Plasma Phthalate Levels In Children With Speech Delay

Speech delay is one of the most common developmental problems. One of the risk factors may be the exposure to environmental chemicals. There is increased environmental exposure to phthalates, an endocrine-disrupting chemical. In this study, we aimed to determine the relationship of phthalates with speech delay. We included 50 children with isolated speech delay and 40 healthy children of similar age. Children were surveyed for risk factors for speech delay and phthalate exposure. Plasma di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP) and dibutyl phthalate (DBP) levels were measured by high pressure liquid chromatography. The DEHP, MEHP and DBP levels in study and control groups were 0.377 [0.003 - 1.224] µg/ml, 0.212 [0.007 - 1.112] µg/ml (p = 0.033), 0.523 [0.031 - 2.477] µg/ml, 0.152 [0.239 - 2.129] µg/ml (p <0.001), and 0.395 [0.062 - 1.996] µg/ml, 0.270 [0.006 - 0.528] µg/ml (p = 0.004), respectively. Multiple linear regression analysis was used to adjust the association between the phthalate levels and factors differing between the two groups in terms of delayed speech risk factors. While there was no signicant difference between the study and control groups in terms of DEHP level (p=0.233), the MEHP and DBP levels were found signicantly higher in the study group (p<0,001). Conclusion: The statistically signicant higher phthalate levels in those with speech delay indicate that these children are more exposed to phthalates and more epidemiological studies are needed to evaluate the association between phthalates and speech delay.


Introduction
Speech delay, one of the most common developmental problems, is considered as child's inability in attaining to the language development they should reach at certain ages and to lag behind their peers [1].
Delay in language acquisition without any identi ed cause is observed at a rate of 2.3-19% in pre-school children aged between 2-5 years [2]. Children with a speech delay can have a higher risk of suffering psychological and behavioral adjustment problems both in preschool and later life [3,4].
Speech delay has been associated with multiple risk factors, such as maternal and pregnancy related problems in prenatal period, prematurity [5], hypoxic birth [6], hearing loss [7], and environmental factors such as low socioeconomic level, caregiver education level and lack of stimuli [8].
Another risk factor for speech delay may be the mother's or baby's exposure to environmental chemicals during prenatal or postnatal periods. Exposure to certain chemicals may negatively impact on the baby's neurological development [9] which in turn may affect language skills. One of the environmental chemicals are phthalates.
Phthalates are semi-volatile synthetic chemicals that are used to soften plastics. Many products used in daily life, such as food packages, cosmetics/personal care products, medical materials and toys can contain certain amounts of different phthalate derivatives [10,11]. Since phthalates are not covalently bound to the plastic matrix, they can easily contaminate the environment. Moreover, humans are exposed to these chemicals by different routes (oral, dermal, inhalation and intravenous) [12,13]. In many studies, phthalates were detected in different body uids such as blood, urine, saliva and breast milk [14,15].
Phthalates were also determined in amniotic uid and this nding shows that they can cross the placental barrier and humans are exposed to these plasticizers even during the fetal period [16].
Phthalates, such as di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP), are the most abundant chemicals in the environment. These chemicals affect many systems, particularly the reproductive system. They are suggested to have anti-androgenic properties [17,18]. Due to their effects on androgens, they may disrupt testosterone-dependent brain development during prenatal period [19].
Although there are some studies that evaluated the association of phthalate metabolites with neurodevelopmental status in the neonatal, infancy and childhood periods [20,21], there is limited research concerning their effects on language development. Published studies showed contradictory and inconclusive results on the relationship between phthalates and speech delay [22,23].
Due to the limited number of studies exploring the effects of phthalate exposure on language development, we aimed to examine the relationship between phthalate levels and speech delay in children aged between 2-6 years in the present study.

