Visual-Spatial Attention Abilities Among School Children Exposed to Low Environmental Lead Level

: Lead (Pb) is an intrusive toxic matter which harms the neurocognitive and behavioral development of children even with doses considered normal. The present paper aims at evaluating the visual-spatial attention function among 142 schoolchildren living in a mining area (cases) in comparison to 177 living in the Chouiter area (control group) in Marrakech. We used the Bell Dam Test (BDT) for the assessment of their visual attentional abilities and unilateral spatial neglect. Interviews with parents were conducted to identify co-variables related to Pb exposure. Agricultural soil, drinking, and groundwater were collected from the surrounding area of the children's school. The determination of Pb content in the environment was performed by ICP-MS. The results demonstrated that cases' scores in the test of visual attentional abilities and in scanning procedures were significantly worse in comparison to the control group (p = 0.05 and p = 0.002, respectively). In cases, soil Pb level was higher (67.14±10.13 g/g) than in controls (41.73±13.6 g/g). Pb levels in groundwater and drinking water were low with a significant difference between the two areas in groundwater. The mean scores of visual attentions were significantly lower in children exposed to environmental Pb contamination compared to those who are not exposed (p = 0.008). These findings suggest that Pb is probably incriminated in the genesis of visual attention deficit.


Introduction
Trace Elements are essential to the state of trace for numerous cellular processes (low proportion) but can become toxic when the concentration exceeds a certain threshold like Copper (Cu), Nickel (Ni), Iron (Fe), Zinc (Zn), etc. The nonessential trace elements have no beneficial effect on the cell. By contrast, they have a polluting character with toxic effects on the live bodies even in low concentrations, such as lead (Pb), Aluminum (Al), Cadmium (Cd), mercury (Hg), and so forth. These micropollutants cause nuisances, even when they are rejected in very low quantities. Their toxicity is developed by bioaccumulation along the food chain (Tchounwou et al., 2012). The inorganic form of Pb which is the oldest and most widely used by humanity is neurotoxic even at rates met in the environment. Children are most vulnerable and most exposed to Pb (NTP, 2012). Moreover, it was reported that, compared to other pollutants, Pb is a more significant predictor of cognitive impairment (Clay et al., 2019).
Surface soil contamination has long been acknowledged to be a major lead exposure in humans and is currently a global health concern (Mielke and Reagan, 1998;Rabinowitz et al., 1991). Additionally, high soil lead concentrations will likely last for centuries and resuspension of Pb-contaminated soil can increase atmospheric Pb levels and spread lead to nearby places (Laidlaw and Filippelli, 2008).
Exposure to Pb levels during development, even if they are weak, could have adverse effects on the neurobehavioral and cognitive performance of children (Finkelstein et al., 1998). Manifest distraction, low frustration tolerance, impulsivity, and organizational inabilities have all been identified as symptoms of attention behavior dose-dependent impairment (Bellinger et al., 1984). The U.S. National Toxicology Program (NTP, 2012) concluded that, in children, blood Pb levels below 5 g/dL are associated with an increase in the diagnosis of behavioral problems related to attention and a decline in cognitive function. In this context, the presence of a heavy metals mine (copper, cadmium, and lead) in Marrakech city suggests targeting this sector. Compared to Lekouch (2004), the average blood lead level decreased from 12 to 3.14 µg/dL (Bouhouch et al., 2016), nevertheless, the risk of lead exposure still exists due to the environmental load in Pb (Barkouch, 2007;Zaida, 2007;El-Fadeli et al., 2014).
In addition, with those low average concentrations of lead measured within the children, chelator therapy didn't change significantly their scores on neuropsychological tests (Bouhouch et al., 2016). Recently, we have found a significant association (p<0.031) between the visual attentional function of schoolchildren living in the mining area and Pb content on their nails (Maidoumi et al., 2017). Whereas, it's interesting to assess at last how environmental Pb content may affect their superior brain functions to understand the Pb exposition pathways and suggest eventual solutions. Therefore, this study aims at evaluating visual attentional function and unilateral spatial neglect among lead-exposed Moroccan children in comparison with the control group.

Study Location
The research was carried out in Marrakech, which is approximately located in the center of Morocco. This study was interested in two suburban areas; one on the West of Marrakesh called «Sâada» and the other the 20 km on the Eastern part of Marrakesh called «Chouiter» far from any sources of contamination ( Fig. 1). "Drâa Lesfer" which is a mining area is located in the Saada region, 13 kilometers west of Marrakech. This mine extracts Pb, Cd, and, Cu. Zn is naturally associated with these metals in the rock.

