POSSIBLE
DELETERIOUS
IMPACT
OF
GLYPHOSATE
AND
ITS
COMMERCIAL
FORMULATION IN REPRODUCTIVE HEALTH
OF FISHES
I. Argou-Cardozo1, F. Zeidán-Chuliá2,3
1Specialization Course in Public Management and Regional Development, Faculty of Administration and Tourism, Federal University of Pelotas (UFPel), 96010-610 Pelotas, RS, Brazil;
2Departamento de Ciencias Biomédicas Básicas, Facultad de Ciencias Biomédicas y de la Salud, Universidad Europea de Madrid, 28670 Villaviciosa de Odón, Spain;
3Faculty of Medicine, University of Turku, FI-20520 Turku, Finland.
Abstract
Glyphosate (GLY) is the active ingredient of Roundup® and it is the most utilized herbicide worldwide in the
maintenance of conventional agricultural crops, and lawns in parks. A growing number of studies have associated environmental GLY to different pathologies such as obesity, diabetes, heart disease, Alzheimer’s disease, depression, and autism. Different fi sh species have been used for a long time as experimental biological models to measure the environmental impact of different substances. Therefore, the present study approached the possible association between the exposure to GLY / Roundup® and the ecotoxicological impact on fi sh reproductive health. With this
goal, we performed a comprehensive analysis of the literature and its content by systematic review of international databases. Two independent electronic searches were performed on Medline / PubMed and Scielo for identifying relevant studies published in English up to September 2019. The application of inclusion / exclusion criteria settled the boundaries for this systematic review and after qualitative analysis of the data; we found evidences that suggest a link between the exposure to GLY / Roundup® with deleterious effects on reproductive health in eight different
species of fi sh. Keywords
Health • Pollution • Pesticides • Commercial herbicides • Agrotoxics; Roundup® • Fish toxicity • Reproduction • Fertility • Indicators • Gene-environment Interactions • Systematic Review
Introduction
Glyphosate (GLY) or N-(phosphonomethyl)glycine has been a widely used herbicide during the last 30 years which destroys plants and microorganisms by inhibiting the shikimate pathway. It was considered as ‘practically non-toxic and not an irritant’ to both animals and humans since this biochemical route only exists in a small number of organisms that utilize the shikimic acid pathway to produce amino acids (e.g., green plants) [1, 2]. With all, a larger and growing body of literature has showed experimental evidence for potential adverse effects of GLY, its major metabolite aminomethylphosphonic acid (AMPA), as well as its commercial formulation Roundup®
in humans. Different groups have studied the potential deleterious effects of GLY / Roundup® in the context of
diverse and apparently unrelated human pathologies such as carcinogenesis and autism spectrum disorders (ASD), with particular interest on data suggesting mechanisms that may
GLY is used for weed control in both cultivated and uncultivated areas and it is highly water-soluble [14]. This characteristic increases the risk for GLY and its adjuvants in their commercial formulation to contaminate surface and ground waters in application areas and therefore represents a serious threat to the ecological balance of the affected regions.
As a model organism, some general characteristics make many fi shes valuable for pathological and toxicological studies with relevance in human health [15] and include: 1) high fecundity with externally fertilized, often transparent eggs, as well as relatively brief generation times (perfect feature for reproductive studies), and 2) easy exposure of aquatic animals to water-soluble compounds (e.g., contaminants, new drugs, and chemicals) through a relatively large epithelial surface area of gills and skin.
Thus, in the present systematic review, we search for literature evidence regarding the potential interrelation between the exposure to GLY / Roundup® and changes in reproductive
health indicators of fi sh species.
Methods
1. Systematic review question
Is there an interrelation between the exposure to GLY / Roundup® and changes in reproductive health in different
fi sh species?
2. Search strategy
The literature was screened to fi nd published articles linking GLY / Roundup® exposure to differences in reproductive health
Indicators of fi shes. Comprehensive and systematic electronic searches were performed on Medline / Pubmed (https://www. ncbi.nlm.nih.gov/pubmed/) and Scielo (http://www.scielo. org/php/index.php?lang=es) databases by two independent investigators (IAC and FZC), started from 16 January 2018 until 20 September 2019. The following terms were used: [“Roundup” or “Glyphosate”]) AND ([Reproduction] or [Reproductive health] or [Fertility] or [Infertility]. Duplicate references were excluded and analyzed by the same two reviewers (IAC and FZC) based on inclusion and exclusion criteria. The authors performed a hand search of the reference lists from the selected published articles (in addition to the electronic search) to identify potentially missing studies / data that may deserve qualitative analysis in the present systematic review.