Study Participants
This study is a case-control study. Ethical approval was obtained from the Ethical Review Board of The study groups applied to Hacettepe University İhsan Doğramacı Children's Hospital were as follows: 1. Study group (n=50): Children between 24-72 months admitted to the Developmental Pediatrics Outpatient Clinic with isolated speech delay were recruited as the study group.
2. Control group (n=40): Healthy children between 24-72 months admitted with acute complaints to the General Pediatrics Outpatient Clinic were recruited as the control group.
Patients who were previously diagnosed with a neurodevelopmental, genetic, or metabolic disorder or living in a stimuli poor environment which was related to cause speech delay were excluded from the study.
During the patients' evaluation, families were informed and written consent was obtained. A questionnaire to evaluate the possible routes of phthalate exposure, demographic information, and risk factors of speech delay was given after obtaining consent. This questionnaire mainly investigated whether mothers' were in contact with products containing phthalate derivatives during pregnancy and the postpartum period and whether children's dietary habits and environment might cause signi cant phthalate exposure.

Data collection
Data were collected between October 2019 and February 2020. Language and other developmental domains such as gross motor, ne motor, problem-solving and personal-social development of the children in the study and control groups were assessed with the Ages and Stages Questionnaire (ASQ).
ASQ is a screening tool widely used in large-scale screening programs and research, and can be lled by parents/other caregivers directly or in the company of a trained professional [24]. We used Turkish version of the Ages and Stages Questionnaires (ASQ-TR). The sensitivity and speci city of ASQ-TR are 0.94 and 0.85, respectively [25]. Children who were below the cutoff scores in the language domain and within the normal range in other domains were included in the isolated speech delay and were followed up. Children whose total scores were within the normal range in all developmental domain evaluations were included in the control group.
From the patient's le, information on the demographic data [child's age, gender, birth order, mother's and father's age and education level, family's socioeconomic status, the place where they lived (urban/suburban)], and breastfeeding status/duration were obtained. The missing data were recorded on the form by questioning the family. The socioeconomic status of the children was determined by the Hollingshead-Redlich Scale [26].
Deplasticization of the glassware All glassware were washed and were kept in 10% nitric acid for 24 h. Later, glassware were rinsed 4 times and then cleaned with n-hexane:tetrahydrofuran (50:50) for 2 h. They were dried at 37°C. High pressure liquid chromatography (HPLC) vials were kept at 400°C for 4 hours to avoid plastic contamination. Aluminum foil was used in order to prevent contact with the plastic material on the lids of all glass materials.

Biological samples
Venous blood samples were taken into heparinized tubes, prepared in 5 ml drip form with a sterile needle tip without plastic structure at the rear end. Samples were centrifuged (3500 rpm for 10 min). Plasma samples were kept at -80°C and stored until analysis. Recovery studies were performed on blank samples of plasma spiked with levels of 9.1 µg/ml of DBP, 9.8 µg/ml of DEHP, and 10.1 µg/ml of MEHP. Within-day precisions were DBP 0.71±0.40% CV, DEHP 3.09±1.29% CV, and 3.27±1.05% CV for MEHP. Between-run precisions were 1.06±0.56% CV for DBP, 9.21±1.19 CV for DEHP and 7.92±2.11% CV for MEHP.

Statistical Analysis
Statistical analysis and documentation were performed using IBM SPSS 21.0 (Chicago, IL). For descriptive statistics, mean and standard deviation or median and smallest-largest values were given in numerical variables, while number and percentage values were given for categorical variables.
Kolmogorov-Smirnov test was used for the assumption of normality. In the comparison of the groups, if the assumption of normality was provided, the signi cance test of the difference between the two means was used. If the assumptions of normality were not provided, the Mann-Whitney U Test was used. Categorical variables were compared by using chi-square test. Multiple regression analysis was used to explain the total change in the dependent variable studied with independent variables differing between groups. p values <0.05 were considered as statistically signi cant.

