## 4 Discussion  
### Toxicity of Biochar Elutriates to Aquatic Invertebrates  
This experiment was conducted to investigate the toxicity on four aquatic invertebrate species exposed to four different BCs. We discovered that the respective BCs, despite complying with international certification standards such as the EBC, can induce adverse effects to non-target organisms in the form of acute toxicity, as was the case with _Lecane quadridentata_, _Moina macrocopa_, and _Daphnia magna_. The ciliate _Paramecium caudatum_, in contrast, did not show any chronic or lethal toxicity when exposed to the BCs. Acute toxicity was only detected if the organisms were exposed to the pure BC elutriate. When the organisms were exposed to elutriate obtained from a BC-soil mixture in ratio 1:8, no chronic and no lethal effects to all tested species were observed. The results show that the application rates have a decisive influence on the soil biota. If users follow standards that regulate BC additions to the soil (e.g., BBodSchG), the potentially harmful effects on rotifers and cladocerans can be most widely diminished. Nonetheless, the compliant use of certified BC does not guarantee 100% safety, particularly near sensitive habitats or with regard to BC utilization in animal feed. In the living environment, the toxicants are mixed, blended and, in many cases, occur at low concentrations that may be regarded as non-adverse. [PERSON] et al. [56] showed that one toxicant, even at no observable effective concentration (NOEC), can cause an increase in the toxicity of the other (synergistic effect). With regard to the present results, it remains unclear if a single hazardous substance evidenced in the BCs or the sum of hazardous substances is responsible for the detected toxicities in rotifers and cladocerans. Based on the concentration of pollutants known to be hazardous to aquatic invertebrates (e.g., PAHs, PCDD/Fs, and heavy metals (especially zinc, copper, and manganese)) [16; 33; 56; 57; 58], the four \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Treatment** & _Lecane_ & _Moina_ & _Paramecium_ \\  & **Mean Value \(r\)** & **Mean Value \(r\)** & **\% Inhibition** \\ \hline Control & 0.371 & 0.408 & 0 \\ BC 1 + soil & 0.356 & 0.400 & 1.99 \\ BC 2 + soil & 0.358 & 0.402 & 0.66 \\ BC 3 + soil & 0.356 & 0.402 & 2.65 \\ BC 4 + soil & 0.359 & 0.402 & 0.66 \\ Soil & 0.358 & 0.406 & 1.32 \\ \hline \hline \end{tabular}
\end{table} 
Table 10: Results of the growth inhibition and reproductive tests with _Lecane quadridentata_ (Lecane), _Moina macrocopa_ (Moina), and _Paramecium caudatum_ (Paramecium) exposed to the BC:soil (1:8) elutriate. \(n=5\). \begin{table}
\begin{tabular}{c c} \hline \hline
**Treatment** & **Mean Value \(r\)** \\ \hline Control & 0.408 \\ BC 1 & 0.401 \\ BC 4 & 0.401 \\ Soil & 0.406 \\ \hline \hline \end{tabular}
\end{table} 
Table 9: Reproductive test with Moina macrocopa (7 days). \(n=5\). BCs could be classified in their potential danger to each other in a direct comparison. The result was the following order and its toxicity potential: BC 3 \(<\) BC 2 \(<\) BC 1 \(<\) BC 4 (from low to high toxicity potential). Thus, BC 4 is expected to generate the potentially lowest LC\({}_{50}\) values, causing the highest toxicity. BC 3 instead is expected to tendentially have the highest LC\({}_{50}\) values, causing the lowest toxicity to the organisms. Interestingly the BC with the highest concentrations of hazardous substances, which undeniably was BC 4, did not provoke the highest toxicological impact on the organisms. Instead, BC 2, which is probably a \"cleaner\" and with regard to EBC thresholds (e.g., PAHs) an uncritical BC, had the lowest LC\({}_{50}\) values both for \(L\). _quadridentata_ and _M. macrocopa_. The EBC analysis did not indicate a very particular high concentration of any specific contaminant for BC 2, nor for the other BCs, which could be an explanation for the incongruuous order of toxicity.