*3.3.1.3. Sample preparation*

The biota samples were dried at 40°C (24 h) and crushed then about three grams of biological sample were dissolved in aqua regia (a mixture of suprapure acids HCl 30 and 65% HNO3 in the report 21–7 mL). The mixture was mineralized using a sand bath until complete dissolution. After cooling, the samples were filtered on paper filter (porosity <45 µm) in a 50‐mL volumetric flask and filled with ultrapure water. The metal content in the samples was determined by inductively coupled plasma optical emission spectrometry. A calibration curve in the range of 0.1–0.5 mg/L (As, Se, Sb, Cd, Cr, Cu, Co, Fe, Mn, Mg, Ni, Pb, Zn) was performed using a Certified Reference Material solution (100 mg/L Multi Element Standard Solution, Certipur, Merck). The quality control of the data was carried out according to Quality Control Standards 21A, 100 mg/L, produced by PerkinElmer. A reagent blank in order to estimate the metal contents from acids was prepared.

The mollusks (two bivalves' species: *Unio pictorum* and *Anodonta cygnea*) and one gastropod species (*Viviparus viviparus*) were selected in this study (**Figure 8**) as they prevail in the total biomass of benthic invertebrate community structure, and they are widely used as bioindica‐ tors for water quality. Their shells were subjected to metals detection, because they are formed throughout mollusks life and their chemical composition is an integral index to describe the composition of the aquatic environment over time [75]. Bioaccumulation factors of metals were calculated for each tested species.

**Figure 8.** The analyzed benthic macroinvertebrates species.

### *3.3.2. Results and discussion*

### *3.3.2.1. Metal accumulation in benthic organisms*

This study included metal analysis results in the bivalves and gastropod shells from Murighiol and Uzlina sampling site. Other researchers [76–79] performed their studies as well using the same biological model, mollusk shells, for the metal accumulation analysis.

The mollusks have the largest representation and are the most valuable groups among the benthic invertebrates' communities due to the fact they are dominant in the total benthic community biomass and represent a basic food for the next trophic level (e.g., fish).

Two types of bivalve species identified at Uzlina and Murighiol were selected for metal analysis, respectively: *U. pictorum, A. cygnea*. Also from Gasteropoda, the species *V. viviparus* were selected (**Figure 8**). For each species, the dry and wet biomasses were determined (**Table 5**).


**Table 5.** Dry and wet biomass values of the selected species.

Certified Reference Material solution (100 mg/L Multi Element Standard Solution, Certipur, Merck). The quality control of the data was carried out according to Quality Control Standards 21A, 100 mg/L, produced by PerkinElmer. A reagent blank in order to estimate the metal

The mollusks (two bivalves' species: *Unio pictorum* and *Anodonta cygnea*) and one gastropod species (*Viviparus viviparus*) were selected in this study (**Figure 8**) as they prevail in the total biomass of benthic invertebrate community structure, and they are widely used as bioindica‐ tors for water quality. Their shells were subjected to metals detection, because they are formed throughout mollusks life and their chemical composition is an integral index to describe the composition of the aquatic environment over time [75]. Bioaccumulation factors of metals were

This study included metal analysis results in the bivalves and gastropod shells from Murighiol and Uzlina sampling site. Other researchers [76–79] performed their studies as well using the

The mollusks have the largest representation and are the most valuable groups among the benthic invertebrates' communities due to the fact they are dominant in the total benthic

community biomass and represent a basic food for the next trophic level (e.g., fish).

same biological model, mollusk shells, for the metal accumulation analysis.

contents from acids was prepared.

74 Water Quality

calculated for each tested species.

**Figure 8.** The analyzed benthic macroinvertebrates species.

*3.3.2.1. Metal accumulation in benthic organisms*

*3.3.2. Results and discussion*

The Biota Sediment Accumulation Factor (BSAFsed) was calculated using the equation: *BSAFsed = Cb/Csed*, where *Cb* is the metal concentration in biota/organism and *Csed* is the metal concentration in the sediment sample [80].

At Murighiol sampling site, in the *U. pictorum*, shells (collected in July) were recorded the highest values for Cu, Ni, and Zn. Moreover, the Cu concentration in sediment was 47 mg/kg d.m. over the set limit. It was estimated that 4% of the Cu concentration, 2% of Zn, and 1% of Ni were found in *U. pictorum* shell species. The BSAFsed values were <0.05 (**Table 6**). Also, various metals were detected in the *U. pictorum* and *V. viviparous* shells from Uzlina, in July (**Table 7**).


\* Average of metal concentrations (for two replicates) expressed in mg/kg d.m.

\*\* Csed 2009–2013—average of the metal concentration detected in sediment from 2009 to 2013 [63, 65].

**Table 6.** Metal concentration (mg/kg d.m) in the shell of *Unio pictorum* at Murighiol in July 2013.


\* Average of metal concentrations (for two replicates) expressed in mg/kg d.m.

\*\* Csed 2009–2013—average of the metal concentration detected in sediment from 2009 to 2013 [63, 65].

**Table 7.** Metal concentration (mg/kg d.m) in the shell of *Unio pictorum* and *Viviparus viviparus* at Uzlina in July 2013.

Concentrations of As, Cd, Cr, Fe, Pb, Se, Sb, Mg did not showed significant values in shells of analyzed benthic organisms. The metals Cu, Ni, and Zn were present in sediment over the set limits of national norms inducing their accumulation in shells. The highest values of Cu and Zn were both in *U. pictorum* and *V. viviparus* (**Table 7**). Similar concentrations of Zn, Cu, Pb, Cd, and Co in *V. viviparus* were found in the River Dnieper in the same gastropod shells [81].

Metal concentrations showed a lower magnitude in mollusk shells than in their bodies, and this result could be explained by the fact that metals were accumulated in shell only after they were absorbed by the body. The bioaccumulation selectivity of metals in gastropod shells follows the next order: Fe > Mn > Zn > Cu > Pb > Co > Cd. Thus, the quantitative distribution of metals in mollusk shells is considered by the level of biochemical involvement, metabolic processes, their toxicity degree as well as the bioavailability to aquatic organisms [81].

Some studies [82] revealed that Fe belongs to metals which play an important role in body metabolism and is not toxic. The Mn, Mg, Co, Cu, Zn, and Ni are involved in growth, devel‐ opment, and reproduction process, but in high concentrations can show toxic effects (see the above section "*Laboratory tests—acute and chronic effects*"). Pb and Cd are not involved in metabolic processes; thus, they are highly toxic at low concentrations and have a great storage capacity in the organisms at long‐term exposure. The results on metal concentrations in *U. pictorum* and *A. cygnea* shells, metal detection in sediment samples (2013), average of metals detection in sediment in period of 2009–2013 at Uzlina and BSAFsed values are presented in


**Table 8**. The metals were determined in both bivalve species *U. pictorum* and *A. cygnea* shells, in September 2013 (**Table 8**).

\* Average of metal concentrations (for two replicates) expressed in mg/kg d.m.

**Metal Cb\***

76 Water Quality

shells [81].

*Unio pictorum*

**BSAFsed Cb\***

\* Average of metal concentrations (for two replicates) expressed in mg/kg d.m.

\*\* Csed 2009–2013—average of the metal concentration detected in sediment from 2009 to 2013 [63, 65].

**Table 7.** Metal concentration (mg/kg d.m) in the shell of *Unio pictorum* and *Viviparus viviparus* at Uzlina in July 2013.

Concentrations of As, Cd, Cr, Fe, Pb, Se, Sb, Mg did not showed significant values in shells of analyzed benthic organisms. The metals Cu, Ni, and Zn were present in sediment over the set limits of national norms inducing their accumulation in shells. The highest values of Cu and Zn were both in *U. pictorum* and *V. viviparus* (**Table 7**). Similar concentrations of Zn, Cu, Pb, Cd, and Co in *V. viviparus* were found in the River Dnieper in the same gastropod

Metal concentrations showed a lower magnitude in mollusk shells than in their bodies, and this result could be explained by the fact that metals were accumulated in shell only after they were absorbed by the body. The bioaccumulation selectivity of metals in gastropod shells follows the next order: Fe > Mn > Zn > Cu > Pb > Co > Cd. Thus, the quantitative distribution of metals in mollusk shells is considered by the level of biochemical involvement, metabolic processes, their toxicity degree as well as the bioavailability to aquatic organisms [81].

Some studies [82] revealed that Fe belongs to metals which play an important role in body metabolism and is not toxic. The Mn, Mg, Co, Cu, Zn, and Ni are involved in growth, devel‐ opment, and reproduction process, but in high concentrations can show toxic effects (see the above section "*Laboratory tests—acute and chronic effects*"). Pb and Cd are not involved in metabolic processes; thus, they are highly toxic at low concentrations and have a great storage capacity in the organisms at long‐term exposure. The results on metal concentrations in *U. pictorum* and *A. cygnea* shells, metal detection in sediment samples (2013), average of metals detection in sediment in period of 2009–2013 at Uzlina and BSAFsed values are presented in

*Viviparus viviparus*

**As** <0.05 0.006 <0.05 0.006 7.75 9.30 **Cd** <0.01 – <0.01 – – 0.51 **Cu** 2.61 0.05 2.60 0.05 54.7 47.0 **Cr** <0.01 0.0003 0.42 0.01 29.6 29.2 **Co** 0.11 0.01 0.19 0.02 10.8 9.84 **Fe** 140 – 279 – – 20987 **Mn** 58.7 – 30.0 – – 614 **Ni** 0.34 0.0085 0.58 0.015 40.0 39.3 **Pb** <0.05 0.002 0.15 0.006 26.7 21.3 **Se** <0.09 – <0.09 – – – **Sb** <0.05 – <0.05 – – – **Zn** 0.90 0.006 3.87 0.02 158 96.9 **Mg** 154 – 211 – – –

**BSAFsed Csed\* Csed\***

**2009–2013\*\***

\*\* Csed 2009–2013—average of the metal concentration detected in sediment from 2009 to 2013 [63, 65].

**Table 8.** Metal concentration (mg/kg d.m) in the shell of *Unio pictorum* and *Anodonta cygnea* at Uzlina in September 2013.

In this case, the highest metal concentration was recorded for Cu, Ni, and Zn. The Cu and Ni concentrations from sediment exceed the allowed limit values both in September 2013 and as well as during 2009–2013 monitoring period. As shown in **Table 8**, the *A. cygnea* were found to have a greater capacity for metal accumulation (especially for Cu, As, Cr, Zn) than *U. pictorum* shells.

The bioaccumulation level varied depending on species, metals type, and sampling sites. No significant differences were observed between bioaccumulation factors of Cu, Zn, and Ni calculated for *U. pictorum* collected in July and September. The BSAFsed values were subuni‐ tary maintained. It was observed a difference considering the sampling points, respectively, at Murighiol the bioaccumulative metals impact (Ni and Zn) was greater compared to Uzlina. This aspect may be explained by the dredging works for the canal enlarging/widening to facilitate navigation, allowing a better water circulation from the branch inside the canal.

This preliminary study for the metal bioaccumulation capacity in the shell mollusks from Danube Delta aquatic system showed that essential metals involved in metabolic processes (such as Fe, Mn, Zn, Cu, and Mg) have a greater storage capacity than those toxic (such as Pb and Cd). The statement was also confirmed in other studies [83–85].

All the biota sediment bioaccumulation factors were subunitary, which indicated a slowly bioaccumulation process occurred in the studied aquatic ecosystems.

### **3.4. Preliminary risk assessment**

Risk characterization is required for all chemicals as an estimation of their exposure and adverse effects on the environmental compartment. Generally, this is based on Predicted Environmental Concentration (PEC) and Predicted No Effect Concentration (PNEC) calcula‐ tion, in terms of exposure and assessment of effects [86].

In order to estimate the current contamination of Danube surface water and sediment with metals, we use the average of the measured environmental concentrations (MEC) as PEC values, for the period 2009–2013 at Murighiol and Uzlina. The PNEC value calculation was made using an assessment factor (AF) of 1000 applied for acute toxicity values—LC50 (96 h) or 10 applied for chronic toxicity values—MATC for *C. carpio* (our laboratory tests), which expresses the degree of uncertainty in the actual environmental extrapolation [87]. The risk quotients (RQs) between MEC values and acute or chronic PNECs were calculated, and the level of risk was expressed as: insignificant risk (RQs <0.1); low risk (RQs <1); moderate risk (RQs <10), and high risk (RQs >10). The estimated RQs for the most detected metals in Danube water and sediment (Ni, Cd, Cr, Cu, Pb, and Zn) were summarized in **Table 9**.


\* Average of concentrations in period of 2009–2013; I—insignificant risk; L—low risk; M—moderate risk; H—high risk.

**Table 9.** Estimated acute and chronic RQs at Murighiol (S7) and Uzlina (S8) for *Cyprinus carpio*.

The results showed different levels of risk in accordance with detected environmental concentration of metals, the acute and chronic toxicity and the environmental compartment (water or sediment). In water, Cr and Pb showed insignificant risk; Ni and Zn showed a low risk; and Cd, Cu, and As highlight a moderate risk considering both acute and chronic effects on *C. carpio*. Variations of the RQs depending on sampling location are not observed.

As we expected, the risk level increases within the sediment compartment. The sediment contamination revealed low‐to‐moderate risk, exception for As and Cu. Therefore, Cr and Pb showed low risk; Ni, Cd, Zn and Cu highlighted moderate risk; and As and Cu could express a high risk on fish *C. carpio*. Cu, Zn, and Ni were constantly present in sediment over the set limits of national norms inducing also their accumulation (see the section "*Field tests bioaccumulation*"). No variation is observed of the RQs depending on sampling location. Using long‐term toxicities in PNECs estimation, the RQs increased for Ni, Cd, and Cr and decreased in case of Cu, Pb, As, and Zn, due to the use of a small applied factor (AF = 10) to chronic toxicities.

The results highlighted a pessimistic view concerning the quality of aquatic ecosystem needed to support the carp fish survival. The concern is related to the constantly presence of metal concentrations especially in sediments (the food provider compartment) which could deter‐ minate the bioaccumulation. The same statement was made in a Romanian study named "*Ecotoxicology of heavy metals in Danube meadow*" [55].
