extracted from the midgut of dissected lamprey larvae and placed in previously weighed, sterile tubes. The midgut content samples were weighed and suspended in 0.5 mL 0.85% normal saline. To homogenise the faeces and release microorganisms into the solution, the samples were twice intensively vortexed for 20 s, sonicated for 2.5 min. in an ultrasonic bath and vortexed for a further 2 min. The sample volume was adjusted to 1 mL, and samples were mixed for 10 s. Serial tenfold (log) dilutions in saline were made in triplicate.

2.2.3. Estimation of TVC of Microorganisms

The TVC test (according to ISO 6222 [32]), which estimates the total number of cultivable aerobic and anaerobic microorganisms, such as bacteria, yeast or mould species, was performed for samples of water and lamprey midgut content. The pour-plate technique was used. Molten Yeast Extract LAB-Agar (BioMaxima S.A., Lublin, Poland), which is a universal medium for a broad range of microorganisms, was cooled to 45 °C and poured into a sterile Petri dish containing water or midgut content samples. A volume of 0.1 mL of log dilutions (10−1, 10−2, 10−3, 10−4, and 10−5) of lamprey midgut content samples was applied. Additionally, 0.5 mL or 0.1 mL of undiluted water samples and 0.1 mL of serially diluted (10−1, 10−2) water samples were applied. Counts of the number of live psychrophiles (at 22 °C for 72 h) and mesophiles (at 37 °C for 48 h) were made and recorded as colony-forming units (CFU) per mL of each sample.

2.2.4. Estimation of the IMs Numbers

The water samples (volumes: 1 mL, 5 mL, 10 mL, or 50 mL, dissolved in sterile distilled water) were filtered using nitrocellulose filters with a pore size of 0.2 µm (Sartorius, Goettingen, Germany). However, when the presumed contamination was high, the water samples were diluted (1:2, 1:10, and 1:20) in sterile distilled water, and 0.1 mL of each dilution was spread on the surface of an agar plate with medium. The midgut content samples were inoculated on the agar media by spreading 0.1 mL of undiluted suspension and each serial dilution (10−1, 10−2, 10−3, and 10−4) on the surface. The EC numbers in water and midgut content samples were determined using Chromogenic Coliform LAB-AGAR (CCA) plates (BioMaxima S.A., Lublin, Poland), according to ISO 9308-1 [33]. EC (dark blue) colonies were counted after 24 h incubation at 37 °C. The FS numbers in water and midgut content samples were determined using plates with Bile Esculin Azide LAB-AGAR (BioMaxima S.A., Lublin, Poland), according to ISO 7899-2 [34]. FS brown colonies with a dark brown halo were counted after a 48 h incubation at 37 °C. EC or FS CFU numbers were estimated in 100 mL of studied water samples or 1 g of midgut content. All experiments were performed in triplicate.

2.3. European Brook Lamprey Larval Condition

To estimate the condition of European brook lampreys, additional electrofishing was carried out in both stretches of the River Gać on each sampling occasion. All (total 54) ammocoetes were anaesthetised with 2.0 mL dm−3 of Propiscin (2% etomidate, i.e., ethyl-3[(1R)-phenylethyl] imidazole-4-carboxylate) (Inland Fisheries Institute, Olsztyn, Poland) [35], measured for TL (mm) and W (g) and released. For each specimen, the Fulton condition factor (FC) [36] was calculated: FC = 100 × W(g) × TL (cm)−3. The relations between W and TL were determined by linear regression (log-transformed data) [37].

