In vivo exposure to electronic waste (e-waste) leachate and hydraulic fracturing fluid adversely impacts the male reproductive system

Abstract

Human health effects can arise from unregulated manual disassembly of electronic waste (e-waste) and/or hydraulic fracturing fluid spills. There is limited literature on the effects of e-waste and hydraulic fracturing wastewater exposure on the male reproductive system. Thus, this proof-of-concept study begins to address the question of how wastewater from two potentially hazardous environmental processes could affect sperm quality. Therefore, three groups of eight-week-old adult mice were exposed (5 d/wk for 6 wks) via a mealworm (Tenebrio molitor and Zophabas morio) feeding route to either: (1) e-waste leachate (50% dilution) from the Alaba Market (Lagos, Nigeria); (2) West Virginia hydraulic fracturing flowback (HFF) fluid (50% dilution); or, (3) deionized water (control). At 24-hours (hr), 3 weeks (wk), or 9-wk following the 6-wk exposure period, cohorts of mice were necropsied and adverse effects/persistence on the male reproductive system were examined. Ingestion of e-waste leachate or HFF fluid decreased number and concentration of sperm and increased both chromatin damage and numbers of morphological abnormalities in the sperm when compared to control mice. Levels of serum testosterone were reduced post-exposure (3- and 9-wk) in mice exposed to e-waste leachate and HFF when compared to time-matched controls, indicating the long-term persistence of adverse effects, well after the end of exposure. These data suggest that men living around or working in vicinity of either e-waste or hydraulic fracturing could face harmful effects to their reproductive health. From both a human health and economic standpoint, development of prevention and intervention strategies that are culturally relevant and economically sensitive are critically needed to reduce exposure to e-waste and HFF-associated toxic contaminants.

1. Introduction

The global shift towards digital economies and lifestyles has been accompanied by a rise in electronic waste (e-waste), defined as any discarded, broken, or obsolete electronic equipment at the end of its lifecycle [1]. A World Economic Forum report states that the global annual production of e-waste currently stands at around 50 million tons and is projected to reach 120 million tons by the year 2050 [1, 2]. E-waste is primarily composed of toxic metals, including lead (Pb), mercury (Hg), arsenic (As), cadmium (Cd), selenium (Se), hexavalent chromium (Cr VI), barium (Ba), nickel (Ni), cobalt (Co), manganese (Mn), gold (Au), silver (Ag), and copper (Cu), along with persistent organic pollutants (POPs) such as dioxins, furans, and polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polyvinyl chloride (PVC), and polycyclic aromatic hydrocarbons (PAHs) [3–5]. Based on estimates from 2019, annual global e-waste generation stands at 53.6 metric tons, of which only 17% is managed properly, while the rest remains unaccounted [6]. Due to poor international regulatory and recycling regimes, e-waste generated in both developed and developing nations typically finds its way into the hands of informal recyclers who use rudimentary methods to extract metals from within the discarded materials for resale [7–9]. E-waste contributes to 2% of total solid waste generation, which amounts to 70% of all hazardous materials found in landfills [2]. A major portion of e-waste is discarded into landfills and can potentially leach hazardous metals and organic compounds [10] into groundwater and nearby rivers. The same toxicants can also be released into the environment by incineration of the waste or run-off from the e-waste piles after heavy rains or floods that can produce significant damage to human and environmental health [11].

Hydraulic fracturing, another potential toxic waste issue, has grown exponentially over the past few decades. Use of hydraulic fracturing in the US has received a mixed reception. Some states (e.g. Pennsylvania, North Dakota and Texas) rely on this practice for unconventional oil and gas (UOG) extraction, while other states (e.g. New York and Vermont) have banned all extraction activity [12]. The process of UOG extraction involves the combined use of water and numerous chemicals to create fracturing fluid that is then applied at high pressure into the ground to create fractures that go first in a vertical direction and then horizontally through the rock for many miles [13]. Ten to seventy percent of all fracturing fluid returns back to the surface in the form of flowback water for about the first two months; flowback water contains contents of the originally-injected fracturing fluid, as well as additional underground minerals such as radionuclides, calcium and magnesium carbonate salts, and iron oxides [14, 15]. Alternatively, produced water (or formation water) is natural water that returns to the surface and is high in oil and gas [16]. Companies are not required to disclose a full list of components that comprise their proprietary fracturing fluid, so it is difficult to conclusively assess the subsequent hazard associated with exposure to fracturing fluids [4]. However, based on publicly available information, fracturing fluid contains a variety of toxic chemicals including hydrocarbons, radionuclides, and biocides, as well as toxic heavy metals including cadmium (Cd), manganese (Mn), strontium (Sr), arsenic (As), and lead (Pb) [17–19]. Although the chemistry of fracturing fluid varies over time, the concentration of these produced/flowback water-associated chemicals tends to increase as acidity increases [20]. The US Environmental Protection Agency (EPA) reports that accidental spills, disposal and storing of fluid, and leakage of chemicals can contaminate the groundwater, leading to human exposure [21]. Additionally, the produced water returning from the fractured shale seam is often recycled [22] because of the relative scarcity and cost of freshwater. This process requires careful handling of the used fluids during which materials can be spilled and solid wastes can be generated for land disposal.

E-waste leachate (a by-product produced from e-waste processing plants and follow-up incineration, usually containing high concentrations of toxic material), and HFF (formed during the process of oil extraction by hydraulic fracturing), are recognized environmental hazards that are known to both contain toxic heavy metals, and a variety of organic pollutants. However, these wastewaters also differ from each other in a number of ways, including disparities in their physicochemical characteristics (Table 1). Moreover, due to the propriety nature of hydraulic fracturing chemicals, there is likely large variations in the physicochemical characteristics of different HFF. Similarly, for e-waste leachate, different factors such as type of electronics, and disposal methods can affect the physiochemical composition of the leachate, making direct comparisons extremely difficult.

Table 1.

Physical parameters of e-waste and HFF [10, 96]

Parameters	E-waste leachate	HFF
pH	6.1 – 9	2.3 – 9.5
Total dissolved solid (TDS) (mg/l)	6.6 – 3230	680 – 261000
Density (g/cm3)	Not studied	1.08 – 1.22
Total organic carbon (mg/l)	10.6 – 2161	6.2 – 99
Electrical conductivity (mS/cm)	11.9 – 3448	15 – 200

According to the World Health Organization (WHO) report, Drinking Water: Equity, Safety and Sustainability [23], more than half of the world’s population is dependent on groundwater extraction for their drinking needs and daily use of water. These natural water resources are susceptible to contamination due to industrial activities and poor waste management. In general, municipal and industrial wastewater contain a range of contaminants (e.g., biowaste, heavy metals, nanoparticles, dyes, pharmaceuticals, radioactive materials), depending on the source. The improper disposal of these wastewaters can cause contamination of natural waters, including rivers and lakes, as well as soils, which could have adverse effects on the male reproductive system, which is a rising global issue [24, 25]. Indeed, there exists a worldwide trend of high male infertility rates over the last decade, specifically in Sub-Saharan countries, such as Nigeria, Sudan, and Cameroon [26]. The frequency of male infertility rates in these countries exceed 30%, compared to the overall global occurrence of male infertility reported at 7% [26]. In a retrospective study from Nigeria, a downward trend in sperm quality (i.e., sperm motility, sperm concentration, and total progressively motile sperm count) was observed from 2010 to 2019 [27]. The high levels of male infertility in Africa have also been associated with high rates of infections, especially those linked with sexually transmitted disease, along with nutritional deficiencies, heavy metal and industrial chemical exposure, and cigarette smoking [26].