Participants
Data were obtained through a cross-sectional study, which includes 319 children aged 6 to 10 years living in Marrakech. Participants were drawn from four schools chosen to represent the two suburban areas "Sâada" and "Chouiter" and enrolled in the study from September 27 th , 2019, to February 5 th , 2020.

Socio-Demographic Variables
Parents filled out a questionnaire on their children's sex, age, parental educational level, father's occupation, and their children eating habits. This information was obtained at the time when the children's cognitive functions were assessed. Anthropometric measurements of children were also noted to understand their nutritional status (BMI).

Cognitive Performance Assessment
We used the bell test, designed by Gauthier et al. (1989), for the evaluation of Visual Attention (VA) and unilateral spatial neglect. The total number of surrounding bells was noted along with the time it took for the patient to run. The maximum score is 35. Forgetting 6 or more bells, at the right or left of the page indicates unilateral spatial neglect. The spatial distribution of omitted bells allows the examiner to assess the severity of visual neglect as well as the laterality of this negligence (right or left). The sequence by which the children completed this task can be figured by connecting the bells surrounded by the following order he performed.

Environmental Assessment
To understand the effect of environmental contamination on the attention performance of these children, measurements of Pb on soil and water were performed in both areas. Since painting, interior dust, and exterior soil or dust are three routes to which children can be exposed directly or indirectly and which contain significant amounts of lead, we then studied the spread of lead in soil. Tap water, being a weak source of lead exposure, was also analyzed.

Soil Sampling
Forty-eight soil samples were collected from the school garden. Soil samples were taken randomly from the upper layer (0-10 cm) and homogenized to form a composite sample. Then, the soil was dried in ambient air and sieved through a mesh whose diameter is less than 2 mm. The soil was sealed in paper envelopes for later analysis.
Mineralization: From each sample, three powder samples were taken and accurately weighed (0.5 g). The soil samples were then placed in crucibles to be gradually heated to 450°C. After cooling, bi-digestion with Hydrofluoric acid (HF) and perchloric acid (HClO4) was performed on a hot plate. Subsequently and to complete the dissolution, Hydrochloric acid (HCl) was added. Finally, distilled water was used to reach a volume of 50 mL (AFNOR, 1997).
Samples analysis: The metal analyses (Pb) of samples were carried out by using Thermo Fisher ICP-MS of the characterization center of the Faculty of Science Semlalia of Marrakesh. Standard solutions (HNO3 1M: 500 ng/mL) made it possible to draw up the calibration curve (10, 100, 500 µL). Then, 100 mL of 1 m nitric acid was added to a volume of 0.5 mL of the mineralized soil solution.

Water Analysis
Water sampling: Two water samples were taken from each school. One was taken from the drinking water and the other from the groundwater next to the target school. Polyethylene bottles pre-washed with nitric acid (1%) were used to preserve the samples. These latter were maintained at 4°C until they were sent to the mineral dosing center at the pharmaceutical UFR in Nantes, France. Lead content in water: Perkin-Elmer Inductively Coupled Plasma Mass Spectrometry (ICP-MS) model NexION 300 X was used to measure Pb concentrations. Doses are expressed in µg/L. Two water-based Nistcertified controls were employed to confirm the analytical procedure's accuracy (SRM 1643f and SRM 1640a). The controls are analyzed at each assay run and are run regularly every 10 samples and at the end of the essay. The calibration standards were made using a stock solution containing 1 g/L of lead. The stock solution was purchased from Carl ROTH. Starting from the 1 g/L solutions, a stock solution S1 (Pb: 500 µg/L) is made by successive dissolutions. The calibration range is created based on the anticipated Pb detection thresholds for water and the S1 stock solution. The element has been calibrated as follows: Pb: 6.25-12.5-25 µg/L. Millipore Direct Q UV3 instrument's Milli-Q ultrapure water was the water used to perform the standards. This is an ultrapure water system coming directly from the tap. The internal standard is Rh (100µg/L). The isotope used is Pb: 207.977. The acidified water samples are directly analyzed and the results are read in µg/L.

