Ecotoxicity Effects of Lead Bullets in Human and Wildlife: The Consequences of Environmental Pollution, Low Intelligence Quotient, Brain Damage and Brain Overclaim Syndrome

Abstract

Bullets from gunshots made of lead are used to kill and arrest criminals, as they are also used by criminals to intimidate or kill innocents for psychosocial gains. So the increased environmental pollution caused by lead from industries, firearms, gasoline, among others is a source of concern for environmental health specialists, clinical toxicologists, experimental toxicologists, industrial toxicologists and ecotoxicologists. Lead can get into body system accidentally via oral, inhalational, epidermal, dermal, intraperitoneal, and intravenous routes. The toxicokinetic data of lead disposition via various routes of administrations are quite inconsistent. Hence the set blood limit concentration has been considered to be incorrect. In view of this, toxicokinetic data analysis of lead was carried out with intent to determine toxic doses of lead in various organs, and its toxicological consequences. Findings have shown that at lower doses, kinetics of lead is linear (first order), and at higher doses the kinetics becomes non-linear (zero-order). Metabolic processes modulated by lead could be either rate limiting or non–rate-limiting causing induction and inhibition of a myriad of metabolizing enzymes in liver, brain, kidney, intestine and lung. The LD50 of lead bullet in human was 450 mg/kg, which caused death in 9.1 days, and penicillamine (18 mg/kg) can be used for treatment. Mean residence time (MRT) and elimination half-life (T12β) were 25.8 and 18 days, respectively.

1. Introduction

Bullets made from lead cause lead intoxication (plumbism), which may be fatal if left unremoved for a period of time [1]. Fragment of lead radiates in target animals [2]. The target doses of lead fragments in Andean condors, Turkey vulture and bald eagle are 45.5–58.2, 20.7–33.8 and 11.5–27.0 mg/kg, respectively [3, 4, 5]. Interference of lead with calcium metabolism can lead to neurological and neuromuscular disorders via signal transduction of protein kinases, neurotransmitter and calcium [6]. Behavioural and learning deficiencies have been linked to interference of lead with signalling of brain cells in human and birds [7, 8]. Bullet fragments can be lodged in body joints and cause anaemia, abdominal colic, nephropathy and neuropathy. The precipitating factors are infection, metabolic stress and alcoholism [2]. Mass spectrometric isotope dilution analysis with chelation therapy was used for the mobilization of lead from bone [1]. Violence can lead to the use of firearms causing lead poisoning characterized in part by changes in behaviour, neurological status and death. Clinical neuronal manifestations are fatigue, malaise, irritability, loss of libido, headache, encephalopathy, delirium, ataxia, convulsions and motor neuropathy [9]. As low as 10 μg/dl interferes with haem synthesis and increases aminolevulinic acid (ALA) that suppresses gamma aminobutyric acid (GABA) neurotransmission [10]. Lead concentration less than 4 μm causes acute encephalopathy [11]. Lead exposure (0.25 μg/g) of brain tissues for 50 days, starting on post-natal day 1, caused abnormal expression of glial-related genes [12]. Lead (19–31 μg/dl) caused decrease in the size of cortical column in somatosensory cortex [13]. Placental blood lead (10 μg/dl) caused cognitive impairment [14]. Bullet lodged in various parts of the body can stay in the body for 3 months to 40 years, and associated neurodiseases are alcoholism, delirium tremens, thyrotoxicosis and shock. Gunshot after 4 years caused depression with detected blood lead of 6.7 μg/dl [1]. Intracellular bullets may result in unwanted long-term complications [15]. After 4 years gunshot, caused malaise and weakness with blood level of lead 62 μg/dl, whereas gunshot after 3 years caused paroxysmal abdominal pain and post prandial emesis [16]. Clinical toxic threshold doses for lead fragments in blood (50–100 μg/dl), liver (6 mg/kg), Kidney (4–16 mg/kg) and bone (>20 mg/kg) have been reported for Anseriformes, Falconiformes and Accipitriformes, respectively [17]. Lead concentration of blood (<0.2 ppm), liver and kidney (>2 ppm) and bone (<10 ppm) have been reported for birds not exposed to lead [18], but the cost of antidotes has increased [19]. Therefore, poisoning severity score can be modified to assess the degree of bullet toxicity [20] in human and wildlife with a view to curtailing consequences of lead poisoning.

2. Materials and methods

Literatures were searched for information on acute and chronic effects of lead bullets in wildlife, young and adult humans, especially in relation to brain damage and criminology. Some established formulas for determination of LD₅₀ were modified for determination of acute and chronic toxicity effects of lead in wildlife and human. The developed formulas were used to determine lethal time fifty (LT₅₀), chronicity factor (CF), dose of lead that can kill one man, toxic dose rate, target concentration, clearance, effective dose of antidote, volume of distribution, area under curve, mean residence time and elimination rate constant [21, 22, 23]. Up and down procedure (UPD) was used to estimate LD₅₀ of bullets, as the initial estimate of the LD₅₀ was within a factor of the authentic LD₅₀ [24], and has been validated [25].

2.1 Kinetic energy of lead bullet

The velocity of bullet is proportionally more influential than the weight of the bullet. Therefore, the kinetic energy transferred to the target animal is presented by the equation given below [26]. Kinetic energy of bullet is the mass of bullet times squared velocity of the bullet.

$$\mathrm{KE}=\frac{1}{2}{\mathrm{mv}}^2$$E1

KE = kinetic energy; m = mass of bullet; v = velocity.

Low velocity = 1200 ft./s; medium = 1200–2500 ft./s; high = >2500 ft./s [27]. Bullets from handguns have velocity of <1000 ft./s, rifles (<2500 ft./s) and bullets of 5.56 mm are small but relatively fast. Outer jacket of the bullet determines margin line of tissue injury which is responsible for bullet moving more than 2000 ft./s [28].