3. Selection criteria
Articles were included only if they dealt with evidence linking the exposure to GLY / Roundup® and changes in reproductive health in fi sh without time restrictions, based on the following inclusion criteria:
a. Only studies published in English;
b. Studies performed in fi sh species (male & female animals);
c. Studies that allow access to their specifi c data; d. The most recent data will be preferred in case of
duplication.
4. Selection of studies
The rigorous screening was independently conducted by two reviewers (IAC and FZC) with the predefi ned eligibility criteria above. Every study was identifi ed and classifi ed by its title and abstract to selection criteria. After initial screening of titles and abstract, the full articles were evaluated by the same reviewers. Situations of disagreement between authors were solved after discussion. For the second screening phase, the complete published article was read, analyzed, and discussed between both reviewers. Data regarding the study design and characteristics, sample size, year of publication, and limitations were collected from each included article.
Results and Discussion
The fi rst electronic screening (see Figure 1 displaying the fl owchart of the study selection) resulted in 148 titles from
Medline / Pubmed and none from Scielo databases, in which no duplicated papers were found. Thus, a total of 148 articles were considered for potential inclusion. From those, 139 articles were excluded based on title and abstract (theoretical reviews and hypothesis with no experimental data were not considered), resulting in 10 full-texts that were identifi ed (1 additional full-text article was included by manual search), according to the inclusion criteria in this systematic review (Table 1). In total, eight different fi sh species corresponding to eight different families were utilized in the selected studies (e.g., Rhamdia quelen, Jenynsia multidentata, Poecilia vivipara, Danio rerio, Astyanax lacustris, Hypomesus transpacifi cus, Astrolebias nigrofasciatus, and Oryzias latipes) displaying changes in reproductive health indicators such as number of copulations, mating and hatching success, sperm quality, embryo mortalities and/or malformations, and number of copulations among others (Figure 2).
In 2007, Soso et al. showed that the eventual entrance of sub-lethal doses of GLY into the ponds may have deleterious effects on R. quelen reproduction; and additionally, that the chronic and/or repeated exposure to this pesticide can negatively alter R. quelen reproduction [16]. Five years later, Hued´s group demonstrated that acute and subchronic exposures to Roundup® induced a diversity of gill and liver histological
sexual activity in male fi sh exposed to the herbicide for 7 and 28 days [17]. Furthermore, sperm quality seems to be also affected by Roundup® exposure. In this sense, a Brazilian study in 2013 showed detrimental effects in plasma membrane integrity and mitochondrial functionality in spermatic cells of P. vivipara
induced by this herbicide at realistic concentrations, and thus capable of generating consequent changes in fi sh populations of P. vivipara [18]. Not only male individuals are affected but female gametocytes were also shown to have an impact after GLY exposure. In particular, the concentration of GLY used by Armiliato and colleagues (2014), which corresponds to the maximal concentration of this herbicide allowed for Brazilian inland waters, was suffi cient to induce ovarian changes in D. rerio females associated with higher steroidogenic factor 1 (SF-1) expression [19]. As for Harayashiki´s study (2013) [18] in P. vivipara, the effect of GLY on sperm quality was also investigated in D. rerio in 2014 [20]. Interestingly, this was the fi rst study that demonstrated the harmful effects of GLY per sé in this fi sh, such as damaged sperm DNA, reduced integrity of the mitochondrial membrane and functionality, as well as decreased sperm motility [20]. In the same year, Uren Webster and collaborators demonstrated that both 10 mg/L Roundup® and 10 mg/L GLY
exert a similar adverse impact on embryo survival and hatching, while 10 mg/L GLY reduces egg production [21]. Further studies focused concentration that is within legal limits in U.S.A and even in the more stringent limit set in Brazilian law and studied the effects of low concentrations of GLY-based herbicide in A. lacustris. Goncalves et al. observed that some spermatic cells submitted to GLY-based herbicides were able to survive and kept alive, but sperm motility was not detected, leading a signifi cant lower sperm quality [22].
