*3.2.1.4. Data processing and statistics*

The acute effect concentration values in the fish and crustacean tests were calculated using probity analysis method, based on exponential regression relationship between cumulative percentages of mortality (expressed as probity units) for each exposure period against logarithmic concentrations of test substance. For each result, standard deviations were calculated.

### *3.2.2. Results and discussion*

### *3.2.2.1. Fish toxicity*

Acute toxicity tests provide a measure of toxicity for a target species under specific environ‐ mental situations and could suggest a rapid and severe effect of contaminants. Acute and chronic toxicity test mimicked the metals accidental release or long‐term accumulation in sediment [67–70]. The carp fish LC50‐96h values showed different responses in direct corre‐ lation with the metals type and concentration. The LC50‐96h values were 0.16, 0.28, 0.31, and 0.40 mg/L for Cd, Ti, Zr, and As (**Figure 4**), 2.17, 12.2, 30.10, and 65.8 mg/L for Cu, Zn, Pb, and Ni (**Figure 5**), 120, and 758 mg/L for Cr and Sb, obtained from two replicates for each metal (**Table 4**).

*3.2.1.1. Sample preparation*

68 Water Quality

6.5 and 8.5 units.

*3.2.1.2. Fish toxicity test procedure*

*3.2.1.3. Crustacean toxicity test procedure*

registered after 24 and 48 h.

*3.2.2. Results and discussion*

*3.2.2.1. Fish toxicity*

calculated.

*3.2.1.4. Data processing and statistics*

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

Using OECD methodologies for acute toxicity, the lethal concentrations for 50% of tested organisms were estimated. Metals' long‐term toxicities on fish were conducted using an in‐ house methodology based on the changes in some physiological indicators such as growth rate, mortality, biomass, production, food use and biochemical indicators, hepatic enzyme

The toxicity test determined the metal concentration that immobilizes or kills 50% (LC50) of *D. magna* crustacean, after chemical exposure at 20°C ± 2°C in the dark for 24 or 48 h. The test procedure was performed according to OECD 202 using the microbiotest Daphoxkit F Magna provided by MicroBioTests Inc., Belgium. Briefly, the test was performed in three replicates, in multiwall test plates (six rinsing wells and 24 wells for toxicant dilutions) using 20 organisms per each concentration (at least five different concentrations for each metal) and control (untreated standard freshwater). The mortality/immobility percentage of organisms was

The acute effect concentration values in the fish and crustacean tests were calculated using probity analysis method, based on exponential regression relationship between cumulative percentages of mortality (expressed as probity units) for each exposure period against logarithmic concentrations of test substance. For each result, standard deviations were

Acute toxicity tests provide a measure of toxicity for a target species under specific environ‐ mental situations and could suggest a rapid and severe effect of contaminants. Acute and chronic toxicity test mimicked the metals accidental release or long‐term accumulation in sediment [67–70]. The carp fish LC50‐96h values showed different responses in direct corre‐

activity, respectively. **Table 3** presents the technical parameters of fish toxicity tests.


**Table 3.** Test conditions of acute and chronic toxicity tests.

**Figure 4.** Acute and chronic toxicity of Ti, Zr, Cd, and As classified in very toxic class for *Cyprinus carpio*.

**Figure 5.** Acute and chronic toxicity of Zn, Cu, Pb, and Ni classified in toxic class for *Cyprinus carpio*.

According to Global Harmonization System for chemical classification and labeling, Cd, Ti, Zr, and As were the most toxic metals for fish. Cd, Ti, Zr, and As showed to be very toxic compared with the other analyzed metals. Research studies revealed similar acute toxicity intervals: 6.16–47.58 mg/L for Ni, 0.15–21.4 mg/L for Zn, 0.28–34.5 mg/L for Cu, 0.005–7.92 mg/ L for Cd and 90 to >139 mg/L for Cr [71]. The maximum acceptable toxicant concentration (MATC) is a value calculated from chronic toxicity tests [72] in order to set water quality norms for aquatic life protection.


a Governmental Decision no. 351/2005 concerning the hazard chemical discharge.

b Directive 2008/105/EC on environmental quality standards in the field of water policy.

c National Plan of River Basin Management (2016-2021)—Annex 6.1.3B.

d According to REACH 1907/2006; Regulation (EC) 1272/2008; Regulation (EU) 286/2011; Global Harmonization System for chemical classification and labeling (GHS) Revision 2011. The toxicity class was decided on the highest toxicity of target organisms.

**Table 4.** In-house toxicity data of metals for fish and crustacean in relation with the national and international norms for metals limits in surface water.