2.4. Statistical Analyses

Seasonal and between-site differences in microbial pollution of the River Gać water were tested using a two-way analysis of variance (ANOVA II) followed by multiple comparisons (the HSD Tukey post hoc test). To meet ANOVA assumptions, TVC and IMs numbers were log-transformed and examined for normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test). The same procedure was used to test bacterial abundance in lamprey midgut content. To detect the relationship between bacterial abundance in water and ammocoete midgut content, cluster analysis with Pearson’s correlation as a criterion of similarity and the Unweighted Pair Group Method (UPGM) as a clustering method were used. Differences in the condition factor (FC) between sites were tested (t-test) [38]. The differences in W-TL regression coefficients between sites were tested using analysis of covariance (ANCOVA) [38]. The slopes (b-values) for sites were tested against isometry, i.e., b = 3, with Bailey’s t-test. Statistical analyses were conducted using STATISTICA 13 (Dell Inc., Round Rock, TX, USA, 2016).

3. Results

3.1. TVC and IM Estimation in Water

In the River Gać, the numbers of TVCs (amounts of mesophiles and psychrophiles) depended on the sample site, season and type of microorganism, i.e., mesophilic or psychrophilic (Table 2), and all possible interactions were significant (Table 2). The numbers of mesophiles and psychrophiles were higher in site 2 than in site 1 in individual months, i.e., 4–58 times and 8–107 times, respectively (only in September, psychrophiles were slightly more abundant in site 1) (Figure 1a,b). The numbers of both types of microorganism varied through the year and were the highest in June (1186 CFU/mL mesophiles and 7667 CFU/mL psychrophiles) and the lowest in January (129 mesophiles and 227 psychrophiles) in site 1, while there was no such pattern in site 2, where values were more balanced (Figure 1a). Psychrophilic bacteria were always more numerous than mesophiles, but the magnitude of their predominance varied with season and site, with the highest value in site 1 (Figure 1b). In site 2, the psychrophile predominance over mesophiles was not as distinct as in site 1 (Figure 1b).
The levels of both IMs (EC and FS) in the River Gać depended on the sample site and season (Table 2), and all possible interactions were significant (Table 2). In site 2, IM numbers were several times higher than in site 1 throughout the whole period of study (Figure 1c). In site 1, FS tended to dominate over EC, contrary to site 2, where EC was always more prevalent. In site 1, the numbers of both IMs reached a maximum in June (1013 CFU/100 mL EC and 1650 CFU/100 mL FS) and minimum in January (46 CFU/100 mL EC and 253 CFU/100 mL FS), while in site 2, seasonal differences between summer and winter were not visible (Figure 1c).

3.2. TVC and IM Estimation in the Midgut Content of Ammocoetes

Similarly to water samples, the TVC levels of mesophiles and psychrophiles in ammocoete midgut depended on the season, site and microorganism type, but only one type of interaction (site and season) was significant (Table 2). The TVC levels were higher in site 2 than in site 1 in individual months, i.e., 3–5 times for mesophiles and 2–15 times for psychrophiles (Figure 2a,b). Multiple comparisons showed that in site 1, the numbers of mesophilic and psychrophilic microorganisms were the lowest in winter (January) (2.01 × 106/g and 4.15 × 106/g, respectively) and increased in warmer seasons, but in site 2, the highest values were noted in April (5.70 × 107/g and 1.31 × 108/g, respectively) (Figure 2a). Although psychrophile abundances were 2–3 times higher than mesophiles in ammocoete midguts, the differences were not significant in each season and site (Figure 2a).
Contrary to TVC, the value of IMs in the midgut of ammocoetes depended on site and season but not on the type of bacteria (EC and FS); all interactions were significant (Table 2). Both types of IMs were higher in site 2 than in site 1 in June (9 times for EC and 5 times for FS) and September (31 times for EC and 6 times for FS), while in April, they were higher only for EC. The lowest levels of IMs in both locations were detected in winter (January) (minimum 6.59 × 103/g for FS in site 1 and maximum 3.50 × 104/g for EC in site 1) and, additionally, in April for EC in site 1 (7.51 × 103/g), and the highest were detected in the warmer seasons, i.e., in June in site 1 (2.40 × 105/g for EC and 7.62 × 105/g for FS) and in site 2 (3.77 × 106/g for FS) and September in site 2 (6.95 × 106/g for EC) (Figure 2c). In site 1, FS predominated over EC in April (25 times) and June (3 times), while in site 2, EC was greater in April (almost 3 times) and September (6 times).
Cluster analysis (Figure 3) revealed that the composition and count of IMs in the water and ammocoete midgut at site 2 were similar (distance only 0.04). The IMs of ammocoete samples from site 1 were similar to those for the above-mentioned groups (distance 0.48). Water IMs at the site 1 cluster were the most different from the other samples (distance 0.88), constituting a discrete branch in the dendrogram (Figure 3).