Air rifle pellet that weighed 8.25 grains and of 0.38 calibre received velocity of 101 m/s, but could damage the brain, whereas that of 113 grains of 58 m/s could damage brain matter [29]. An impactor (bullet) of 200–297 mm² could exert force of 980–1334 N on parietal bone [30]. Therefore Head Injury Criterion (HIC) is used as protective measure for skull. It is a function of the period of acceleration at the head centre of gravity, bearing in mind that head is a one-mass structure:

$$\mathrm{HIC}=\left\{\left(t^2-t^1\right){\left[\frac{1}{t^2-t^1}{\int }_{t^2}^{t^1}a\left(t\right)\mathrm{d}t\right]}^{2.5}\right\}max$$E2

t₁ = initial time (s); t₂ = final time (s); a(t) = acceleration at the centre of gravity of the head; t₂-t₁ = acceleration window (15–36 ms) [31].

2.2 Determination of calcium-lead concentration in erythrocytes

Erythrocyte volume $\left(V\right)=\frac{4}{3}{\mathit{\pi a}}^2\mathit{xb},$ where a = larger axis; b = minor axis; $\pi$= 3.1415. Surface area and volume of a single red blood cell are 155 ${\mathrm{\mu m}}^2$ and 87 ${\mathrm{\mu m}}^3$ respectively [32]. Calcium concentration (mg/100 ml) to per cent decrease of lead erythrocytes content is equal to;

$$\frac{\%\text{decrease}\text{erythrocytes lead content}}{\text{concof}{\mathrm{Ca}}_2\text{mixture}}=\frac{40-10}{8-4}=1:7.5$$E3

2.3 Determination of acute toxicity of lead in rodents

The detection limit of lead (0.04 μg/dl) at 5% level of significance, blood level lead (0.2–37 μg/dl) and urine lead (9–930 μg/dl) as well as less than 1% of lead transport from sink to plasma have been considered for calculations [21, 22, 23, 33, 34].

$$\text{Chronicity factor}\left(\mathrm{CF}\right)=\frac{\acute{{\mathrm{LD}}_{50}}}{90\text{days}{\mathrm{LD}}_{50}}$$E4

$$\text{Dose rate}\left(\mathrm{DR}\right)=\text{Target concentration}\left(\mathrm{TC}\right)\times \text{Clearance}\left(\mathrm{Cl}\right)$$E5

$$\begin{array}{l}\text{Also median lethal time fifty}\left(\mathrm{LT}50\right)=\frac{{\mathrm{LD}}_{50}}{D^P}\text{whereas}D=\mathrm{LD};\\ P=\text{Power coefficient}1/3\end{array}$$E6

$$\text{Note that}\mathrm{ED}50=\frac{{\mathrm{LD}}_{50}}{3}x{W}_{a}x{10}^{-4}$$E7

$$\frac{{\mathrm{LD}}_{50}}{3}=\frac{{\mathrm{ED}}_{50}}{W_a\times {10}^{-4}}$$E8

$${\mathrm{LD}}_{50}=\frac{3\left({\mathrm{ED}}_{50}\right)}{W_a\times {10}^{-4}}$$E9

$${\mathrm{LD}}_{50}={\mathrm{LT}}_{50}\times D^P=\frac{3\left({\mathrm{ED}}_{50}\right)}{W_a\times {10}^{-4}}$$E10

$$D^P=\frac{3\left({\mathrm{ED}}_{50}\right)}{W_a\times {10}^{-4}}\times \frac{1_{}}{{\text{LT}}_{50}}$$E11

Therefore:

$${\mathrm{ED}}_{50}=\frac{D^P\times W_a\times {10}^{-4}\times {\text{LT}}_{50}}{3}$$E12

$$\text{Also the volume of distribution}\left(\mathrm{Vd}\right)=\frac{\text{Dose}}{\text{Concentration}}$$E13

$$\text{Clearance}=\frac{\text{Dose}}{\mathrm{AUC}}$$E14

2.4 Calculation of pulmonary oxygen toxicity of lead

The cumulative pulmonary toxic dose (CPTD) of lead expressed as OTU (oxygen toxicity units) is calculated thus, partial pressure of oxygen (PO₂) remains constant [35].

$$\mathrm{OTU}=t_{x}x{\left(\frac{0.5}{\mathrm{P}{\mathrm{O}}_2-0.5}\right)}^{\frac{-5}{6}}.$$E15

Where as $t_x$ = time of exposure; $\mathrm{P}{\mathrm{O}}_2$ = constant; $\frac{-5}{6}$= exponent (−0.8333).

$$\text{Exposure time}{t}_x=\text{Segmenttime}\mathrm{x}\left(\frac{\text{MaxP}{\mathrm{O}}_2-\text{lowP}{\mathrm{O}}_2}{\text{MaxP}{\mathrm{O}}_2-\text{MinP}{\mathrm{O}}_2}\right)$$E16

2.5 Calculation of central nervous system oxygen toxicity of lead

CNS oxygen toxicity is calculated thus,$\mathrm{P}{\mathrm{O}}_2$ remains constants [36]. Therefore

$$\text{CNSfraction}=\frac{\text{TimeatP}{\mathrm{O}}_2}{\text{TimelimitforP}{\mathrm{O}}_2}=\frac{\text{TimeatP}{\mathrm{O}}_2}{m\mathrm{P}{\mathrm{O}}_2+b}$$E17

where as m = slope of the time; b = intercept for the given range of $\mathrm{P}{\mathrm{O}}_2$.

