Biomonitoring of radioactive contamination using benthic invertebrate communities in Manzala Lake, Egypt

Manzala Lake is located on the northeastern coast of Egypt and is considered the largest lake in Egypt (Fig. 1). The lake lies between 31°45', 32°15' E and 31°00', 31°35' N with an area of approximately 52611 ha. It extends along the Mediterranean Sea, separated from the sea by a sandy margin, except at three outlets that supply the lake with saline seawater from the Mediterranean Sea. These outlets allow the movement of organisms between the lake and the sea: Boughaz El-Gamil, Boughaz El-Boughdady, and Boughaz New El-Gamil (Abdel Mola and Abd El-Rashid 2012). Manzala Lake has been exposed to various threats in the past six decades, chiefly from agricultural drainage, sewage, and industrial wastes. Annually, it receives approximately 650 and 1.7 million m³ of untreated sewage water from Bahr El-Baqar and Hadous Drains, respectively. It also receives the industrial discharge from Industrial Compound in Port Said City. Freshwater (mainly from agricultural drainage) flows yearly into Manzala Lake from the main drains and canals (Faraskur, El-Serw, Matariya, Ramsis, Hadous, and Bahr El-Baqar) (Elshemy et al. 2016). A large amount of wastewater is transported into the lake from a heavily populated area of the Eastern Delta (Qalyubia, Sharkia, Ismailia, and Port Said Governorates), which significantly contributes to the deteriorating water quality of the lake (Zahran et al. 2015).

Figure 1

Samples were collected from ten sites (M1–M10) during the winter and summer of 2020 (Fig. 1, Table 1). The three samples were taken from each site to represent that specific location. M1, M2, and M3 are influenced by seawater, sewage from the El-Gamil, New El-Gamil, and El-Boughdady outlets, and the Alhuria drain. Various sewage discharges influence M6 and M7 from the Bahr El-Baqar drain. M8 is affected by sewage and agricultural discharges from the new Bahr El-Baqar, and the Hadous and Ramsis drains. Agricultural emissions from the El-Matariya drain influence M9. M10 is affected by agricultural layoffs from the Inania, Faraskur, and El-Serw drains. Direct discharges do not influence M4 and M5.

Water samples were collected with a water sampler and placed in plastic bottles (2 l). Water temperature (°C), pH, total dissolved solids (TDS), water transparency (cm), and dissolved oxygen (DO) were measured, as discussed in the American Public Health Association-defined methods (APHA 2005) (supplementary information [SI]). The sediment and benthos were sampled using an Ekman grab sampler with a sample area of 225-cm² (15 × 15 cm) and separately packed in plastic bags. All samples were preserved in an icebox until they were placed in the lab. In the lab, the benthos samples were sieved using a net with a mesh diameter of 500 µm. The reserved materials were individually preserved in labeled bottles with 10% formalin. The samples were sorted in the laboratory, and the spotless benthic invertebrates were fixed with 75% ethanol. Relevant identification guides were used to identify the species or genus-level specimens (Day 1956, Walker 1959, Brinkhurst, and Jamison 1971, Flemming 1983, Brown et al. 1984, Hussein et al. 1988, Ibrahim et al. 1999). The sediment and benthos samples were dehydrated in an air-circulated oven at 74°C. The dried samples were weighed and crushed.

Gamma spectrometry based on a high-purity germanium (HpGe) detector was used to determine the specific activity concentration of natural and artificial radionuclides. The detector, coupled to standard electronics attached to a multichannel analyser card (MCA) installed in a PC, has a relative efficiency of approximately 50%, compared with the 3 × 3 NaI(Tl) detector. The detector was protected using a lead liner (10 cm thick) covered with a 2 mm copper foil. Data and spectrum analysis were performed using the ORTEC software MAESTRO. The energy calibration of the spectrometer was performed with point sources ²⁴¹Am (59.6 keV), ¹³⁷Cs (661.6 keV), and ⁶⁰Co (1172 and 1332.3 keV). For the spectrometer efficiency calibration, three well-known reference materials (RGU-1, RGTh-1, and RGK-1) were applied (IAEA 1987, Anjos et al. 2005, El Aassy et al. 2012). The water, sediment, and benthic invertebrate samples were placed in Marinelli beakers pre-dried and washed with dilute H₂SO₄ to avoid contamination. The Marinelli beakers were firmly closed for at least 4 weeks to ensure that the levels of Ra and their daughters were in secular equilibrium. For the sediment and benthic invertebrate samples, the specific activity concentration of radionuclides is expressed as Bq kg⁻¹; for the water samples, Bq l⁻¹is used. The time accumulation of the spectra was at least 48 h, depending on the activity of the samples. The activity concentration of ²²⁶Ra (²³⁸U-series) was determined via gamma-energy lines of 295.2 and 351.9 keV for ²¹⁴Pb and 609.3 and 1120.3 keV for ²¹⁴Bi. The gamma-energy lines of 338.4 and 911.2 keV for ²²⁸Ac, 583 and 2614.4 keV for ²⁰⁸Tl, and 727.3 keV for ²¹²Bi were used to represent the activity concentration of the ²³²Th series. The activity concentration of ⁴⁰K and ¹³⁷Cs was determined via gamma lines of 1460.8 and 661.7 keV, respectively (Chieco et al. 1990). The activity concentration of the radionuclides was calculated using the reported equation (El Afifi et al. 2006).