3.3. Condition of Lamprey Ammocoetes

In the relatively unpolluted stretch of the River Gać (site 1), the Fulton condition index of European brook lamprey ammocoetes was higher compared to polluted site 2, i.e., FC = 1.626 ± 0.156 and 1.147 ± 0.099 in site 1 and 2, respectively (t52 = 5.307, p < 0.001). A similar pattern was also shown by differences in weight–length regression parameters (Table 3). ANCOVA showed no differences in slope (b) coefficient values (F1, 50 = 0.034, p = 0.854). The common slope was bc = 2.880 (±0.0986) and did not differ from b = 3 (t25 = 1.216, p = 0.115). Significant differences were found for the intercept (a) (F1, 51 = 30.177, p < 0.001). These results indicate that both regression lines are parallel and that the size of ammocoetes from the relatively unpolluted stretch of the river (site 1) is higher than the polluted stretch (site 2). The adjusted average weight (±95% C.L.) of lamprey larvae was 4.33 ± 0.15 in site 1 and 3.80 ± 0.13 in site 2.

4. Discussion

Our results support the prediction of an impact of domestic pollution on brook lamprey larvae condition. The operation of WWTP and, consequently, the influx of faecal pollutants into the River Gać has contributed to the identification of two zones downstream and upstream from WWTP discharge: polluted (site 2) and relatively unpolluted (site 1) stretches of the river, and we ultimately determined the presence and seasonal fluctuations of the microbial content of the river. The influx of pollutants detected as IM composition and count has affected the natural conditions in the water column and the midgut content of European brook lamprey ammocoetes.

