Visual Pathway Alterations after Severe Acute Occupational Elemental Mercury Poisoning: Report of a Series of 29 Cases

BACKGROUND: There are almost no available data on retinal involvement after acute exposure to high concentrations mercury and the available reports are on a small number of patients that suffered chronic expposure. The purpose of this paper is to report visual ndings in 29 workers exposed to very high concentrations of mercury vapor in a factory in northern Spain in 2012. METHODS: Twenty-nine patients and 16 controls were evaluated in a comparative case series. Fifteen of the 29 workers underwent belated chelation for heavy metal intoxication, only 3 in a prompt way. The mercury levels in blood and urine samples, visual acuity (VA), contrast sensitivity (CS), visual eld (VF), color discrimination and optical coherence tomography (OCT) data were recorded. The pattern reversal visual-evoked potentials (PRVEP), full-eld and multifocal electroretinography (ffERG/mfERG), pattern electroretinography (PERG), systemic symptoms, presence of erethism, and electromyography (EMG) were also gathered. A descriptive analysis was performed. The evolution of patients who did not undergo chelation (group 1), those who underwent late chelation (group 2), and those with deep VF defects (group 3) were compared with a control group. The correlations between variables also were studied. RESULTS: The VA was affected slightly. The loss of CS in at least one of four spatial frequencies and color vision alterations occurred in 96.5% (n=28) and 44.8% (n=13), respectively, in the entire group. VF alterations were identied in 72.4% (n=21). No morphologic changes were seen in the OCT scans. Latencies over 100 milliseconds and reduced amplitudes of P100 were found in the PRVEP. The ffERG and PERG results suggested that both the outer and inner retinal processes were involved. The mfERG indicated reduced parafoveal retinal function. Twenty-six workers exhibited symptoms of erethism. The EMG showed sensorimotor polyneuropathy and multiple mononeuropathy alterations. Signicant negative correlations among blood mercury levels, VA, and ffERG were observed. comparison. No differences seen between G1 G2. of P50 in the PERG, suggesting that the cone cells and ganglion macular cells can be affected by These ndings reinforce the idea that both the outer and inner retina visual processes are involved. the the photoreceptor in mercury since amplitudes showed loss of retinal 50


Background
Acute or subacute poisonings as the result of exposure to elemental mercury in the workplace are uncommon. However, these episodes may have profound effects on the visual pathway due to the neurotoxicity of this metal [1,2]. Controversy exists on the magnitude of the possible damage in the retinal structures. Some experimental studies have reported the presence of mercury in the retina and choroid after systemic administration [3,4], but others have limited the presence of the metal to the retinal pigment epithelium and external neuroretinal layers [5,6].
The introduction of electrophysiology tests and current clinical examination techniques such as auto uorescence or optical coherence tomography (OCT) have suggested that besides central nervous system (CNS) poisoning, which is widely accepted, the retina also is involved and that not all visual functional alterations are caused by high visual pathway damage [6][7][8].
Although physiologic and morphologic retinal changes resulting from mercury toxicity have been demonstrated in animal models; there are few reports of human retinal effects resulting from occupational poisoning in the literature; the last long series on mercury poisoning in humans was published before the latest retinal diagnostic techniques, i.e., spectral-domain OCT (SD-OCT) or multifocal electroretinography (mfERG), became available in clinic. Only one group has published OCT studies but on patients who had been exposed chronically [7,8], and only one study has been published on mfERG results in patients with color vision loss who also were exposed chronically to mercury [6]. To our knowledge, the current study is the rst that includes both techniques and pattern ERG (PERG) to evaluate morphologic and functional retinal ndings after acute exposure.
In the current study, we describe the visual pathway alterations in 29 workers inadvertently exposed to very high concentrations of mercury vapor for 14 consecutive days during maintenance work in a factory in northern of Spain at the end of 2012, one of the most severe incidents of acute elemental mercury intoxication that occurred in the European Union.
The opportunity to study those patients and the improvements in the retinal evaluation techniques have provided essential information on the degree of the mercuric effects at the intraocular level.