The precision and accuracy of the analytical results provided in this study were verified using quality control procedures in IAEA reference materials (IAEA-414, IAEA-446). Using the equation proposed by Rihs and Condomines (2002), the uncertainty of the radionuclide concentration in samples with gamma spectrometry was calculated, and the confidence interval for counting instruments was 90%. The detector's lower limits of detection (LLDs) were required to estimate a minimum detection level for the radionuclides in each sample. The LLDs were defined by Imtiaz et al. (2005). The LLD values obtained for the sediment were 9.35, 0.45, 1.34, and 0.2 Bq kg⁻¹ for ⁴⁰K, ²²⁶Ra, ²³²Th, and ¹³⁷Cs, respectively, and those for water were 0.84, 0.1, 0.1, and 0.02 Bq l⁻¹.

The ordinary index of benthic species richness (SR), the diversity index (H'), and evenness or equitability (E) were calculated for all the sampled sites using the Primer software (version 5) (Clarke and Gorley 2006). Hierarchical cluster analysis (CLUSTER) was performed to analyse the community structure using PC-ORD 5.0. Redundancy analysis (RDA) was employed to study the benthic invertebrate species in relation to the environmental and radioactive element variables. RDA was appropriate for data analysis as in the preliminary detrended correspondence analysis; the length of the gradient for the first axis was 2.33 SD ˂ 3 SD (Lepš and Petr Smilauer 2003). Log transformation was performed to standardise the parameters before the examination. Monte Carlo permutation test parameters were set to 999 permutations to identify the significant environmental and radioactive element variables influencing the abundance and distribution of the benthic invertebrates (p ˂ 0.05). The RDA was conducted using the Canoco 4.5 software (Ter Braak and Smilauer 2002).

The environmental parameters of water samples in Manzala Lake are shown in Table 2. The annual average water temperature fluctuated from 15.6°C in winter to 32.4°C in summer, with averages of 16.27 and 31.17°C during winter and summer, respectively. The pH of the Manzala Lake was alkaline, ranging from 7.56 to 8.97, with averages of 8.27 and 8.29 during winter and summer, respectively. The water transparency fluctuated between 10 and 70 cm. The lowest value (10 cm) was recorded at stations M6 and M9 during winter, whereas the maximum value was recorded in the east-west of the lake. The DO results showed a wide variation, with decreased DO in the southern section due to the effect of drains. Station M6 exhibited a noticeable impact of domestic waste from the Bahr El-Baqar drain, indicating a complete DO depletion in summer, and low DO (0.7 mg l^-1) in winter. Conversely, the maximum value was 14.7 mg l^-1, recorded at station M1 during the summer. The water salinity ranged from a relatively high 21.9% at station M3 (Boughaz El-Boughdady) to a relatively low 1.25% at station M8 (Mahmoud et al. 2020).

Regarding Manzala Lake, the salinity fluctuated substantially during recent decades, with the average value reported (17.24) in 1967 declining to (5.37) in 1975, and (2.53) in 2001, before progressively increasing to 4.26 in 2015 and 5.02 in 2020. Although prior studies found substantial geographical variability in DO levels, the lake is typically oxygenated, except for places near the inlets of diverse wastewaters, where the DO is fully depleted (Table S1). Long-term variations in the physicochemical properties of Manzala Lake water revealed the spatial and temporal variances in most parameters based on the quantity and quality of the various wastes discharged into the lake and the amounts of seawater that enter the lake through boughazes.