4.1. Water Microbiological Quality

Among the microorganisms in natural water reservoirs, the psychrophilic group dominates because of their capacity to tolerate the relatively low-temperature conditions in Polish rivers and lakes. However, mesophilic bacteria may also be detected in lower numbers in addition to these autochthons, as their temperature preferences suggest their allochthonous origin. In drinking water, levels of 100 CFU/mL for psychrophiles and 20 CFU/mL for mesophiles are accepted in Poland and the European Union, while in France, levels of 100 CFU/mL are accepted for psychrophiles and 10 CFU/mL for mesophiles, so the psychrophiles/mesophiles ratio in clean water is 5:1 or 10:1, respectively [39,40]. In faecally polluted water, the psychrophiles/mesophiles ratio may be lower due to the influx of faecal allochthonous microorganisms.
In both study sites, psychrophiles dominated over mesophiles. The dominance of psychrophiles over mesophiles in the relatively unpolluted river section (site 1) seems to be a typical phenomenon of natural waters where allochthonous microorganisms remain in a minority. The prevalence was as significant as 6–11 times in almost all seasons. The increase in abundance of both microbial groups was observed from winter to summer and is caused by natural changes in water temperature throughout the year (in the section of the river upstream from WWTP—site 1). However, excessive increases in mesophiles, especially in relation to psychrophiles, may indicate their allochthonous origin, as due to higher temperature requirements, mesophilic bacteria may be zoonotic, being primarily a component of natural microbiota, e.g., intestinal flora of warm-blooded organisms, including humans [22,26,41]. In the river stretch affected by the continuous inflow of sewage from Spała WWTP—site 2, psychrophiles predominated over the mesophiles by only two to three times in almost all seasons. The less prominent difference in this location confirm the constant inflow of a large load of pollutants, including the faecal contamination of water, affecting the ratio of organisms dependent on temperature. It was also reflected by the higher TVC of both psychrophiles and mesophiles in all seasons and the lack of seasonal variation in the TVC for both types of bacteria in site 2. We suggest that the highest TVC of psychrophiles and mesophiles at the polluted site below WWTP was obtained in June because of the synergistic effect of water temperature and intensity of tourist activity. High temperatures in summer favour the multiplication of all microorganisms (both meso- and psychrophilic), while large amounts of organic and inorganic pollutants from the treatment plant intensify the growth and expansion of heterotrophic microflora [26]. Elevated amounts of microorganisms in water correspond with the increased tourist traffic in Spała, which is usually observed from May and throughout the entire summer until late September [42].
The microbial indicators of water faecal pollution (EC and FS) confirmed the above water quality results for TVC in both locations. In site 2, downstream from the WWTP, the faecal contamination was higher than in site 1, where it did not exceed the standards for recreational outdoor swimming waters [40] (for EC 1000 cfu/100 mL, for FS 400 cfu/100 mL), with the exception for FS numbers in September and June, when the standards were exceeded 4 times and 1.5 times, respectively. EC and FS tended to occur in greater numbers in water along the section of the River Gać polluted by the inflow of WWTP and exceeded the standards [40] many times each season, from 12 to 76 times for EC and from 6 to 84 times for FS, in September and April, respectively. Both IMs are allochthonous in open waters, and higher EC readings than FS reflect the relationships present in human intestines [43,44]. Such a dominance of EC bacteria over FS was obtained for water sampled at site 2. It suggests a constant and large inflow of faecal-polluted water into the river because EC is sensitive to environmental conditions and is usually the first to die in river water, particularly during winter. In contrast, the greater amounts of FS than EC detected at site 1 indicate less and occasional sewage inflow to this river section. FS are known to survive longer in water [26,41], and thus, the FS number was still high, while the number of EC decreased with time from a possible sewage leak. As in the vicinity of site 1, there was no official source of pollution, and it was likely an illegal inflow of domestic sewage from unknown sources, i.e., rural domestic pollution, agricultural non-point sources, pollutants from residential sites, etc.
The typical changes in levels of both IMs in the relatively unpolluted river stretch during the whole period of the study confirm a significant impact of water temperature [45]. In summer, relatively warm water allows faecal microorganisms to survive longer; the opposite is true in winter—low temperatures reduce their survival. In site 2, the constant and intense inflow of sewage influenced IM levels in all seasons. Even the low temperatures in January did not diminish this effect. Moreover, the constant wastewater effluent into aquatic environments could be a source of nutrients and facilitate the proliferation or extended survival of enteric bacteria (IMs) [26,46].
The poor microbial quality of water in site 2 is a consequence of WWTP effluents representing the primary source of faecal contamination along rivers [47] with the primary and secondary treatment methods removing only a portion of faecal bacteria, exposing aquatic animals to a variety of stressors [48]. The highest densities of coliform indicators (lactose-fermenting EC-like rods belonging to the order Enterobacterales) in water were found at the untreated sewage effluent and downstream sites from the effluent [49]. However, the sediment in aquatic environments is generally considered a main derivative reservoir of IMs [50], since the accumulation levels are 100–1000 times higher than in the water column. Furthermore, the sediment may provide a favourable environment for IMs to survive for up to two months [46], which may then be termed “naturalised” IMs, and they may be able to occupy the intestines of poikilothermic organisms such as fish [51]. Lamprey larvae burrowing in sediment and feeding on suspended organic matter must be significantly exposed to IMs.