Methods
Pattern reversal visual-evoked potentials (PRVEP) and ERG recordings were assessed using a computerized Optoelectronic Stimulator Vision Monitor MonPack 120 Metrovision (Pérenchies, France) according to the International Society for Clinical Electrophysiology of Vision (ISCEV) protocols [14,15]. We recorded full-eld ERGs (ffERGs), PERGs, and multifocal mfERGs from both eyes of each patient with scaled hexagons stimulating 61 zones. Four patterns of abnormal mfERG amplitude responses were assessed: paracentral loss, foveal loss, peripheral loss, and generalized loss [16]. The technical data used for the electrophysiologic tests are shown in Table 1. For statistical analyses, participants were divided in two groups: group 1 (G1) (n = 14) was comprised of workers who did not undergo belated chelation treatment and group 2 (G2) (n = 15) included those who underwent chelation after 8 to 12 months after the mercury poisoning. In addition, for the electrophysiology function assessment, we included two additional subgroups. Another subgroup (G3) included 11 of 29 patients who had the deeper and more extensive defects in the VF tests, i.e., concentric constriction and hemi eld defect patterns. Finally, we included an age-matched healthy control group (n = 16).
Acquired dyschromatopsia, especially in the blue-yellow range of the Roth 28 Hue Test, occurred in 13 (44.8%) patients. The mean CCI was 1.642 ± 1.183 (normal value, 1.0 and higher values indicate worse hue discrimination [11,12]. No signi cant difference in the CCI was seen between G1 and G2 (Table 3).
Twenty-eight (96.5%) patients showed changes in the achromatic CS in at least one of the four spatial frequencies, but mainly in the higher ones; the difference between G1 and G2 did not reach signi cance (Table 3, Fig. 1).
The OCT results showed no signi cant differences when the CRT and RNFLT measurements were compared to values in the normative SD-OCT databases [17,18] between G1 and G2 (Table 3).
Correlation analyses between the blood mercury levels (BML) and all variables showed only a signi cant negative correlation with the BCVA (Table 4).

Electrophysiology Function Assessment
The ffERGs were recorded in 28 patients and 16 controls. No signi cant differences were found between G1 and G2 ( Table 5). The ERG amplitudes from all mercury-contaminated patients (G1 plus G2) did not differ from those of controls for the 30-Hz icker and singleash cone response (SFCR). The a-and b-wave amplitudes did not differ signi cantly between patients and controls in the scotopic rod response (SRR) and maximal scotopic response (MSR), respectively (Table 5). Statistical differences were found in all patients (G1 plus G2) and controls for the b-wave in SRR, a-wave in MSR, and the sum of oscillatory potential (OP) protocols (Table 5).
Because about half of the patients had no visual impairment, the electrophysiologic measurement averages from the global sample (G1 plus G2) could have been falsely near normal. Thus, we focused on the results obtained from patients with relevant impaired VF tests (G3) compared to the control group. These data showed lower amplitudes in subgroup G3 compared to the global sample in all ERG parameters reported previously [6]. These differences were signi cant for MSR and 30-Hz icker for the a-and b-waves and for the bwave in the SFCR (Table 5).
A negative signi cant correlation was seen in G1 between the BML and ERG amplitudes of the b-wave in SRR, MSR, sum of the OP, and in 30-Hz icker responses and sum of the OP in G2 (Table 6).  However, this trend seen in all patients became signi cant when patients with impaired VFs (G3) were compared with the control group (Table 7), which agreed with previous studies [19].
No signi cant differences were seen when the implicit times in both P50 and N95 components were compared. There was no correlation between the PERG values and the BML.
PRVEP PRVEP was recorded in 29 workers and 14 controls. The average implicit times of P100 and amplitudes did not differ between all patients and the control group for the 60-and 30-minute checkerboard stimuli except in the implicit times of P100 for both 60 and 30 for the left eyes (Table 8); but signi cant differences were seen when G3 was compared to the control group (Table 8). However, no signi cant differences were seen when G1 and G2 were compared (Table 8); and no correlation was seen between the PRVEP and BML. and > 15° when G3 was compared to controls. These results agreed with previous studies [16,20]. No signi cant differences were seen when the mfERG patterns in G1 and G2 were compared (Table 9) and no correlations were seen between the mfERG values at rings 2-5º, 5º-10º, 10 o to 15º, and > 15º and the BML. Amplitude P1/N1 ratio (nV/deg 2 ).
*Group 3 is a subgroup of patients with evident visual disturbances in their visual eld test. Patients with concentric constriction (17 eyes) and hemi-eld defects (5 eyes) patterns.
Although comparability between the mfERG and perimetry test was limited, we assessed the VF defects patterns with the mfERG dysfunction patterns obtained in the three-dimensional plots. With limitations, we subjectively assessed these patterns based on the comparative methods and approaches used previously [21,22]. The data showed that 14 (48.3%) patients showed different patterns between the mfERG defects and the total deviation of visual sensitivities less than 5% recorded in their VF tests. Eight (27.5%) patients had similar peripheral pattern defects in both tests, and four (13.8%) patients had mixed patterns; in three (10.3%) we could not establish any comparison. No differences were seen between groups G1 and G2.
EMG EMG, performed in 27 of 29 workers, showed different abnormality patterns and decreased nerve conduction velocity in most patients (Table 2). There were no signi cant differences between G1 and G2. No correlation was seen between the BML, nerve conduction velocity, and the P100 component in PRVEP in the entire sample.