The application of herbicides has also been considered effective for limiting the growth of invasive aquatic plants. With all, the putative adverse effects of these herbicides on non-targeted organisms are not well studied and thus, the possible impact on aquatic ecosystems. With this porpoise, Jin et al. (2018) evaluated the effect of four herbicides (penoxsulam, imazamox, fl uridone and GLY) on Delta Smelt´s reproductive and detoxifi cation systems, showing that both acute exposure to fl uridone and GLY exert the most detrimental effects when compared to penoxsulam and imazamox in H. transpacifi cus
[23]. It is not clear yet how GLY and other herbicides may affect ephemeral wetlands but the literature provides data highlighting the effect of Roundup® exposure not only in
fi sh reproduction, but also in embryonic diapause and the embryonic upper thermal tolerance of other species such as
A. nigrofasciatus [24]. In particular, 96 hours of exposure to the low and environmentally relevant Roundup® concentration
of 0.36 mg a.e./L can signifi cantly decrease fi sh fertility and its embryonic thermal tolerance [24]. Most of these reports [16-24] show evidence for direct toxicity and the question remaining would be whether exposures with low doses of
GLY / Roundup® may lead to adult onset toxicity through
epigenetic-based mechanisms. At least one recent study from Smith and collaborators (2019) supports this idea and explains how Roundup® and its active ingredient GLY may
induce developmental, reproductive, and epigenetic effects such as changes in the expression of reproductive genes in the brain, genes Involved in sex differentiation and gonad development of Japanese medaka (O. latipes) [25].
For humans, sources of exposure to GLY / Roundup® are
numerous and affect a large segment of the general population. Those ones include dermal exposure from residential and commercial use, as well as inhalation through spray drift in high farming areas and through food consumption [26, 27]. On the other hand, fi sh models provide a number of exceptional advantages for biomedical research: 1) they require small laboratory infrastructure and can be bred and maintained in large numbers easily and at low cost, 2) they offer the chance to combine the analytical clarity of developmental biology with the power of genetics, and fi nally 3) that transgenic lines can be quickly generated [28]. We believe that the toxicological data in fi sh models presented in this systematic review suggest the need for further studies exploring the effects of GLY / Roundup®
on reproductive health in humans.
Conclusion
The results of the present systematic review demonstrate an association between GLY / Roundup® exposure and
deleterious effects in fi sh reproduction refl ected in dramatic changes in indicators of reproductive toxicity, and also provides a signifi cant insight into ecological risks rendered by GLY and Roundup® exposure. Most importantly, we believe that
further studies should be performed to elucidate any potential parallelism in the context of human reproductive health.
Acknowledgments
FZC is grateful to FINDOS Turku Grant for Completing a Doctoral Degree (University of Turku), University of Turku Joint Research Grant Fund, and Selma and Maja-Lisa Selander´s Fund (Minerva Foundation, Helsinki) for supporting this work.
Confl ict of Interest Statement
References
1. Bai SH, Ougbourne SM. Glyphosate: environmental contamina-tion, toxicity and potential risks to human health via food con-tamination. Environ Sci Pollut Res Int. 2016;23(19):18988–9001. https://doi.org/10.1007/s11356-016-7425-3
2. Gomes MP, Smedbol E, Chalifour A, Hénault-Ethier L, Labrecque M, Lepage L, et al. Alteration of plant physiology by glyphosate and its by-product aminomethylphosphonic acid: an overview. J Exp Bot. 2014;65(17):4691–703. https://doi.org/10.1093/jxb/eru269 3. Davoren MJ, Schiestl RH. Glyphosate-based herbicides and
