A Multibiomarker Approach to Assess the Health State of Coastal Ecosystem Receiving Desalination Plants in Agadir Bay, Morocco

Marine pollutants such as heavy metals (HMs) are considered among the most copious oxidative stress (OS) inducers in marine organisms which leads to reactive oxygen species (ROS) formation. Complementary to our previous bioassays studies, the present research focuses on Catalase (CAT), Glutathione S-transferase (GST) and Malondialdehyde (MDA) as oxidative stress biomarkers and the integrated biomarker response (IBR) indexes (IBR1 and IBR2) as an ecotoxicological assessment tool in Mytilus galloprovincialis using central composite face centered (CCF) design. The oxidative stress biomarkers were measured in adult mussels (45-55 mm) on 3 days-exposed under different sub-lethal concentrations of cadmium (Cd), zinc (Zn), and copper (Cu). Using multiple regressions, ANOVA analysis revealed that experimental data fitted to second-order (quadratic) polynomial equations. The results showed that types, concentrations and metals combinations has a direct effect in CAT and GST activities, MDA level and IBR indexes. Additionally, metal-metal interactions were found synergistic (supra-additive), antagonistic (infra-additive) or zero interaction in the toxicological effect. As necessary, the optimization of the experimental results was done in order to determine the optimal conditions for the oxidative stress responses and IBR indexes. It was demonstrated that the CCF design combined with the multi-biomarker approach and IBR index can be used as an appropriate tool in ecotoxicological modulation and prediction of oxidative stress and antioxidant status by heavy metals in the mussels Mytilus galloprovincialis.

Elazzaoui, A.; Kalla, Y.; Lefrere, L.; Bouhaimi, A., and Moukrim, A., 2022. Using Mediterranean mussel bio-indicators to assess the initial health state of ecosystems receiving desalination plant effluents in Agadir Bay (Morocco). Journal of Coastal Research, 38(1), 154–167. Coconut Creek (Florida), ISSN 0749-0208. Mussels are often used as a biological bio-indicator for the quality assessment of marine ecosystems. Adopting this approach, the present work aims to study the initial health status of two ecosystems in Agadir Bay, Morocco expected to receive seawater desalination plant effluents. This study was conducted between February 2015 and March 2017 in two stations, Tifnit-Douira and Cap Ghir. In addition to the gametic development, variations of sex ratio and gonadal index were also determined. The reproductive cycle of Mytilus galloprovincialis is spread throughout the year. Under natural conditions, three collective spawning periods were observed. The main one was during the spring and the two other periods were optional and were in winter and summer. Variations of seawater temperature can make some changes in mussel spawning, with one principal period lasting during spring and summer and the optional one during winter. In both studied stations, sexual rest was manifested by a small percentage of the population particularly during summer, and could not reach the whole population at the same time. This finding is consistent with the gonadal index that never reached the value of 1. A unique hermaphroditism case was revealed in Tifnit-Douira mussels, and it remained statistically insignificant. The sex ratio was perfectly balanced in both stations and was in favour of males. The gonad histology did not show any cellular abnormalities in the germinal tissue. These results are of great importance since they characterize the current state of the studied stations area before the installation of the desalination plant. This state must be maintained after installation in order to preserve the typical halieutic resources of these ecosystems. The present findings confirm the good environmental health status and the aquaculture potential of Agadir Bay, especially in the north ecosystems (Cap Ghir) due to upwelling-nutritional intake and optimum seawater temperature.

Mediterranean mussel (Mytilus galloprovincialis Lmk) has a great environmental and economic importance for Morocco. This work studies the genetic structure and impact of chemical pollution on three different marine populations of Mytilus galloprovincialis that live within Agadir bay. Three collections were made at two clean sites (Cape Ghir and Cape Aglou) and at an impacted site exposed to intense boating and industrial activities (Anza). A 300-bp portion of the mitochondrial DNA coding-region Cytochrome C Oxidase subunit 1 (COI) was studied by polymerase chain reaction (PCR) and DNA sequencing reactions to assess and evaluate amounts of polymorphism in each site. Genetic analysis using COI for 64 individuals showed no significant differentiation between the three subpopulations. AMOVA demonstrated that only 2.83% of variation exists between populations. Besides the genetic evidence presented herein, mussel’s adaptation mechanisms and strategies to marine pollution are also discussed.