Discussion
This study focused especially on retinal and visual function examinations because of the possibility of evaluating patients using new OCT and ERG techniques.
Mercury vapor is a signi cant source of mercuric load in occupational exposure because it is odorless and colorless and tends to accumulate in poorly ventilated areas. Once the lungs have absorbed the inhaled vapor, the mercury can reach different tissues via the bloodstream, with the primary target the CNS and eyes because of proximity [2,23]. When it is oxidized, it cannot penetrate the bloodbarrier again and remains for prolonged periods in tissues [2,7,8,23].
As mentioned, the neurologic and thus the visual pathway effects resulting from mercury toxicity have been described widely [2,23,24].The long-term exposure effects can include symptoms from tremor, neuropathy, personality changes referred to as mercurial erethism, speech disruption, delirium or rigidity to symptoms of VF defects, reduced VA, color and night vision, or decreased CS [2,23,25,26]. However, the introduction of electrophysiology testing has established the presence of primary retinal involvement and that not all alterations of the visual pathway are due to CNS poisoning [6].
As mentioned, the rst patient complaints were attributed to a viral infection, which delayed the diagnosis. Thus, when diagnosed correctly, the mercuric values in urine (mean, 302.86 µg/g Cr) and blood (mean, 392.93 µg/L) signi cantly exceeded the maximal accepted level for occupational exposure (< 30 µg/g Cr and 10 µg/L, respectively) [9,10]. In such cases, the mainstay of treatment is chelation therapy; however, only three patients underwent early chelation, which was stopped prematurely because of severe adverse reactions. Fifteen workers underwent delayed chelation (8 to 12 months after the initial incident). However, late chelation did not result in signi cant symptom relief.
Twenty-six workers exhibited symptoms related to erethism. Some also showed typical symptoms associated with cognitive mercury poisoning such as memory and attention disturbances [23,24]. Tremor of the hands, head, and eyelids, a late symptom of mercury poisoning, also occurred in some patients. EMG showed signs of mixed sensorimotor polyneuropathy and multiple mono-neuropathy alterations 12 to 18 months after exposure.
In this series, the VA decreased minimally and occurred in nine patients from G1 and ve from G2; however, advanced visual functions were impaired signi cantly apparently independently of the mercury levels since signi cant negative correlations were seen only among the BML, BCVA, and ffERG.
Color vision and CS impairment at high spatial frequencies also were found, with the most frequently observed color vision alteration in the blue-yellow range. These ndings agreed with previous studies [25,[27][28][29].
The most prevalent VF pattern was concentric constriction (17 eyes, 29.3%), which agreed with previous studies [30,31]. This visual impairment may have a central origin (calcarine cortex), as it has been reported previously [32]. In addition, the increased implicit time of The ffERG showed changes in the SRR and OP of the ISCEV protocol, suggesting that rod cells are impaired by acute mercury-vapor intoxication. We did not nd differences in the MSR, 30-Hz icker, and SFCR data, which are key for assessing macular cone function, in the global sample compared to controls; however, signi cantly lower measurements were found in the ffERG in G3 compared to the controls, together with a lower amplitude of P50 in the PERG, suggesting that the cone cells and ganglion macular cells can be affected by mercury poisoning. These ndings reinforce the idea that both the outer and inner retina visual processes are involved. Finally, the mfERG results are further evidence of damage to the photoreceptor pathway in mercury poisoning, since the amplitudes showed loss of the retinal response within the central 50 degrees, as reported previously [6].
A discrepancy was observed between the dysfunction patterns observed in the VF and the mfERG, with less involvement in the mfERG.
This nding suggested retinal damage (detected by the mfERG) in addition to that in the visual pathway.