cancer risk: a post-IARC decision review of potential mecha-nisms, policy and avenues of research. Carcinogenesis. 2018;39(10):1207–15. https://doi.org/10.1093/carcin/bgy105 4. Argou-Cardozo I, Zeidán-Chuliá F. Clostridium Bacteria and
Au-tism Spectrum Conditions: A Systematic Review and Hypotheti-cal Contribution of Environmental Glyphosate Levels. Med Sci (Basel). 2018;6(2):E29. https://doi.org/10.3390/medsci6020029 5. Zeidán-Chuliá F, Argou-Cardozo I. Are Gene–Environment
In-teractions Underpinning the Development of Creative Polyma-thy? Interchange. 2018; 49(3):343–52. https://doi.org/10.1007/ s10780-018-9329-2
6. Lovely C, Rampersad M, Fernades Y, Eberhart J. Gene-environ-ment interactions in developGene-environ-ment and disease. Wiley Interdiscip Rev Dev Biol. 2017;6(1). https://doi.org/10.1002/wdev.247 7. Zeidán-Chuliá F, Rybarczyk-Filho JL, Salmina AB, de Oliveira
BH, Noda M, Moreira JC. Exploring the multifactorial nature of autism through computational systems biology: calcium and the Rho GTPase RAC1 under the spotlight. Neuromolecular Med. 2013;15(2):364–83. https://doi.org/10.1007/s12017-013-8224-3 8. Argou-Cardozo I, Cano Martín JC, Zeidán-Chuliá F. Dental
amal-gam fi llings and the use of technological devices as an environ-mental factor: Updating the cumulative mercury exposure-based hypothesis of autism. Eur J Dent. 2017;11(4): 569–70. https://doi. org/10.4103/ejd.ejd_222_17
9. Choudri BS, Charabi Y, Ahmed M. Pesticides and Herbicides. Water Environ Res. 2018;90(10):1663–78. https://doi.org/10.21 75/106143018X15289915807362
10. Foster WG. Environmental toxicants and human fertility. Minerva Ginecol. 2003;55(5):451–7.
11. McCue K, DeNicola N. Environmental Exposures in Reproduc-tive Health. Obstet Gynecol Clin North Am. 2019;46(3):455–68. https://doi.org/10.1016/j.ogc.2019.04.005.
12. May H, He X, Qi K, Wang T, Qi Y, Cui L, et al. Effects of en-vironmental contaminants on fertility and reproductive health. J Environ Sci (China) 2019;77:210–17. https://doi.org/10.1016/j. jes.2018.07.015
13. Mattison DR, Plowchalk DR, Meadows MJ, al-Juburi AZ, Gandy J, Malek A. Reproductive toxicity: male and female reproduc-tive systems as targets for chemical injury. Med Clin North Am. 1990;74(2):391–411.
14. Marques MN, Passos EA, da Silva MT, Correia FO, Santos AM, Gomes SS, et al. Determination of glyphosate in water samples by IC. J Chromatogr Sci. 2009;47(9):822–4.
15. Schmale MC, Naim RS, Winn RN. Aquatic Animal Models of Human Disease. Comp Biochem Physiol C Toxicol Pharmacol. 2007;145(1):1–4.
16. Soso AB, Barcellos LJ, Ranzani-Paiva MJ, Kreutz LC, Quevedo RM, Anziliero D, et al. Chronic exposure to sub-lethal concentra-tion of a glyphosate-based herbicide alters hormone profi les and affects reproduction of female Jundiá (Rhamdia quelen). Environ Toxicol Pharmacol. 2007;23(3):308–13. https://doi.org/10.1016/j. etap.2006.11.008
17. Hued AC, Oberhofer S, de los Ángeles Bistoni M. Exposure to a commercial glyphosate formulation (Roundup®) alters normal gill and liver histology and affects male sexual activity of Jenyn-sia multidentata (Anablepidae, Cyprinodontiformes). Arch Envi-ron Contam Toxicol. 2012;62(1):107–17. https://doi.org/10.1007/ s00244-011-9686-7
18. Harayashiki CA, Varela AS Jr, Machado AA, Cabrera Lda C, Primel EG, Bianchini A, et al. Toxic effects of the herbicide Round-up in the gRound-uppy Poecilia vivipara acclimated to fresh water. Aquat Toxicol. 2013;142-143:176–84. https://doi.org/10.1016/j.aqua-tox.2013.08.006
19. Armiliato N, Ammar D, Nezzi L, Straliotto M, Muller YM, Nazari EM. Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafi sh Danio rerio exposed to glypho-sate. J Toxicol Environ Health A. 2014;77(7):405–14. https://doi. org/10.1080/15287394.2014.880393
20. Lopes FM, Varela Junior AS, Corcini CD, da Silva AC, Guazzelli VG, Tavares G, et al. Effect of glyphosate on the sperm quality of zebrafi sh Danio rerio. Aquat Toxicol. 2014;155:322–6. https://doi. org/ 10.1016/j.aquatox.2014.07.006
