Bioaccumulation of trace metals in Mediterranean mussels (Mytilus galloprovincialis) from a fish farm with copper-alloy mesh pens and potential risk assessment

Figures & data

Figure 1. Location of the copper alloy cage site (40°03′42″N–26°20′36″E, 40°03′51″N–26°20′45″E, 40°03′45″N–26°20′55″E, 40°03′36″N–26°20′.

Table 1. Analyses of copper-alloy* material of the mesh used for the fish pen (means ± standard deviation).

Contents	Min (%)	Max (%)	mean ± SD (%)
Cu	70.00	73.00	71.50 ± 2.12
Zn	29.18	25.57	27.38 ± 2.55
Sn	0.80	1.20	1.00 ± 0.28
P	0.02	0.10	0.06 ± 0.06
Pb	N/A	0.07	0.07
Fe	N/A	0.06	0.06

An antimicrobial wrought copper-zinc brass alloy with the Active standard ASTM designation of C44500, data from German Copper Institute:

https://www.kupferinstitut.de/en/arbeitsmittel/kupferschluessel.html

N/A: not available.

Table 2. Physico-chemical water quality parameters in the experimental site.

Parameter	Unit	US	DS	CF	CB
Oxygen (O2)	mg/L	7.2–9.1	7.4–9.1	7.0–10.6	7.1–10.8
Temperature	°C	11.6–20.5	12.0–20.1	10.2–22.9	10.9–23.0
Salinity	‰	28.0–35.0	26.1–35.1	22.8–32.5	24.0–32.7
pH		8.3–8.5	8.1–8.5	8.1–8.5	8.2–8.4
Ammonia (NH4-N)	μmol L^−1	0.261–1.871	0.078–1.928	0.035–1.697	0.072–1.701
Ammonia (NH4-N)	mg/L	0.0047–0.034	0.0014–0.035	0.0006–0.031	0.0013–0.031
Phosphate (PO4-P)	μmol L^−1	0.21–6.213	0.101–2.913	0.102–7.564	0.100–6.811
Phosphate (PO4-P)	mg/L	0.0199–0.590	0.0096–0.277	0.0097–0.718	0.0095–0.647

US: upstream area, DS: downstream area, CF: cage frame, CB: cage bottom.

Unit conversions for ammonium nitrogen (NH₄-N): 1 μg NH₄ L⁻¹ = 0.055437 μmol NH₄ L⁻¹;

for phosphate phosphorus (PO₄-P): 1 μg PO₄ L⁻¹ = 0.010529 μmol PO₄ L⁻¹.

Table 3. Metals (mg/kg) in muscles tissue, digestive gland, gills and total edible part of mussels from different locations around copper alloy cage system. Values (means ± SD, n = 50) with different superscripts in same column are significantly different at 5% level.

Location		Cu	Zn	Mn	Fe
Muscle tissue
Upstream
Dry		0.14 ± 0.03^a	13.15 ± 1.61^a	0.10 ± 0.04^a	10.6 ± 3.97^a
Wet		0.018	2.16	0.017	1.69
Downstream
Dry		0.16 ± 0.08^a	17.55 ± 7.21^ab	0.17 ± 0.07^a	12.7 ± 1.53^a
Wet		0.027	2.89	0.031	2.09
Cage Frame
Dry		1.89 ± 0.36^b	21.77 ± 2.58^b	0.71 ± 0.08^c	21.7 ± 5.8^b
Wet		0.30	3.23	0.13	3.36
Cage Bottom
Dry		2.72 ± 0.57^b	40.45 ± 18.6^b	1.78 ± 0.43^b	26.4 ± 9.77^b
Wet		0.51	6.74	0.29	4.41
Digestive gland
Upstream
Dry		0.50 ± 0.25^a	25.20 ± 8.44^a	0.17 ± 0.08^b	7.91 ± 2.16^a
Wet		0.096	4.23	0.032	0.134
Downstream
Dry		0.71 ± 0.12^a	35.16 ± 10.6^a	0.67 ± 0.22^c	15.2 ± 6.34^b
Wet		0.12	5.91	0.124	2.620
Cage Frame
Dry		3.17 ± 2.16^b	51.72 ± 11.9^b	0.04 ± 0.01^d	16.2 ± 3.99^b
Wet		0.59	8.57	0.007	2.720
Cage Bottom
Dry		4.89 ± 1.18^b	57.11 ± 19.9^b	1.34 ± 0.32^a	26.2 ± 7.44^b
Wet		0.87	9.62	0.246	4.39
Gills
Upstream
Dry		0.08 ± 0.02^a	5.46 ± 1.61^a	0.13 ± 0.05^a	5.78 ± 1.87^a
Wet		0.013	0.94	0.023	0.98
Downstream
Dry		0.16 ± 0.09^a	6.30 ± 2.33^a	0.10 ± 0.01^a	5.62 ± 1.27^a
Wet		0.029	1.10	0.016	0.96
Cage Frame
Dry		2.63 ± 0.81^c	32.15 ± 10.6^b	0.60 ± 0.12^c	15.5 ± 4.42^b
Wet		0.50	5.30	0.118	2.66
Cage Bottom
Dry		5.26 ± 1.70^d	35.92 ± 20.5^b	1.68 ± 0.46^b	29.4 ± 4.48^c
Wet		1.01	5.89	0.313	4.95
Total Edible Part
Upstream
Dry		0.72	43.81	0.40	24.29
Wet		0.127	7.330	0.072	2.804
Downstream
Dry		1.03	59.01	0.94	33.52
Wet		0.176	9.900	0.171	5.670
Cage Frame
Dry		7.69	105.6	1.35	53.40
Wet		1.390	17.10	0.255	8.740
Cage Bottom
Dry		12.87	133.5	4.80	82.00
Wet		2.390	22.25	0.849	13.75