Toxicants associated with both e-waste leachate and hydraulic fracturing flowback/produced fluid can potentially enter the environment, leading to a risk for human exposure [8, 28]. In epidemiological and animal studies, it has been observed that exposure to heavy metals such as Pb [29], Cd [30], Cr [31], As [32, 33] and organic pollutants such as PAHs [34] can cause a decline in sperm count and quality. Such waste products are also known to cause endocrine disruption and modifications in mechanisms of action in the testes of a variety of animals [35]. However, there is currently insufficient information available to establish quantitative dose-response relationships and no-adverse-effect exposure thresholds, particularly for chemical mixtures, on male reproduction [36, 37].

This proof-of-concept in vivo toxicological study was conducted to evaluate the potential toxicity of two waste fluid products, e-waste leachate collected from several sites near the Alaba International Market in Lagos, Nigeria and pooled “flowback/produced fluids” from hydraulic fracturing sites in West Virginia on male reproductive parameters in a mouse model.

The worm feeding protocol, [38] used to expose mice to e-waste leachate and HFF was employed, rather than a drinking water or diet scenario due to mouse avoidance as a result of its unpleasant taste/smell. Such evasion can lead to a less accurate dosing regimen among and between treatment groups. Oral gavage was also not chosen due to its invasive nature, lack of human relevancy, and undue stress to the animals and, in turn, potential health outcomes unrelated to the treatment, itself.

2. Materials and Methods

2.1. Experimental Design

Three groups of 15, eight-week-old male B6C3F1 mice were fed (5d/wk; total of 6-wk) a diet of mealworms (Tenebrio molitor or Zophobas morio) [38] that were injected with either: (1) e-waste leachate (50% dilution with deionized water) from the Alaba International Market in Lagos, Nigeria; (2) hydraulic fracturing fluid (50% dilution with deionized water), collected and pooled from Marcellus Shale drilling sites in West Virginia; or (3) deionized water (control). The 5d/wk dosing regimen used in this study is reflective of most occupational settings in the US. Meal/Superworm-fed mice were euthanized at 24-hr, 3-wk, or 9-wk following the end of exposure to evaluate cumulative effects on the male reproductive system and on germ line persistence. The experimental design of the study is shown in Figure 1.

Figure 1.

Experimental design. Three exposure groups were exposed to either: e-waste leachate (n=15); HFF (n=15); or deionized water controls (n=15). Necropsy and tissue collection were performed at three post-exposure timepoints (24-hr, 3-wk, and 9-wk). The right cauda epididymis was used to collect sperm for determining sperm quality (i.e., sperm count, density, viability, morphology, maturity and chromatin integrity). Blood was collected at the time of necropsy for differential blood counts, and serum recovered for assessing circulating testosterone levels (Figure created with BioRender.com)

2.2. Animal Care

Upon arrival to New York University (NYU)’s Department of Environmental Medicine (Sterling Forest, NY), eight-week-old B6C3F₁ male mice (The Jackson Laboratories, Bar Harbor, ME, USA) were maintained on a 12-hr light/12-hr dark cycle at ambient temperature (22°C) with a relative humidity of 55%. Mice, two per cage, were allowed to acclimate for one week prior to the start of ingestion exposures; filtered and acidified water and pelleted lab chow (Purina 5001 lab chow, Indianapolis, IN) were provided ad libitum prior to and after daily feeding of worms. All mouse procedures for this study were conducted under approval of the NYU Institutional Animal Care and Use Committee (IACUC).

2.3. E-waste Leachate and Hydraulic Fracturing Fluid Collection

Leachate samples, in the form of fluid and suspended particles, were collected from the Alaba International Market in Lagos, Nigeria using a 10 cm³ water sampler. The collected fluid was stored in 250 ml dark glass bottles and filtered using 15 cm Whatman filter paper before being shipped from Lagos, Nigeria to NYU’s Department of Environmental Medicine (Sterling Forest, NY).

Concentrations of PAHs and PBDEs were analyzed at the Nigerian Institute for Oceanography and Marine Research Central Laboratory (Lagos, Nigeria) using gas chromatography coupled to a flame ionization detector (GC-FID Agilent 7890A and GE-ECD Agilent 780A, respectively). Metals were analyzed at the same location using Atomic Absorption Spectrophotometry and Inductive Coupling Plasma Atomic Absorption Spectrophotometry (ICP-AAS) (PyeUnicam model 969; Limit of detection (LOD) = 0.2 ppb).

The HFF samples used for this study consisted of pooled flowback and produced water collected from a wastewater truck (pre-treatment) that serviced several wells in West Virginia. Metals content in HFF fluid was analyzed at US Geological Survey (USGS), Reston, PA using ICP-MS (LOD = 0.001 ppb).

At NYU, both e-waste leachate and HFF fluid samples were stored at 4°C in dark bottles until used for experiments. At the time of use, aliquot samples were allowed to reach room temperature (about 15 min) before injection of the worms.

2.4. Mealworms/Superworms and Mouse Feeding Procedure

Giant Mealworms, Tenebrio molitor (yellow mealworms: 1 to 1.5 inches long), and Zophobas morio (Superworms: 1.7 to 2.2 inches long) were purchased online from Fluker Farms (Port Allen, LA) and stored in dry, oatmeal-filled plastic bins with ventilated tops. Superworms and giant mealworms are both larvae of two different species of the darkling beetle (i.e., Zophobas morio, and Tenebrio molitor, respectively [39]. Giant Mealworms and Superworms were used specifically to deliver e-waste leachate and HFF to mice in a non-invasive manner, yet one assuring an accurate delivery dose of the fluid. The biochemical/nutritional make-up of both larvae are similar (Table 2), and therefore mice receiving either worm species consumed similar nutrient value from their supplemental larvae “treat”.

Table 2.

Comparison of nutrient content of Zophobas morio and Tenebrio molitor worms (adapted from Finke, 2002) [40].

Nutritional content	Zophobas morio larva	Tenebrio molitor larva
Weight (mg/insect)	610	304
Protein (g/kg)	197	184
Fat (g/kg)	177	168
Neutral detergent fiber	39	29
Acid detergent fiber	27	25
Ash (g/kg)	10	12
Metabolizable energy (kcal/kg)	2423	2252

Mice were provided standard mouse chow (Purina 5001) ad libitum throughout the entire study, including before, during and after exposure. Importantly, mouse chow diet represented their major source of nutrition, ensuring they received at least the recommended daily nutrition throughout this study. Worms were offered only as “treats,” and not as the primary source of nutrition in these studies. Based on data reported by the manufacturer, mice consume about 5g of chow on average every day. In comparison, the worm treat provided about 0.6g of food to each mouse [40]. The nutritional content of Purina 5001 chow is as follows: protein (240 g/kg), fat (100 g/kg), fiber (50 g/kg), ash (70 g/kg), and metabolizable energy of about 3000 kcal/kg [41]. Based on a 5g daily consumption of this standard chow diet, about 1.2 g of protein, 0.5 g of fat, 0.25 g of fiber and 0.35 g of ash was consumed daily by each mouse. In comparison, 0.6 g of the worm(s) treat was ingested daily by each mouse, which is equal to about 0.12 g protein, 0.1 fat, 0.04 g fiber, and 0.006 g ash. Thus, nutrient content from the worm treat was about 10% of protein, 20% of fat, 16% of fiber, and 2% of ash consumed daily by each mouse, yielding about 10 – 12% of the daily food intake per mouse.

The mealworms were fed a diet of apples and potatoes as both a food and water source. Prior to injection, e-waste or HFF fluid samples were vortexed for at least two minutes. Twenty minutes prior to injection, worms were placed at 4°C to slow their movement and then injected between body segments with either 0.25 mL (Z. morio) or 0.125 mL fluid (T. molitor), depending on the worm type, by pushing the needle fully into the ventral body, starting in the caudal region in the direction of the rostral area; the needle was slowly removed while dispensing the fluid to ensure minimal external leakage. New worms were injected at about the same time each day with the pre-selected treatment fluid, and the treated worms were immediately stored at −18°C to prevent any metabolism of the injected fluid until used for feeding to mice. Mouse bedding and worm containers were changed every three to four days [38].

