The M. aquaticum test organisms were taxonomically identified by using a specific key for aquatic macrophytes. They were cultivated in the emersed state under the same conditions regarding sediment, light, and temperature as the test conditions. They were irrigated with a Steinberg medium diluted 1:1 with deionized water (see Table 5). The two test substances atrazine and 2,4-D were tested in three different M. aquaticum biotests. The first test used emersed test organisms with no adaptation to the submerged state. The second test used plants, which were 7 days adapted to the submerged state before the test started. The third test used plants, which were 28 days adapted to the submerged state before the test started. For the adaptation phase, the head whorls of the plants (length 8 cm) were put in artificial sediment in a 40-mL beaker and were submerged into the Steinberg medium diluted 1:1 with deionized water. Similarly for the tests, three head whorls (length 6 cm) in 40-mL beakers with 50 g of artificial sediment were put in a high 2,000-mL beaker with 1,500 mL Steinberg medium diluted 1:1 with deionized water (see Figure 4). To avoid floating of the head whorls, the artificial sediment was weighed with 2 g of quartz sand. The artificial sediment was composed of OECD sediment, saturated with the Steinberg medium according to. The tests were performed in climate chambers with a constant temperature of 22 ± 2°C and a light/dark rhythm of 16/8. The light intensity was about 6,000 Lux. The atrazine application was performed with 0.009% ethanol as a solvent. The 2,4-D application was performed without a solvent. The nominal concentrations of atrazine were 10, 40, 160, and 640 μg/L. The nominal concentrations of 2,4-D were 7, 31.25, 125, 500, and 2,000 μg/L. Three 2,000-mL beakers each with three test organisms were used for one treatment group, control group (C), or solvent control group (SC). Thus, each treatment group consists of three replicates, and in total, nine individual plants are respectively pseudoreplicates. The duration was 7 days for all six tests.
The growth rates of the following endpoints were determined:
fresh weight (g)
shoot length (cm)
root length by the longest root (cm)
The average specific growth rate for fresh weight and shoot length is calculated according to the following formula:
Because for root length Ns would be 0, only a linear growth rate calculation is possible according to the formula:
μ is the average specific growth rate; Ns, the fresh weight or shoot length at the start of the test; and Ne, the fresh weight, shoot length, or root length at the end of the test.
Measurements of the quantum yield
At the start and the end of the atrazine tests, measurements of the quantum yield of the PSII were performed using a Mini-PAM (Walz GmbH, Effeltrich, Germany) with a leaf clip extension. The principles of this method are described in[38–40]. The quantum yield of the PSII correlates with photosynthetic carbon assimilation and indicates inhibition of photosynthetic activity by a stressor[41, 42]. For these measurements, the test organisms had to be taken off the medium.
The average specific quantum yield of the PSII is calculated according to the following formula:
is the average specific quantum yield of the PSII; Fm/, the maximum fluorescence, and Ft, the steady state fluorescence.
The percentage inhibition of growth rate regarding fresh weight, shoot length, and root length is calculated for each treatment group with the following formula:
%IGR is the percentage inhibition of growth rate; μSC, the average specific growth rate of the solvent control group; μC, the average specific growth rate of the control group; and μTG, the average specific growth rate of the treatment group.
Percentage inhibition of the quantum yield of PSII is calculated for each treatment group with the following fomula:
where %IQY is the percentage inhibition of the quantum yield of PSII;, the average specific quantum yield of PSII of the solvent control group; and, the average specific quantum yield of PSII of the treatment group.
The lowest observed effect concentrations (LOEC) and the no observed effect concentrations (NOEC) were determined using Dunnet’s multiple comparison test (one-way ANOVA). The EC50 estimations were conducted by non-linear regression analysis of the concentration-response curve. The data analysis in this study was performed with the software Graphpad Prism 5.0.
Analysis of atrazine and 2,4-D was based on and. Samples were taken 1 h after 2,4-D and atrazine application of the test and were refrigerated at −20°C. In the laboratory, 0.5 mL of the water samples was mixed with 0.5 mL methanol. The stock solutions and the highest test concentration of the test substances atrazine and 2,4-D were analyzed by high performance liquid chromatography, Agilent 1200, Santa Clara, CA, USA) with a Merck Supersphere C18E column (Darmstadt, Germany). As the mobile phase for the 2,4-D measurements, water, acetonitrile, and an acetonitrile buffer (pH 2.01) were used in a gradient of 40 to 70% acetonitrile in 15 min. The flow rate was 0.35 mL/min. The oven temperature was set at 40°C. The detection of 2,4-D was carried out at a wavelength of 227 nm. The mobile phase for the substance atrazine consisted of 68.5% water and 31.5% acetonitrile. The flow rate was 1 mL/min. The oven temperature was set at 40°C. The detection of atrazine was carried out at a wavelength of 225 nm. All concentration calculations were based on external standard samples. The measured samples were in the range of the calibration curves.