4.2. TVC and IMs in Lamprey Ammocoete Midgut Content

In accordance with the presence of microorganisms in water, the total number of mesophiles and psychrophiles in lamprey midguts was greater for animals living in site 2 that was constantly polluted by WWTP than in site 1, which was relatively unpolluted. The seasonal variation in the numbers of both bacterial types reflects changes in TVC in water, especially in the unpolluted section of the river, and it is visibly associated with the feeding activity of European brook lamprey ammocoetes [16]. In site 1, in winter (January), when the animals do not feed, the level of psychrophiles and mesophiles in their gut was as much as seven times lower than in September. Also, in site 2, the level of psychrophiles and mesophiles in the gut decreased two-fold in January, even though the number of bacteria in the water was still as high as in September. The ratio of psychrophiles/mesophiles present in all ammocoete midguts was 2–3 only, i.e., the same as in the polluted river section, which was characterised by the better survival of mesophilic bacteria derived from faeces [26].
The influence of water faecal contamination on the ammocoete midgut content of both IMs was evident, as more EC or FS were present in water at site 2, and higher levels of these IMs were collected from this water and estimated in midgut samples. However, for a total number of mesophiles and psychrophiles, seasonal variation depended on the lamprey feeding activity throughout the year and mode of foraging. Our results indicate that during the feeding season, lamprey ammocoetes enrich the intestinal microbiota abundance by filtration from water. Thus, they may play an essential role in the purification of the water environment, absorbing the allochthonous bacteria, as was shown for E. tridentatus ammocetes, decreasing the EC level and improving water quality [29]. In winter, the bacteria are not absorbed from water, and their numbers decrease due to the lack of nutrients and low temperature inside the body of these ectothermic animals.
The diet of lamprey ammocoetes summarised by Docker (2015) [52] consists mainly of suspended organic matter; however, at low temperatures, large quantities of undigested algae and other food can pass through their guts [53]. In the case of larval Petromyzon marinus, Hayden et al. (2019) [54] assessed, via stable isotope analysis, that up to 75–85% of consumed detritus may be allochthonous and terrestrially derived. Similarly, for larvae of the Lampetra appendix, feeding mainly on organic detritus (>85%), gut fullness, although variable, was highest during spring, and the assimilation of the organic fraction of the diet averaged >65% across streams and seasons [55]. The contents of ash-free dry mass of larval food constituted, on average, 0.09% bacteria, but its seasonal changes were evident for both P. marinus and Ichthyomyzon fossor with the highest peaks over May/June and September. Taking into account typical habitats of ammocoetes [1,15,16,56] and their feeding habits [16,53], it is likely that abundant EC and FC in sediments can also be ingested and therefore present in gut, which is in agreement with the observations that the diet has a significant influence on the diversity (and function) of the gut microbiota [57].
Studies on the bacterial community of lamprey tissues are scarce [58,59,60]. An earlier study with conventional culture-dependent methods revealed a strong similarity of the gut microbial flora of Geotria australis ammocoetes to that of the environment [58], supporting our findings. There is substantial information on the influence of microbial water quality, including faecal pollution, on the fish gut microbiome, which might be relevant for lampreys. Although EC is not a typical inhabitant of fish or lamprey microbial flora, it has been isolated from the intestinal tract of several fish, as have other human faecal bacteria [30,61,62,63]. This observation indicates that the bacterial flora of fish may reveal the bacteriological conditions of their environment. The occurrence in fish of faecal bacteria of warm-blooded organism origin varies in terms of their composition and concentration in different tissues and organs and depends on the fish species [30,63]. For example, the presence of faecal bacteria was recorded in the intestinal tracts of 14 freshwater fish species from moderately polluted river sections, but the densities of faecal coliforms obtained from a culture-based approach differed by 50,000 times between the highest and lowest scores. Such discrepancies can be explained by their life history, diet and foraging habits. Filter-feeding, detritivorous or benthophagous species living close to the substrate or burrowing in it are more exposed to faecal contamination and bacteria than nektonic predatory species. In particular, the contamination rate of various tissues evaluated experimentally for fish exposed to EC introduced into the ambient water showed that the highest bacterial concentrations were recovered from the digestive tract [61,62,63]. Thus, the intake with food of faecal bacteria derived from warm-blood organisms is the primary pathway for their acquisition by fish. Consequently, linear relationships were found between concentrations of faecal bacteria in polluted water and their recovery from several fish tissues [63,64].