Ethical Considerations
The Mohammed VI University Hospital Center's ethics committee approved the study protocol on October 29, 2019.
The parents of the children submitted written consent in advance concerning their children's privacy rights and protection of their data. The Ministry of Health and Social Protection provided support for the study. The Ministry of the Interior and the Ministry of Education both approved of it as well.

Statistical Analysis
SPSS (20 th edition) was used to analyze the collected data. For a univariate study, centrality and dispersion parameters were assessed (mean, minimum, maximum, and standard deviation). Compliance analyses were performed using kurtosis, Kolmogorov-Smirnov, and skewness. Variances' homogeneity was analyzed using Levene's test. After that, bivariate analysis was conducted using parametric tests (student test, Anova). The significance level is retained for a p<0.05.

Population-Related Variables
The demographic characteristics are detailed below ( Table 1) and show that the two groups of children have roughly the same average age. The sex ratio was 0.77 for both populations. The participants were matched by gender and age. We find that each educational level (CE1 and CE2) is almost half of the population of children whether for cases or control. Approximately half of the mothers breastfed their children and the average breastfeeding between both groups showed that there was no significant variance in duration (t = -1.575; p = 0.116). Furthermore, 37.4% and 29.2% of children do not eat their breakfast regularly (always and often) for the cases and controls respectively. The Body Weight index (BWI) was 14.84±1.25 (Kg.m-2) in children from mining areas compared to the control 22.9±4.41. This disparity is statistically significant (t = -6.075; p = 0.001).
Compared to the control group, the proportion of children with two educated parents is much lower in the cases (21.5% versus 48.7%) (X 2 = 22.687; p = 0.000***).

Visual Attentional Scores and Unilateral Spatial Neglect
The mean score of exposed children (cases) on the VA test was 16.63±7.07 out of 35 for the CE1 and was significantly lower (t = -2.999, p = 0.003) compared to the controls (19.85±5.3 out of 35). In the same way, a significant difference in mean VA scores was elucidated (t = -2.562, p = 0.011) between the two groups enrolled in CE2 (Cases: 21.23±6.36; Control: 23.57±5.36), Fig. 2. Moreover, the mean score, for both population and the two levels (CE1, CE2), was significantly lower compared to the reference standard ODEDYS (T-test/ p = 0.000). The reference means the score is 28 out of 35 for the CE1 and 29out of 35 for the CE2 (Berthelot et al., 2005).
As a result, we found that both groups of children have low visual attentional skills. When these scores were compared to those from another research, Aboussaleh et al. Regarding the distribution of omissions, we concluded that the majority of children omitted 6 or more bells independently (X 2 = 6.039, p = 0,110) from their living site (Fig. 3). In fact, the omission of 6 or more bells indicates unilateral spatial neglect (Gauthier et al., 1989).
The second step is to investigate the severity of this neglect by identifying the laterality of the distribution of omitted bells. Indeed, the majority of omitted bells are located on right and left of the paper for cases and controls. For the exposed children (cases), about 3.5% of them have right visual-spatial neglect compared to only 2.3% in control (Fig. 4).
However, 3.4% of control children had central neglect. Since omissions in more centered columns of the sheet suggest greater neglect (Gauthier et al., 1989), this could have an impact on their learning quality.
The way by which the children encircled the bells indicates that the majority of children didn't use the same scanning strategy ( Table 2).
The scanning method chosen is disorganized in 47.7% of the exposed children and significantly different (p = 0.002) from those of non-exposed ones (35%). This result corroborates the findings of Gauthier et al. (1989) which demonstrated that the strategy of scanning left hemiplegic is disorganized compared to that of normal. Subjects with VA deficiency exhibit a disorganized scanning strategy and remission of visual neglect syndrome may lead, in brain-damaged subjects, to a reorganization of the scanning strategy.
The DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4 th edition, 1994) classification describes this attention deficit as relating to a young child with a lack of attention to detail. Indeed, the child has difficulty maintaining his attention on a particular task or game, has difficulty in organizing tasks, and has frequent forgetfulness during daily activities with easy distraction to external stimuli. The bells test requires maintaining attention (selective attention) while performing a conflicting task, as well as visual processing (visualspatial scanning) (Azzano et al., 2011). Therefore, a deficiency in VA may be associated with a dysfunction in the parietal and temporal lobes, especially the posterior associative cortex. Indeed, the Inferotemporal cortex contains a whole region dedicated to visual object recognition and the temporal lobe regions are crucial for understanding the spatial relationships among objects in a visual field (Purves et al., 2004).
At molecular level, it is known that cognition, attention, memory, mood, and reward behaviors are mediated by monoaminergic neurotransmitters such as serotonin and dopamine. Pb suppresses neurotransmitter levels at lower concentrations, whereas, during prolonged exposure or at higher concentrations, Pb mimics Ca and thus promotes the release of neurotransmitters, which increases their concentration in the synaptic cleft (Malavika et al., 2021). By altering synaptosome production, release, uptake, receptors, and transporters, as well as the quantities of second and third messengers like PKC, Ca, transcription factors, etc., it is also considered that Pb affects the availability of neurotransmitters (Sadiq et al., 2012).
Based on these results that describe visual attentional disorders in schoolchildren from the mining area, we observed that visual attention was affected. Although, the laterality of spatial neglect didn't show any differences between the two groups or the two sides of spatial neglect, the scanning strategy organization was disturbed. The results corroborate those of El Azmy et al. (2014) on children in the Mrirt area (Middle Atlas, Morocco) which showed that 64% of children, especially those with VA issues, do not adopt an organized scanning method to look for bells.

