Case-study and risk management of dioxins and ...

Environ Sci Pollut Res DOI 10.1007/s11356-015-4146-y

RESEARCH ARTICLE

Case-study and risk management of dioxins and PCBs bovine milk contaminations in a high industrialized area in Northern Italy Luigi Bertocchi & Sergio Ghidini & Giorgio Fedrizzi & Valentina Lorenzi

Received: 27 August 2014 / Accepted: 19 January 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Milk supplied to a dairy plant in Brescia City (Northern Italy) was found to be contaminated by dioxin like PCBs at levels above the European (EU) action limit (2 pg WHO-TEQ/g fat). As a consequence, 14 dairy farms were sampled individually, in order to identify and possibly eliminate the source of contamination. All the farms were located in Brescia or just nearby, an area that is characterized by a strong industrialization. Four out of the 14 farms showed contamination levels above the legal maximum limit set by European Commission at 5.5 pg WHO-TEQ/g fat for the sum of dioxins and DL-PCBs. Concentrations of 8.16, 6.83, 5.71 and 5.65 pg WHO-TEQ/g fat were detected. In the three most polluted farms, cow ration was substituted with feed coming from uncontaminated areas and the time needed to reduce milk pollution was evaluated. In all the three farms, contamination levels dropped below the EU legal limit after only 1 month from the removal of the pollution source. In each sampled farm, DLPCBs were the major contributors to the total WHO-TEQ level, with percentages up to 87 % in the most contaminated one. PCB 126 WHO-TEQ value explained by itself large part of this contamination, and its decrease was fundamental for the reduction of milk contamination levels. This study

Responsible editor: Roland Kallenborn L. Bertocchi : V. Lorenzi Primary Production Department, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna BBruno Ubertini^, Via Bianchi, 9, 25124 Brescia, Italy S. Ghidini (*) Department of Food Science, Parma University, Via del Taglio, 10, 43126 Parma, Italy e-mail: [email protected] G. Fedrizzi Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Via Fiorini, 5, 40127 Bologna, Italy

provides an example of an on-field successful emergency intervention that succeeded in decontamination of dairy cows, allowing a fast restart of their production activity. Keywords PCDD/Fs . PCBs . Cow . Milk . Italy . Brescia

Introduction Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are included in the BDirty Dozen^ of the Stockholm Convention (UNEP 2001). They are toxic chemical compounds of great public health concern. They persist in the environment, accumulate in the food chain and they have been associated with serious human health effects (EFSA 2012). In 2013, the International Agency for Research on Cancer (IARC) has classified dioxin-like PCBs (DL-PCBs) and non-dioxin-like PCBs (NDL-PCBs) as carcinogenic to humans (Lauby-Secretan et al. 2013), together with 2,3,4,7, 8-pentachlorodibenzofuran and 2,3,7,8-tetrachlorodibenzo-pdioxin, already included in group 1 (IARC 2012). Dioxins (PCDDs and PCDFs) and PCBs are lipophilic chemicals that bioaccumulate and biomagnify in the food chain, ending up in the fatty tissue of animals and humans. Dietary intake is responsible of almost 90 % of the total human exposure. Foods of animal origin, especially fish, dairy products and meat, are the main contributors to human body burden (EFSA 2012). In particular, milk and dairy products are considered the major sources (27.5–49.6 %) of PCBs and dioxins exposure in most infant and toddler population groups across Europe. Also in Italy, milk and dairy products are the main culprit for infants’ and toddlers’ exposure to PCDD/Fs