21. Uren Webster TM, Laing LV, Florance H, Santos EM. Effects of glyphosate and its formulation, roundup, on reproduction in ze-brafi sh (Danio rerio). Environ Sci Technol. 2014;48(2):1271–9. https://doi.org/10.1021/es404258h
22. Gonçalves BB, Nascimento NF, Santos MP, Bertolini RM, Yasui GS, Giaquinto PC. Low concentrations of glyphosate-based her-bicide cause complete loss of sperm motility of yellowtail tetra fi sh Astyanax lacustris. J Fish Biol. 2018;92(4):1218–24. https://doi. org/10.1111/jfb.13571
23. Jin J, Kurobe T, Ramírez-Duarte WF, Bolotaolo MB, Lam CH, Pandey PK, et al. Sub-lethal effects of herbicides penoxsulam, imazamox, fl uridone and glyphosate on Delta Smelt (Hypome-sus transpacifi cus). Aquat Toxicol. 2018;197:79–88. https://doi. org/10.1016/j.aquatox.2018.01.019
of the threatened annual fi sh Austrolebias nigrofasciatus. Che-mosphere. 2018;196:260–9. https://doi.org/10.1016/j.chemo-sphere.2017.12.196
25. Smith CM, Vera MKM, Bhandari RK. Developmental and epi-genetic effects of Roundup and glyphosate exposure on Japa-nese medaka (Oryzias latipes). Aquat Toxicol. 2019;210:215–26. https://doi.org/10.1016/j.aquatox.2019.03.005
26. Annett R, Habibi H, Hontela A. Impact of glyphosate and glyphosate-based herbicides on the freshwater environment.
J Appl Toxicol. 2014;34(5):458–79. https://doi.org/10.1002/ jat.2997
27. Damalas CA, Eleftherohorinos IG. Pesticide Exposure, Safety Issues, and Risk Assessment Indicators. Int J Environ Res Public Health. 2011;8(5): 1402–19. https://doi.org/10.3390/ ijerph8051402
Figure Legends
Table Captions
Table 1. Literature evidence for the possible deleterious impact of GLY / Roundup® in reproductive health indicators of fi shes. The table summarizes the main results of ten studies included in this systematic review. The table is organized by year of publication.
TÍTLE AUTHOR /
YEAR
COUNTRY PMID MATERIAL FISH
SPECIES
CONCLUSIONS
Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profi les and affects reproduction of female Jundiá (Rhamdia
quelen)
Soso et al., 2007
Brazil 21783773 Ovaries and blood
samples taken from 8 females of each treatment immediately before or at 1, 10, 20, 30, and 40 days after exposure
Rhamdia quelen
The author's data indicated that the presence of glyphosate in water was deleterious to Rhamdia
quelen reproduction, altering steroid profi les and
egg viability
Exposure to a commercial glyphosate formulation (Roundup®) alters normal gill
and liver histology and affects male sexual activity of Jenynsia
multidentata (Anablepidae,
Cyprinodontiformes)
Hued et al., 2012
Argentina 21643816 Adult male and female
individuals of the com-mon native species exposed to a sublethal Roundup®
concentra-tion (0.5 mg / l) for 7 and 28 days
Jenynsia multiden-tata
Roundup® also altered the normal histology of
the organs studied and caused a signifi cant decrease in mating number and mating success in male fi sh exposed to the herbicide
Toxic effects of the herbicide Roundup in the guppy Poecilia
vivipara acclimated to fresh
water
Harayas-hiki et al., 2013
Brazil 24036434 Tissue sample (brain,
muscle, gills, and liver) and adult fi sh sperm
Poecilia vivipara
Male Guppies exposed to Roundup® had lower
sperm quality, measured as reduced plasma membrane integrity, mitochondrial functionality, DNA integrity, motility, motility period, and sperm cell concentration than those maintained under control (without water addition)
Changes in Ultrastructure and Expression of Steroidogenic Factor-1 in Ovaries of
Zebraf-ish Danio rerio Exposed to
Glyphosate
Armiliato et
al., 2014 Brazil 24617544 Ovaries (n = 18 per triplicate) were exposed to glyphosate for 15 days
Danio
rerio A signifi cant increase in oocyte diameter was observed after glyphosate exposure (65 μg / L) and the presence of concentric membranes, appearing as myelin-like structures, associated with mitochondrial outer membranes and with calf granules. In addition, higher expression of steroidogenic factor-1 (SF-1) was found in oo-cytes, suggesting a relationship between oocyte growth and SF-1 expression.