Average weight of the mice at the start of the experiment was 25 grams. In order to achieve a 10 mL/kg body weight dose concentration for each mouse (selected from unpublished preliminary dose-response studies performed in this laboratory), mice were either fed one Superworm injected with a volume of 0.25 mL of either wastewater fluid, or two giant mealworms injected with 0.125 mL per worm. Mice were acclimated to worm-feeding for one week prior to the start of the experiment. For the feeding process, each mouse was placed into an individual cage with fresh bedding and fed the appropriately dosed mealworm. Visual monitoring revealed that mealworms were completely consumed by each mouse within 10–15-minutes of presentation. After ingestion, mice were transferred back to their housing cage, where they had easy access to food for the rest of the day. To prevent bias, both Superworms and Giant Mealworms were selected for feeding in a random manner for mice in all treatment groups. Body weights of each mouse were recorded daily before feeding to evaluate any weight changes compared to control-fed mice. After 6-wk of treatment, all mice were weighed weekly for the duration of the 3- and 9-wk recovery periods; mice were weighed again immediately prior to euthanasia.

2.5. Biological Parameters

At the time of sacrifice, mice were euthanized via intraperitoneal (IP) injection of sodium pentobarbital (175 mg/kg; Sleepaway, Fort Dodge Laboratories, IA). Blood was collected from each mouse from the descending aorta and spread on glass slides for differential blood cell counts. Two slides (100 cells per slide) were counted for each mouse from each treatment group. The remaining blood was allowed to clot at 4°C for 24-hr and then centrifuged for 15 min at 2,000 x g (at room temperature) to separate serum from packed cells. Serum was aliquoted into 1 mL tubes and stored at −20°C until needed.

The following organs were collected, trimmed of fat, and weighed immediately after removal: thymus, spleen, liver, kidneys, heart, left and right testes, left and right caudal epididymides, prostate, and seminal vesicles. The seminal vesicles with coagulating glands and fluid were weighed together. Relative organ weights were calculated by dividing selected organ weight (mg) by body weight (g).

2.6. Sperm Counts, Density and Viability

The right cauda epididymis from each mouse was placed into each well of a six-well plate (Corning Costar, Corning, NY) containing 2 mL of M16 medium. Sperm were collected by mincing the cauda (using microsurgery scissors) and incubating the plate for 15 min (at 37°C) to allow sperm to swim into the medium [42]. Sperm were counted twice using a hemocytometer and bright light microscopy (40X). Total sperm number was calculated by multiplying mean sperm count by the dilution factor and volume of M16 media. Sperm density was calculated by dividing total sperm number (x 10⁶) by cauda epididymis weight. Sperm viability/cell injury was determined using Trypan blue exclusion; 75 μL of sperm suspension (in M16 media) was vortexed with 25 μL of 0.4% Trypan blue. Sperm were counted using a hemocytometer and bright light microscopy (40X). Sperm heads that stained dark blue were considered non-viable. The percentage of viable sperm was determined by dividing the number of viable sperm (with unstained heads) by the total number of sperm counted [43].

2.7. Sperm Morphology

Sperm morphology was determined by placing 20 μL of M16 sperm suspension onto a glass slide, fixed with a 1:1 methanol:water mixture and stained with Coomassie Blue (0.22%) in acetic acid (10%). Excess stain was removed by rinsing each slide under gently running tap water; slides were dried overnight before cover-slipping with a single drop of Permount (Fisher Scientific International, Inc., Hampton, NH) [44]. Morphological abnormalities were evaluated by examining 200 sperm per mouse using bright light microscopy and oil immersion (100X). Sperm alterations were calculated by averaging the number of abnormal sperm per slide (100 sperm examined per slide; two slides per mouse per treatment group). Criteria used for scoring abnormal sperm included: (1) heads lacking normal hooks; (2) amorphous heads; (3) sperm without heads; (4) misfolded sperm; (5) sperm with corrugated tails; (6) sperm without tails; (7) sperm without heads; and (8) the presence of a cytoplasmic droplet (proximal or distal) in the midpiece [44].

2.8. Sperm Maturity and Chromatin Integrity

Two slides per mouse per treatment group were fixed in 3% glutaraldehyde (in phosphate-buffered saline) for 30 minutes (at room temperature) and the sperm then stained with a 5% solution of aqueous aniline blue. Immature sperm (which contain compacted DNA due to residual histones) were identified by complete stain absorption as described by [45]. One hundred sperm were counted per slide using bright light microscopy at 100X magnification.

Sperm chromatin integrity was assessed by fixing 20 μL of sperm suspension (on two glass slides/mouse) in a 96% ethanol-acetic acid-deionized water solution for 30 min at 4°C. The slides were hydrolyzed in 0.1 N HCl for 5 min and rinsed several times in distilled water at room temperature; fixed sperm were stained with 0.05% toluidine blue in McIlvain’s buffer (0.2 M sodium phosphate and 0.1 M citric acid, pH 3.5). Cells with damaged chromatin absorbed stain with shades of light to deep blue (any blue shade indicated chromatin damage), whereas sperm with intact chromatin integrity showed no staining in the allotted 5-min time period [46]. One hundred sperm were evaluated per slide (two slides per mouse per treatment group) using bright light microscopy at 100X magnification.

2.9. Serum Testosterone Levels

Serum testosterone levels were measured according to the manufacturer’s instructions using a commercially available ¹²⁵I radioimmunoassay kit (MP Biomedicals, Costa Mesa, CA, USA) [47]. Samples were assayed in duplicate using 50 μL of serum per tube, and resulting values were compared with a standard curve consisting of increasing testosterone standards ranging between 0 ng/mL to 50 ng/mL testosterone.

2.10. Hematoxylin and Eosin (H&E) Staining of Circulating White Blood Cells

Blood slides were allowed to air dry overnight at room temperature and fixed by immersing each slide for 1 min into 100% methanol. Fixed cells were stained with Hematoxylin and Eosin (H&E) (Thermo Fisher Scientific Inc., Waltham, MA) by dipping each slide five times into hematoxylin and six times into eosin stain, followed by a gentle rinse in tap water and air-drying at room temperature overnight; slides were cover-slipped and permanently mounted using several drops of Permount (Fisher Scientific Inc, Hampton, NH). The percentages of neutrophils and lymphocytes were determined by differential counting of 100 cells per slide; a total of two slides per mouse per treatment group were evaluated using bright field microscopy and oil-immersion (100X).

2.11. Statistical Analysis

One-way analysis of variance (ANOVA) followed by post-hoc testing (Least Significant Difference) when appropriate were used to assess the effect of treatment and post-exposure time using SAS Statistical software (SAS v9, Cary, NC, USA). Means were considered as different between groups when the p-value of the comparison was <0.05. Data are presented as mean + standard error. Evaluated outcomes that were compared by treatment and time post-exposure included sperm viability, sperm morphology, sperm maturity, sperm chromatin integrity, and serum testosterone levels, which were sorted by treatment and time post-exposure. Testosterone values for each of the three treatment groups were evaluated for each individual post-exposure timepoint and also pooled across time and averaged, and the mean values compared among the treatment groups.

3. Results

3.1. Physiochemical Characterization of E-waste Leachate and HFF fluid

To begin to gain information concerning some of the individual contaminants found in the E-waste leachate (pH 5.45) and HFF (pH 4.33) used in this study, wastewaters were analyzed for either metals and/or organic compounds. E-waste leachate contained As, Cd, Fe, Hg, and Pb at levels exceeding standards set by the EPA, WHO, and Nigeria Standard for Water Quality (NSDWQ) (Tables 3 and S1 [Supplementary Material]). E-waste leachate also included measurable levels of several polycyclic aromatic hydrocarbon (PAH) congeners, including benzo(a) anthracene, chrysene, benzo(b) fluoranthene, and benzo(a) pyrene, at levels above the EPA maximum contaminant level [MCL] of 0.2 ppb. Only two PBDE congeners (i.e., 2,2’,4,4’,5,6’-Hexabromodiphenyl ether (BDE154) and 2,2’,3,4,4’,5’,6-heptabromodiphenyl ether (BDE183) were at levels above the instrument detection limit; no MCL currently exists for PBDE compounds. Other analyzed PBDE congeners (i.e., BDEs 28, 47, 99, 100, 153, and 209) were either not present or were found at levels below the instrument detection limit. In the case of HFF fluid, only beryllium (Be; 100 ppb) and selenium (Se; 300 ppb) exceeded the EPA MCL of 4 and 50 ppb, respectively. Most other metals measured, including As, Cd, total Cr, Cu, Fe, Hg, and Pb, were below the action levels of governmental agencies (as shown in Table 3). Due to financial constraints, HFF fluid samples were only analyzed for metal content.