Environ Sci Pollut Res

and DL-PCBs (31.30–36.40 %), and the second food category, after fish and fish products, contributing most to the total exposure of adolescents (18.28 %), adults (14.68 %), elderlies (14.96 %) and very elderlies (23.00 %) (EFSA 2012). Moreover, referring to the Tolerable Weekly Intake (TWI) of 14 pg WHO-TEQ/kg body weight, established by the Scientific Committee on Food in 2001 (SCF 2001), cow milk is one of the foods most frequently identified as the main contributors to the TWI in Europe (EFSA 2012). In Northern Italy, bovine milk contamination by dioxins and PCBs is an important and current issue, particularly in Lombardy Region, a high-risk area where industrial and agricultural activities still coexist. In this region, around 31,092 t of PCBs were produced from 1958 to 1983 by the only Italian PCB-producing plant (Caffaro company), located in Brescia (Breivik et al. 2002; Turrio-Baldassari et al. 2008). This is a highly industrialized city, where there are also many metallurgical plants and foundry activities for metallic scraps recycling (Lerda et al. 1996; Abballe et al. 2013). Almost 40 % of the metal scraps produced in Italy are reprocessed in Brescia Province. This activity can generate air emissions containing dioxins, PCBs and other pollutants (Lerda et al. 1996; Buekens et al. 2001; Corsaro et al. 2012). Moreover, in Brescia Province, industrial activities coexist with about 2000 dairy farms, with a total production of around 10 million tons of bovine milk per year, the highest among Lombardy Provinces (Confagricoltura 2010; BDN 2013). Due to the strong industrialization, PCDDs, PCDFs and PCBs widely contaminate the environment of Brescia. Several studies conducted in this area reported high level of these substances in soil, ambient air, forage, food and humans (CTS 2003; Donato et al. 2006; Turrio-Baldassarri et al. 2007, 2008, 2009, Colombo et al. 2013; Zani et al. 2013). In particular, high levels of PCB contamination were found in soil, food and people living in the area close to Caffaro factory (CTS 2003; Turrio-Baldassarri et al. 2007, 2009). Chemicals produced by Caffaro polluted water, irrigation channels and, as a consequence, the soil of a nearby agricultural area, thus entering the food chain (La Rocca and Mantovani 2006). Donato et al. (2006) demonstrated a strong association between high levels of PCBs in Brescia citizens and consumption of food produced in the area immediately south of the plant. Due to the strong PCB contamination, this area was declared Bsite of national interest for remediation (SIN)^ by the Italian authorities and all the farming activities in it were prohibited (Donato et al. 2006; Abballe et al. 2013). Following the finding of PCBs and dioxins contamination in Brescia soils also in areas outside the SIN boundary (Donato et al. 2006), where many dairy farms are located, the attention was drawn to the risk of chemical contamination of food of animal origin produced in these areas. However, to date, there are very few published data regarding the contamination of food of animal origin produced outside the SIN

(Donato et al. 2006). In the present study, an investigation was undertaken to determine the level of contamination in milk of dairy farms located in Brescia City or close to the town border. At the end of 2007, the milk collected by four tank trucks of a Brescia dairy plant was analysed for PCB and dioxin contamination. Milk delivered to the dairy plant was produced by farms located in Brescia City or nearby. Two out of the four milk collection tanks showed contamination values over the action level of 2.0 pg WHO-TEQ/g fat for DL-PCBs (Bertocchi et al. 2008), set by the European Commission Recommendation 2006/88/EC (Official Journal of the European Union 2006a). As established by the EU Recommendation, investigations were done in order to identify and eliminate the source of contamination, and the presence of NDL-PCBs was also checked. In the present study, contamination levels of the milk produced by the suppliers of the dairy plant are presented and analysed in order to assess the degree of diffusion of PCBs and PCDD/Fs pollution in Brescia, to compare the contamination profile with those found in the SIN area (TurrioBaldassarri et al. 2009) and to evaluate the safety of the food produced in this high-risk municipality. In addition, an example of an on-field successful emergency intervention is provided, including the time required to reduce contamination values under the European legal limits, once the source of pollution has been removed. The obtained decontamination results offer a useful tool for risk managers and decision makers in the case of milk contamination. Actually, a rapid compliance is fundamental to guarantee food safety, to reduce consumer exposure and to avoid economic collapse of the dairy farms involved.

Materials and methods Sampling In December 2007, the 14 farms, represented in the two tank trucks found over the action level (Bertocchi et al. 2008), were sampled individually in order to identify and eliminate the source of contamination. Eight out of the 14 investigated farms were located in Brescia City, 3 were situated 5–8 km west of the city, 1 near the south-west border and 2 close to the south border (Fig. 1). In the area covered by the study, there were also several steel plants, landfills and one waste-toenergy plant, in addition to Caffaro factory. The size of the dairy herds ranged from 22 to 444 Holstein Friesian cows. The produced milk was sold to a Brescia dairy plant for direct selling or transformation. Cows diet was made of 90–100 % local products, and the total mixed ration consisted of 20–24 kg of corn silage, 5–8 kg of hays and 10–12 kg of concentrates.

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Fig. 1 Sampling area and location of the 14 farms. The SIN area where Caffaro plant (black dot) is located is highlighted in grey