Experimental exposure of fish for 60 days to different concentrations of metals revealed different long-term effects. The final results showed no effects concentrations on target organisms, assessment of environmentally safe concentrations, respectively. The calculation of MATC values started by multiplication of the LC50-96h of each metal with an application factor of 0.1 (**Table 2**). The monitored physiological parameters from chronic test revealed that Cd is non-toxic at 0.001 mg/L, Ti, Zr, and As were safety to 0.005 mg/L, Cu at 0.05 mg/L, Sb at 0.06 mg/L, Ni at 0.10 mg/L, Zn at 0.60 mg/L, Cr and Pb at 1.00 mg/L, comparative with the controls (**Figures 4** and **5**, **Table 4**). Similar values for Cu (0.012 mg/L) and Zn (0.5 mg/L) were also obtained in other studies [73].

### *3.2.2.2. Crustaceans toxicity*

**Figure 4.** Acute and chronic toxicity of Ti, Zr, Cd, and As classified in very toxic class for *Cyprinus carpio*.

**Figure 5.** Acute and chronic toxicity of Zn, Cu, Pb, and Ni classified in toxic class for *Cyprinus carpio*.

for aquatic life protection.

70 Water Quality

According to Global Harmonization System for chemical classification and labeling, Cd, Ti, Zr, and As were the most toxic metals for fish. Cd, Ti, Zr, and As showed to be very toxic compared with the other analyzed metals. Research studies revealed similar acute toxicity intervals: 6.16–47.58 mg/L for Ni, 0.15–21.4 mg/L for Zn, 0.28–34.5 mg/L for Cu, 0.005–7.92 mg/ L for Cd and 90 to >139 mg/L for Cr [71]. The maximum acceptable toxicant concentration (MATC) is a value calculated from chronic toxicity tests [72] in order to set water quality norms

Toxicity tests on *D. magna* crustaceans showed various toxicities of metals; the LC50-48h showed 0.14 mg/L for Cd, 0.81 mg/L for Cr, 5.56 mg/L for Ti, 91.2 mg/L for Zr, and 148 mg/L for Sb. Cd and Cr showed the highest toxicities and were classified in very toxic chemicals class for *Daphnia* sp. (**Figure 6**). Similar literature values were reported for Cr between 0.02 and 0.05 mg/L [71] and for Cd between 0.024 and 0.355 mg/L [45].

**Figure 6.** Acute toxicity of Ti, Zr, Cd, Cr, and Sb for *Daphnia magna*.

The surface water quality norms require specific limits only for few very toxic and toxic metals. For example, Ti, Zr, Cd, and Pb norms are not established by the National Plan of River Basin Management—Annex 6.1.3B, despite of their acute toxic effects at very low concentrations (**Table 4**). Also the Directive 2008/105/EC on environmental quality standards in the field of water policy sets limits only for Ni, Cd, and Pb. The present limits assure the protection of aquatic organisms, especially for fish and planktonic crustaceans.

### **3.3. Field test: bioaccumulation**

In order to assess the impact of metals in the field, the following sections present some preliminary data concerning the metal bioaccumulation into benthic invertebrates (mollusks).

### *3.3.1. Materials and methods*

### *3.3.1.1. Studied area characterization*

The studied area was focused on a highly sinuous channel, located on the southeast area of the Danube Delta (Sf. Gheorghe Branch) receiving 22% of Danube's water flow. The Sf. Gheorghe Branch has a width varying between 150 and 550 m, and the water depth varies between 3 and 27 m. The sampling sites location was selected taking into consideration the changes in the Sf. Gheorghe Branch morphology as a result of the pressure from anthropic and environmental factors. Iron Gates I dam construction on Danube River led to a 10% decrease in the suspended sediment amount at Isaccea station. Moreover, the Iron Gates II dam building induced a 50% decrease in suspended sediment at Isaccea. These constructions alongside meander modification (during the years 1984–1988) have produced major changes in sediment distribution. The establishing of space location was performed using GPS type system map 60CSx—Garmin [74].

In addition, the anthropic activities undertaken to strength the banks against coastal erosion led to meanders cutoff, which in turn caused continuous biotope degradation. These changes negatively impacted the ecosystem functions by reducing the structure of the main and constant ecological communities, the benthic invertebrates. So that, to characterize metal bioaccumulation (in benthic invertebrates), two representative sampling sites were selected considering the pressure resulted from anthropic and environmental factors (Murighiol and Uzlina)—**Figure 7**. At temporal scale, this study was conducted during summer and autumn of 2013.

**Figure 7.** Location of sampling sites in Danube Delta (Sfantu Gheorghe Branch) (St 1—Murighiol; St 2—Uzlina).
