## 2 Materials and Methods
### Testing  
Nominal concentrations for the 3,5-dichlorophenol reference toxicant were selected based on LC50 data from ISO 14669:1999 [33].  
The study includes several treatment groups (Table 1), each with specific goals to assess the toxicological impact of the reference toxicant on _A. tonsa_.  
* Control (0.00 mg/L): Establishes baseline conditions using seawater, serving as a reference to compare toxicant effects.
* 0.10 mg/L: Tests the minimum concentration where toxicant effects begin, aiming to identify the sensitivity threshold.  
* 0.32 mg/L: Assesses moderate toxicant exposure, expecting noticeable biological impacts.
* 1.00 mg/L: Evaluates higher exposure levels, anticipating significant adverse effects or increased mortality.
* 3.20 mg/L: Tests high concentrations, expecting severe impacts, including near-complete mortality.
* 10.00 mg/L: Determines the maximum impact, likely resulting in 100% mortality.  
Collectively, these groups allow for a detailed analysis of the toxicant's effects across concentrations, supporting the research objective of understanding its environmental impact and establishing safe exposure levels.  
Following the ISO guidelines [33], each experimental replicate (\(N=4\)) consisted of 5 organisms, leading to a total of 20 organisms per treatment group.  
A reference stock solution was prepared one day before testing. To make this solution, 0.1 g of 3.5-dichlorophenol was added to SW in a 100 mL calibrated glass flask and mixed on a stirrer plate at 500 RPM for 10 min, resulting in a stock solution of 1 g/L 3,5-dichlorophenol.  
A dilution series of the reference toxicant (0 mg/L, 0.1 mg/L, 0.32 mg/L, 1 mg/L, 3.2 mg/L, and 10 mg/L) was prepared by diluting the stock solution with SW in six 500 mL calibrated flasks, labeled from C0 to C5. The flasks were filled with 0 mL, 0.5 mL, 1.6 mL, 5 mL, 16 mL, and 50 mL of stock solution, respectively (Table S1).  
The test chambers consisted of 50 mL cell culture flasks made of polystyrene, closed with screw caps made of polypropylene to minimize contamination, evaporation, and volatilization. Five copepods, at copepites V and adult stages, were transferred via pipette (Figure S7) to each chamber containing 50 mL of the test solution with headspace.  
Test chamber positions in the test area and organism assignments were determined using a randomized [60] block design (Figure S8). Each test chamber was labeled with species initials, treatment group, and replicates (Figure S8).  
Before the experiment began, light intensity was checked using an HD 2302.0 Delta OHM light meter.  
The exposure duration was 48 \(\pm\) 1 h. Observations for immobilization were performed and recorded every 24 \(\pm\) 1-h. Immobilization was defined as the lack of swimming ability within 10 sec after gentle agitation of the test chamber.  
Temperature and lighting conditions were monitored daily. A photoperiod of 16 h of light and 8 h of dark was maintained in the exposure area, provided by LED lamps.  
Water quality parameters, temperature, pH, salinity, and dissolved oxygen were measured at the beginning and end of the experiment.  
The results will be considered as valid if the following conditions are satisfied together:  
(a) the dissolved oxygen concentration at the end of the test is greater than or equal to 4 mg/L;  
(b) the percentage mortality of the controls is less than or equal to 10%;  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Treatment** & **Concentration** & **Description** & **Number of** \\
**Group** & **mg/L** & & **Organisms * \\ \hline
1 & 0.00 & SW & 20 \\ (Control) & & & 20 \\ \hline
2 & 0.10 & & 20 \\ \hline
3 & 0.32 & SW & 20 \\ \hline
4 & 1.00 & and & 20 \\ \hline
5 & 3.20 & reference toxicant & 20 \\ \hline
6 & 10.00 & & 20 \\ \hline \hline \end{tabular}  
* Five organisms per replicate.  
\end{table}
Table 1: Test design.  
3. the toxicity of the reference chemical is within the documented range [33]. Upon completion of the study, all test organisms were discarded, and the entire experimental process was stopped.