**2.2 Chemicals and reagents**

194 Recent Advances in Plasticizers

The project aimed at determining the potential health risks that may be associated with using river water and treated effluent from wastewater treatment plants in Cape Town. Since phenols and phthalate esters were placed on the United State Environmental Protection Agency list as priority pollutants, both phenols and phthalate esters congeners were analyzed in water samples. However, emphasis is more on the phthalate esters congeners. The derivatization of the phenolic congeners did not in any way affect the intensity of the phthalate esters congeners included in this study (Olujimi et al., 2011b).

Influents and effluents from six wastewater treatment plants namely; Athlone, Bellville (which consist of the Old and New plants), Kraaifontein, Potsdam, Stellenbosch and Zandvliet) were investigated for the occurrence of seventeen organic compounds (eleven priority phenols and six phthalate esters). Five of these wastewater treatment plants (WWTPs) were located in the City of Cape Town, while one is located in Stellenbosch. Rivers associated with each treatment plant are: Athlone - Vygekraal River; Bellville - Kuils River; Kraaifontein -Mosselbank River; Potsdam - Diep River; Zandvliet - Kuils River and Stellenbosch -Veldwachters River. Five of the WWTPs and associated rivers investigated are presented in Figure 1. Samples were taken at the point of discharge, as well as upstream and downstream from point of discharge (about 1-2km) to evaluate the possible impact of effluent on organic compounds load on the aquatic environment. The geographical location, population equivalent and treatment processes of the investigated treatment plants are

Industrial

Industrial

Industrial

Industrial

Industrial

Abbreviations: S = Screening; G = Grit removal; Sed = Sedimentation; AS = Activated Sludge; EAAS = Extended Aeration Activated Sludge; N = Nitrogen; BNR = Biological nutrient removal; Chl = Chlorination; UVdis = UV disinfection; AD = Anaerobic digestion; FB = Filter bed; N/K = Not known;

E18.70442 o 133,000 Domestic S + G + Sed + AS (N) +

equivalent Source Treament Process River

S + G + Sed + AS (BNR) + Sed + Chl + AD + Dew

S + G + EAAS (N) +

Sed + Chl + AD + Dew

S + G + Sed + AS (N) +

S + G + Sed + FB + AS (BNR) + Sed + Chl + AD + Dew

S + G + Sed + AS (N) +

Sed + UVdis + Dew Kuils River 1

Sed + Chl + AD + Dew Diep River

Sed + Chl + AD + Dew Kuils River 2

Vygekraal River

Mosselbank River

Veldwatchers River

**2. Materials and method** 

**2.1 Study areas** 

presented in Table 1.

WWTPID Geographical

A S33.5709o

B S33.5923o

C S33.82539o

D S33.5070 o

E S33.94345 o

F S34.0312 o

Location of plant

People

E18.3048 o 900,000 Domestic

E18.4332o 591,000 Domestic

E18.3108 o 385,000 Domestic

E18.82492 o N/K Domestic

E18.4259 o 400,000 Domestic

Table 1. Description of the six wastewater treatment plants investigated.

WWTP ID = Wastewater treatment plant identification.

Analytical grade phenol (PH) 99.9 %, 2-nitrophenol (2-NP) 99 %, 4-nitrophenol (4-NP) 99 %, 2,4-dinitrophenol (2,4-DNP) 99.7 %, 4,6-dinitro-2-methylphenol (DNMP) 98 %, 2,4 dimethylphenol (2,4-DMP) 98 %, 2-chlorophenol (2-CP) 99.8 %, 4-chlorophenol (4-CP) 99 %,

Health Risk Assessment of Plasticizer in Wastewater Effluents and Receiving Freshwater Systems 197

where a is the slope and Sb is the standard deviation of the y-intercept (De Sousa et al.,