We recognised 30 taxa of the benthic invertebrates across all the sampling sites (Table S2, Fig. 2): six Arthropoda species (48%), seventeen Mollusca species (43%), and seven Annelida species (9%). The total average abundance of benthic invertebrates was 4330 ind. m-2. Abdel Gawad et al. (2012) recorded 19 taxa for benthic invertebrates in Ashtum El-Gamil, Lake Manzala, where Mollusca was the largest group, forming approximately 68.5% of the total population density of benthic invertebrates, followed by Arthropoda and Annelida. Fishar et al. (2015) identified nineteen macrobenthos species from the Mollusca, Annelida, and Arthropoda groups. The maximum number of species was reported for Mollusca, while Annelida and Arthropoda had the lowest number of species. El Komi (2021) also recorded 16 species of benthic communities in Lake Manzala. He stated that Ostracoda was the most numerous species found in the lake's sandy bottom sediments and was reported at most sample locations. The highest (22 species, 4823 ind. m^-2) and lowest (17 species, 3838 ind. m^-2) abundances and species numbers were recorded in the winter and summer, respectively. Arthropoda comprised one Insecta and five Crustacea; it recorded the highest average density and lowest number of species (2056 ind. m^-2, six species). This high density was due to the dominance of one species (Ostracods sp.) that accounted for 1038 ind. m^-2. It constituted 24% of the study area's population, which agrees with El Komi (2021). Abdel Gawad et al. (2012) stated that Ostracoda dominated the meiofauna in Lake Manzala and exhibited the highest peak in summer. Freshwater and marine mollusk species were included in the present investigation. It comprised three Bivalvia and fourteen Gastropods and recorded the highest species number and second highest density (17 species, 1866 ind. m^-2). Gyraulus ehrenbergi was the dominant species of molluscs (43%). Annelida comprised three Polychaeta and four Clitellata and had the lowest density (408 ind. m^-2). M3 In Lake Manzala recorded the highest density and diversity of species (13 species and 13089 individuals m^-2). M7 had the highest SR (1.5) and the highest H '(2), while M6 had the lowest SR (0.18) and the lowest H '(0.3). There was a noticeable decrease in the number of species (2 species) and individuals (275 individuals m^-2) at M6. The results showed that the study area recorded a widespread oxygen variation representing high hypoxia at M6. DO is an essential vital biotic impact on the aquatic ecosystem. The solubility and availability of nutrients are affected by DO, which releases nutrients from sediments accelerated under hypoxia, thereby influencing production in aquatic ecosystems (Abdelmongy and El-Moselhy 2015). At M8, the number of species was reasonably high, with Gyraulus ehrenbergi constituting 62% of the total individuals. Therefore, the minimum evenness (E = 0.53) of M8 was mainly due to the unequal distribution of individuals among the species (Table 3). The CLUSTER results showed that the sites could be divided into four groups (Fig. 3). Group 1 comprised M1, M2, and M4; group 2 represented only M3 (in front of Boughaz El-Boughdady) because the number and abundance of its marine origin species was greater than other sites; group 3 included M5, M7, M8, and M9; and group 4 included M6 and M10. Group 1 is the northern section of the lake and is categorised by a high abundance of Arthropoda and marine species. Contrarily, group 3 is the southern section of the lake and is categorised by the high diversity and density of Mollusca. Hussein et al. (2011) stated that freshwater snails show a high degree of tolerance and plasticity in a specific range of physicochemical variation. Group 4 exhibited the lowest benthos density, owing to high sewage contamination and agricultural discharge from the Bahr El-Baqar and El-Serw drains.

Figure 2

Figure 3

The CF (Table S3), described as the ratio of the concentration of a contaminant substance in the organisms (Bq kg⁻¹ wet weight) to the concentration in the surrounding water environment (Bq l⁻¹), first appeared in aquatic toxicology in (Rand 1995). It is necessary to quantify the active concentrations of radioactivity in ecosystems and biota to describe the intake and accumulation of radionuclides (Eisenbud and Gesell 1997). The range of CF values of 226Ra in the benthic invertebrates varied (0.7 × 102 to 3.4 × 102 and 0.7 × 102 to 5.2 × 102 Lkg−1), with average values of 1.5 × 102 and 1.7 × 102 Lkg−1 during winter and summer, respectively. The CF of 232Th varied from 0.5 × 102 to 1.3 × 102 and 0.66 × 102 to 1.5 × 102 Lkg−1 with average values of 0.9 × 102 and 1.2 × 102 Lkg−1 during winter and summer, respectively. The highest CF of 40K was 1.4 × 103 and 1.9 × 103 Lkg−1 at M5, with average values of 0.5 × 103 and 0.6 × 103 Lkg−1 during winter and summer, respectively. The CF of 137Cs varied (0.2 × 102 to 0.31 × 102 and 0.2 × 102 to 0.26 × 102 Lkg−1) with average values of 0.23 × 102 and 0.21 × 102 Lkg−1 during winter and summer, respectively. The reported values for ²³²Th and ¹³⁷Cs were lower than the recommended values listed in the technical report on CFs in the marine environment (IAEA 2005). According to the IAEA, the permissible limits of CFs for ²²⁶Ra, ²³²Th, and ¹³⁷Cs were 0.65 × 102, 0.51 × 103, and 0.65 × 102 Lkg−1, respectively. The CF for 226Ra exceeded the recommended level, indicating that Ra had accumulated to high levels in mollusk shells, which agreed with the finding in mollusks in certain locations along the coast of Syria (Ammar et al. 2009). The CFs in this study agreed with reported values (Holtzman 1966, Davy and Conway 1974, Iyengar 1990, Ammar et al. 2009). According to studies on freshwater environments, mollusks and crustaceans can accumulate Ra to different degrees (Hameed et al. 1997, Hosseini et al. 2008, Ammar et al. 2009).