Table 3.

Analysis of heavy metals in e-waste leachate (ICP-AAS) and HFF fluid (ICP-MS) samples

Heavy metal	E-waste Leachate (ppb)	HFF Fluid (ppb)	USEPA MCL* (ppb)	WHO Standard (ppb)	NSDWQ (ppb)
As	10	5	10	10	10
Cd	48	0.01	5	3	3
Total Cr	33	8	100	50	50
Cu	41.2	0.2	1300	N/A***	N/A***
Fe	81,730	10	300	30	30
Hg	21	0.2	2	6
Pb	50	0.01	5	10	10
Be	<LOD**	100	4	N/A***	N/A***
Se	<LOD**	300	50	N/A***	N/A***

^*MCL = Maximum Concentration Limit;

^**LOD = Limit of Detection;

^***N/A = Not Applicable

3.2. Effects of Exposure to E-waste Leachate and HFF fluid on Body and Organ Weights

In this study, body weight measured at the end of the exposure in relation to that determined prior to the start of exposure is defined as body weight gain [Supplementary material S2]. Body weight gain over time failed to show a consistent trend. Although not significant, mice at the 3- and 9-wk post exposure timepoint had a larger weight gain, compared to those weighed at the 24-hr timepoint. Overall, exposure treatment had no effect on body weight, compared to age-matched control mice [48].

The effects of a 6-wk daily ingestion exposure to either e-waste or HFF fluid on organ weight, measured at the time of sacrifice, are shown in Tables 4, and 5. Significant organ weight changes were observed in the liver, spleen, heart, seminal vesicles, and right testis following treatment with both liquid waste mixtures, compared to controls at all three post-exposure timepoints. In contrast, weights of the kidney, prostate, thymus, left testis and cauda were similar to those of the time- and organ-matched controls, in both exposure groups [Tables 4 and 5].

Table 4.

Mean ± standard error (SEM) for absolute weights (mg) of liver, spleen, heart, seminal vesicle, and right testis at 24-hr, 3-wk and 9-wk post-exposure.

Organ	Treatment	24-hr	3-wk	9-wk
Liver	Control	1379.4±54.3	1702.4±45.2	1406.4±67.9
	E-waste	1384.2±28.7	1626±37.5	1604.6±53.9*
	HFF Fluid	1344.2±51.7	1533.4±50.6*	1296.6±35.9*
Kidney	Control	474.6±13.8	497.4±8.4	486.2±14.4
	E-waste	472.4±13.7	491.2±10.5	536.8±15.6
	HFF Fluid	440.2±22.0	472.4±7.8	490.6±23.1
Spleen	Control	90±2.7	94.6±3.4	90±1.1
	E-waste	87.2±3.1	92.6±1.7	106.2±6.3*
	HFF Fluid	94.6±7.7	92.2±1.7	92.8±6.6
Thymus	Control	40±6.4	43.8±7.4	48.2±4.7
	E-waste	39.4±3.7	33±1.8	57.2±10.4
	HFF Fluid	43.6±2.0	32.4±1.5	65.8±11.1
Heart	Control	148.4±6.9	162.8±2.8	140.8±3.0
	E-waste	145.8±5.3	157.6±5.7	152.8±4.3
	HFF Fluid	139±3.4	142.6±4.3*	139.4±65.8
Seminal Vesicle	Control	285±21.1	277.2±21.8	311.8±16.3
	E-waste	254±17.1	313±5.2*	263.8±27.8
	HFF Fluid	235±10.6	281.2±15.2	279.6±11.4
Prostate	Control	24±4.6	25.2±2.5	25.2±2.0
	E-waste	19.6 ± 2.2	16.2±0.9	30±2.2
	HFF Fluid	23±5.4	19±2.9	30.6±3.6
Left testis	Control	106±3.4	108±3.4	107.8±2.9
	E-waste	102.8±1.7	109.2±2.1	113±2.3
	HFF Fluid	105.4±1.3	101.4±2.1	111.4±2.4
Right Testis	Control	109.2±3.7	110.8±1.8	108±2.9
	E-waste	132.2±20.8	109.2±1.7	114.8±1.6
	HFF Fluid	107.6±1.7	105.8±1.4	113.4±0.8
Left cauda	Control	19.6±1.3	17.8±1.0	18±0.7
	E-waste	15.8±1.4	19.6±1.3	20.2±0.5
	HFF Fluid	16.6±1.4	14.4±1.1*	19.6±1.2
Right cauda	Control	16.6±1.6	16.6±1.2	19±1.3
	E-waste	17.8±2.1	16.8±0.7	22.6±0.87
	HFF Fluid	19±0.8	16.4±0.5	18.8±0.97

^*Significantly different from control at p<0.05 at each timepoint

Table 5.

Mean ± standard error (SEM) for relative weights (mg/g) of liver, spleen, heart, seminal vesicles, and right testis at 24-hr, 3-wk and 9-wk intervals.

Organ	Treatment	24-hr	3-wk	9-wk
Liver	Control	45.9±1.1	52.5±0.7	43.9±1.4
	E-waste	46.7±1.2	49.1±0.8	44.8±2.1
	HFF Fluid	46.1±0.8	47.6±0.9*	40.7±1.8
Kidney	Control	15.8±0.5	15.4±0.3	15.2±0.3
	E-waste	15.9±0.1	14.8±0.4	14.9±0.9
	HFF Fluid	15.1±0.3	14.7±0.1	15.3±0.3
Spleen	Control	3.0±0.1	2.9±0.1	2.8±0.1
	E-waste	2.9±0.1	2.8±0.1	2.9±0.1
	HFF Fluid	3.2±0.2	2.9±0.1	2.9±0.2
Thymus	Control	1.3±0.2	1.3±0.2	1.5±0.1
	E-waste	1.3±0.1	0.9±0.1	1.5±0.2
	HFF Fluid	1.5±0.1	1.0±0.04	2.0±0.3
Heart	Control	4.9±0.2	5.1±0.2	4.4±0.1
	E-waste	4.9±0.1	4.8±0.2	4.3±0.3
	HFF Fluid	4.8±0.1	4.4±0.1*	4.4±0.1
Seminal Vesicle	Control	9.5±0.7	8.6±0.7	9.7±0.4
	E-waste	8.6±0.5	9.4±0.1	7.3±0.7*
	HFF Fluid	8.1±0.3	8.8±0.5	8.8±0.4
Prostate	Control	0.8±0.1	0.8±0.07	0.8±0.05
	E-waste	0.7±0.1	0.5±0.03	0.8±0.05
	HFF Fluid	0.8±0.2	0.6±0.09	1.0±0.1
Left testis	Control	3.5±0.1	3.3±0.1	3.4±0.1
	E-waste	3.5±0.1	3.3±0.1	3.2±0.1
	HFF Fluid	3.6±0.1	3.2±0.1	3.5±0.1
Right Testis	Control	3.6±0.1	3.4±0.1	3.4±0.1
	E-waste	4.4±0.6*	3.3±0.1	3.2±0.1
	HFF Fluid	3.7±0.3	3.3±0.1	3.6±0.3
Left cauda	Control	0.6±0.04	0.6±0.04	0.6±0.03
	E-waste	0.5±0.04	0.6±0.04	0.6±0.02
	HFF Fluid	0.6±0.04	0.4±0.03	0.6±0.05
Right cauda	Control	0.6±0.04	0.5±0.04	0.6±0.04
	E-waste	0.6±0.1	0.5±0.02	0.6±0.02
	HFF Fluid	0.7±0.05	0.5±0.01	0.6±0.03