C18-E cartridges (strata, 500 mg/ 6 ml) from Separations Limited were used for the extraction of phenols and phthalates from water samples based on recoveries obtained for phenols using HPLC (Olujimi et al., 2011a). Prior to the sample processing, the cartridges were fitted onto a vacuum manifold (Supelco) connected to pump. The cartridges were conditioned with 5 ml of n-hexane:acetone (50:50, v/v), followed sequentially by 5 ml of methanol and 10 ml of Milli-Q purified water (purified by Milli-Q System, Millipore, Bedfore, MA, USA). Prior to extraction of each 500 ml, water samples were filtered on vacuum using a 0.22 µm filter to remove suspended particulate matter that might block the SPE cartridges. Hydrochloric acid (37 %) was used to adjust the pH of the water sample to pH ≤ 3 before passing it through the conditioned cartridge. The cartridge were then rinsed with 5 ml of Milli-Q water and left on the vacuum manifold for 30 min to dry (-70 Kpa). The retained analytes of interest were eluted with 3.5 mL of methanol followed by 3.5 ml of nhexane:acetone (50:50, v/v) into 10 ml glass vials. This was blown to dryness on hot plate at 70 oC under gentle flow of nitrogen gas. The retained analytes were then derivatized

1 ml of standard mixture/ extracted analytes

Blow to dryness on hot plate at 70 oC under gentle flow of nitrogen

50 µl each of Acetonitrile and MTBSTFA

Vortex mix (90 s)

Derivatized at 90 oC for 20mins in GC oven cool to room temperature

Analyze on GC-MS

**2.5 Solid phase extraction (SPE) for water samples** 

according to the procedure described in section 2.3 (Figure 2).

Fig. 2. Derivatization procedure for silylation.

2003).

2,4-dichlorophenol (2,4-DCP) 100 %, 4-chloro-3-methylphenol (4-C-3MP) 99 %, pentachlorophenol (PCP) 99.6 %, dimethyl phthalate (DMP), diethyl phthalate (DEP) 99 %, benzybutyl phthalate (BBP) 98 %, dioctyl phthalate (DOP) 99 %, diethylhexylphthalate (DEHP) 99 %, dibutyl phthalate (DBP) 99 % were purchased from Superlco (Bellefonte, PA USA). Helium (99.999 %) is supplied by Afrox gas, South Africa, Potassium Carbonate, acetic anhydride were supplied by Separations (South Africa). The solvents (methanol, nhexane, acetone and acetonitrile) were of analytical grade from Sigma Aldrich and were further purified by distillation. Separate stock solutions (1000 mgl-1) of individual congeners were prepared in methanol. A working mixture containing each compound at 10 mgl-1 was also prepared and stored at 4°C in the dark. Milli-Q water used was from apparatus Millipore (Bedford, MA, USA).

#### **2.3 Derivatization procedure**

Some EDCs such as phenols with hydroxyl group within the molecule have to be derivatized with N-Methyl-N- (Tert-Butyldimethylsilyl) trifluoroacetamide (MTBSTFA), which results in the formation of tert-butyltrimethylsilyl (TBMS) derivatives. The high polarity of the phenolic compounds gives rise to poor chromatographic performance and as a consequence derivatization was carried out. The phenol-silylate is more volatile and affords better detection limits when using gas chromatography (GC). The standard mixture was derivatized according to Olujimi *et al.* (2011b). Briefly, 1 ml of the standard mixture (phenols and phthalate esters) was measured into sample vial and blown to dryness under gentle flow of nitrogen gas. The dried standard mixture was reconstituted with 50 µl acetonitrile and 50 µl silylating reagent N-Methyl-N- (Tert-Butyldimethylsilyl) trifluoroacetamide (MTBSTFA) and mixed in a vortex for 90 s. The solution was derivatized at 90 oC for 20 min in a GC oven. The sample was cooled down to room temperature and 1 µl was injected into the GC-MS for analysis. The stepwise derivatization procedure is shown in Figure 2. The GC-MS parameters used for the analysis is presented in Table 2 after initial optimization studies.