4.3. Lamprey Condition in Polluted Water

European brook lamprey ammocoetes from the highly polluted section of the River Gać had a lower condition index. The Fulton condition factor, used as a general index of fish physiological status, indicated that fish are heavier at comparable lengths, which is presumably due to better individual growth. In several studies, the values of this condition index were found to be significantly greater in fish exposed to municipal sewage effluents [28]. Therefore, such an increase in fish condition and growth rate would be explained by enhanced productivity in urban effluent waters rather than by its toxic effects. However, a link between greater condition (and thus, growth) and higher contaminant concentration is not apparent, because sewage wastewater effluent appears to affect the size distribution of fishes with greater productivity and larger individuals predominating at sewage-impacted sites compared to other sites downstream [27]. Moreover, effluent water usually has a higher temperature, also increasing the growth rate of fish.
On the other hand, raw and treated sewage are complex mixtures of substances of various degrees of toxicity that act on aquatic organisms in a synergistic/antagonistic way. Chronic exposure to such substances causes physiological dysfunction in fish. Among such effects are significant changes in some blood parameters, specifically a decrease in the numbers of red blood cells, granulocytes and lymphocytes, the activity of phagocytic cells, and the concentration of Hb and plasma protein [65]. Numerous environmental pollutants have been shown to impact organismal health via, at least in part, oxidative stress [49,66,67]. Water pollutants may disorient animals that use chemical signals from ambient water. For example, some species of lamprey possess a peripheral olfactory pit organ on their heads, used for conspecific recognition, communication, and habitat evaluation, such as the selection of patches of suitable habitat [68].
Our findings aligned with several observations indicating a negative impact of water pollution on lamprey populations [14,52,69,70]. Although still abundant and widespread in Great Britain, L. planeri is often found in the absence of the two anadromous British species (P. marinus and L. fluviatilis) above a pollution source or physical barrier that prevents the anadromous species reaching that part of the river [1]. The importance of large loads of IMs found in the guts of water-filtering ammocoetes buried in contaminated sediments, apart from potentially affecting their condition, remains unknown and requires further study. High IM abundance warrants investigation, since it may result from direct accumulation due to intake or, as Coxon et al. (2019) [71] demonstrated for poikilothermic hosts, may result from replication in the gut.
The cluster analysis indicating the relationship between the microbial water quality and microbial content of ammocoete faeces placed the samples of lampreys and water taken in a polluted section of the River Gać into one cluster separate from the sample of relatively unpolluted water. Thus, it supports the impact of water faecal pollution on lamprey microbial midgut content. Unexpected results from in situ observations by Boeker and Geist (2016) [5] and experimental work by Shirakawa et al. (2013) [4] also revealed side effects of lamprey (e.g., Eudontomyzon sp.) foraging and burrowing behaviour on water quality. The influx of water into sediment improved oxygen availability for the microbial community and led to a shift toward aerobic communities.

5. Conclusions

To conclude, our study highlights the significant impact of domestic pollution and low microbial water quality on European brook lamprey ammocoetes in a lowland river. The presence of microbial faecal pollution indicators, such as E. coli and faecal streptococci, in the water samples from the polluted downstream site (site 2) consistently exceeded recreational water standards, emphasising the severity of the pollution. Furthermore, the composition and abundance of the studied groups of microorganisms in ammocoete midguts closely mirrored the presence and numbers of the indicator microorganisms in water, underscoring the importance of the aquatic environment for the intestinal contents of this threatened species and, possibly, their health and condition. The differences in body condition indices between lampreys inhabiting polluted and relatively unpolluted river stretches highlight the negative consequences of faecal pollution on their overall condition. Our study emphasises the critical need for effective environmental management practices in aquatic ecosystems, especially in small streams often used as wastewater receivers; their water quality is seldom monitored, while they remain important reservoirs of endangered species, like the European brook lamprey. Reduction in faecal pollution in river ecosystems is crucial in this context. Regarding the tendency toward population decline of European brook lamprey in many countries, the discharge of domestic pollution represents an additional threat to this species.