Environmental Assessment
In Table 3 we have shown the concentrations of Pb in soil and water in the two areas. We sum up by saying that the average Pb levels in the soil were lower than the limit values for soil (100 µg/g) in France (MATF, 1998) but higher than the normal levels in uncontaminated soil (35 µg/g) (Bowen, 1979). Additionally, a highly significant difference was elucidated between the mining area and the control one (t = 1.563; p = 0.000). This disparity can be explained by the exposure of 35.9% of children from the mining area to Pb through their father's occupational clothing.        The Pb content in water was very low in both areas and remains far from the Moroccan limit value (10 µg/L) determined by the standard 03.7.001 (OMS, 2017). However, the groundwater content of Pb in the mining area showed a very significant difference from that of the control (p = 0.000). Nevertheless, the water of both zones is far from being declared contaminated. This result contradicts that of Zaida et al. (2007) indicating lead contamination of the water of the mining area. The installation of a drinking water distribution network about ten years ago could be an explanation for the drop in lead levels in the water in this area.

Association of Environmental Pb Content and Visual Attention Function
The significant difference in mean scores between children exposed to lead contamination and those who are not (p = 0.008) suggests that lead is probably incriminated in the genesis of this cognitive deficit ( Table 4). The study of (Ruebner et al., 2019) corroborates this finding.
Indeed, the risk of attention deficit disorder (sustained attention and attention regulation) has been raised by environmental lead exposure. The results of Bellinger et al. (1994) state that, early exposure to lead in children and adolescents resulted in difficulties focusing and shifting attention, which most likely contributed to overall cognitive impairments.
Learning problems can be expected and could hinder their academic success. This conclusion was confirmed by several studies that reported a link between visual processing and academic success (Dhingra et al., 2010;Goldstand et al., 2005).
Thus, early detection of lead exposure and associated neurocognitive developmental issues may allow for the adoption of certain interventions, such as remediation, to inhance cognitive trajectories.
Finally, to minimize early contamination, precautionary measures should be taken to prevent environmental lead exposure. Lifestyle changes are advised, including washing hands before meals, decreasing the frequency of eating roadside food, reducing the use of kohl-based cosmetics, streamlining the use of conventional medicine, and the choice of accommodation away from heavy traffic.

Conclusion
Despite the existence of a proven association in this study between children's visual attention abilities and the risk of environmental lead exposure, however, it is not yet clear whether this is a causal association or not. Other factors could likely affect the children's academic achievement as well as the deterioration of their cognition.
Validation of these results could be considered using impregnation data such as blood lead. Thus, a comparison could be established with children from municipalities with similar population density and socio-economic parameters. In addition, other environmental factors should be investigated, such as children's diet and parents' occupation. This would strengthen the study conclusions and would allow the establishment of Pb exposure reduction strategies targeted at young children.

Acknowledgment
This study would not have been possible without the collaboration of the Mineral Element Dosing center of the Pharmaceutical Science University of Nantes and the Characterization Center of the Faculty of Science Semlalia of Marrakesh. Hence, the authors recognize the effort of all the work teams for their assistance during the analysis.

Funding Information
The resources made available to us by the Mineral Elements Dosing Center at the Pharmaceutical department of the University of Nantes (France) were used to support this work.