#### **2.4 Determination of limits of detection and quantification GC-MS**

Lower concentration standards were prepared through serial dilution of individual standard of phenols and phthalate esters as well as the mixture standards. 1 µl aliquots of each of the standard was injected into GC, to determine the lowest concentration. Different procedures for the determination of limits of detections (LODs) and limit of quantifications (LOQs) are reported in the literature. These limits can be experimentally estimated from the injection of serially diluted standard solutions or extracts of fortified water samples until the signal-to-noise ratio (s/n) ratio reaches a value of three. LOD was estimated as three times the noise level of the baseline in the chromatogram, while the limit of quantification (LOQ) is set at three times the LOD. For this study, LOD and LOQ were calculated using the equations below:

$$\text{LOD} = 3.3 \times \text{Sb/a} \tag{1}$$

and

$$\text{LOQ} = 10 \times \text{Sb/a} \tag{2}$$

where a is the slope and Sb is the standard deviation of the y-intercept (De Sousa et al., 2003).

#### **2.5 Solid phase extraction (SPE) for water samples**

196 Recent Advances in Plasticizers

2,4-dichlorophenol (2,4-DCP) 100 %, 4-chloro-3-methylphenol (4-C-3MP) 99 %, pentachlorophenol (PCP) 99.6 %, dimethyl phthalate (DMP), diethyl phthalate (DEP) 99 %, benzybutyl phthalate (BBP) 98 %, dioctyl phthalate (DOP) 99 %, diethylhexylphthalate (DEHP) 99 %, dibutyl phthalate (DBP) 99 % were purchased from Superlco (Bellefonte, PA USA). Helium (99.999 %) is supplied by Afrox gas, South Africa, Potassium Carbonate, acetic anhydride were supplied by Separations (South Africa). The solvents (methanol, nhexane, acetone and acetonitrile) were of analytical grade from Sigma Aldrich and were further purified by distillation. Separate stock solutions (1000 mgl-1) of individual congeners were prepared in methanol. A working mixture containing each compound at 10 mgl-1 was also prepared and stored at 4°C in the dark. Milli-Q water used was from apparatus

Some EDCs such as phenols with hydroxyl group within the molecule have to be derivatized with N-Methyl-N- (Tert-Butyldimethylsilyl) trifluoroacetamide (MTBSTFA), which results in the formation of tert-butyltrimethylsilyl (TBMS) derivatives. The high polarity of the phenolic compounds gives rise to poor chromatographic performance and as a consequence derivatization was carried out. The phenol-silylate is more volatile and affords better detection limits when using gas chromatography (GC). The standard mixture was derivatized according to Olujimi *et al.* (2011b). Briefly, 1 ml of the standard mixture (phenols and phthalate esters) was measured into sample vial and blown to dryness under gentle flow of nitrogen gas. The dried standard mixture was reconstituted with 50 µl acetonitrile and 50 µl silylating reagent N-Methyl-N- (Tert-Butyldimethylsilyl) trifluoroacetamide (MTBSTFA) and mixed in a vortex for 90 s. The solution was derivatized at 90 oC for 20 min in a GC oven. The sample was cooled down to room temperature and 1 µl was injected into the GC-MS for analysis. The stepwise derivatization procedure is shown in Figure 2. The GC-MS parameters used for the analysis is

Lower concentration standards were prepared through serial dilution of individual standard of phenols and phthalate esters as well as the mixture standards. 1 µl aliquots of each of the standard was injected into GC, to determine the lowest concentration. Different procedures for the determination of limits of detections (LODs) and limit of quantifications (LOQs) are reported in the literature. These limits can be experimentally estimated from the injection of serially diluted standard solutions or extracts of fortified water samples until the signal-to-noise ratio (s/n) ratio reaches a value of three. LOD was estimated as three times the noise level of the baseline in the chromatogram, while the limit of quantification (LOQ) is set at three times the LOD. For this study, LOD and LOQ were calculated using the

LOD = 3.3 × Sb/a (1)

LOQ = 10 × Sb/a (2)

Millipore (Bedford, MA, USA).