Author Contributions

Conceptualisation, M.P. and G.Z.; in microbiological part M.M. and D.D.; methodology, in microbiological part M.M. and D.D.; formal analysis, M.P.; field sampling and lab investigation, G.Z., M.M., D.D., K.P. and J.G.; writing—original draft preparation, J.G., G.Z., M.M. and D.D.; writing—review and editing, G.Z., M.M., D.D., M.P., K.P. and J.G.; visualization, M.P. and K.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data will be available from the corresponding author upon reasonable request.

Acknowledgments

The Authors would like to thank Iwona Grzejdziak from the Department of Biology of Bacteria and Marta Leśniak from the Department of Ecology and Vertebrate Zoology, Faculty of Biology and Environmental Protection, University of Lodz, for their kind technical assistance in experiments. Thanks to Carl Smith and Richard Bailey from the Department of Ecology and Vertebrate Zoology, Faculty of Biology and Environmental Protection, University of Lodz, for English correction and helpful suggestions on an earlier version of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 02349 g001
Figure 1. Seasonal variation in total viable count (TVC) of mesophile and psychrophile abundance (a) and proportion (b) and indicator microorganisms E. coli and faecal streptococci abundance (c) in water samples from two sites of the River Gać. Log-transformed data (number of colony-forming units, CFU/mL—TVC; CFU/100 mL—IM) are represented as the mean ± SD. Multiple comparisons significant differences p: ns > 0.05, * < 0.05, ** < 0.01; *** < 0.001.
Figure 1. Seasonal variation in total viable count (TVC) of mesophile and psychrophile abundance (a) and proportion (b) and indicator microorganisms E. coli and faecal streptococci abundance (c) in water samples from two sites of the River Gać. Log-transformed data (number of colony-forming units, CFU/mL—TVC; CFU/100 mL—IM) are represented as the mean ± SD. Multiple comparisons significant differences p: ns > 0.05, * < 0.05, ** < 0.01; *** < 0.001.
Water 16 02349 g001
Water 16 02349 g002
Figure 2. Seasonal variation in total viable count, i.e., mesophile and psychrophile abundance (a) and proportion (b) and indicator microorganisms, i.e., E. coli and faecal streptococci abundance (c) in ammocoete midgut content from two sites of the River Gać. Log-transformed data (number of colony-forming units CFU/g) are represented as the mean ± SD. Multiple comparisons and significant differences p: ns > 0.05, * < 0.05; *** < 0.001.
Figure 2. Seasonal variation in total viable count, i.e., mesophile and psychrophile abundance (a) and proportion (b) and indicator microorganisms, i.e., E. coli and faecal streptococci abundance (c) in ammocoete midgut content from two sites of the River Gać. Log-transformed data (number of colony-forming units CFU/g) are represented as the mean ± SD. Multiple comparisons and significant differences p: ns > 0.05, * < 0.05; *** < 0.001.
Water 16 02349 g002
Water 16 02349 g003
Figure 3. Clusters (the UPGM method and Pearson correlation as a similarity index) of bacteria abundance in water and lamprey midgut content from two sites in the River Gać.
Figure 3. Clusters (the UPGM method and Pearson correlation as a similarity index) of bacteria abundance in water and lamprey midgut content from two sites in the River Gać.
Water 16 02349 g003
Table 1. Comparison of water quality parameters for the River Gać during sample collection.