2.6 Calculation of exposure dose of lead from contaminated environment

The equation for calculation of exposure dose of lead from contaminated environment [35, 36] is given as follow:

$$D=C\times \mathrm{IR}\times \mathrm{AF}\times \frac{\mathrm{EF}}{\mathrm{BW}},$$E18

whereas D = exposure dose; C = contaminated concentration; IR = intake rate of contaminated medium; AF = bioavailability factor; EF = exposure factor; BW = body weight, but the exposure factor is calculated as follow:

$$\mathrm{EF}=\frac{\left(F\times \mathrm{ED}\right)}{\mathrm{AT}},$$E19

whereas F = frequency of exposure (days/year); ED = Exposure duration (years); AT = averaging time (ED × 365 days/year).

2.7 Calculation of water dermal contact dose of lead

Dose of water concentration of lead that can penetrate dermal layer of skin [35, 36] is calculated as follow:

$$D=\frac{\left(C\times P\times \mathrm{BSA}\times \mathrm{ET}\times \mathrm{CF}\right)}{\mathrm{BW}},$$E20

whereas D = dose (mg/kg); C = contaminant concentration (mg/l); P = permeability coefficient (cm/h); BSA = exposed body surface area (m²); ET = exposure time; CF = conversion factor (1 l/1000 cm³); BW = body weight.

2.8 Calculation of soil ingestion exposure time of lead

Dose of lead ingested via soil can be calculated [35, 36] using the following formula:

$$D=\frac{\left(C\times \mathrm{IR}\times \mathrm{ET}\times \mathrm{CF}\right)}{\mathrm{BW}},$$E21

where D = exposure dose (mg/kg); C = contaminant concentration (mg/kg); IR = intake rate of contaminated soil (mg/day); EF = exposure factor; CF = conversion factor (kg/mg); BW = body weight.

2.9 Calculation of dose of lead particles present in food

Quantity of lead fragments present in the ingested food can be calculated as follow:

$$D=\sum _{n=i}^n\frac{C\times \mathrm{Cri}\times \mathrm{EF}}{\mathrm{BW}},$$E22

whereas D = exposure dose (mg/kg/day); C = contaminant concentration (mg/g); Cri = consumption rate of incriminating food (g/day); EF = exposure factor; BW = body weight (kg); n = total number of incriminating food group [30].

2.10 Toxicokinetic scaling of lead fragments

Steady state volume of distribution (Vss) of lead fragments is calculated thus:

$$\mathrm{Vss}=1.22W^{0.68}\text{where}W=\text{body weight of animal}$$E23

$$\mathrm{Cl}=0.91W^{0.5}\text{where}\mathrm{Cl}=\text{clearance}$$E24

Vss and Cl are based on plasma concentrations of blood and free lead. However, the Vss and Clu for plasma free concentration of lead are presented below.

$$\mathrm{Vss}=247W^{0.93}$$E25

$$\mathrm{Clu}=186W^{0.76}$$E26

Maximum tolerated dose (MTD) = $47.5e^{-0.51}$, e = 2.718 [37].

Relationship between respiratory minute volume and body weight is given by the equation.

$$V_m=0.518W^{0.802}$$E27

A value of 15.6 l/min has been calculated for 70-kg weighed human [38].

Apparent volume of distribution (Vd) related to absolute oral bioavailability is given as follow:

$$\frac{\mathrm{Vd}}{F}=\frac{\text{Dose}}{\mathrm{AUC}\times \mathrm{Kel}},$$E28

where F = bioavailability; AUC = area under curve; Kel = elimination constant.

$$\mathrm{Cl}/F\times \mathrm{MLP}=\beta \times W^a$$E29

where Cl = clearance; MLP = maximum lifespan potential; W = body weight; a = exponent.

$$\text{Dose}\left(\mathrm{mg}\right)=\text{Animal}\mathrm{AUC}\times \text{Scaled Human}\frac{\mathrm{Cl}}{F}$$E30

AUC = lowest value among species [39].

$$\text{Blood}/\text{Plasma concentration ratio}\left(\mathrm{Pp}\right)=1+\mathrm{H}\times \left(\mathrm{fu}–1\right)$$E31

whereas fu = fraction unbound in plasma; H = hematocrit (human, 0.44; rat,0.46; mouse, 0.45; rabbit, 0.36; monkey, 0.36).

$$\mathrm{Cl}=33.35\mathrm{ml}/minx\frac{{\left(a\right)}^{0.77}}{\mathrm{Rfu}}$$E32

$\mathrm{Rfu}$ = ratio of unbound fraction in plasma between rats and humans; $a$ = coefficient of surface area.

$$\text{Conc}.H=\text{Conc}.\mathit{Ax}\frac{{\text{Dose}}_H}{\mathrm{Dos}{\mathrm{e}}_A}x{\left(\frac{W_A}{W_H}\right)}^c$$E33

$${\text{Time}}_H={\text{Time}}_{A}x{\left(\frac{W_A}{W_H}\right)}^b$$E34

where b and c are exponents of simple allometry of $\mathrm{Cl}$ and ${\mathrm{Vd}}_{\mathrm{ss}}$, Percent (%) error between observed clinical concentration and predicted concentration of lead is calculated thus [40].

$$\%\text{error}=\frac{\text{Observed}-\text{Predicted}}{\text{Observed}}\times 100$$E35

The experiment for elimination half-life has allometric exponent of 0.25 [41].