**2.3 Derivatization procedure** 

equations below:

and

presented in Table 2 after initial optimization studies.

**2.4 Determination of limits of detection and quantification GC-MS** 

C18-E cartridges (strata, 500 mg/ 6 ml) from Separations Limited were used for the extraction of phenols and phthalates from water samples based on recoveries obtained for phenols using HPLC (Olujimi et al., 2011a). Prior to the sample processing, the cartridges were fitted onto a vacuum manifold (Supelco) connected to pump. The cartridges were conditioned with 5 ml of n-hexane:acetone (50:50, v/v), followed sequentially by 5 ml of methanol and 10 ml of Milli-Q purified water (purified by Milli-Q System, Millipore, Bedfore, MA, USA). Prior to extraction of each 500 ml, water samples were filtered on vacuum using a 0.22 µm filter to remove suspended particulate matter that might block the SPE cartridges. Hydrochloric acid (37 %) was used to adjust the pH of the water sample to pH ≤ 3 before passing it through the conditioned cartridge. The cartridge were then rinsed with 5 ml of Milli-Q water and left on the vacuum manifold for 30 min to dry (-70 Kpa). The retained analytes of interest were eluted with 3.5 mL of methanol followed by 3.5 ml of nhexane:acetone (50:50, v/v) into 10 ml glass vials. This was blown to dryness on hot plate at 70 oC under gentle flow of nitrogen gas. The retained analytes were then derivatized according to the procedure described in section 2.3 (Figure 2).

Fig. 2. Derivatization procedure for silylation.

Health Risk Assessment of Plasticizer in Wastewater Effluents and Receiving Freshwater Systems 199

Human exposure to toxic effects are expressed in terms of average daily dose (ADD) which is the amount of substance taken into the body on daily basis during the exposure period

For risk of carcinogens for exposures that last less than lifetime, the dose is adjusted using

 *LADD* = *ADD* x (*ED/Lft*) (6)

For agents that cause non-cancer effects, a Hazard Quotient (H.Q) was calculated, comparing the expected exposure to the agent to an exposure that is assumed not to be

For oral or dermal exposures, the Average Daily Dose (ADD) was compare to a Reference

For chemicals that may cause cancer if ingested, risk is calculated as a function of Oral Slope Factor and can was calculated by using the formula:Risk = Oral Slope Factor \* Lifetime

The formulae used to generate the contaminant exposure concentration in water were those described by the US-EPA (1990) for water to fish; vegetables; dairy and meat concentrations. The formula for the consumption of recreationally caught fish and shellfish-water to edible

Any Hazard Quotient less than 1 is considered to be safe for a lifetime exposure.

**2.8.3 Cross-media transfer equations used to generate exposure estimates** 

���� � ��� ∗ ����

H. Q. = Average Daily Dose / Reference Dose (7)

� � ∗ ����� (9)

BCF =[0.79 ∗ log �Kow)] – 0.40 (10)

*ADD* = average daily dose *Cmedium* = concentration in the contaminated water

*ADD = (Cmedium x IR x ED x Fc) /BW x AT* (mg/kgd) (5)

calculated

where:

*IR* = daily intake rate *ED* = exposure duration *Fc*, = the fraction contaminated

*BW* = body weight

where: *Lft* is lifetime

the formula:

Dose (RfD):

**2.8.2 Cancer risk** 

Average Daily Dose (8)

tissue is presented in equations below:

*AT* = lifetime averaging time

associated with toxic effects.

**2.8.1 Non-cancer toxic effects (Hazard Quotient)** 


Table 2. Gas Chromatography and Mass Spectrometer Parameters.