From the bulk tanks of the 14 dairy farms, milk samples (1 L each) were collected in chemically clean glass bottles, stored at −20 °C and shipped on dry ice to the laboratory. The sampling was carried out in agreement with the European Commission Regulation 1883/2006/EC (Official Journal of the European Union 2006b). Milk analysis Milk analysis was carried out using high-resolution gas chromatography coupled with high-resolution mass spectrometry (HRGC-HRMS). The laboratory performing the analysis was certified under UNI CEI EN ISO/IEC 17025. For determination of the 17 2,3,7,8-substituted PCDD/Fs, the 12 DL-PCBs congeners (PCB 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169 and 189) and six NDL-PCBs (PCB indicators, PCBs 28, 52, 101, 138, 153 and 180), the US EPA Method 1613/B 1994 and US EPA Method 1668/A 2003 were applied. Milk samples were homogenized, freeze-dried and lyophilized (Freeze Dryer Martin Christ, Germany). Before fat extraction, samples were mixed with silica and spiked with two mixtures containing 15 13C12-labelled 2,3,7,8-substituted PCDD/F internal standards (Cambridge Isotope Laboratories, USA) and 14 13C12-labelled PCB internal standards (Cambridge Isotope Laboratories, USA). Then, the samples were transferred into the accelerated solvent extraction (ASE) cells (Dionex, USA). The extraction was carried out with two cycles at 135 °C, using toluene as extraction solvent. The solvent was evaporated with rotary evaporation, and the extract was dried overnight in the oven. The fat content was determined gravimetrically. The lipid fraction was dissolved with 5 mL of hexane/ dichloromethane solution (1/1, v/v) and spiked with a clean-

up standard solution mixture containing three 13C12-labelled PCBs (Cambridge Isotope Laboratories, USA). The following acid purification was performed using a multi-layer glass column packed from bottom to top with respectively: granular anhydrous sodium sulphate, silica gel, Extrelut powder soaked with sulphuric acid, silica gel and granular anhydrous sodium sulphate. The column was eluted with n-hexane. The solution was concentrated in a TurboVap evaporator (Zymark Corp., USA), and an automated multi-column cleanup was performed on the Power-Prep system (Fluid Management Systems, USA). This system was controlled by a personal computer and uses disposable pre-packed multi-layer silica columns, basic alumina and carbon columns to separate analytes of interest from matrix interferences. At the end of the Power-Prep process, two fractions containing PCDD/Fs and PCBs congeners were collected separately in two different evaporation tubes. PCDD/Fs fraction was eluted with toluene, while PCBs fraction was eluted with hexane/dichloromethane solution (92/8, v/v). The eluates were taken to complete dryness in a TurboVap evaporator and in a vacuum concentrator (Genevac, USA). Then, the PCDD/F and PCBs (DL and NDL) fractions were dissolved respectively with a solution containing two 13C12-labelled 2,3,7,8-substituted PCDD/F injection standards (Cambridge Isotope Laboratories, USA) and 5 13C12-labelled PCB injection standards (Cambridge Isotope Laboratories, USA). PCDD/F and DL-PCB congeners were identified and quantified using HRGC (Agilent Technologies, USA) and HRMS (Waters Micromass Autospec Ultima, USA), equipped with DB-5MS 60 m×0.25 mm×0.25 μm capillary column (Agilent Technologies, USA) and helium as carrier gas. The mass spectrometer operated in the selected ion monitoring (SIM) mode at a resolution over 10,000. The concentration of congeners was quantified using isotope dilution method.

Quality assurance and quality control Method performance and accuracy were assessed continually taking part to inter-laboratory studies, in particular, in 2007 the laboratory participated to FAPAS proficiency tests obtaining a z-score=−0.1 for PCDD/Fs and a z-score=−0.3 for DL-PCBs analysis. Method specificity and sensitivity were secured using highresolution equipment (≥10,000) according to US EPA official methods (US EPA Method 1613/B 1994; US EPA Method 1668/A 2003). Isotope dilution technique was used for the determination of 15 PCDD/F congeners and DL-PCBs; instead, internal standard technique was applied for determination of the two remaining PCDD/F congeners (1,2,3,7,8,9HxCDD and OCDF) and the six NDL-PCB indicators. The limits of detection (LOD) and the limits of quantification (LOQ) for each congener were in agreement with US EPA

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official methods and with European legislation (Official Journal of the European Union 2006b). The concentration of the congeners below the LOQ was calculated using the Bupper-bound approach^: All the values of the non-detected congeners were assumed to be equal to the respective LOQ (Official Journal of the European Union 2006b). Procedural blanks were analysed with each sample batch to demonstrate freedom from contamination. Blank analysis allowed to verify the absence of interferences. According to US EPA methods, the accuracy in respect to the calibration curves for any compound was verified in each set of analysis by examining the calibration verification (VER) standard (US EPA Method 1613/B 1994; US EPA Method 1668/A 2003). For each analysis, the internal standards recovery was evaluated to monitor method performance. The internal standards were the C-labelled compounds spiked into the samples. The percent recovery of each labelled congener was within the limits given by the US EPA methods for PCDD/Fs and PCBs. Certified reference material and/or laboratory reference materials were regularly analysed by the laboratory in order to continually monitor the performance and assure analytical reliability. Calculation of TEQ for dioxins and DL-PCBs TEQ values were calculated using the measured concentrations and the WHO-toxic equivalency factors (TEFs) 2005 values (Van den Berg et al. 2006). All values are reported as upper-bound.