Error involved in prediction of clearance is in most cases >30% [42]. Since inhaled lead particles can be distributed immediately the following equation can be used to calculate lead concentration in the blood.

$$C_t=C_o{e}^{-\mathit{kt}}$$E36

$C_t$= lead concentration at time t; $C_o$= theoretical lead concentration obtained if it had been inhaled at time t = 0; k = elimination rate constant [43, 44, 45].

$$\text{Ratio of urinary clearance to total body clearance}=\frac{{\mathrm{Cl}}_{\mathrm{u}}}{{\mathrm{Cl}}_{\mathrm{b}}}$$E37

$$\text{Ratio of plasma free lead to total body lead}=\frac{\mathit{PL}}{\mathit{Bl}}$$E38

2.11 Application of the formulas for calculation of toxicokinetic parameters of lead

The reported LD50 of lead in human is 450 mg/kg body weight, but LD1 = 450/50 = 9 mg/kg body weight

1. LT₅₀(d) $\frac{{\mathrm{LD}}_{50}}{D^P}=\frac{450}{9\frac{1}{3}}=\frac{450}{2.06}=\frac{218.4}{24\mathrm{h}}$ 9.1 days

   However, for an adult man weighing 60 kg, the ED50 for lead antidote is calculated as follows:

2. (ED50) $\frac{{\mathrm{LD}}_{50}}{3}\times W_a\times {10}^{-4}$

   (ED50) $\frac{450}{3}\times \mathrm{60,000}\times {10}^{-4}$

   (ED50) $150\times 6=900$

   ED₁$\frac{90}{50}=$18 mg/kg of antidote

3. Using dose rate (DR) = TC × Cl

   1080 = 0.037 × Cl

   Cl = $\frac{1080}{0.037}$= 29189.2 kg/h.

   $\frac{29189.2}{1000}\times \frac{1}{24}=$ 1.24 l/kg/h.

   At concentration of lead = 525 μg/dl.

   If LD₁ = 9 mg/kg.

   Dose of lead for 60 kg man = 60$\times$9 = 540 mg

4. (Vd=)$\frac{\text{Dose}}{\text{Concentration}}$

   Vd = $\frac{540}{525}$= 1.028 = 1.03 l/kg

5. AUC = $\frac{\text{Dose}}{\text{Clearance}}$

   $\frac{450}{1.24}$= 362.9 mg/kg/h

6. β = $\frac{\mathit{Clb}}{\mathit{Vd}}$= $\frac{1.24}{1.03}$= 1.20 h.

7. MRT = $\frac{\mathit{Vd}}{\mathit{Clb}}$ = $\frac{1.03}{1.24}$ = 0.83 h.

8. T½β = $\frac{0.693}{\beta }$ = $\frac{0.693}{1.20}$= 0.58 h.

3. Results

LD₅₀, ED₅₀, clearance, LD₁, and volume of distribution of lead bullet in adult human, Red grouse, Mallard, Partridge, pheasant, Woodpigeon and Woodcock are presented in Tables 1 and 2. However indices of lead poisoning at various concentrations are presented in Table 3. Concentrations of lead (≤ 5 − > 100 mg/dl) are injurious to haematological, neurological, cardiovascular, reproductive, renal, immunological and respiratory systems (Table 3). The calculated maximum tolerated dose of lead for 70 kg weighed man was 337.1 mg/kg. Bioavailability was 82.3%, whereas 62% of lead was cleared in the urine and 42% sequestrated in various tissues of 70 kg weighed man. Inhaled lead (525 μg/dl) would translate to 622 μg/dl over a period of 5 months with elimination rate of 1.2 h.

Weight (kg)	LD50 (mg/kg)	ED50 (mg/kg)	CL (kg/h)	LD (mg/kg)	Vd (L/kg)	AUC (mg/kg/h)	β (h)	MRT (h)	T½β
60	450	18	1.24	9.0	1.03	0.01875	1.16	0.86	0.6

Table 1.

Toxico-therapeutic and pharmacokinetic parameters of lead in adult human.

Keys: LD₅₀ = Median lethal dose; ED₅₀ = Effective dose fifty; Cl = Clearance; LD₁ = Lethal dose for 1 human; Vd = Volume of distribution; AUC = Area under curve; β = Elimination rate constant; MRT = Mean residence time; T½β = Elimination half-life.

Name of bird	Weight (kg)	LD50 (μg/kg)	ED50 (μg/kg)	Cl (kg/h)	LD (μg/kg)	Vd (I/kg)
Red grouse	0.600	384	7.6	5.49	8	0.07
Mallard	1.063	274	9.7	4.65	5	0.06
Partridge	0.488	267	4.3	4.85	5	006
Pheasant	1.163	360	14	3.68	7	0.10
Woodpigeon	0.453	120	1.8	6.16	2	0.02
Woodcock	0.300	126	1.3	1.23	3	0.10

Table 2.

Toxicokinetic parameters of lead bullet fragments in birds.

Keys: LD₅₀ = Median lethal dose, ED₅₀ = Effective dose fifty; Cl = Clearance; LD₁ = Lethal dose for 1 human; Vd = Volume of distribution.

Systemic	Range of toxic doses of plasma blood lead (μg/dl) and their toxicity signs
	≤5–14	15–24	25–34	35–44	45–54	55–100	>100
Neurological	Depression, loss of 4–7 IQ, damage D1 receptors, Reduction of glutamine synthetase, positioning range, bad mood, headache, memory loss, drowsiness, trembling, tingling of limbs, penetration of brain by lead, Kills birds and humans or birds and humans are severely poisoned, 10 μg/dl exceeds EU limit	Reduction of glutamine synthetase	—	Abnormal oligodendrocytes	Increased D1 in T = striatum, decreased D1 in nucleus accumbens	Acute encephalopathy neurological signs Acute death	Epilepsy death
Cardiovascular	Hypertension	—	—	—	—	—	Death
Haematological	Ratio of blood lead to erythrocytes lead for 6 month- old child (1/2–2/5), infants (7/10–7/11), adult (5/12) irrespectively. Increased delta amino levulinic acid, haemolysis, anaemia, curvilinear relationship between plasma lead and whole blood is within the range (2.13–3.97 μg/dl), chelation is not recommended at >45 μg/dl	—	—	—	—	—	—
Reproductive	Low birth weight, premature birth, anomaly, abortion	—	—	Decreased libido	—	—	—
Renal	—	—	Renal insufficiency	—	—	—	—
Immunological	—	—	—	—	Humoral and cellular immunity is affected	—	—
Respiratory	All the toxic doses can affect respiration

Table 3.