Upstream (un-affected distant Area: 50 m upstream distance from cage, 6 m below surface (Individual mean weight of mussels = 29.58 ± 7.79 g).

Downstream (affected distant area: 50 m downstream distance from cage, 6 m below surface (individual mean weight of mussels = 39.04 ± 9.9 g).

Cage Frame: HDPE cage frame, 30 cm below surface (individual mean weight of mussels = 6.75 ± 1.09 g).

Cage Bottom: Bottom panel of copper-alloy net pen, 6 m below surface (mussel growth on hydrozoa attached on horizontal bottom panel of copper-alloy mesh, individual mean weight of mussels = 41.75 ± 8.21 g).

Table 4. Permissible upper limits of trace element in marine products according to various international standards.

Elements (mg/kg wet weight)
Standards	Cu	Zn	Mn	Fe
FAO/WHO (1989)	30	40	—	—
WHO (1989)	30	100	1	100
MAFF (1995), TFC (2008)	20	50	—	—

FAO: Food and Agriculture Organization–UN; WHO: World Health Organization; MAFF: Ministry of Agriculture, Fisheries and Food-United Kingdom; TFC: Turkish Feed Codex.

Table 5. Mean levels of Cu, Zn, Mn and Fe (mg/kg dry basis) reported in mussel (M. galloprovincialis) from different regions of the Mediterranean, and in the present study.

Location	Cu	Zn	Mn	Fe	References
Mediterranean Area
Adriatic Sea (Venice Lagoon)	15.8–35.5	100.2–241.6	14.1–18.6	260–458	Giusti and Zhang (2002)
Tyrrhenian Sea (Gulf of Gaeta)	5.5–11.5	123–180			Conti and Cecchetti (2003)
NW Mediterranean (River Paillon)	4.7–5.9	128–205			Roméo et al. (2003)
NW Mediterranean (Harbor of Nice, HN)	7.3–8.3	238–264			Roméo et al. (2003)
NW Mediterranean (East of HN)	3.6–5.2	154–264			Roméo et al. (2003)
NW Mediterranean (Entrance of HN)	3.1–5.4	195–394			Roméo et al. (2003)
Marmara Sea (NW coasts)	6.7–9.5	208–320	4.5–11.7	120–415	Topcuoğlu et al. (2004)
Adriatic Sea (Croatian coasts)	5.3–6.9	105–111	4.5–7.0	85.8–261.3	Orescanin et al. (2006)
Adriatic Sea (Portonovo)	3.3–8.0	37.5–184.3	2.1–18.4	59.9–64.3	Gorbi et al. (2008)
Aegean Sea (Izmir Bay, inner bay)	5.2–5.3	26.9–28.5	4.2–4.7	48.6–49.9	Kucuksezgin et al. (2008)
Aegean Sea (Izmir Bay, middle bay)	3.5–3.8	17.8–17.9	2.6–2.7	49.0–49.9	Kucuksezgin et al. (2008)
W Mediterranean (Baleari Island)	4.2–5.2	154–289			Deudero et al. (2009)
Adriatic Sea (Croatian coasts)	10.5	158.0			Kljaković-Gaspić et al. (2010)
Adriatic Sea (Montenegro coasts)	4.6–17.2	132–345	7.3–85.0	128–603	Joksimovic et al. (2011)
Adriatic Sea (Montenegro coasts)	6.2–14.5	135–210			Stanković et al. (2011)
Adriatic Sea (Apulian coasts)	4.66–19.2	75.3			Spada et al. (2013)
Other Areas
Black Sea (Turkish coasts)	11.7–23.3	312–396	46.9–73.0		Bakan and Özkoç (2007)
Black Sea (Turkish coasts)	6.3–7.9	182–296			Türk-Çulha et al. (2008)
N Atlantic (Spanish Gallician coasts)	3.9–9.7	159–351			Besada et al. (2011)
Black Sea (Turkish coasts)	2.4–4.8	79–163			Bat et al. (2012)
Island of Gossa (W coast of Norway)	1.3–1.8	13.3–15.2		11.0–11.7	Brooks et al. (2012)
N Aegean Sea (Strait of Canakkale)	0.7–12.9	43.8–133.5	0.4–4.8	24.3–82.0	Present study

Table 6. Dietary reference intake (DRI) values of metals and estimated daily consumption (EDC) and individual risk rate (IRR) through the consumption of mussel grown around copper-alloy cage systems.