Results and discussion Milk contamination level and profile Samples lipid level (fat %) and concentrations of PCDD/Fs (pg g−1 fat), DL-PCBs (pg g−1 fat) and NDL-PCBs (ng g−1 fat) are listed in Table 1. Among PCDDs, the higher chlorinated molecules (1,2,3,4,6,7,8-HpCDD and 1,2,3,4,6,7,8,9-OCDD) were more abundant than the lower chlorinated ones. Among PCDFs, it was 2,3,4,7,8-PeCDF the congener which showed the highest concentration in all milk samples, as also found by Turrio-Baldassarri et al. (2009) in milk collected in Brescia SIN area. The 2,3,4,7,8-PeCDF has been reported to be the most abundant PCDFs congener in milk of farm located near waste incinerators, chemical and metallurgical plants (Ramos et al. 1997). A similar PCDD/Fs pattern has been found also in milk samples collected from industrial areas of Tuscany and Sardinia (Ingelido et al. 2009; Storelli et al. 2012). Regarding DL-PCBs content in all the milk samples, the mono-ortho congeners were more abundant than the non-ortho ones, in

Table 1 Results of the analytical determinations expressed as pictogram per gram fat for PCDDs, PCDFs and DL-PCBs Mean±st. dev.

Min–max

Fat (%) PCDD/Fs (pg g−1 fat) 2,3,7,8-TCDF 1,2,3,7,8-PeCDF

4.11±0.23

3.80–4.60

0.08±0.02 0.08±0.03

0.06–0.12 0.06–0.15

2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF 1,2,3,4,6,7,8,9-OCDF total PCDFs 2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,8,9-OCDD total PCDDs total PCDFs+PCDDs

1.23±0.46 0.62±0.32 0.44±0.14 0.39±0.14 0.12±0.01 0.24±0.07 0.18±0.01 0.30±0.02 3.70±1.10 0.08±0.03 0.23±0.08 0.13±0.01 0.25±0.09 0.13±0.02 0.37±0.18 0.37±0.13 1.57±0.46 5.27±1.51

0.57–2.07 0.26–1.51 0.23–0.65 0.19–0.70 0.11–0.13 0.18–0.39 0.16–0.20 0.27–0.33 2.14–5.77 0.06–0.14 0.10–0.42 0.11–0.16 0.12–0.46 0.11–0.20 0.18–0.65 0.27–0.71 0.99–2.59 3.13–7.62

ratio PCDFs/PCDDsa

2.38±0.37

1.85–3.16

850±561 72.9±44.9 3004±1809 54.8±32.1 412±241 99.7±55.7 212±122 45.2±25.6 4751±2865

240–1951 19.3–153 840–6748 17.9–110 113–830 29.6–199 62.8–427 14.2–86.8 1338–10492

4.74±3.87 1.91±1.43 29.8±17.0 3.17±1.55 39.6±22.7 4790±2886

1.19–16.5 0.48–5.87 10.8–66.4 1.38–6.14 14.4–82.7 1353–10574

0.02±0.01 0.01±0.01 0.03±0.03 4.97±3.08 6.14±3.36 3.23±2.09

0.01–0.06 0.00–0.04 0.01–0.11 1.48–10.9 1.98–11.5 0.67–6.80

DL-PCBs (pg g−1 fat) Mono-ortho DL-PCBs PCB 105 PCB 114 PCB 118 PCB 123 PCB 156 PCB 157 PCB 167 PCB 189 Total mono-ortho DL-PCBs Non-ortho DL-PCBs PCB 77 PCB 81 PCB 126 PCB 169 Total non-ortho DL-PCBs Total DL-PCBs NDL-PCBs (ng g−1 fat) PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180

Environ Sci Pollut Res Table 1 (continued)

Total NDL-PCBs

Mean±st. dev.

Min–max

14.4±8.40

4.16–29.0

Concentrations of NDL-PCBs are expressed as nanograms per gram fat. Milk lipid levels are also included. Mean, standard deviation and minimum-maximum range are referred to the 14 bulk tank milk samples analyzed a