Toxicological indices of lead poisoning in human.

Note: All the reported threshold limits set for blood lead are wrong; − = System is affected by the dose.

3.1 Effects of lead on intelligence quotient

Lead in the brain affects intelligence quotient (IQ) of school age child. IQ measured at 3.8 years is affected by 5 μg/dl of lead concentration in the brain [44]. The established Benchmark limit doses of 1% extra risk are for intelligence quotient (IQ- BMDL₀₁) (12 μg/dl), systolic blood pressure, SBP-BMDL₀₁ (36 μg/dl) and chronic kidney disease, CKD- BMDL₀₁ (15 μg/dl) respectively. Increased blood lead of 1–6.7 μg/dl is associated with mortality from ischaemic heart disease and cardiovascular disease [45] This may likely affect 600,000–900,000 population of Sweden that consume game meat [46].

4. Discussion

4.1 Median lethal effects of lead bullet in human

The reported LD₅₀ (450 mg/kg) of lead in human indicates that lead is very dangerous to life. The concentration, 100 mgpb/m³ is unsafe [33]. However, blood concentration of lead (525 μg/dl) after 5 months caused epilepsy and death, but 10–60 μg/dl for 21 days caused neurological signs [47], as 6.25 μg/dl (0.3 μM) damaged and reduced dopamine uptake [48] and up to 97.2 μg/dl can cause acute death [49]. The concentration of pb (>50 μg/dl) at 60-day increased D₁ receptors in the striatum, but decreased D₁ receptors in the nucleus accumbens [50]. Lead (5.2–20.8 μg/dl) reduced glutamine synthetase activity [51], as 38.2 μg/dl caused abnormal oligodendrocytes [52]. Where blood lead level is below 45 μg/dl, chelation is not recommended [53]. The calculated 1080 mg of lead poisoning antidote agrees with the report indicating that, 500–1000 mg of penicillamine can be used for treatment of plumbism in man [54]. Accumulation of lead in astrocytes altered neurotransmitter release, receptor density, impaired development and function of oligodendrocytes, caused abnormal myelin function, neurotrophic factor expression, abnormal dendritic branching patterns, disruption of blood brain barrier, thyroid hormone transportation to brain, lowered IQ, impaired neuropsychological function and impaired academic achievement [55]. Interspecies comparison of lead LD₅₀ between rat and mouse using regression analysis, showed high correlation of 0.8 and 0.9, respectively. LD₅₀ variability showed 90% probability; 54% in one category, and 44% in adjacent category suggesting the possibility of an alternative method to conventional in-vivo acute oral toxicity test [56]. Blood lead concentration was significantly higher among Australian consumers of meat contaminated with lead-based ammunition (≤18.1 μg/dl daily), as compared to non-consumers of lead-based ammunition meat (≤7.4 μg/dl rarely) [57].

4.2 Clinical implication of delayed lead bullet elimination

The lead clearance of 1.2 l/kg/h agrees with the reported value of 1.18 l/kg/h [58]. Our findings agree with the report indicating that, other methods can complement the application of LD50 for discovery of potential toxicants. Such methods could aim at encouraging replacement, refinement and reduction [23]. Many scaling factors are determined experimentally, as all scaling factors have uncertainty associated with them. Mathematics of translation from one species to another requires multiple experimentally estimated scaling factors [59]. The developmental effects of lead occur at the age (>2 years). Low level of lead (>10 μg/dl) is associated with adverse effect in the developing child [60] and inversely proportional to neuropsychological development in the first 7 years of life [61]. The reported average blood lead concentration in child aged 1 and 5 years are 0.03 mg/l (3 μg/dl) and 0.11 mg/l (11 μg/dl), respectively. The elimination half-life in adult is 1 and 10 months in children [62]. Circulation of lead after absorption is 30 days. It diffuses into soft tissues including brain, and after 2 min diffuses into bone with blood half-life of 30 days and bone half-life of 20–30 years [63]. Also, half-life of lead in blood is 1–1.5 months and 25–30 years in bone respectively. However, Center for Disease Control (CDC) has defined poisoning level of lead equals or greater than 5 μg/dl [64]. Blood lead concentration of 9.1 μg/dl causes bad mood, headache, memory loss, daylight drowsiness, trembling, tingling of limb among others. Hence there is no known level of lead exposure considered safe [65]. This confirms that lead can enter brain because, it has a molecular weight of 207.2 g [66]. The total elimination half-life of lead is greater than 18 months. The primary route of elimination is urine [67], suggesting that urine is the most important sample in forensic toxicology of lead poisoning. Therefore, governments at various levels and law enforcement agents should curtail the use of lead bullet, so as to avoid damage to physical and intellectual capacity of affected humans [68]. Blood lead concentration (391 μg/dl) requires the use of calcium EDTA, but lead (49 μg/dl) can be neutralized using 600 mg succimer, three times daily for 14 days [69].