Results
Sociodemographic characteristics and risk factors for speech delay in both groups are shown in Tables 1 and 2. The number of boys (p = 0.036) and history of speech delay (p<0.001) was higher in the study group. The father's education level (p= 0.049) and smoking during pregnancy (p = 0.008) was higher in the control group. There was no signi cant statistical difference between the two groups in terms of other characteristics and factors.    The median values of detectable DEHP, MEHP and DBP levels were signi cantly higher in the study group compared to the control group (Mann-Whitney U test, p = 0.033, p <0.001 and p = 0.004, respectively) ( Table 3). Multiple linear regression analysis for risk factors including gender, smoking during pregnancy, father's education level, and family history of speech delay between the study and control groups showed no signi cant difference in terms of DEHP levels (p=0.233). In contrast, MEHP and DBP levels were found to be signi cantly higher in the study group (p<0.001) ( Table 4). Since the data were skewed in multiple linear regression models, square root transformation for DEHP and DBP and logarithmic transformation for MEHP were performed.   The questionnaire results applied to evaluate the possible phthalate exposure routes of children and their mothers in the study and control groups are shown in Tables 5 and 6.   When phthalate levels of children in the control group were examined according to phthalate exposure routes, no statistically signi cant difference was found in terms of DEHP and DBP levels according to the mothers' phthalate exposure pathways (at any time and during pregnancy). In childhood, plasma DBP levels were found to be higher in those who used plastic storage containers for food (0,284 [0,076 -0,468] µg/ml) than those who did not (0,062 [0,006 -0,528] µg/ml) (p = 0.049). While plasma MEHP levels of children in the control group were higher than those whose mothers used fabric softeners at any time (0,313 [0,063 -2,129] µg/ml) and during pregnancy (0,267 [0,063 -2,129] µg/ml) compared to those who did not (0,137 [0,024 -0,348] µg/ml and 0,124 [0,024 -0,348] µg/ml, respectively) (p = 0.010, p = 0.011, respectively). There was no difference in MEHP results in terms of phthalate exposure routes in childhood.