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

The present study aimed to assess the oxidative stress potential of Mytilus galloprovincialis across four sites along the Algerian west coast from March 2022 to June 2022. Among these sites, three were previously identified as contaminated, while the Barbadjanie site was considered isolated and distant from pollution sources. We measured glutathione (GSH) and malondialdehyde (MDA) levels, as well as the specific activities of glutathione S-transferase (GST) and catalase (CAT) in the digestive glands of the mussels. Our findings revealed a significant decrease in GSH levels in mussels from the contaminated sites, indicating reduced antioxidant capacity. In contrast, MDA levels, GST, and CAT activities exhibited significant increases (P< 0.05) in the same mussels, suggesting heightened oxidative stress and increased cellular damage. To further evaluate the impact of pollution, we applied the Integrated Biomarker Response (IBR) index to the four biomarkers measured. The IBR analysis showed the lowest score for mussels from Barbadjanie, confirming its relatively low pollution impact, and the highest score for mussels from Beni Saf Port, indicating severe pollution effects. This multi-biomarker approach was effective in demonstrating oxidative stress responses in mussels, highlighting its reliability in assessing the impacts of environmental pollution on aquatic organisms. The study underscores the importance of using comprehensive biomonitoring tools to evaluate ecological health and pollution effects in marine environments.

Electrical and electronic equipment reaching the end of its useful life is currently being disposed of at such an alarmingly high pace that raises environmental concerns. Together with other potentially dangerous compounds, electronic waste contains the rare-earth element gadolinium (Gd), which has already been reported in aquatic systems. Additionally, the vulnerability of aquatic species to this element may also be modified when climate change related factors, like increase in temperature, are taken into consideration. Thus, the present study aimed to evaluate the toxicity of Gd under a scenario of increased temperature in Mytilus galloprovincialis mussels. A multi-biomarker approach and Gd bioaccumulation were assessed in mussels exposed for 28 days to 0 and 10 μg/L of Gd at two temperatures (control – 17 °C; increased – 22 °C). Results confirmed that temperature had a strong influence on the bioaccumulation of Gd. Moreover, mussels exposed to Gd alone reduced their metabolism, possibly to prevent further accumulation, and despite catalase and glutathione S-transferases were activated, cellular damage seen as increased lipid peroxidation was not avoided. Under enhanced temperature, cellular damage in Gd-exposed mussels was even greater, as defense mechanisms were not activated, possibly due to heat stress. In fact, with increased temperature alone, organisms experienced a general metabolic depression, particularly evidenced in defense enzymes, similar to the results obtained under Gd-exposure. Overall, this study underlines the importance of conducting environmental risk assessment taking into consideration anticipated climate change scenarios and exposures to emerging contaminants at relevant environmental concentrations.

The aim of our study is to evaluate the impact of Bousfer desalination plant brine discharges on the Algerian west coast, on a natural population of the marine gastropod mollusc Patella rustica. The effects of a chronic exposure to such discharges are complex to understand due to the combined effects of environmental physico-chemical parameters (e.g., high salinity) and different pollutants that can modulate the physiological responses of this species to stress. In this context, we assessed the biological effects in a marine species P. rustica, by a multibiomarker approach that provided information on the health status of organisms in response to such an environmental stress. We measured biomarkers in the whole soft tissues of limpets as indicators of neurotoxicity (AChE activity), oxidative stress (CAT, SOD, GR, and GPx activities), biotransformation (GST), oxidative damage (LPO through TBARS levels), and genotoxicity (CSP 3-like activity). In parallel, hydrological parameters were measured in the Bay of Oran, at four selected sites: site H considered as a "hotspot," located at Bousfer desalination plant; two other sites E and W, at the east and the west of H respectively; finally, site R "reference" located in Madragh, which is considered as a remote clean site. Our analyses revealed that the activities of antioxidant defense enzymes reached the highest levels in P. rustica collected from site H. The activation of antioxidant defense system in these organisms translated the alteration of their status health, reflecting a level of environmental disruption generated by the desalination plant brine discharges and the high salinity in this area. We also observed that the tissues of limpets collected from site H as well as the two other sites, E and W, had undergone molecular damage, confirmed by the high levels of CSP 3-like activity. This damage resulted from chronic exposure to environmental conditions, potentially genotoxic, due to the desalination plant discharges. The present results indicate the adverse impact of brine effluents from desalination plants on marine fauna and suggest the need for a more consistent approach to environmental management of brine discharges.