4.3 Relationship between brain damage caused by lead, low intelligence quotient and renal dysfunction

Acute encephalopathy occurs in children of blood lead concentration of ≥80 g/dl [70], therefore low threshold of 30–70 g/dl has been suggested [71]. Every increase of 10 g/dl in blood lead caused a loss of 4—7IQ points, hence it is difficult to identify a threshold for decrement in IQ [72, 73]. Renal dysfunction was caused by lead concentration of blood at <40–70 g/dl, and 10 g/dl was associated with 9% reduction in creatinine clearance [74, 75]. Nevertheless, 85 g/dl blood lead caused increased susceptibility to cold and >50 g/dl caused significant immunological changes [76], such as increase in lymphocytes, abnormal T-cell subsets and cellular immune function [77, 78]. Lead concentration of >62 g/dl inhibited conversion of vitamin 2 into 1.25—dihydroxy vitamin 2 which was reduced in children with severe renal insufficiency, at blood lead concentration of 33–55 g/dl [79]. Nevertheless, blood lead concentration of 14 g/dl caused low birth weight, premature birth and increased risk of developmental abnormalities [80]. Threshold level for hypohaemoglobinaemia is 50 g/dl in adult and ≤40 g/dl in children, respectively [38]. Regulatory bodies such as Control of Lead at Work (CLAW) and Scientific Committee on Occupational Exposure Limit (SCOEL) have recommended 30 g/dl for female workers of child-bearing capacity and 60 g/dl for men and others. However, European Lead Association has recommended 30 g/dl for men and 10 g/dl for women with reproductive capacity, respectively [81, 82]. Mean blood lead concentration of pigs that fed on venison was 2.29 g/dl which is 3.6 times higher than that of pig that did not feed on venison (0.63 g/dl). The venison-fed pig eliminated lead within 6 days of last ingestion [45]. Blood concentration of lead >5 g/dl is associated with high risk of spontaneous abortion in woman and the concentration > 40 g/dl is associated with decreased libido, sperm count and abnormal morphology of sperm cells [83, 84].

4.4 Effects of lead bullet on human and wildlife, diagnosis and therapeutic benefit of plumbism

About 0.4 million water birds of 33 species die every year from lead shot in European Union wetlands, and it cost European Union 105 million euros to replace 0.7 million captive-bred birds for killed ones. Restriction of water birds hunting cost greater than 100 million euros [85, 86, 87]. The milk plasma concentration ratio for lead is 50–100:1 after 24 h in mice, indicating a higher efficient concentration of lead milk of lactating mice as compared to that of non-lactating mice [88]. Linear biokinetic model of prehistories/preindustrial children’s blood of 0.06–0.12 g/dl was calculated for two lead intakes, which was lower than CDC threshold limit value of 10 g/dl. Toxicokinetics of bone lead that causes resorption with metabolic stimuli is of great concern for baby growth [89]. However, plumbism in birds cause death in 3 weeks [90]. The metabolizing enzymes that play very great role in the toxicokinetics of lead are d-amino-levulinate dehydratase d–ALA–d and porphobilinogen synthetase [91]. The activity of d-ALA-d, an allosteric enzyme with 28 thiol group is inhibited by lead [92], leading to accumulation of d-amino levulinic acid (d-ALA), whose concentration in urine of human and other animals is used to diagnose lead poisoning [93]. Sublethal dose of lead (0.2–0.5 ppm) has been reported with ≥0.5 ppm showing a significant decreased of d-ALA-d, causing brain damage which can be reversed by zinc. Haemosynthetase, ALA-dehyrotase and ferrochelatase have antidotal effects [94]. The latter enzyme binds iron to protoporphyrin, an indicator of blood lead, 40 ppm of the protoporphyrin is a clear proof of plumbism, over 500 pm affects neuromuscular activity with a consequent change in the motor functions [95, 96, 97]. Dimercaprol, a diethylene triamine pentaacetic acid, D-penicillamine, thiamine and calcium disodium ethylene diamine tetraacetate chelate lead for elimination. Highly hunted birds such as red grouse, mallard partridge, pheasant, woodpigeon, woodcock and deer could have lead fragments that exceeded threshold values of 100–10,000 ppb [98]. Many lead fragments in the carcasses of killed animals weighed >12–25 mg each, concentration of blood lead (5.9–18.1 g/dl) has been reported for consumers of game meats in Greenland and Switzerland and >4.1 millions shots have been reported against macropods, deer, red foxes, feral pigs, European rabbits and feral goats in Australia, annually [74], signifying that Australia may have high incidence of plumbism among wildlife. One million wild fowl estimated to have been killed by lead poisoning and ≥3 million sublethally poisoned [17], including Anseriformes, Falconiformes and Accipitriformes. They were severely poisoned with lead blood concentration of (>100 g/dl) [18]. Lethal lead concentration range (56–120 g/dl) has been reported for bearded vulture, Cape vulture and golden eagle [99, 100, 101, 102, 103] suggesting variation in susceptibility to lead poisoning among wild birds. However, concentration range of 10–47 g/dl was survived, and associated with different isotopes of lead [100, 104, 105]. Clinical threshold limit values for lead toxicosis of blood (>0.5 mg/kg), liver and kidney (>6 mg/kg) have been reported. Fragment sample size of 0.5 to >5 mm radiate from the wound channel [2]. Concentration of lead in the liver (28.9 ppm) for bald eagles and 19.4 ppm for golden eagles are sources for concern [106]. However reduced circulatory erythrocyte volume reduce uptake of lead by blood [107].

4.5 Exposure to environment lead

Exposure to lead arises from air and surfaces, and absorption occurs via ingestion, inhalation, percutaneous and transdermal routes. The first two are relevant to firing ranges, 0.1–5 μm lead can be inhaled, absorbed through the lung and 50% gets distributed to various parts of biological system. Absorption of ingested lead is >8–10% [108]. However, 12.5 g/m³ of airborne lead particles of ≤1 μm is of public health significance and the particles higher than >1 μm are deposited in the upper respiratory tract [109]. About 70% of workers exposed to lead (50 g/m³) had blood lead of 405 g/dl and 6% had >505 g/dl respectively [110]. However, 94% of samples of deer killed with bullets contained fragments of lead, which portend very high risk for scavengers [111]. Blood lead levels increased with time after injury up to 3 months with fragments and increasing age, which is 30% higher in the patient whose torsos are affected. Hence, blood level could be higher (11.8%) at 3 months and 2.6% at 12 months, respectively. Therefore, there is need for continued surveillance after gunshot [112]. The uncertainty in predictive power of algometric scales remains a concern in plumbism caused by lead bullet. Hence, the scales can only apply for exploratory research [113]. The uncertainty and availability, both in terms of inter-subject and application associated can be significant [114]. Natural isotopes of lead; 204 pb, 206 pb, 207 pb, and 208 pb constitute manufactured lead in various percentages, and their measurements in human and wildlife could be compared with their potential sources for environment risk assessment [2].