Effect of glyphosate on the sperm quality of zebrafi sh
Danio rerio
Lopes et al., 2014
Brazil 25089920 Adult fi sh sperm Danio
rerio
The authors investigated sperm quality in Danio rerio after 24 and 96 h of glyphosate exposure at concentrations of 5 and 10 mg / L. Their results showed a decrease in sperm motility. Mitochon-drial functionality, membrane and DNA integrity were also reduced at the highest concentra-tion during both exposure periods. These data showed that glyphosate may induce detrimental effects on reproductive parameters in D. rerio
and that this change would reduce the fertility rate of these fi sh.
Effects of glyphosate and its formulation, Roundup®, on
reproduction in zebrafi sh (Danio rerio)
Uren Webster et al., 2014
UK 24364672 Embryos from 21 day
rearing exposure of zebrafi sh (Danio rerio) at 0.01, 0.5 and 10 mg / L (glyphosate acid equivalent) Roundup®
and 10 mg / L glypho-sate
Danio rerio
Both 10 mg / L Roundup® and glyphosate
Table 1 (continued)
TÍTLE AUTHOR /
YEAR
COUNTRY PMID MATERIAL FISH
SPECIES
CONCLUSIONS
Low concentrations of glypho-sate-based herbicide cause complete loss of sperm motility of yellowtail tetra fi sh Astyanax lacustris
Gonçalves et al., 2018
Brazil 29488225 Adult fi sh sperm (10
individuals)
Astyanax lacustris
The authors showed that relevant environmental concentrations of glyphosate-based herbicides (50 μg l - 1, 300 μg l – 1, and 1800 μg l – 1) can
affect the sperm quality of Astyanax lacustris fi sh. Sperm viability was impaired by 300 μg l - 1,
a concentration that is within legal limits in US water bodies, while motility was impaired by 50 μg l - 1, which is the strictest limit established by
law.
Sub-lethal effects of herbicides penoxsulam, imazamox, fl uri-done and glyphosate on Delta Smelt (Hypomesus
transpaci-fi cus)
Jin et al.,
2018 USA 29448126 Liver and brain tissues of adult male and female fi sh
(5 females and 5 males) in triplicate per
treatment
Hypome-sus trans-pacifi cus
The authors found that 17β-estradiol concentra-tions were signifi cantly increased in female and male fi shes exposed to 0.21 μM fl uridone and in male fi shes exposed to 0.46, 4.2, and 5300 μM glyphosate. GSH concentrations decreased in males exposed to 2.8 μM of fl uridone and higher concentrations, and 4.2 μM glyphosate
A glyphosate-based herbicide reduces fertility, embryonic upper thermal tolerance and alters embryonic diapause of the threatened annual fi sh
Austrolebias nigrofasciatus
Zebral et al., 2018
Brazil 29306198 Embryos from a total
of 12 mating pairs of
A. nigrofasciatus
Aus-trolebias nigrofas-ciatus
The results showed that fi sh couples exposed to 0.36 mg a. Roundup® e./L produced fewer but larger embryos. In addition, embryos exposed to 3.62 mg a. Roundup® e./L had a reduced proportion of pigmented embryos 30 days after exposure. In addition, embryos exposed to 0.32 mg a. Roundup® e./L had a critical maximum thermal reduction of 2.6 °C and was more sensitive to smaller increases in heating rates. Their data indicated that Roundup® has nega-tive results on fi sh reproduction and embryonic development
Developmental and epigenetic effects of Roundup and glypho-sate exposure on Japanese medaka (Oryzias latipes)
Smith et al., 2019
USA 30875550 Embryos were
exposed to 0.5 mg/L glyphosate, 0.5 mg/L and 5 mg/L Roundup® (glyphosate acid equivalent) for the fi rst 15 days of
their embryonic life and then allowed to sexually mature with-out further exposure
Oryzias latipes
Relevant concentrations of both Roundup® and glyphosate caused developmental abnormali-ties and