Abundance ratio between PCDFs and PCDDs

particular the molecule that showed that the highest concentration was PCB 118 (accounting for 63 % of the sum of the 12 DL-PCBs), followed by PCB 105 and PCB 156. Among nonortho DL-PCBs, PCB 126 displayed the highest values (representing the 75 % of the sum of the four non-ortho PCBs). A similar DL-PCBs profile was found by Turrio Baldassarri et al. (2009), in milk collected in three small dairy farms located in the SIN area. Considering NDL-PCBs levels, the sum of the six indicators ranged from 4.16 to 29.00 ng g−1 fat. None of the samples exceeded the limit of 40 ng g−1 fat set by EU Regulation No. 1259/2011. In all the samples, the NDL-PCBs profile was characterized by the prevalence of PCB 153, followed by PCB 138 and PCB 180, while the remaining congeners (PCB 28, PCB 52 and PCB 101) were present at lower abundance, as also found by Turrio-Baldassarri and colleagues (2009). The average PCB 153 contribution to the sum of the six indicators was 44 % (39–51 %). WHO-TEQ values for PCDD/Fs and DL-PCBs were calculated (Van den Berg et al. 2006), and the results are reported in Table 2. For each farm, total WHO-TEQ levels of PCDFs, PCDDs and their sum, WHO-TEQ values of mono-ortho and non-ortho DL-PCBs, WHO-TEQ DL-PCBs total levels, and the sum of PCDD/Fs and DL-PCBs (total WHO-TEQ) are shown. PCDFs/PCDDs and DL-PCBs/(PCDDs+PCDFs) ratios are also included.

Table 2

Total WHO-TEQ values (PCDD/Fs+DL-PCBs) were in the range of 1.78–8.16 pg WHO-TEQ/g fat, with an average value of 4.13 pg WHO-TEQ/g fat. The contamination level of four samples (farms 6, 8, 10, 14) was over the 5.5 pg WHOTEQ/g fat maximum limit established by EU Regulation No. 1259/2011 for the sum of dioxins and DL-PCBs. The noncompliant farms were all located in Brescia City, 1–5 km far from the boundary of Brescia SIN area. In particular, farm 10 (5 km east to the SIN boundary) was the most contaminated one, followed by farm 6 (1 km west to the SIN boundary, the closest one), farm 14 (5 km south-east to SIN boundary) and farm 8 (2.5 km south to SIN boundary). Milk samples of farms 5, 9, 11, 12 and 13 exceeded the EU action limit of 2.0 pg WHO-TEQ/g fat for DL-PCBs. All these farms were situated in Brescia City, with the exception of farms 12 and 13, located few kilometers south of the city. None of the samples exceeded the action limit set for PCDD/Fs. Farms 1, 2, 3, 4 and 7 showed contamination levels below the EU legislative values, both action and maximum limits. Farm 7 was the only one situated in Brescia City (close to the south-west border), and the other farms were all located some kilometres west/south-west to the city. The trend of reduction of food contamination going from the SIN to the east or to the north-west of the city, as reported by Donato et al. (2006), was not evident in our study. In fact, the three most contaminated farms were located in these areas (two east and one west). This likely explains the findings of Donato and colleagues (2006), who underlined that consumers of food produced in these areas had higher serum levels of PCBs than non-consumers. In fact, it seems that in the past, some soil from area surrounding the chemical plant had been transported to other part of the city, causing pollution of these areas (Donato et al. 2006). Focusing on both the analytical value and the WHO-TEQ PCDFs/PCDDs ratios, their results were always higher than 1, in agreement with previous findings (Turrio-Baldassarri et al. 2009). Analytical value of PCDFs/PCDDs ratio ranges from

WHO-TEQ levels

Farm

1

2

3

4

5

6

7

8

9

10

11

12

13

14

PCDFs (TEQ pg g−1 fat) PCDDs (TEQ pg g−1 fat) PCDFs+PCDDs (TEQ pg g−1 fat) PCDFs/PCDDs (TEQ pg g−1 fat) Mono-ortho DL-PCBs (TEQ pg g−1 fat) Non-ortho DL-PCBs (TEQ pg g−1 fat) Total DL-PCBs (TEQ pg g−1 fat) PCDD/Fs+DL-PCBs (TEQ pg g−1 fat) DL-PCBs/(PCDDs+PCDFs) (TEQ pg g−1 fat)

0.32 0.27 0.59 1.19 0.05 1.13 1.18 1.78 1.99

0.34 0.24 0.59 1.41 0.05 1.29 1.34 1.93 2.28

0.34 0.27 0.62 1.27 0.04 1.12 1.16 1.78 1.88

0.43 0.32 0.75 1.32 0.07 1.45 1.52 2.27 2.04

0.71 0.40 1.12 1.77 0.12 3.03 3.15 4.27 2.83

0.56 0.38 0.94 1.45 0.24 5.65 5.89 6.83 6.24

0.45 0.32 0.77 1.40 0.08 1.86 1.94 2.71 2.52

0.75 0.64 1.39 1.17 0.20 4.07 4.27 5.65 3.08

0.48 0.31 0.79 1.56 0.20 3.78 3.98 4.76 5.05

0.66 0.36 1.02 1.81 0.31 6.82 7.14 8.16 6.96

0.26 0.19 0.46 1.35 0.09 2.08 2.17 2.63 4.75

0.76 0.53 1.29 1.42 0.21 3.51 3.72 5.01 2.89

0.59 0.46 1.05 1.28 0.12 3.14 3.26 4.31 3.10

0.91 0.44 1.35 2.08 0.21 4.15 4.36 5.71 3.23

In bold, the values above the maximum limit set by EU regulation No. 1259/2011

Environ Sci Pollut Res Fig. 2 Percentage contribution of PCDDs+PCDFs and DL-PCBs to the total WHO-TEQ (PCDD/Fs+DL-PCBs) for each farm