4.6 Lead causes cognition impairment

Lead causes impairment of neurodevelopment, cognition and behavioural development in the foetus and young child. The source of plumbism from wild birds killed by ammunition is significant among 5 million people in European Union countries [45]. The half-life of lead in blood and bone is 30 days and decades, respectively [115]. Unfortunately, maximum level (ML) for lead in game animals has not been set in the Codex Alimentarius General Standard for contaminants and toxicants [116], and by European Union [117], leading to concentration of 690 μg of lead fragments in wild-shot moose carcasses in Finland, Norway and Sweden [45]. Daily blood lead (12 μg/l) and lead intake (0.5 μg/kg) can affect intelligence quotient (IQ) of a child [118]. Bullet position may preclude surgical removal in order to avoid exacerbation of neurologic damage. The complication of the removal may be due to immediate migration of the fragment [119]. Retention of lead fragments in joint space is associated with increased risk of lead poisoning, and joint disruption leading to synovial metaplasia [120]. Good radiography and clinical findings are highly essential, for identification and complete surgical removal of bullet fragments, that may have high potential of distribution to various parts of biological system [121]. Injuries from bullet are most severe in brain and liver, causing temporary cavitation far from the actual bullet track. Bone and subcutaneous fats are highly resistant to bullet injury [122, 123]. Toxic leads widely used to hunt game animals and varmints are a source of environmental pollution. Lead and bismuth are highly frangible [26] and about 90% of the total burden of lead is found in bone and 5% in plasma, which pass through the cell membrane and cause toxic effect in brain, red blood cells, liver among others. In view of this, the knowledge of lead kinetics is of prime importance to a greater and better understanding of lead toxicity, as the risk of its adverse effects is very high [124]. Substitution of toxic lead bullets for non-toxic bullets such as steel, bismuth and tungsten may be possible alternative of curtailing lead poisoning from firearms, and phasing-out period of lead bullets could reduce cognition impairment [125]. Rationales used to remove lead from paints, gasoline and household items should be applied to lead-base ammunition globally, an issue that regularizes international intervention [126]. About 45% of surveyed states and provinces in the USA and Canada have non-toxic short regulation above federal water fowl regulations [127].

4.7 Forensic implication of lead poisoning

Regulatory toxicology consists of collection, processing and evaluation of epidemiological and experimental toxicological data, that end up in a decision for protection of health against toxic substances [128]. Clinicians treating plumbism from gunshot must be aware of potential incompatibilities between drugs and lead [129]. Mathematical modelling is becoming increasingly relevant for drug development [130]. One in every five injuries caused by firearm is fatal [131]. Ballistic weapon can move with a velocity of 915 m/s, whereas bullet can move up to 610 m/s [132]. Hence there is need for anatomical mapping of the target organs. Neck injuries caused by bullet are described according to the anatomical zones (1–111). Zone I extends from clavicle to cricothyroid membrane, zone II from cricothyroid membrane to the mandibular angle and zone III from mandibular angle to base of the skull [133]. But advanced neurotrauma research can improve the quality of life of patients that suffer from traumatic brain injuries [134].

4.8 Relationship between lead poisoning and brain over claim syndrome

Brain over claim syndrome (BOS) is about the relationship between neuroscience and criminal responsibility, distinguishing between internal and external critiques based on neuroscience [135]. The brain holds the key to mind and behaviour, which is useful to the law [136]. Criminal behaviour and violence are worldwide public health problem, since criminal behaviour has neurobiological basis with judicial implication. The affected brain pathways are genetic (foetal neural development), hormones and neurotransmitters (cortisol and testosterone), psychophysiology (e.g., low resting heart rate, low electroencephalography), brain imaging and neurology (reduced frontal lobe function) with attendant legal context (punishment, prediction and prevention) [137], respectively. Morality is part of human judgment, behaviour and mind. Frontal, temporal and cingulate cortex mediate between emotion and reasoning, amygdala, hippocampus and basal ganglia play vital role in morality. Therefore, genetic polymorphism, endocrine and environmental factors could modify the psychology of morality. Hence, abnormal behaviour can arise from structural brain stimulation [138], especially the anterior cingulate which causes empathy, orbital prefrontal cortex (causes regret), ventromedial prefrontal cortex (PFC) (ethical discussion), ventrolateral PFC (inhibits behaviour) and dorsolateral PFC is for reasoning [139]. Relationship between lower intelligence, crime and custodial outcomes has been established [140]. Also established is a very strong association between preschool lead and subsequent crime rate trends over decades in USA, Britain, Canada, France, Australia, Finland, Italy, West Germany and New Zealand [141]. Nevertheless, removal of lead from petrol since 1975 has led to the decline of crimes in the USA [142], because inhalation of lead content of petrol from 2 g to 0.5 g per gallon between 1975 and 1980 was highly reduced [143]. Atmospheric exposure could cause aggressive crime in children [144].