Discussion
In studies on speech delay, male gender [29,30], education level of parents [8,30] and family history of speech delay [8] are some of the well-known risk factors. In our study, the males were diagnosed with isolated speech delay three times more. Moreover, the family history of speech delay was higher and the father's education level was lower in the study group compared to the control group.
In numerous studies, it is reported that exposure to smoking in prenatal and postnatal periods have adverse effects on children's neurological development process and thus on language development [31,32]. However, contrary to expectations, smoking rate during pregnancy was reported to be higher in the control group. However, there was no difference between the two groups in terms of maternal smoking in the postnatal period and smoking in the home.
Many epigenetic factors may play a role in the etiology of speech delay [33]. Among these factors, genetics, lifestyle, and environmental pollutants are suggested to be the main factors [34]. The development of the central nervous system in children is sensitive to the environment during the intrauterine period and rst years of life [35]. Considering that language development is one of the primary markers of the neurodevelopmental process, studies investigating the role of phthalates, one of the environmental pollutants, in the etiology of speech delay attract more attention day by day.
Phthalates have been reported to impair testosterone-induced brain development. Exposure to these chemicals, in prenatal period may have substantial impact on speech. Moreover, they may also interfere with the sex-related neurodevelopmental stages and males are suggested to be more susceptible to such effects [36].
In our study, median plasma levels of DEHP, its metabolite MEHP, and DBP were signi cantly higher in the study group compared to control group. After correcting for speech delay risk factors, which differed between the two groups with direct regression analysis, there was no signi cant difference between the two groups in terms of DEHP levels, while MEHP and DBP levels were found to be signi cantly higher in the study group.
In a prospective study conducted in Denmark, the researchers examined the relationship between prenatal phthalate exposure and language development of children aged 20-36 months. In the study, high urinary diethyl phthalate (DEP), butyl benzyl phthalate (BBP), and DEHP metabolite levels in the third trimester of mothers were associated with low language scores (word count and complex language use) in boys, but this relationship was not observed in girls [22].
In a two-centered cohort study from Sweden and the United States, which examined the relationship between language development and urinary phthalate metabolites, in corrected analyses, doubling the prenatal DBP and BBP metabolite exposure signi cantly increased the estimated relative risk (odds ratio, OD) for language delay in the Swedish group by approximately 25-40%. However, no association with any DEHP metabolite with language delay was observed in either cohort [23]. Unlikely, we found an inverse relationship between MEHP and speech delay.
In a study published in Singapore, a large number of metabolites of different environmental chemicals were measured by gas chromatography-mass spectrometry in hair samples taken from mothers at 26-28 weeks of gestation and their relationship with the developmental areas of 24-month-old children were examined. The researchers found that high phthalic acid levels were associated with low expressive language scores (univariate p value=0.022) [37].
In our study, in which possible exposure routes from the intrauterine period to early childhood were questioned, the plasma DEHP levels were found to be higher in the study group whose mothers reported using hair dyes and conditioners at any time and those who used deodorant during pregnancy. Plasma MEHP levels of children in the control group whose mothers used softeners (at any time and during pregnancy) were higher. Although it is stated that oral and respiratory exposure is more prominent for phthalates [14], these results also show the importance of dermal exposure. In the control group, plasma DBP levels were higher in those whose mothers used plastic storage containers for food. DBP is a phthalate predominantly found in personal care and cosmetic products, and enteric-coated drug tablets [14], and no difference was found in both the study and control groups in the inquiries made in this regard. Therefore, more research should be done with DBP-related exposure sources and their possible undesired outcomes.
Our study has some limitations and strengths. Firstly, although the number of participants in the study and control groups was determined using the G-power 3.0.10 program with 0.80 effect size, 80% power, and 5% margin of error based on previous studies, repeating a similar study with larger groups will increase the strength of the study. There was no gender match between the groups, but this was adjusted when evaluating the results, along with other confounding risk factors that differed between the two groups. Although a single blood sample was taken in our study and a cross-sectional evaluation was made, it should be kept in mind that exposure routes mostly continue in the same environment and through similar routes in pregnancy. However, prospective follow-up studies should be conducted in terms of half-lives and long-term effects of endocrine disruptors. Although questioning about possible intrauterine and childhood exposure routes in our study is one of the advantages of this study, it should be considered that the results may be affected by the possibility of incomplete or incorrect recall in the retrospective responses of the questionnaire studies. In our study, in order to prevent possible contamination during the collection of blood samples and experimental procedures, the use of plastic materials was avoided, and other materials used were pre-treated as described in the method section. In addition, although the early development inventory we used in our study was a parent-centered and easyto-use screening questionnaire, each child in our study was evaluated by the same clinician, taking into account the parents' views. Thus the results achieved provided a more objective evaluation.

Conclusion
Plasma DEHP, MEHP, and DBP levels were found to be signi cantly higher in children with isolated speech delay compared to healthy controls. After the adjusted analysis of the factors differing between the two groups in terms of delayed speech risk factors, there was no signi cant difference in terms of DEHP levels, while MEHP and DBP levels were markedly higher in the study group. The statistically signi cant higher phthalate levels in those with speech delay indicate that these children are more exposed to phthalates in whatever form in different ways.
In conclusion, endocrine-disrupting agents in the etiology of speech delay and their mechanism of action are still not fully explained. Our study contributes to the limited number of studies in this area, but more epidemiological and pathophysiological studies with higher sample sizes are needed to con rm this cause-effect relationship. Con icts of Interest/ Competing interests: The authors have no con icts of interest to disclose.
Data availability: It can be shared if desired (data transparency).
Code availability: N/A (software application or custom code) Author's Contribution: Dr Yaman conceptualized and designed the study and the data collection instruments, collected data, drafted the initial manuscript, and reviewed and revised the manuscript.
Prof Erkekoğlu planned and supervised the plasma phthalate measurement experiments, and critically reviewed and revised the manuscript.
Dr Özkemahlı and Msci Yirun performed the plasma phthalate measurement experiments, worked out the technical details and performed the numerical calculations for the suggested experiments.
Drs Akkus and Bahadur, and child development specialist Özdemir took part in developmental assessment and data collection.
Prof Özmert conceptualized and designed the study, coordinated and supervised data collection, and critically reviewed and revised the manuscript for important intellectual content.