^*Significantly different from control at p<0.05 at each timepoint

Exposure of mice via daily ingestion (6-wk) of HFF fluid-injected worms significantly decreased the absolute (by 10%) and relative liver weight (by 9%) at 3-wk post-exposure, compared to time-matched control mice. In contrast, ingestion of e-waste leachate injected mealworms significantly increased absolute liver weight by 14%, but only at 9-wk post exposure, compared to time-matched controls. However, significant differences in liver weight did not persist when liver weights were determined relative to body weight at the 9-wk post-exposure timepoint. Moreover, no significant differences in liver weight were observed for HFF-treated mice between either treatment groups at 24-hr or 3-wk post-exposure. In comparison to those effects observed following HFF exposure on liver weight, e-waste leachate-exposed mice demonstrated a significant (p = 0.007) increase in liver weight of 18% (absolute weight) at 9-wk post-exposure, compared to time-matched controls. Unlike HFF-induced liver changes observed in exposed mice, relative spleen weight was unchanged by any exposure treatment, at any timepoint post exposure. Heart weight was also impacted by repeated ingestion of worm-injected HFF. At 3-wk post-exposure, HFF-exposed mice demonstrated a significant decline in absolute (by 12%; p = 0.03) and relative heart weight (by 14%; p = 0.007), compared to those in time-matched controls.

No significant treatment-induced effects were observed on seminal vesicle absolute weights in e-waste leachate- or HFF-exposed mice, compared to control mice at any post-exposure timepoint. Alternatively, relative weights of the seminal vesicles in mice exposed to e-waste leachate were reduced significantly (p = 0.005) by 25% at 9-wk post-exposure, compared to time-matched, post-exposure controls; no significant differences in relative seminal vesicle weights were observed at any other post-exposure time points in the e-waste exposure group. In contrast, exposure of mice to HFF fluid-injected worms revealed no significant effect on relative or absolute seminal vesicle weight compared to control mice. While absolute weight of the right testis in e-waste exposed mice did not show any significant difference between exposure groups, a significant (p = 0.04) increase in relative right testis weight (by 22%) was observed 24-hr post-exposure, compared to time-matched controls.

3.3. Effects of Exposure to E-waste Leachate and HFF fluid on Sperm Quality

3.3.1. Sperm Counts

Sperm counts in both e-waste- and HFF fluid-exposed mice were unchanged at 24-hr post-exposure, compared to time-matched, control-fed mice (Figure 2a). However, at 3-wk post-exposure, sperm counts were significantly (p <0.0001) reduced by 26% in HFF fluid-exposed mice, compared to their control counterparts. Sperm counts in both the e-waste- and HFF fluid-fed groups of mice were significantly (p <0.0001) reduced by 33% and 37%, respectively, compared to control-fed mice at 9-wk post-exposure. In control-fed mice, sperm counts increased significantly (p = 0.003) by 23% at 3-wk post-exposure compared to the 24-hr timepoint and remained relatively stable for up to 6-wk later. Sperm counts in e-waste-fed mice followed the same trend as control mice from 24-hr to 3-wk post-exposure; in this case, sperm counts at 3-wk post-exposure increased significantly (p = 0.003) by 25% compared to those levels observed after 24-hr. In contrast, sperm counts in e-waste-fed mice dropped significantly (p <0.0001) between 3- and 9-wk post-exposure. Moreover, HFF-fed mice exhibited stable sperm counts from 24-hr to 3-wk post exposure, followed by a significant (p = 0.0008) decline of 28% at 9-wk compared to the 3-wk timepoint.

Figure 2.

Mean of (a) Mean sperm count (x 106) (b) percent sperm viability (% of control) and (c) sperm density in mice exposed by ingestion of injected mealworms with de-ionized water (control), e-waste leachate and HFF fluid exposed at 24-hr, 3-wk and 9-wk post a 6-wk exposure. Data are means ± standard error (SE) for the control, e-waste leachate, and HFF fluid groups, respectively. SE is shown with error bars. Significantly different groups at p<0.05 compared to control at each timepoint are indicated by *.

3.3.2. Sperm Viability

Sperm viability in control-fed mice remained at or close to 100% over all post-exposure times (Figure 2b). In contrast, sperm viability in both the e-waste- and HFF fluid-exposed groups at 24-hr post-exposure dropped significantly (p <0.0001, and p =0.001, respectively) below the time-matched, control-fed values (by 14% and 9%, respectively). At 3-wk post-exposure, the percentage of viable sperm in the e-waste-exposed mice began to increase above that seen at 24-hr, but viability remained significantly (p <0.0001) below time-matched controls (i.e., by 14% of time-matched controls). Sperm viability from mice fed mealworms injected with HFF fluid remained significantly (p <0.0001) below (by 15%) levels observed in the time-matched control mice at 3-wk post-exposure. Sperm viability in both e-waste and HFF fluid-treated mice began to rise at 9-wk post-exposure, reaching those seen in control levels.

3.3.3. Sperm Density

Sperm density in water control-fed mice remained consistent over all three post-exposure timepoints (Figure 2c). No significant differences (p>0.05) were observed in sperm density between the control- and e-waste leachate-exposed treatment group at the 24-hr timepoint. However, sperm density in HFF-exposed mice decreased significantly (p = 0.01) at 24-hr and 3-wk post-exposure by 27% and 24%, respectively, compared to time-matched controls. In contrast, sperm density was unaffected (compared to time-matched controls) by the 6-wk treatment with e-waste leachate at the same 3-wk post-exposure timepoint. At 9-wk post-exposure, sperm concentrations decreased significantly for both the e-waste leachate (p = 0.0008) and HFF fluid (p = 0.004) treatment groups by 45% and 37%, respectively, compared to their time-matched control groups. Both e-waste- and HFF-exposed mice showed significant declines in sperm density levels at 9-wk post-exposure compared to the earlier time-points (i.e., 24-hr and 3-wk post-exposure) and control values.

3.4. Sperm Morphological Abnormalities

Total number of sperm abnormalities remained relatively stable in control mice over time, ranging between 10 and 13 abnormalities per 200 sperm (Figure 4a). However, the total number of morphological abnormalities in sperm collected from either e-waste leachate- or HFF fluid-exposed mice remained significantly (p<0.05) elevated across all three post-exposure time points, compared to time-matched control levels (Figure 4a). At 24-hr post-exposure, sperm from e-waste-exposed mice exhibited a 41% greater number (p = 0.03) of abnormalities compared to time-matched controls. Treatment with e-waste increased (p = 0.0008) the number of sperm abnormalities by 74% above control values (9 abnormalities per 200 sperm) at 3-wk post-exposure. At 9-wk post-exposure, the total number of abnormalities in the e-waste-treated group remained relatively stable compared to that observed at 3-wk, but was nonetheless significantly elevated by 40% (p =0.03) compared to its time-matched control counterpart. Sperm recovered from HFF fluid-exposed mice at 24-hr post-exposure did not show significant change in the numbers of abnormalities than those measured in time-matched control samples. However, at both 3- and 9-wk post-exposure, HFF fluid-exposed mice exhibited sperm morphological abnormalities significantly (p = 0.02 and p =0.01, respectively) above by 48% of that observed in the matched controls (albeit, not significantly different compared to the e-waste group).

Figure 4.

Mean of sperm: (a) total, (b); head, (c); tail and (d); midpiece abnormalities (per 200 sperm) in mice exposed by ingestion of injected mealworms with either deionized water (control), e-waste leachate or HFF fluid at 24-hr, 3-wk and 9-wk post-exposure timepoints. Data are shown as means ± standard error (SE) for control, e-waste leachate, and HFF fluid groups, respectively. *Significantly different p<0.05 compared to controls at each timepoint.