Climate change (CC) induces significant worldwide alterations in salinity and temperature, impacting ecosystems and their services. Marine organisms, susceptible to these changes, may experience modified vulnerability to anthropogenic contaminants, including rare-earth elements (REEs) such as yttrium (Y) derived from electronic waste. This study investigated the influence of temperature and salinity changes on the impacts of Y in Mytilus galloprovincialis mussels. Organisms were subjected to Y (0 and 10 μg/L) for 28 days under three salinity scenarios (20, 30 (control), and 40, at a control temperature of 17 °C) or to two temperatures (17 and 22 °C, at the control salinity of 30). Under these conditions, Y bioaccumulation and different biomarkers were evaluated. Results showed that salinity and temperature did not affect Y accumulation, indicating effective detoxification mechanisms and physiological adaptations in the exposed organisms. However, in Y-exposed mussels effects were intensified under decreased salinity, evidenced by increased metabolism, defense enzyme activities, and acetylcholinesterase (AChE) levels. Similar responses occurred under heat stress with enhanced metabolic capacity, AChE activity, and activation of defense mechanisms such as glutathione S-transferases. These defense mechanisms mitigated cellular damage caused by Y, but under the highest temperature and especially lower salinity, Y-exposed mussels exhibited increased oxidative stress and decreased efficiency of activated defense enzymes, resulting in cellular damage compared to their uncontaminated counterpart. The present study sheds light on the effects that interactions between temperature, salinity, and the presence of emerging contaminants like REEs may have on marine organisms. Such assessments are crucial for developing effective strategies to mitigate the impacts of CC and protect the long-term health and resilience of marine ecosystems.

Integrated use of antioxidant enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain)

Assessment of organochlorine pesticide pollution in Donax trunculus from Agadir Bay (Center of Morocco): Utilization of integrated biomarkers

This study examined the influence of increasing temperatures in spring and summer on biochemical biomarkers in Mytilus galloprovincialis mussels sampled from Bizerte lagoon (northern Tunisia). Spatial and seasonal variations in a battery of seven biomarkers were analyzed in relation to environmental parameters (temperature, salinity, and pH), physiological status (condition and gonad indexes), stress on stress (SoS), and chemical contaminant levels (heavy metals, polycyclic aromatic hydrocarbons (PAHs), and PCBs) in digestive glands. Integrated biological response (IBR) was calculated using seven biomarkers (acetylcholinesterase (AChE), benzo[a]pyrene hydroxylase (BPH), multixenobiotic resistance (MXR), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA), and metallothioneins (MT). Seasonal variations in biological response were determined during a critical period between spring and summer at two sites, where chemical contamination varies by a factor of 2 for heavy metals and a factor 2.5 for PAHs. The analysis of a battery of biomarkers was combined with the measurement of physiological parameters at both sites, in order to quantify a maximum range of metabolic regulation with a temperature increase of 11 °C between May and August. According to our results, the MT, MDA, CAT, and AChE biomarkers showed the highest amplitude during the 11°C rise, while the BPH, GST, and MXR biomarkers showed the lowest amplitude. Metabolic amplitude measured with the IBR at Menzel Abdelrahmen-the most severely contaminated station-revealed the highest metabolic stress in Bizerte lagoon in August, when temperatures were highest 29.1°C. This high metabolic rate was quantified for each biomarker in the North African lagoon area and confirmed in August, when the highest IBR index values were obtained at the least contaminated site 2 (IBR = 9.6) and the most contaminated site 1 (IBR = 19.6). The combined effects of chemical contamination and increased salinity and temperatures in summer appear to induce a highest metabolic adaptation response and can therefore be used to determine thresholds of effectiveness and facilitate the interpretation of monitoring biomarkers. This approach, applied during substantial temperature increases at two sites with differing chemical contamination, is a first step toward determining an environmental assessment criteria (EAC) threshold in a North African lagoon.

Seasonal environmental parameters influence biochemical responses of the fiddler crab Minuca rapax to contamination in situ

A multibiomarker approach helps assess environmental health as it provides a complete tool to understand the effects of environmental stressors on ecosystems and human health. We applied this approach in the central Atlantic Ocean of Morocco, an area subjected to the impact of many types of pollutants, threatening the durability of its resources. In this study, four biomarkers acetylcholinesterase (AChE), glutathione-s-transferase (GST), metallothioneins (MTs), and catalase (CAT) were measured in the digestive gland of the mussel Mytilus galloprovincialis collected from four sites: Imsouane (S1), Cap Ghir (S2), Imi Ouaddar (S3), and Douira (S4). These sites were chosen due to the diversity of impacts ranging from industrial to agricultural and touristic. We also assembled all the enzymatic responses (AChE, GST, CAT, and MTs), using the integrated biomarker response (IBR), to estimate the degree of impact of pollutants at the prospected sites to reveal all the complex interactions between biomarkers and to classify sites via the integrated approach. Results show a seasonal change in biomarker responses with variability between sites. We also recorded the highest levels of AChE inhibition and GST induction in S1, higher levels of catalase activity in S4, and a significant impact on metallothionein concentration in S1 and S3. This project highlights the interest in using a multibiomarker approach to ensure accurate interpretation of biomarker variation to protect the Moroccan coast and its resources.

Biochemical responses of the marine mussel Mytilus galloprovincialis to petrochemical environmental contamination along the North-western coast of Portugal

Season specific influence of projected ocean changes on the response to cadmium of stress-related genes in Mytilus galloprovincialis