1.85 to 3.16, indicating a prevalence of PCDFs over PCDDs. In addition, the contribution of PCDFs to the total WHO-TEQ PCDD/Fs was always found to be higher than 50 % (range 54–68 %). Moreover, DL-PCBs/(PCDDs+PCDFs) WHO TEQ ratio was always above 1 (range 1.88–6.96). Taken together, this data suggested that the cause of pollution is a PCB contamination. Wakimoto and colleagues (1988) documented an higher presence of PCDF impurities than PCDD residues, in PCB industrial mixtures. On the other hand, the presence of detectable levels of PCDDs in milk could be attributed to the strong industrialization of the investigated area and concurrently to the persistence of these molecules; in fact, the more chlorinated PCDD congeners were the most present in the analysed samples. Table 3

In Fig. 2, the contribution of DL-PCBs to the total WHOTEQ value (PCDD/Fs+DL-PCBs) is reported for each herd. DL-PCB contribution ranges from 65 to 87 %, underlining once again the presence of a more important PCB contamination compared to the dioxins pollution. Farms 6, 8, 10 and 14, the most contaminated ones, show percentages of 86, 75, 87 and 76 %, respectively. In particular, in the farm with the highest total WHO-TEQ value (farm 10), DL-PCB contribution was the greatest one (87 %). Analysing the percentage contribution of each congener (Table 3) to the total WHO-TEQ levels (PCDD/Fs+ DL-PCBs), PCB 126 resulted as the greatest responsible of the contamination, ranging from 61 to 81 %. Farm 10, the most polluted one, showed the highest PCB 126

Percentage contribution of each congener to total WHO-TEQ (PCDD/Fs+DL-PCB)

% Total WHO-TEQ PCDFs 2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF PCDDs 2,3,7,8-TCDD 1,2,3,7,8-PeCDD Mono-ortho DL-PCBs PCB 118

1

2

3

4

5

6

12.15 11.91 12.24 12.67 11.71 1.73 1.89 2.25 2.06 1.70 1.64 1.48 1.96 1.55 1.35 1.37 1.30 1.66 1.28 1.20

7

8

9

10

11

12

13

14

5.71 10.86 0.86 1.85 0.63 1.47 0.56 1.41

9.09 1.37 1.15 1.04

6.75 1.11 0.82 0.85

5.76 1.05 0.55 0.40

6.52 10.11 0.98 1.76 0.87 1.30 0.72 1.39

9.52 10.87 1.30 2.64 1.20 1.13 1.02 0.86

3.56 9.27

3.09 7.30

3.23 9.42

2.79 9.38

2.40 5.42

1.30 3.66

2.90 6.92

2.55 7.48

1.36 3.99

0.75 3.06

2.26 3.71

2.39 6.48

2.68 6.81

1.71 4.88

1.70

1.71

1.42

2.14

1.76

2.25

1.82

2.23

2.52

2.48

2.21

2.71

1.78

2.25

Non-ortho DL-PCBs PCB 126 61.31 64.32 60.66 61.45 69.03 80.81 66.44 68.95 77.13 81.31 77.01 67.40 70.37 70.48 PCB 169 2.57 2.60 2.32 2.35 2.03 1.81 2.25 2.96 2.20 2.26 2.17 2.64 2.35 2.13 Total WHO-TEQ PCDD/F+DL-PCB 1.78 1.93 1.78 2.27 4.27 6.83 2.71 5.65 4.76 8.16 2.63 5.01 4.31 5.71 (pg WHO_TEQ/g fat) Only the congeners showing a percentage of >1 % are reported

Environ Sci Pollut Res Table 4 Comparison of contamination levels in bovine milk from farms of the SIN area16 and farms outside the SIN boundary (this study)

n.a. not available data a

Data available only for one farm

WHO-TEQ PCDDs+PCDFs WHO-TEQ PCDDs/PCDFs WHO-TEQ DL-PCBs Total WHO TEQ ∑6 indicators NDL-PCB (ng g−1fat)

contribution (81 %). This finding has been stressed correlating the concentrations of PCB 126 and the total WHO-TEQ values of the 14 milk samples. The correlation coefficient reveals an excellent relation (R2 =0.98) between PCB 126 congener and total WHO-TEQ. A same strong positive correlation has been reported in an Irish survey on bovine milk collected in farms adjacent to chemical industrial plants (O’Donovan et al. 2011). These results would support the use of PCB 126 as an effective proxy measure for total WHO-TEQ or DL-PCBs WHO-TEQ in bovine milk and could be useful as an indicator in large-scale screenings in a PCB contamination scenario (O’Donovan et al. 2011).

SIN area farms (Turrio-Baldassari et al. 2009)

This study

Mean

Range

Mean

Range

6.7 2.7 40.1a 44.8a 263

4.7–8.5 2.2–3.4 n.a. n.a. 183–321

0.90 1.46 3.22 4.13 14.4

0.46–1.39 1.17–2.08 1.16–7.14 1.78–8.16 4.16–29.00

source: the release of PCB products in the environment by Caffaro plant. In the present study, farms situated in Brescia City generally showed a highest level of contamination, with the exception of farms 7 and 11, located close to the south border, which revealed a lower pollution. In farms 1, 2, 3 and 4 (5–8 km west/south-west to the SIN), the level of contamination was found below all the EU legislative limits, and the percentage contribution of DL-PCB to the total WHO-TEQ was the lowest one, ranging from 65 to 70 %. Farms 12 and 13 located in a little town immediately south to the city showed contamination values comparable to the levels found in Brescia.

Comparison with previous data Decontamination experience A comparison between our data (14 farms located outside Brescia SIN) and the previous report about PCDD/F and PCBs in bovine milk collected in three small farms in the SIN area (Turrio-Baldassarri et al. 2009) was carried out (Table 4). The levels of contamination highlighted in the 14 farms analysed in the present study were much lower than those reported for the SIN area (Turrio-Baldassarri et al. 2009); however, they revealed a spreading of the pollution also outside the SIN boundary. The similarity of the congeners pattern (Figs. 3 and 4) suggests a common contamination Fig. 3 DL-PCBs congener profiles in samples from this study and from the survey of Turrio-Baldassarri et al. (2009). The mean percentage contribution of each congener to the total concentration of the 12 DL-PCBs is reported. Standard deviation is also provided. Data by Turrio-Baldassarri et al. (2009) available only for one farm*

In this survey, analysis of the bulk tank milk allowed the identification of four farms (6, 8, 10 and 14), in which the total WHO-TEQ level was above the maximum limit imposed by EU Regulation No. 1259/2011. These farms were placed under seizure, and produced milk was destroyed until recovery within the limits, in agreement with EU Regulation 1069/2009. Due to the economic consequences, farm 8 closed down. In the other three farms (6, 10 and 14), animal feed, the most probable source of contamination, was removed and

Environ Sci Pollut Res Fig. 4 NDL-PCBs congener profiles in samples from this study and from the survey of Turrio-Baldassarri et al. (2009). The mean percentage contribution of each congener to the sum of the six indicators is reported, including standard deviation

WHO-TEQ/g fat to 1.83 pg WHO-TEQ/g fat, in 1 month. Levels below the action limits were obtained in farms 6 and 10 after 2 months (1.39 pg WHO-TEQ/g fat and 1.55 pg WHO-TEQ/g fat respectively). Brambilla et al. (2008) calculated for PCDD/Fs a half-life of 17±3 days on WHO-TEQ basis after contamination suspension in lactating cows (on-field study). Moreover, a survey on lactating goats suggested a 20-day period as sufficient to obtain a total WHO-TEQ level in milk below the regulatory value, after a decontamination process (Fournier et al. 2013). Data obtained from this study were consistent with these results. The present findings also agree with the data on decontamination of ruminants through milk excretion, recently reported by Rychen G. et al. (2014), who suggested that lactating ruminants may be decontaminated in few weeks. Milk soundness was achieved both in the farm in which animal ration was totally substituted and in the two farms in which only local hay was replaced. An initial exclusion of suspect hay in cow ration could be a useful first-aid action for the management of on-field contamination emergencies,

replaced, and bulk tank milk was monitored for dioxins and PCBs. In farm 10, the one with the highest contamination, feed was completely substituted with an equivalent total mixed ration whose ingredients came from an area with a nonpollution story. In herds 6 and 14, only the locally produced hay was removed and replaced with a non-contaminated product. After 1 and 2 months, the bulk tank milk of the three farms was collected and analysed for dioxins and PCBs. Results are presented in Table 5. Given the values of month 1 analysis, in farm 14, month 2 test was not made. The obtained results clearly show that, after only 1 month of decontamination, all the farms reached values below the maximum limit set by EU. However, the first month was not sufficient for farms 6 and 10 to reduce DL-PCB contamination below the action limits of 2.0 pg WHO-TEQ/g fat, starting from a DL-PCBs level of 5.89 pg WHO-TEQ/g fat and 7.14 pg WHO-TEQ/g fat respectively. On the other hand, DL-PCB contamination of herd 14 dropped from 4.36 pg Table 5