Sperm lacking normal hooks (i.e., those with amorphous heads) or sperm without heads were scored as abnormal sperm heads (Figure 3). In both the control- and e-waste leachate-treated mice, sperm head abnormalities decreased over time, while abnormal heads in HFF fluid-exposed mice increased with post-exposure time (Figure 4b). The number of abnormal sperm heads in both treatment groups remained elevated (albeit, not significantly) across all time points, compared to control values. However, at 9-wk post-exposure, abnormal sperm head numbers increased significantly (p = 0.03; by threefold) in the HFF fluid exposure group compared to the control group.

Figure 3.

Examples of normal sperm and morphological abnormalities in mice exposed by ingestion of injected mealworms for 6-wk to e-waste leachate or HFF fluids. (a) Normal mouse sperm, (b) Misfolded tail, (c) Detached head, (d) Missing tail, (e) Cytoplasmic droplet, (f) Broken tail, and (g) Amorphous head. Coomaassie blue stained images observed by light microscopy using oil immersion (100X magnification)

Sperm tail abnormalities were identified as misfolded tails, sperm possessing corrugated tails, and sperm without tails (Figure 3). Number of tail abnormalities in the control-treated mice were relatively constant (roughly four sperm abnormalities per 200 sperm counted) over time (Figure 4c). Exposure to both e-waste leachate and HFF fluid increased the number of sperm tail abnormalities, compared to the time-matched control group across all time points. At the earliest post-exposure timepoint (24-hr), the number of tail abnormalities from e-waste leachate-exposed mice increased by 60% (p = 0.03) compared to controls. At 3-wk post exposure, the incidence of e-waste leachate and HFF fluid-induced tail abnormalities doubled significantly (p =0.0003, and p =0.003) with respect to time-matched control values. The number of sperm tail abnormalities in e-waste leachate and HFF fluid-exposed mice dropped at 9-wk post-exposure compared to controls, but remained significantly (p =0.0003, and p = 0.03) above time-matched controls.

Midpiece abnormalities consisted of sperm folded onto themselves, as well as proximal or distal cytoplasmic droplets (Figure 4d). In control mice, sperm midpiece abnormality numbers dropped by half from 24-hr to 3-wk post-exposure. At 9-wk post-exposure, the number of midpiece abnormalities in control mice returned to levels seen at 24-hr. Only HFF fluid-exposed mice demonstrated significant (p <0.05) differences in midpiece abnormalities compared to control values. The incidence of midpiece sperm abnormalities in mice from the HFF fluid treatment group increased threefold (p =0.01) at the 3-wk post exposure timepoint, compared to time-matched control mice.

3.5. Sperm Chromatin Integrity and Maturity

The number of sperm with chromatin damage in the control group remained stable (~5 cells per 200 sperm) over time. At 24-hr post-exposure, incidence of chromatin damage increased significantly in e-waste leachate (p = 0.009) and HFF fluid-exposed (p = 0.02) mice by 48% and 44% respectively, compared to the time-matched controls (Figure 5a). A similar trend persisted at 3-wk following exposure, leading to a significant (p <0.0001, and p = 0.0002, respectively) increase in chromatin damage in both the e-waste leachate- (86%) and HFF fluid- (69%) exposed mice, compared to control values. At the final post-exposure time point, levels of chromatin damage in both treatment groups dropped, but remained significantly elevated above control values i.e., 64% (p =0.0005) and 43% (p = 0.02) greater levels for e-waste leachate and HFF fluid, respectively). Temporal patterns revealed that sperm chromatin damage in mice from both the e-waste leachate- and HFF fluid-treated groups increased from 24-hr to 3-wk post-exposure by 35% (p = 0.005) and 26% (p = 0.04), respectively. Alternatively, from 3-wk to 9-wk post-exposure, chromatin damage declined (albeit not significant) by 15% and 18%, respectively, in both the e-waste leachate- and HFF fluid-exposed groups.

Figure 5.

(a) Sperm chromatin damage (no. of sperm with damage/200 sperm) and (b) sperm maturity (no. of sperm with residual histones/200 sperm) in mice exposed by ingestion of mealworms injected with deionized water (control), e-waste leachate or HFF fluid and examined 24-hr, 3-wk and 9-wk post-exposure. Data are shown as means ± standard error for the control, e-waste leachate, and HFF fluid groups, respectively. *Significantly different at p<0.05 compared to controls at each timepoint.

The number of mature sperm from the control-treated mice remained constant (i.e., ~9 sperm each with residual histones per 200 sperm) over all three post-exposure timepoints (Figure 5b). However, exposure to e-waste and HFF fluid increased (although, not significantly) the number of sperm with residual histones by 24% and 18%, respectively, at 24-hr post-exposure, compared to time-matched control mice. At 3-wk post-exposure, the number of sperm with residual histones in the HFF fluid-exposed group dropped to levels comparable to the earlier timepoint. Alternatively, histone levels increased (compared to the 24-hr timepoint) in the e-waste leachate-exposed group at 3-wk post-exposure. At 3-wk post-exposure, the number of sperm with residual histones in the e-waste leachate-exposed mice were significantly (p =0.02) elevated by 29% compared to controls. At the final post-exposure timepoint, residual histone levels in sperm from both wastewater treatment groups were similar to those observed six weeks earlier. However, only histone levels in sperm collected at 9-wk post-exposure from the e-waste leachate-treated group reached statistical significance (p =0.04) i.e., 27% decline in sperm maturity compared to the time-matched control group.

3.6. Effects of Exposure to E-waste Leachate and HFF fluid on Serum Testosterone Levels

Mice exposed to e-waste leachate and HFF fluid exhibited lower serum testosterone levels at each post-exposure time point compared to time-matched controls (Figure 6b). While there were no significant differences in testosterone levels between treatment groups at 24-hr post-exposure, but testosterone levels of mice exposed to both HFF fluid and e-waste leachate dropped significantly below control values by 68% (p = 0.004) and 81% (p = 0.0008), respectively at 3-wk post-exposure. At 9-wk post-exposure, serum testosterone levels of mice exposed to e-waste leachate began to increase, while levels in mice exposed to HFF fluid remained significantly reduced (p = 0.01) by 70% compared to controls. Data pooled across post-exposure timepoints also revealed that treatment of mice with either e-waste leachate or HFF fluid significantly decreased serum testosterone levels compared to control levels by 59% (p = 0.0003) and 70% (p <0.0001), respectively (p <0.05; Figure 6a).

Figure 6.

(a) Total testosterone level (ng/ml); (b) temporal variation of testosterone level (ng/ml) in mice exposed by ingestion of wastewater fluid-injected mealworms with deionized water (control), e-waste leachate and HFF fluid exposed for and examined at 24-hr, 3-wk and 9-wk post-exposure. Data are shown as mean ± standard errors (n = 13–15) mice per each treatment group. *Significantly different (p<0.05) at each timepoint compared to controls.

3.7. Effects of Exposure to E-waste Leachate and HFF fluid on Differential Blood Counts

Percentages of circulating neutrophils in both e-waste leachate- (71%) and HFF fluid- (72%) exposed groups were significantly (p =0.01) elevated, compared to control mice, at 3-wk post-exposure (Figure 7a). At the 9-wk post exposure timepoint, percentages of neutrophils remained similar to that observed at the 3-wk post-exposure timepoint, in the HFF fluid-exposed group. Compared to the time-matched controls and time-matched e-waste exposure groups, neutrophil levels remained elevated, although failed to reach statistical significance. In contrast, circulating neutrophil values in the e-waste exposure group were significantly increased (p <0.01) by 71% at 3-wk post-exposure (compared to time-matched controls), but dropped to control levels by 9-wk post-exposure.

Figure 7.