The RDA results for benthic species and radioactive elements in the sediment (Fig. 7A) indicated that the first two ordination axes accounted for 22.2% and 13.8% of the variation in the benthic assemblages, respectively. The species-radionuclides correlations were as high as 0.903 for axis 1 and 0.977 for axis 2, which explained 44.8% and 27.6% of the cumulative variation in species and radionuclides, respectively. The total cumulative percentage of the relationship between species and radionuclides for the first two axes was 72.4%, demonstrating that the associations could be better reproduced using an ordination map (Table 3). According to the permutation test, the primary radioactive element that could affect the community structure of the benthic invertebrates in the study area was the ¹³⁷Cs radionuclide (F-ratio = 1.85, P-value = 0.012). The RDA results revealed a strong correlation between ¹³⁷Cs and Annelida species (Nereis diversicolor, Limnodrilus udekedmianus, and Ficopomatus enigmaticus), which was recorded in M1 and M2 that were influenced by seawater and sewage from the El-Gamil, New El-Gamil, and Alhuria drains. This may be because the soft tissues of the Annelida species absorbed the ¹³⁷Cs solute in the water, and ¹³⁷Cs is a radioactive element introduced into the lake via the sea. It is soluble in seawater and is mobile. The findings correlated with Saniewski et al. (2022), who indicated that organisms without shells (soft tissues) exhibited the highest average ¹³⁷Cs activity.

Moreover, annelids are deposit feeders, which are more accompanied by Cs uptake. Rowan et al. (2013) stated that ¹³⁷Cs concentrations in deposit-feeding benthic invertebrates were significantly greater in coastal biota and were highly connected with ¹³⁷Cs concentrations in associated sediments. Corbicula fluminalis, Theodoxus niloticus, and Branchiura sowerbyi strongly correlated with ²³²Th. Mollusc species (Gyraulus ehrenbergi, Biomphalaria alexandrina, Physa acuta, Bulinus truncates, Pila ovata, and Lymnaea natalensis) exhibited a transparent relationship with ²²⁶Ra and ⁴⁰K. The high abundance of these species at M8 and M7 indicated that they increase in polluted waters. Archna et al. (2015) stated that the genus Physa, which belongs to Pulmonata, is a biological marker of organic pollution and eutrophication. Furthermore, Gyraulus sp. dwelled in polluted waters (Gallardo and Prenda 1994). Additionally, these stations, located in the southern section of the lake, receive agricultural discharges containing phosphate fertilisers, which enhance the K and Ra levels (Hassan et al. 2016).

The RDA results for benthic species and radioactive and environmental variables in the water (Fig. 7B) illustrated that axes 1 and 2 constituted 31.5% and 15.1% of the total variation, respectively. TDS had significant effects on the distribution of marine arthropod species. Khalil (1990) and Abdel Gawad et al. (2012) stated that the species distribution and biodiversity in Manzala Lake are mainly defined by salinity. In addition, TDS is significant in explaining the population size and distribution. The water clarity has a considerable impact on available light, a crucial variable in the vertical distribution of benthos, a significant food source for many benthos groups (Denis and Galtsova 2012). Pirenella conica and Melanoides tuberculate exhibited a strong relationship with ²³²Th. Ficopomatus enigmaticus indicated a clear relationship with ⁴⁰K. The strong correlation between ²²⁶Ra, ²³²Th, and TDS in water revealed the similarity of the chemical properties of Ra and Th. However, there was a difference in solubility; Ra and Th are highly insoluble in natural waters. Furthermore, a strong correlation existed between 40K and 137Cs, whereas living organisms accumulated Cs and K (Moon et al. 2021), which have similar chemical characteristics. In addition, ⁴⁰K has a higher solubility and is more mobile than the other radionuclides.

Figure 7