TITLE: Assessing Fluoranthene Impact on Initiation of Feeding Response: A Time-to-Event Experiment with Parhyale hawaiensis
AUTHORS: Ibrahim Lawan

[DESCRIPTION]
This protocol delineates a controlled laboratory experiment employing Parhyale hawaiensis, an amphipod crustacean, as a model organism to assess the influence of Fluoranthene, a polycyclic aromatic hydrocarbon (PAH), on the dynamic chemosensory responses of P. hawaiensis. Using a time-to-event (TTE) methodology, we examine how Fluoranthene exposure affects the timing of crucial behavioural responses essential for organism survival and reproduction. This protocol offers comprehensive step-by-step instructions for conducting TTE experiments, providing valuable insights into the behavioural ecotoxicological impacts of Fluoranthene on aquatic organisms.

[STEPS]
SECTION: Introduction
1. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants originating from diverse anthropogenic sources like oil spills and combustion processes (Diamond et al., 2003; Hylland, 2006). Fluoranthene, a notable PAH, has garnered attention due to its persistence, bioaccumulation potential, and adverse effects on aquatic organisms (Behera et al., 2018). However, the influence of Fluoranthene on the chemosensory behaviour of aquatic organisms, particularly epibenthic amphipods, remains poorly understood. Specifically, the influence of environmentally relevant PAHs, like Fluoranthene, on chemosensory systems and potential alterations in ecological fitness are areas requiring further investigation. Chemosensation plays a pivotal role in various behaviours of crustaceans, including amphipods, such as food location, predator detection, mate finding, and physiological indicators (Hardege et al., 2022). We conducted a controlled laboratory experiment to address this knowledge gap using P. hawaiensis as our model organism. Employing a time-to-event (TTE) methodology, which provides a robust framework for examining the effects of environmental stressors on aquatic organism behaviour and physiology, we aimed to elucidate the impact of Fluoranthene on the chemosensory responses of P. hawaiensis and develop a protocol for TTE analysis in behavioural ecotoxicology. TTE experiments offer crucial insights into how pollutants influence the timing of specific behavioural responses, such as feeding or mating activities, which are vital for the survival and reproduction of organisms in aquatic ecosystems. Thus, this protocol outlines step-by-step procedures for conducting TTE experiments using P. hawaiensis as a model organism and Fluoranthene as the toxicant of interest. 

Objective:
To investigate the impact of Fluoranthene on the dynamic chemosensory responses of P. hawaiensis and establish a protocol for time-to-event (TTE) analysis in behavioural ecotoxicology.
SECTION: 1.	Collection and Sexing:
2. 
Collect adult P. hawaiensis individuals from controlled laboratory culture tanks.
Sex the organisms under a stereomicroscope to ensure precise identification of males and females.
 

SECTION: 2.	Acclimation:
3. 
Acclimate the organisms separately for 48 hours to establish consistent baseline conditions prior to the experiment.
SECTION: 3.	Exposure Setup:
4. 
Prepare stock solutions of Fluoranthene at 5g/L concentration  
Prepare four different environmentally relevant concentrations of Fluoranthene (0, 10, 50, and 250 µg/L) in 1-litre glass beakers containing 500 ml of exposure medium.
Ensure continuous aeration to maintain uniform exposure conditions.
Conduct exposures for one week in a static renewal system without feeding to stimulate appetite.
SECTION: 4.	Mortality Assessment:
5. 
Monitor mortality daily over the exposure period by visually inspecting each experimental container and removing dead individuals.
Record mortality counts for each treatment group.
 
 
 
SECTION: 5.	Y-maze Experiment Setup:
6. 
After the exposure period, select at least twelve (12) surviving amphipods from each treatment group.
Transfer the selected organisms individually to clean, reconstituted seawater in 70 ml Y-maze Petri dishes.
SECTION: 6.	Acclimation to Y-maze:
7. 
Allow the organisms to acclimate for at least three (3) minutes in one arm of the maze.
SECTION: 7.	Feeding Response Recording:
8. 
Introduce the freshly prepared feed tab to the other arm of the Y-maze.
Record the time each organism responds to the feed within a 30-minute window.
NB: Response is actively visiting and starting feeding on the provided feed within the allotted timeframe.
Censor organisms failed to respond within the 30-minute period from the analysis.
 
 

SECTION: 8.	Data Collection:
9.   
Record mortality counts, feeding response times, toxicant concentrations, and sex for each organism.
 
 
 a. Experiment_ID: This column identifies the experiment number or batch in which the data was collected. Each unique experiment is assigned a specific ID.
b. Treatment: This column describes the treatment condition applied to the organisms. It typically includes labels such as "Control" or specific treatment names, indicating whether the organisms were exposed to a toxicant or kept as controls.
c. Organism_ID: This column assigns a unique identifier to each individual organism involved in the experiment. It helps in tracking the responses of each organism across different conditions.
d. Sex: This column indicates the sex of the organism, categorised as "Male" or "Female."
f. Toxicant_Conc_µg/L: This column specifies the toxicant concentration (Fluoranthene) the organisms were exposed to.
g. Start_Time: This column records the start time of the observation period for the time-to-event (TTE) experiment.
h. End_Time: This column notes the end time of the observation period or the event of interest, such as feeding response.
i. Response_Time: This column calculates the time the organism takes to respond to the feed, which is the difference between the End_Time and Start_Time.
j. Status: This column indicates the status of the response, typically coded as binary values where "1" represents an event occurrence (e.g., successful initiation of feeding) and "0" represents a censored event (e.g., no response within the allotted time).
SECTION: 9.	Data Analysis:
10. 
To assess the effects of fluoranthene exposure on P. hawaiensis, perform appropriate statistical analyses, such as Kaplan-Meier estimates or Cox proportional hazards models time-to-event (TTE) analysis for feeding response time and Poisson logistic regression for mortality count.
Explore relationships between toxicant concentrations, sex, mortality, and feeding response times using appropriate statistical software, such as R.
Discuss any sex-specific differences observed in the responses.
SECTION: 10.	General Considerations:
11. a. Standardise all experimental conditions, including temperature, lighting, and water quality parameters, to minimise variability.
b. Conduct pilot experiments to optimise the protocol and troubleshoot any potential issues.
c. Ensure the ethical treatment of organisms throughout the experiment, following institutional guidelines and regulations.
SECTION: Conclusion
12. This protocol provides a comprehensive framework for conducting time-to-event experiments in aquatic ecotoxicology, specifically focusing on the behavioural responses of P. hawaiensis to Fluoranthene exposure. By following these standardised procedures, this protocol contributes to advancing robust analysis of behavioural ecotoxicology data and gaining insights into the impacts of pollutants on aquatic organism behaviour and survival.