Table 1. Comparison of water quality parameters for the River Gać during sample collection.
Sampling LocationSite 1
(Relatively Unpolluted)		Site 2
(Polluted)
Sampling date28 September 202016 January 202110 April 202128 September 202016 January 202110 April 2021
Water temperature [°C]12.81.46.110.91.56.5
Dissolved oxygen [mgL−1]5.1511.6111.3512.712.9712.5
Specific conductance [µSL−1]304314313291331331
pH8.617.547.346.196.987.02
Table 2. The effects of site, season and type of microorganisms (mesophiles and psychrophiles; indicator microorganisms (Escherichia coli and faecal streptococci) on microbial numbers in water and ammocoete midgut content (ANOVA III, log-transformed data).
Table 2. The effects of site, season and type of microorganisms (mesophiles and psychrophiles; indicator microorganisms (Escherichia coli and faecal streptococci) on microbial numbers in water and ammocoete midgut content (ANOVA III, log-transformed data).
BacteriaEffectFdfp
waterTVCsite1034.201, 68>0.0001
season81.853, 68>0.0001
TVC300.571, 68>0.0001
site × season88.913, 68>0.0001
site × TVC13.271, 68>0.0001
season × TVC5.943, 68>0.001
site × season × TVC12.463, 68>0.0001
IMssite3510.291, 80>0.0001
season65.593, 80>0.0001
IMs0.481, 800.4921
site × season212.063, 80>0.0001
site × IMs228.911, 80>0.0001
season × IMs23.653, 80>0.0001
site × season × IMs5.963, 80>0.001
ammocoetesTVCsite15.222, 23>0.0001
season48.051, 23>0.0001
TVC7.071, 230.0140
site × season9.242, 230.0011
site × TVC0.532, 230.5958
season × TVC0.271, 230.6086
site × season × TVC0.332, 230.7234
IMssite114.381, 31>0.0001
season81.933, 31>0.0001
IMs0.671, 310.4203
site × season5.683, 310.0032
site × IMs13.811, 310.0008
season × IMs17.333, 31>0.0001
site × season × IMs3.743, 310.0210
Table 3. Weight-length regression parameters (a = intercept; b = slope) and their standard errors of European brook lamprey ammocoetes in two studied sites of the River Gać (log-transformed data).
Table 3. Weight-length regression parameters (a = intercept; b = slope) and their standard errors of European brook lamprey ammocoetes in two studied sites of the River Gać (log-transformed data).
Locationase abse br2np
Site 1−5.5270.2632.8780.1220.95727<0.001
Site 2−5.5940.2342.8830.1100.96527<0.001
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Zięba, G.; Moryl, M.; Drzewiecka, D.; Przybylski, M.; Pyrzanowski, K.; Grabowska, J. Effects of Domestic Pollution on European Brook Lamprey Ammocoetes in a Lowland River: Insights from Microbiological Analysis. Water 2024, 16, 2349. https://doi.org/10.3390/w16162349

AMA Style

Zięba G, Moryl M, Drzewiecka D, Przybylski M, Pyrzanowski K, Grabowska J. Effects of Domestic Pollution on European Brook Lamprey Ammocoetes in a Lowland River: Insights from Microbiological Analysis. Water. 2024; 16(16):2349. https://doi.org/10.3390/w16162349

Chicago/Turabian Style

Zięba, Grzegorz, Magdalena Moryl, Dominika Drzewiecka, Mirosław Przybylski, Kacper Pyrzanowski, and Joanna Grabowska. 2024. "Effects of Domestic Pollution on European Brook Lamprey Ammocoetes in a Lowland River: Insights from Microbiological Analysis" Water 16, no. 16: 2349. https://doi.org/10.3390/w16162349

APA Style

Zięba, G., Moryl, M., Drzewiecka, D., Przybylski, M., Pyrzanowski, K., & Grabowska, J. (2024). Effects of Domestic Pollution on European Brook Lamprey Ammocoetes in a Lowland River: Insights from Microbiological Analysis. Water, 16(16), 2349. https://doi.org/10.3390/w16162349

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