Circulating (a) neutrophil percentage; and (b) lymphocyte percentage in mice exposed by ingestion of injected mealworms with de-ionized water (control), e-waste and HFF fluid and examined 24-hr, 3-wk and 9-wk post-exposure. Data are shown as means ± standard error (SE). *Significantly different at p<0.05 for each timepoint compared to controls.

Percentages of circulating lymphocytes in the control group remained consistent (from 72%−76%) at all three post-exposure timepoints; lymphocyte levels followed a trend directly opposing that of neutrophil percentages in the time-matched controls and in both wastewater treatment groups. A significant (p <0.05) decrease in lymphocyte levels was observed in e-waste leachate and HFF fluid-exposed mice, compared to time-matched controls (Figure 7b). While lymphocyte percentages in the e-waste groups were similar to control levels at 24-hr post-exposure, values dropped significantly (p =0.002) by 23% at the 3-wk post-exposure timepoint and returned to control levels by 9-wk post-exposure, compared to time-matched controls. Following exposure to HFF, lymphocyte levels were significantly reduced (p =0.002; 14%) at 3-wk, compared to the time-matched control group. Unlike that observed following e-waste exposure, lymphocyte numbers in the HFF fluid treatment group failed to recover by 9-wk post-exposure and remained at the same decreased level observed at 3-wk post-exposure.

4. Discussion

Over the past several decades, e-waste and unconventional hydraulic fracturing fluid have emerged as potential risks for both environmental and human health. However, there are limited studies evaluating the toxicology and health outcomes of these industry-related waste products, possibly due (in part) to the complexity of analyzing mixtures and chemical interactions [9]. To address this gap, laboratory studies in mice were performed to better understand the health implications of two wastewater products, e-waste leachate and HFF fluid, on the male reproductive system, that has been shown to be a sensitive target for the effects of both chemical mixtures [49, 50]. Many of the constituents found in both e-waste and HFF have been shown to directly damage male reproductive organs through oxidative stress-related injury, or to act indirectly on the reproductive system through endocrine disruption [51, 52]. The concentrations of certain heavy metals measured in both the e-waste leachate and HFF fluid used in this study exceeded safe limits set for drinking water set by the EPA, WHO, and NSDWQ. Of particular concern in the e-waste leachate are toxic/carcinogenic heavy metals, including Cd, Hg and Pb. In HFF, levels of Be and Se were also above the maximum concentration levels set by these same agencies [53–55]. It is difficult to replicate exactly the human exposure scenario, as populations living in the vicinity of- or working with- e-waste or hydraulic fracturing activities are usually exposed to pollutants through multiple exposure pathways (i.e., drinking water, air/dust, dermal or ingestion) [56, 57]. Thus, a non-overtly toxic, 50% dilution level was selected for this study, based on unpublished preliminary data from our laboratory.

Unlike many other in vivo studies in this research field, our investigation employed a relevant oral ingestion exposure route, used by other investigators [38, 58, 59] and reflective of a major human exposure pathway for both e-waste and hydraulic fracturing-related pollution, either in the form of contaminated drinking water or through the consumption of food grown in contaminated soil [60]. Oral ingestion of worms injected with specific amounts (i.e., 0.25 mL total wastewater) of either e-waste leachate or HFF equivalent to 7.5 mL of wastewater consumption by each mouse during the entire 6-wk exposure period. This particular exposure duration was selected because it encompassed the entire period of mouse spermatogenesis. A single mouse spermatogenesis cycle occurs approximately every 36 days [61]. As sperm proceed through spermatogenesis, they travel up through the epithelium and are replaced by another set of cells; thus, four generations of cells develop in synchrony during this 6-wk time period [62]. After completion of a 6-wk spermatogenesis cycle, epididymal transit of sperm takes an additional 1–2-wk [63]. Therefore, three different post-exposure timepoints were selected to demonstrate effects at different stages of spermatogenesis; early at the end of exposure (24-hr post-exposure); middle to the end of spermatogenesis (i.e., 3-wk post-exposure); and after completion of spermatogenesis, and the beginning of epididymal transit (i.e., 9-wk post-exposure).

The rising incidence of male infertility is of international concern [64]. Currently, about 15% of all couples worldwide, of reproductive age are affected by infertility, of which approximately 50% are attributed to female complications, while 20 to 30% of all cases are attributed to male issues. The remaining 20 to 30% of infertility cases are due to a combination of both male and female factors [65]. Frequent reports of declining sperm quality and counts have emerged over the past 50 years, as well as other disturbing reproductive trends including increased prevalence of testicular cancer, rising frequencies of undescended testis (cryptorchidism), malformation of the penis (hypospadias), as well as increased demands for assisted reproduction [66]. Human and animal studies report that environmental contaminants (including those identified in e-waste leachate and/or UGO), acting mainly via genetic alterations, are a major cause of observed defects in male reproductive function [67]. Indeed, dismantled e-waste and contamination from hydraulic fracturing practices contain a variety of toxic chemicals, including heavy metals and organic contaminants (i.e. PAHs, and PCBs) that can alter fertility [68].

Male infertility is an issue of particular importance in Nigeria; it has been associated with both environmental and biological factors, including infections, nutritional deficiencies, cigarette smoking, heavy metals and industrial chemicals) [26]. However, association of male infertility with e-waste related exposures is not well-defined. A study from Nigeria reported that male reproductive hormones (i.e., testosterone, and follicle stimulating hormone [FSH]) were reduced in e-waste workers, compared to unexposed non-e-waste workers [69]. Though studies on e-waste and male reproduction in Nigeria are somewhat limited, multiple studies from China [70, 71] clearly reveal that exposure to e-waste chemicals (e.g., organochlorine, polybrominated diphenyl ethers, heavy metals) are associated with poor semen quality (i.e., sperm count, motility, and morphology).

Oral exposure to e-waste leachate and HFF in this study revealed a variety of adverse effects in critical parameters related to male reproduction, including sperm count, viability, maturity, density and morphology, chromatin integrity of sperm, and a decline in serum testosterone levels, which persisted long after exposure ceased. The unfavorable impacts observed in this study on sperm parameters are consistent with previous studies demonstrating that oral exposure to e-waste leachate fluid-associated metals and organic chemicals alter male reproductive endpoints in rabbits [72], mice [73, 74], and rats [75]. The findings in our study are also supported by epidemiological evidence, wherein e-waste-exposed individuals (either through occupational or community exposure) show a decline in semen quality and genetic damage of the sperm [51, 70, 71]. While no studies were performed to assess the particular wastewater constituent(s) specifically associated with the observed effects, a major class of e-waste constituents, heavy metals (Cr, Cu, Fe, Mn, Pb, and/or Zn), is known to induce secretory malfunction of the testes, which can ultimately manifest in decreased sperm concentration in the ejaculate, decreased percentage of motile gametes, and an increase in the proportion of morphologically abnormal sperm formed in sexually mature male rats [76, 77]. Although there is limited toxicology literature available for HFF-associated constituents, a systematic review by Elliot and colleagues [78] showed that chemicals generally present in HFF and its wastewater are associated with reproductive and developmental toxicity.

In this study, exposure of mice to either e-waste or HFF for 6-wk resulted in increased numbers of sperm abnormalities (compared to time-matched controls) in the form of amorphous or missing heads, misfolded, doubled, or missing tails, and increased cytoplasmic droplets. These findings are supported by another murine study [79] demonstrating that IP injections of different dilutions (1–25% of e-waste in saline) of e-waste leachates from the Nigerian Orita-Aperin and Oworonsoki landfills, over a period of two weeks, acted to induce dose-dependent increases in total number of abnormal sperm morphologies. In that study, percentages of abnormal sperm determined in control (or unexposed) mice were about 4%, similar to that observed in control mice in our current study. Interestingly, Bakare et al, (2005) observed that abnormal sperm morphology in mice was in the range of 9.7%−13%, five-weeks after oral exposure to 25% e-waste leachate; the percentage of altered sperm morphology in this study are similar to those values observed in our current study (i.e., 10% sperm abnormalities 3-wk after exposure to 50% e-waste leachate). Heavy metals such as Cd, Cr, and Cu (which were present in e-waste leachate samples used in this study), have been associated with oxidative stress leading to genetic toxicity [80]. Moreover, DNA damage in spermatozoa, either in the form of errors in differentiation, or chromosomal aberrations, could be responsible for aberrations in sperm morphology including those observed in the tail, head, or midpiece in the current study [81, 82].