The latency and amplitude of PRPEV were not correlated with the BML; however, patients in G3 had latencies signi cantly over 100 milliseconds and signi cantly reduced P100 amplitudes. Although these results typically occur in optic neuropathies and visual cortex abnormalities, they also can be associated with maculopathies, especially when they are interpreted in conjunction with retinal function tests (PERG, mfERG, and ffERG). These results agree with the results reported by Ventura et al. and da Costa et al. in patients with mercury poisoning [6,19].
Despite the functional retinal involvement and in contrast to the results obtained by Ekinci et al [7,8], OCT did not reveal structural changes in the RNFL, macular CRT, and choroid thickness when results were compared to the normalized reference values [17,18]. These differences might be related to the intensity and the manner of poisoning, as the current patients reached higher levels of mercury in a short time compared to the long exposure times of workers examined by Ekinci et al [7,8].
This study has some limitations. There were no environmental measurements of mercury either before the accident or during the occupational incident. In addition, probably only the most affected patients were evaluated at the IOBA-Eye Institute, and the time that elapsed after the acute accident and the assessment at the IOBA likely was not the most appropriate for adequate follow-up over time.
Most of the identi ed visual alterations, in our opinion, were attributable to the occupational exposure to mercury vapor, but we do not know the ophthalmologic baseline status before the accident. In addition, because of the lack of programmed follow-up, we had no information about the current clinical situation or about the evolution of most patients. Regarding the electrophysiologic tests, since half of the contaminated patients presented with no VA alterations, the results obtained from the global sample probably are affected by this patient subgroup. For this reason, our attention was focused on the subgroup G3. In addition, caution should be exercised when interpreting the results obtained from the comparisons between the control group and subgroup G3, because the samples size might be small for both groups in some comparisons. Finally, the OCT technology has evolved so rapidly that it is possible that with OCTs based on swept-source or ultra-high resolution it would have been possible to detect changes in the retinal or choroidal structures.
Even so, this study presents some relevant ndings from a very rare and extremely serious event, for which references are scarce. First, the VA is affected slightly and there is more VF involvement. However, other visual function assessment tests seem to behave independently of the mercury levels. The most prevalent VF alteration is decreased VFs, but central involvement also was found. This nding could be of retinal and/or neurologic origin considering the mfERG results. No anatomic retinal changes were identi ed in this series, but it is possible that the new OCT systems allow establishing the structural bases of these alterations. Delayed chelation apparently did not bene t the patients.
In summary, despite its limitations, this series of patients affected by the same event contributes to the information obtained about mercury poisoning for future similar situations.

Conclusions
This is one of the largest series of mercury poissoning reported in the last years in which patients could be analyzed with new diagnostic techniques. Besides it is the only one reporting data on OCT or multifocal ERG after acute exposure to high concentrations mercury, as the current reports are on patients suffering chronic expposure. Finally, ndings in the multifocal ERG allowed us to demonstrate that visual impairment after acute events is not only due to high visual pathway neurologic damage, but to retinal damage too at least in patients with the worst visual eld outcomes.

Declarations
Con ict of interest: All authors certify that they have no a liations with or involvement in any organization or entity with any nancial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-nancial interest (such as personal or professional relationships, a liations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
Ethical approval: All procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent: Informed consent was obtained from all individual participants included in the study.
Funding sources: No funding was received for this research.
Availability of data and material: All data and materials are available upon request.
Author Contributions: