**3.1. Occurrence of metals in surface water and sediments**

Several monitoring studies performed by INCD ECOIND Bucharest researchers during 2003–2013 in the Danube Delta—Sfantu Gheorghe Branch (sampling points: Mahmudia, Murighiol, and Uzlina) emphasized some heavy metal concentration patterns in the study area (**Table 2**). The metal concentrations in water were within the limits of Romanian legisla‐ tions, for class I and class II quality (according to the EU‐WFD and the requirements set by the Romanian Law 310/2004 which amends the Law 17/1996). Cu and Ni showed the highest total concentration (**Table 2**, marked lines) among the determined metals.


Min—the minimum detected concentration; Max—the maximum detected concentration; SD—standard deviation.

**Table 2.** Occurrence of metals in water of Danube Delta—Sf. Gheorghe (2003–2013) in µg/L [63, 64].

The studies revealed that metals Cu, Pb, Zn, Cr, Ni, Cd, Mn, and Fe were the most abundant in the sediments of the Danube Delta—Sf. Gheorghe Branch sampling sites. The concentra‐ tions of these metals ranged with the sampling location and seasonal or natural events, as follows: Cu 4.65–194 mg/kg d.m (dry matter), Pb 4.76–51.3 mg/kg d.m., Zn 17.7–218 mg/ kg d.m., Cr 7.5–61.9 mg/kg d.m., Ni 10.8–111 mg/kg d.m., Cd <0.01–1.5 mg/kg d.m. (**Figure 3**). The average value in the period 2009–2013 for Mn was 614.03 mg/kg d.m. and for Fe, it was 20 987 mg/kg d.m. [64, 65].

Alongside metal concentration, several chemical (nutrients, oxygen and pH regime, pesticides, petroleum products, polychlorinated biphenyls) and biological (phytoplankton, zooplankton, and benthic macroinvertebrates) elements were investigated, showing that the organochlorine pesticides and petroleum products exceeded the maximum allowed limits [63, 66].

In addition, other studies performed along Romanian rivers showed that the mining activities had a great impact on sediment ecosystems due to metal pollution. For instance, a study performed during 2003 in Baia Mare (in North Vest of Romania) mining area after a pollution accident showed a high content of heavy metals in Somes River sediment (Cu 104–339 mg/kg, Pb 59–465 mg/kg, Zn 56–2060 mg/kg, Cd 0.05–14.14 mg/kg, CN 0.33–15.86 mg/kg). The detected concentration affected the aquatic ecosystem where the microalgae species disap‐ peared and the number of fish species decreased dramatically compared to the period before the incident. Also many species of mollusks disappeared because their capacity to accumulate large amount of heavy metals was exceeded [67]. In addition, in Rosia Montana area (in the West part of Romania), significant water contamination with heavy metals occurred due to the mining acidic waters from area on two water courses: Rosia and Corna stream. The results showed exceedances of Cu, Cd, Fe, Ni, and Cr, in particular in the Rosia Montana water stream [68]. Along Jiu River (in south of Romania) sediments, heavy metal pollution in most sampling points was recorded according to the pollution load index (PLI) [69].

**Figure 3.** Occurrence of metals in sediments of Danube Delta—Sf. Gheorghe (2003–2013) (average values). S1—Mah‐ mudia, S2—Murighiol, and S3—Uzlina [65].

In the above context, in the following sections will be presented some data concerning the metals effects on freshwater organisms (fish, planktonic crustacean, and mollusks), obtained through laboratory testing or by biological samples collected from contaminated fields.

### **3.2. Laboratory tests: acute and chronic effects of metals**

### *3.2.1. Materials and methods*

Murighiol, and Uzlina) emphasized some heavy metal concentration patterns in the study area (**Table 2**). The metal concentrations in water were within the limits of Romanian legisla‐ tions, for class I and class II quality (according to the EU‐WFD and the requirements set by the Romanian Law 310/2004 which amends the Law 17/1996). Cu and Ni showed the highest

**Min Max Average SD Min Max Average SD Min Max Average SD Ni** <1.0 24.0 **4.02** 5.79 <1.00 68.1 **12.4** 18.6 <1.00 10.3 **2.60** 2.85 **Fe** <20 880 **300** 270 112 3400 **710** 750 80.0 1040 **350** 280 **Mn** <2.0 30.0 **10.0** 10.0 3.00 290 **70.0** 80.0 5.00 50.0 **20.0** 10.0 **Cd** 0.40 0.40 **0.40** 0.00 <0.10 0.50 **0.36** 0.14 <0.10 0.50 **0.37** 0.13 **Cr** <0.5 6.00 **2.09** 1.74 <0.50 21.0 **5.38** 6.00 <0.50 21.0 **3.52** 5.33 **Cu** 2.50 10.5 **5.74** 2.67 0.012 55.3 **12.9** 17.8 0.03 123 **14.5** 26.7 **Pb** <2.0 3.20 **2.08** 0.31 <2.00 5.00 **2.15** 1.29 <2.00 5.00 **2.17** 1.27 **As** <2.0 2.20 **2.01** 0.05 <2.00 3.90 **1.82** 0.88 <2.00 2.64 **1.73** 0.59 **Hg** <0.1 0.24 **0.32** 0.06 <0.10 0. 77 **0.15** 0.20 <0.10 0.14 **0.22** 0.10 **Zn** <2.0 24.7 **10.1** 7.07 <2.00 56.0 **9.58** 11.4 <2.00 57.0 **8.15** 11.7 **Co** <0.5 1.30 **0.66** 0.33 <0.50 5.00 **1.17** 1.65 <0.50 5.00 **1.18** 1.65 Min—the minimum detected concentration; Max—the maximum detected concentration; SD—standard deviation.

total concentration (**Table 2**, marked lines) among the determined metals.

**Metal Mahmudia (2009–2013) Murighiol (2003–2013) Uzlina (2003–2013)**

**Table 2.** Occurrence of metals in water of Danube Delta—Sf. Gheorghe (2003–2013) in µg/L [63, 64].

was 20 987 mg/kg d.m. [64, 65].

66 Water Quality

The studies revealed that metals Cu, Pb, Zn, Cr, Ni, Cd, Mn, and Fe were the most abundant in the sediments of the Danube Delta—Sf. Gheorghe Branch sampling sites. The concentra‐ tions of these metals ranged with the sampling location and seasonal or natural events, as follows: Cu 4.65–194 mg/kg d.m (dry matter), Pb 4.76–51.3 mg/kg d.m., Zn 17.7–218 mg/ kg d.m., Cr 7.5–61.9 mg/kg d.m., Ni 10.8–111 mg/kg d.m., Cd <0.01–1.5 mg/kg d.m. (**Figure 3**). The average value in the period 2009–2013 for Mn was 614.03 mg/kg d.m. and for Fe, it

Alongside metal concentration, several chemical (nutrients, oxygen and pH regime, pesticides, petroleum products, polychlorinated biphenyls) and biological (phytoplankton, zooplankton, and benthic macroinvertebrates) elements were investigated, showing that the organochlorine

In addition, other studies performed along Romanian rivers showed that the mining activities had a great impact on sediment ecosystems due to metal pollution. For instance, a study performed during 2003 in Baia Mare (in North Vest of Romania) mining area after a pollution accident showed a high content of heavy metals in Somes River sediment (Cu 104–339 mg/kg, Pb 59–465 mg/kg, Zn 56–2060 mg/kg, Cd 0.05–14.14 mg/kg, CN 0.33–15.86 mg/kg). The detected concentration affected the aquatic ecosystem where the microalgae species disap‐

pesticides and petroleum products exceeded the maximum allowed limits [63, 66].

The assessment of metals acute and chronic effects was based on fish (*C. carpio*) and planktonic crustacean (*Daphnia magna*) laboratory data. The tested organisms were those recommended by the international ecotoxicology protocols (OECD or ISO), and they are frequently found in Romanian surface waters, easily to acclimatize in laboratory and sensitive to various contam‐ inants. *C. carpio* are in particular the most affected organisms due to the fact they ingest both planktonic and benthic organisms, respectively, and thus, they especially accumulate the contamination from water and sediment. The tests were performed on the following metals: Ni, Zn, Cu, Cd, As, Cr, Pb, Sb, Mn, Ti, and Zr, which were usually detected in the aquatic systems.

### *3.2.1.1. Sample preparation*

For stock solution preparation, a known quantity of metals test as NiSO4, ZnSO4, CuSO4, CdCl2/CdSO4, As2O3, K2Cr2O7, Pb(NO3)2, SbCl5, MnCl2x4H2O, TiO2, ZrCl4 was dissolved into the specified volume of dilution water or growth medium. No added solvents have been used, and all substances have been tested under their maximum solubility. The solutions were stirred for 24 h, in the dark at 25°C. The testing solutions were prepared by mixing the appropriate volumes of stock solution with dilution water or growth medium in order to obtain the final concentrations used for testing. Finally, the pH values of tested solutions were situated between 6.5 and 8.5 units.