WHO-TEQ values at month 0 (first sampling), month 1 and month 2

Farm

PCDFs (TEQ pg g−1 fat) PCDDs (TEQ pg g−1 fat) PCDFs+PCDDs (TEQ pg g−1 fat) PCDFs/PCDDs Mono-ortho DL-PCBs (TEQ pg g−1 fat) Non-ortho DL-PCBs (TEQ pg g−1 fat) DL-PCBs (TEQ pg g−1 fat) PCDD/Fs+DL-PCBs (TEQ pg g−1 fat) DL-PCBs/(PCDDs+PCDFs)

6

10

14

Month 0

Month 1

Month 2

Month 0

Month 1

Month 2

Month 0

Month 1

0.56 0.38 0.94 1.45 0.24 5.65 5.89 6.83 6.24

0.54 0.43 0.96 1.26 0.17 3.28 3.45 4.41 3.58

0.37 0.29 0.66 1.30 0.04 1.35 1.39 2.05 2.11

0.66 0.36 1.02 1.81 0.31 6.82 7.14 8.16 6.96

0.35 0.19 0.54 1.85 0.22 4.03 4.25 4.79 7.88

0.37 0.29 0.66 1.27 0.05 1.50 1.55 2.21 2.34

0.91 0.44 1.35 2.08 0.21 4.15 4.36 5.71 3.23

0.36 0.28 0.64 1.30 0.05 1.78 1.83 2.47 2.86

Results of month 1 and month 2 are referred to the first and second month of decontamination (substitution of local contaminated feeds with clean ones)

Environ Sci Pollut Res Fig. 5 Decontamination curve of farm 10 following the complete substitution of local feeds with a non-polluted total mixed ration. Depletion trend of total WHOTEQ, DL-PCBs (WHO-TEQ), PCB 126 (WHO-TEQ) and PCDFs+PCDDs (WHO-TEQ)

in order to rapidly achieve milk compliance. In fact, hay has been demonstrated to have a major role in delivering dioxinlike compounds to dairy cows by animal feed (Lorber and Winters 2007). A successful feed-mitigation strategy, by hay replacement, could also be a long-term solution, as it happens in the farms of Brescia area. In fact, to date, this is the only way that allows a safe production in this high-risk municipality. Analysing the decontamination curve of farm 10 (Fig. 5), it was clear that a large part of the total WHO-TEQ depends on DL-PCB contamination. As a consequence, after complete feed substitution, the reduction of the total WHOTEQ value was strictly linked to DL-PCBs depletion, in particular to the decrease of PCB 126. The same situation has been seen also in farm 6 (Fig. 6), despite the substitution of the local hay alone. Once again, data analysis underlined the importance of PCB 126 trend in the determination of the final WHO-TEQ level and the possibility of using this congener alone to obtain an approximate indication of the contamination level, when DL-PCBs are the main cause of pollution (O’Donovan et al. 2011). Fig. 6 Decontamination curve of farm 6 following the substitution of just the local contaminated hay with a non-polluted one. Depletion trend of total WHOTEQ, DL-PCBs (WHO-TEQ), PCB 126 (WHO-TEQ) and PCDFs+PCDDs (WHO-TEQ)

Conclusions The sampling of 14 dairy farms located in Brescia City and surroundings revealed a clear PCB contamination of the food produced in this high-risk area. In fact, despite that Caffaro plant stopped PCBs production in 1983, this source of contamination is still polluting the area and it is spreading outside the SIN. Further investigations are necessary to understand the contamination spreading mechanism. This survey provided also an on-field example of a correct risk management that succeeded in bringing contamination levels below the legal limits, in 1 month. For an effective action, the key factor seems to be the removal of locally produced contaminated feed from animal diet, in particular hay. In addition, PCB 126 reveals to be a good indicator of total WHO-TEQ level, in areas with a PCB contamination story. These findings could be a useful tool for risk managers and decision makers in the case of PCB contamination incidents in bovine milk, in particular when the contamination is mainly due to PCB 126. Other on-field studies will be carried out in order to confirm these results.

Environ Sci Pollut Res

A constant check of the levels of food contamination and the availability of a fast intervention plan, which would allow a good management of non-compliant cases, have been demonstrated to be important in order to guarantee the safety of food produced in high-risk areas. Acknowledgments The work is part of PASTORIPOPS project. Conflict of interest There are no conflicts of interest or any financial or personal relationship with other people or organizations that could distort the findings of this study.

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