Increases in chromatin damage observed in the current study over the 9-wk timeframe suggests that exposure to e-waste leachate and HFF damages sperm DNA. In an epidemiological study, Yu and colleagues [70] observed that individuals living near an e-waste dismantling site had higher levels of DNA damage and apoptosis in sperm compared to the control cohort; in this case, the authors concluded that sperm damage was associated with inhaled PBDEs from house dust generated from within an e-waste area. Similarly, in adult males working in e-waste recycling sites, occupational exposure to PCBs and heavy metals (i.e., Pb) resulted in increased chromatin damage in spermatozoa and decreased serum antioxidant enzymes (i.e. glutathione and superoxide dismutase) [51]. As e-waste and HFF constituents including metals can elevate reactive oxygen species (ROS) levels (i.e., superoxide anions and hydroxyl radicals), which can then attack biochemical machinery including DNA, lipids, and proteins, it is likely that such activity can result in genetic damage during spermatogenesis that can adversely impact sperm quality [83, 84].

In the current study, serum testosterone levels in exposed mice declined dramatically at all post-exposure timepoints following exposure to e-waste leachate and HFF. Some constituents of e-waste leachate and HFF, including heavy metals and organic compounds (e.g., PBDEs, PCBs, PAHs, and dioxins), are known for their endocrine disrupting properties [85]. Findings from Akintunde and colleagues [86] demonstrating that adult male rats exposed to increasing concentrations of leachate (from the Elewi Odo municipal battery-recycling site) for 60 days had reduced serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels, support our findings and reveal the endocrine disrupting activity of e-waste leachate. Similarly, e-waste recyclers working in an e-waste site in Nigeria showed significant decreases in levels of testosterone, LH, and FSH compared to non-electronic waste workers. The authors of that study concluded that the observed decrease in hormone levels was associated with the presence of Cd, Cr, Hg, and As [69]. The effects of wastewater from both sources on sperm quality parameters (i.e., sperm count and density, sperm abnormalities, and chromatin damage), and the observed decline in testosterone levels persisted 9-wk past the end of exposure. These persistent changes could arise from a variety of mechanisms, including (but, not limited to) accumulation in the testes of reproductively-toxic metals [87], toxicant-induced damage directly to the testes or Sertoli cells [36], or to wastewater-induced persistent changes in hormonal levels.

In normal mice, peripheral blood cell profiles lean toward higher lymphocyte counts, with about 75–90% lymphocytes and 10–25% neutrophils [88]. Control mice in the current investigation revealed neutrophil/lymphocyte ratio (NLR) levels similar to historical controls, described above. In contrast, exposure of mice to e-waste leachate or HFF in this study led to elevated percentages of circulating neutrophils, suggestive of an inflammatory response; a concurrent drop in lymphocyte percentages were also observed over the same time period. The increase in NLR in the current study could be attributed to oxidative stress induced by individual wastewater constituents, such as heavy metals, VOCs, or PAHs [89–91]. In addition, the observed wastewater-induced decrease in lymphocytes could be indicative of immunosuppression.

Results in the current study are consistent with a study by Xu and colleagues [92], who observed significant increases in circulating neutrophils in 15- to 65-year-old individuals living in close proximity to an e-waste dismantling site in Luqiao, China; in this study, changes in human blood profiles were positively correlated with PCB and PBDE levels [92]. Alternatively, a decrease in lymphocyte values were also observed in the same study population, which was negatively associated with PCB levels present in the e-waste. Alternatively, a study of preschool children living in the vicinity of an e-waste site in Guiyu, China, revealed no differences in blood neutrophil levels in e-waste-exposed individuals, compared to a non-exposed group from Haojiang, China. However, the percentages of lymphocytes in the same study were significantly lower in the e-waste exposure group, compared to the control cohort [93]. In contrast to the substantial number of published studies evaluating the effects of e-waste exposure on blood profiles, literature related to the effects of hydraulic fracturing on the inflammatory response in animals or humans is dramatically lacking.

Taken together, results from our studies reveal that exposure to wastewaters recovered from dramatically different operations, but consisting of relatively similar constituents, leads to a decline in sperm quantity and quality, testosterone levels, and induces a systemic inflammatory response. Based on the wastewater-induced outcomes observed in this toxicology study, changes in fertility and quality of life could be adversely affected and should be examined in future studies.

Limitations:

The dosing regimen of 5 d/wk could have led to a partial recovery in organ responsiveness occurring over the weekend. Thyroid hormones, vital to the physiological regulation of testicular basal metabolic activity [94], influence testicular functions of steroidogenesis and spermatogenesis [95]. Although not queried in these studies, examination of thyroid parameters could have resulted in a clearer understanding of underlying mechanisms associated with the observed adverse changes in sperm parameters. The authors strongly recommend that future studies examining the health effects of HFF and/or e-waste leachate include effects on thyroid regulation as part of their investigations. We recognize that the differences in pH values between the two wastewater products could have contributed to the observed effects. While the pH of control water was ~7.0, the HFF and e-waste leachate were more acidic (5.4 and 4.3, respectively). Exactly what contributions the differences in pH and/or water chemistry (e.g., hardness due to salt concentrations) might have had on the observed changes in reproductive factors remain to be seen. While other endpoints related to reproductive toxicology, such as a dose-response effect and histopathology could have added to the persuasiveness of the study, this proof-of-principle investigation has succeeded in laying the groundwork for future (more mechanistic) studies in this understudied scientific field. It is critical that follow-up toxicological and human studies be performed to assess effects of HFF and/or e-waste leachate on fertility and future generations. We note that oral exposure is a highly-relevant exposure route for HFF, due primarily to accidental entry into the groundwater and aquifers, as well as for residents living close to e-waste sites and use nearby rivers as a source of drinking water. However, effects caused by other exposure routes i.e., dermal and inhalation exposure routes, particularly for oil and gas employees and residents living in close proximity to intentionally-set burning waste piles, also require investigation.

Conclusions

Ethical implications of exposure to e-waste and HFF lie not only in the destruction of the environment, but also in the hazards from potential exposure to those living and/or working near contaminated sites. Results from this proof-of-principle mouse study demonstrated that oral exposure to wastewaters from two environmentally precarious processes, unconventional hydraulic fracturing and e-waste processing adversely, and persistently affects male reproductive functions essential for fertility. Findings from this study are highly significant for public health, given the ease with which e-waste dumpsite leachate can contaminate drinking water and soil, and HFF fluid can leak or breach from well casings or storage pond liners and contaminate local surface and groundwater sources. As wastewater from both processes produced dramatic and persistent adverse effects on sperm parameters and testosterone levels, it is difficult to conclude which product was most harmful. However, given the global extent of these processes and their potential to produce devastating outcomes on male reproductive parameters, there exists an urgent need for additional animal and epidemiological studies to understand the health implications and molecular mechanisms associated with such outcomes.

Highlights:

• E-waste and hydraulic fracturing (HFF) related chemicals cause toxic effects

• Exposure to e-waste leachate and HFF in male mice caused decline in sperm quality

• Serum testosterone levels decreased in both exposed groups

• Neutrophil/lymphocyte ratio increased in mice exposed to e-waste leachate and HFF

• Most of the changes in mice persisted for 3-wk and 9-wk after the end of exposure

Data Availability Statement:

All data generated or analyzed during this study are with the corresponding author, and, if necessary, she is available for taking any questions about the datasets and these can be acquired by reasonable request.