	Cu	Zn	Mn	Fe
Dietary reference intakes (DRI, mg day^−^1 person)
EAR	0.7	9.4	BDL	6.0
RDA	0.9	11.0	2.3^*	8.0
TUL	10.0	40.0	11.0	45.0
Estimated daily consumption (EDC, mg day^−^1 person)
Upstream	0.001	0.034	0.0003	0.013
Downstream	0.001	0.046	0.001	0.026
Cage Frame	0.007	0.080	0.001	0.041
Cage Bottom	0.011	0.104	0.004	0.064
Individual risk rate (IRR)
Upstream	0.0001	0.0009	0.0000	0.0003
Downstream	0.0001	0.0012	0.0001	0.007
Cage Frame	0.0007	0.0020	0.0001	0.001
Cage Bottom	0.0011	0.0026	0.0004	0.001

DRI (dietary reference intakes; IOM, 2006), EAR (estimated average requirement; IOM, 2006), RDA (recommended daily allowance; IOM, 2006); TUL (tolerable upper limit; IOM, 2006),

* AI (adequate intake; IOM, 2006),

EDC (estimated daily consumption) = (metal in mussel (mg/kg) × daily mussel consumption rate (kg/day)) / Bw (kg); (daily mussel consumption of a European: 0.0047 kg/day).

IRR (individual risk rate) = individual exposure (mg/kg/day) / TUL (mg/kg/day);

Individual exposure = (C_m × IR) / BW.

Distant Area: 50 m upstream distance from cage, 6 m below surface.

Cage Area: 50 m downstream distance from cage, 6 m below surface.

Cage Frame: HDPE cage frame, 30 cm below surface

Cage Bottom: Bottom panel of copper-alloy net pen, 6 m below surface (mussel growth on hydrozoa attached on horizontal bottom panel of copper-alloy mesh).

BDL: below detectible limits; Calculations performed using metal concentration of total mussel tissue, considered as the edible part.

Table 7. Maximum allowable daily mussel consumption limit (MADCL), maximum allowable mussel consumption rate (MACR), and target hazardous quotients (THQ) of metals by consuming mussels grown around copper alloy mesh pens. For the estimations, total mussel tissues were considered as the edible part.

	Cu	Zn	Mn	Fe
Maximum allowable daily mussel consumption limit (MADCL, kg day^−^1 person)
Upstream	78.74	5.46	152.78	16.05
Downstream	56.82	4.04	64.33	7.94
Cage Frame	7.19	2.34	43.14	5.15
Cage Bottom	4.18	1.80	12.96	3.27
Maximum allowable mussel consumption rate (MACR, meal/week person)
Upstream	2,428.1	168.3	4,711.2	494.9
Downstream	1,752.1	124.6	1,983.7	244.7
Cage Frame	221.9	72.1	1,330.2	158.8
Cage Bottom	129.0	55.4	399.5	100.9
Target hazardous quotients (THQ)					THI
Upstream	0.00006	0.00085	0.00003	0.00029	0.00123
Downstream	0.00008	0.00115	0.00007	0.00059	0.00189
Cage Frame	0.00065	0.00199	0.00011	0.00091	0.00365
Cage Bottom	0.00111	0.00259	0.00036	0.00142	0.00549

MADCL (maximum allowable daily mussel consumption limit, kg per day) = (TUL × BW_a) / metal concentration in mussel.

MACR (maximum allowable mussel consumption rate, meal per week) = MADCL × time in days / meal size (0.227 kg mussel/meal).

THQ (target hazard quotient) = ((exposure frequency (365 days/year) × exposure duration (year) × mean daily mussel consumption rate (g/day) × metal concentration in mussel (mg/kg)) / (recommended daily allowance × body weight of the adult human consumer (70 kg) × (exposure frequency × exposure duration)) × 10⁻³

THI (total hazard index) = a sum of all calculated THQ values of heavy metals, Σ_THQ).

Distant Area: 50 m upstream distance from cage, 6 m below surface.

Cage Area: 50 m downstream distance from cage, 6 m below surface.

Cage Frame: HDPE cage frame, 30 cm below surface.

Cage Bottom: Bottom panel of copper-alloy net pen, 6 m below surface (mussel growth on hydrozoa attached on horizontal bottom panel of copper-alloy mesh).

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