4.9 Arsenic can mar litigation caused by lead bullet

Since lead decreases arsenic bioavailability [145], it can reduce the chance of arsenic poisoning and vice versa. Hence there is need for knowledge of toxicokinetic changes of lead for better evaluation and interpretation of preclinical safety and clinical hazard [146]. Also the dose of bullets can be used to deduce lead concentration in the body, and the area under curve (AUC) base line should be compared with lead AUC response to eliminate uncertainty and variability [147]. This may be highly beneficial in biopsy [148] and autopsy of living or dead person tissues, affected by lead bullets. Under this condition, statistical moment theory instead of the compartmental model may be used to calculate mean residence time [149]. Elimination and absorption rate constants and volume of distribution may vary with age, gender, weight, clinical status, genetic variability and co-administration of lead with another drug or toxicant. Hence therapeutic monitoring of plumbism is very important [150]. Therefore, Pharmacokinetic/Pharmacodynamic (PK/PD) relationship is very important guide for identifying susceptibility and treatment potential of poisoning from lead bullets [151]. Lead distribution and clearance were 2.5 and 4 times higher in lactating and non-lactating animals respectively [152], and 10 μg/dl limit issued by Centre for Disease Control (CDC) is relatively high [89]. Relationship between blood lead concentration and lead intake is non-linear, [153], but at low concentration the kinetic is linear and at 60 μg/dl in erythrocytes which corresponds to blood level of 25 μg/dl, the kinetic becomes non-linear [153], indicating that lead kinetics is Michaelis–Menten. Accessibility pool parameters (Vd, Clb, β, MRT) can be used compartmentally and non-compartmentally [154]. Gunshot residue and shooting distance could be used to determine contact and near contact wound in putrefied or charred bodies [155, 156]. In conventional rifles and gun bullet projection, the projectile requires high altitude and glides with subsonic speed and a good ratio lift/drag. This is applicable with bullets having projectiles of 2–5 times [157]. Threshold level (25 μg/m³) in air is dangerous to health [158]. Velocity of 150–170 fps is required by bullet to penetrate skin [121]. But bones change the speed of bullets greatly, by changing their course and slowing them down [159]. Origin of bullet can be determined using the major/minor axis which determines the impact angle, although modern laser methods provide better results. Semi-automatic gun and bullet system (7.62 mm) is more effective [160]. Toxicosis from lead ammunition in predatory and scavenging birds have been reported in Argentina, Chile, Swiss Alps, Namibia, South Africa, Botswana, Israel, French Pyrenees, Spain, Iberian Peninsula, Sweden, USA, Granada, UK, Japan, Poland, Finland, Ireland, Portugal, Italy and Canada [17]. International suspension limit of blood lead is 10–70 μg/dl in females and 20–70 μg/dl in males, respectively. However, 5–80 μg/dl and above can cause hypertension, kidney dysfunction, neurocognitive deficits, colic, gout, sperm abnormalities, anaemia, peripheral neuropathy and encephalopathy (Table 3). Hence, there is narrow margin of safety between blood lead suspension and subclinical effects [161]. Therefore, the 1899 Hague Declaration is about abstinence from the use of bullets; an effective treaty applied for over 100 years, but may likely face modern challenges [162]. At short range of shooting bullets, the amount of energy absorbed is increased which is proportional to striking velocity [163]. Jauhari experimented on bullet crochet, defined as deflection of a bullet from its course by firing low velocity handgun cartridges on targets [164].

4.10 The importance of ban on the use of lead ammunition

Gun at 0.436^o hit the centre of a target 1 m off the ground and 1 km away with an air temperature of 27.2${}^{°}\mathrm{C}$. Hence hollow bullet has more drag and hit the centre of the target at a different angle [165]. Bullets from high velocity modern rifles produce lead fragments, and lead shot (6.1 μg/g) could kill birds, when the meats from such birds were consumed, blood concentration (15–128 mg/l) was detected in the blood of human consumers, as well as 24–50 and 9–180 μg/l were detected in mothers and newborns, respectively [166]. Low blood lead concentration (LBLC) is associated with decreased intelligent quotient (IQ) in children [167]. Hence the use of lead ammunition has been banned in the USA, Canada, Mexico, Oceania, Austria, Japan, South Africa, Bennin, Guinea-Bisau, Sudan, Denmark, Norway, Netherland, Finland and Sweden on some categories and species of birds [168]. But 3 mm lead pellet and 3.4 mm steel pellet each weighed 250 mg. Increasing diameter by 0.5 mm compensates for steel’s lower density [169]. Over 90% of ammunitions manufactured in the world contain lead [170]. Pattern density is the primary factor that influences types of ammunition performance [171]. A high lead load (45–52 mg) per 100 g wet weight and the embedded pellet per body mass (1.21/100 g) in woodcock portend high health risk to consumers [172]. Exit wound caused by bullets are very rare at close range [173]. But sabot bullet made from lead alloy and plastic tends to be the most dangerous in soft and medium density materials. However, the effect of lead ammunition in non-quarry species of animals is unknown [174]. All these are very specific in forensic toxicology that can be relied upon in justice delivery, and provision of solution for criminal cases [175]. Hence forensic scientists play critical role in the determination of various risk factors in litigations related to the medical and legal cases [176], which are related to complete penetration of bullet into brain, that may cause death or incomplete penetration that may cause death or survival [177]. Hence, there is need to reduce level of lead exposure in overall population with a view to having healthy and safe environment [161], >0.1 mg/kg concentration has been reported for bovine, sheep, pigs and poultry, a dose that exceeds European Union Maximum level of 100 ppb [178], fulfilling the expectations of the community and justice [179], bearing in mind that wildlife could serve as source of potential lead bullet poisoning [3, 5, 173, 180, 181, 182, 183, 184, 185]. Nevertheless renal damage could be confirmed legally by applying the reported investigation protocol [186]. European Chemical Agency (ECHA) has proposed a restriction on lead use in sports shooting, hunting and fishing. The restriction could reduce lead emissions by 630,000 tonnes over 20 years, representing 72% lead reduction [187].

5. Conclusion

Lethality of lead bullet is dependent on the body weight, median lethal time, doses of lead fragments released per unit time, concentration of lead in the brain, volume of distribution, elimination rate constant, mean residence time and elimination half-life of the lead fragments, which could be detected within 100 days after gunshots. Brain damage caused by lead fragments appears to be associated with low level of intelligence and brain over claim syndrome. Hence, forensic pathologists, police and the concerned judges should take note of prevailing conditions during gunshot attack.