TITLE: Standardised Methods for Feed Tabs Preparation to Assess Feeding Rate and Exposure to Insoluble Substances in Parhyale hawaiensis
AUTHORS: Ibrahim Lawan, Dominique MJ Anderson, Theodore, B. Henry

[DESCRIPTION]
This protocol outlines standardised procedures for preparing feed tabs to assess feeding rates and conduct dietary toxicity testing of insoluble substances in Parhyale hawaiensis. By addressing the need for reliable testing methodologies, particularly those for evaluating the effects of insoluble pollutants on aquatic organisms, this protocol offers a robust framework for researchers. It facilitates comprehensive ecotoxicological studies, contributing to a better understanding of the environmental risks of toxicants (soluble or insoluble) using feeding metrics in marine ecosystems. This protocol uses benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), as a model toxicant because of its ecotoxicological concerns and physicochemical properties (e.g. low water solubility [0.00162 mg/L at 25°C], high lipid–water partition coefficient [log Klipw 7.35], and high octanol-water partition coefficient [log Kow 6.13]). Because BaP has low solubility, toxicity investigations must include assessment via diet as a more realistic and reliable method to assess its effects on aquatic organisms rather than just aqueous exposure.

[STEPS]
SECTION: Introduction
1. Dietary exposure and toxicological assessments of insoluble substances represent emerging frontiers in aquatic ecotoxicology, necessitating the development of standardised bioassays that accurately reflect real-world exposure scenarios (Connon et al., 2012). Current toxicity testing methodologies primarily rely on soluble substances introduced into the aqueous phase, which inherently limits their applicability for evaluating the effects of insoluble compounds (Gotz et al., 2021). Unlike their soluble counterparts, insoluble substances exhibit distinct chemical properties that lead to heterogeneous distribution and non-uniform uptake routes within aquatic environments (Hartmann et al., 2015). Therefore, there is a critical need to adapt test scenarios to address these challenges effectively. Dietary uptake has emerged as the predominant route of exposure to insoluble substances in aquatic organisms; however, this pathway is often overlooked in conventional testing protocols (Bundschuh et al., 2019). To address these limitations, we propose a standardised protocol utilising decomposition and consumption tablets (DECOTABs) (Gotz et al., 2021; Kampfraath et al., 2012) as a reliable food source for Parhyale hawaiensis, thereby facilitating systematic and rigorous toxicity testing via the oral pathway.

Parhyale hawaiensis, a tropical marine amphipod, has significant ecological importance and is a valuable model organism in various scientific disciplines (Paris et al., 2022). Recognised for its sensitivity to environmental stressors, P. hawaiensis has emerged as a promising candidate for tropical marine studies and laboratory investigations. Previous studies have established protocols for laboratory culturing, acute toxicity testing in water and sediment, internal dose determination, gene expression analysis, and assessment of DNA damage and chromosomal alterations in P. hawaiensis (Dos Santos et al., 2022). Leveraging these advancements, our objectives are twofold: 1) to develop a protocol for chronic toxicity evaluation with a particular focus on feeding rate by modifying the decomposition and consumption tablets (DECOTABs), and 2) to standardise a protocol for preparing feed tabs tailored for the toxicity testing of insoluble substances, with benzo(a)pyrene (BaP) serving as a model toxicant. The physicochemical properties of BaP, including its low water solubility (0.00162 mg/L at 25°C), high lipid–water partition coefficient (log Klipw 7.35), and octanol-water partition coefficient (log Kow 6.13), underscore its profound insolubility and facilitate its bioaccumulation and toxicity in aquatic organisms (Hylland, 2006; Latimer & Zheng, 2003; Pampanin & Sydnes, 2013). In addition to addressing the necessity for reliable testing methodologies to evaluate the effects of insoluble pollutants in aquatic organisms, our protocol is poised to make significant contributions to ecotoxicology, especially when assessing the effects of both soluble and insoluble substances using feeding metrics.
SECTION: Step I – Preparing Standard Feed Tabs
2. Prepare feed tabs with a total weight of five (5 g). All measurements below can be scaled up or down based on the target feed weight.
SECTION: Step I – Preparing Standard Feed Tabs
2.1. a.	Mix 30 mL Milli-Q water and 1.2 g agar (Fisher Scientific) in a 300-ml glass beaker (NB: use larger beaker when a higher mass of agar is used as it foams and spills during heating). 
 

SECTION: Step I – Preparing Standard Feed Tabs
2.2. b.	Heat the mixture in a microwave to about 100 °C to dissolve the agar for approximately45 s to 1 min until it foams.

SECTION: Step I – Preparing Standard Feed Tabs
2.3. c.	Add 1.08 g cellulose (Sigma-Aldrich) and 2.72 g small granular feed supplement (52% crude protein and 12% lipid) and homogenise for 1 min.

 

 

SECTION: Step I – Preparing Standard Feed Tabs
2.4. d.	Before cooling, pour the mixture into 36 cylindrical moulds of 1.1 cm diameter and 0.6 cm height (custom-built aluminium device). 

 
(NB: Aquatic animals, such as amphipods and fish, do not utilise cellulose because it is indigestible in their gut (Ighwela et al., 2012). Therefore, cellulose is nutritionally unavailable to these aquatic organisms and is used as a dietary binder, non-nutritive filler, anti-caking agent, bulking agent, and stabiliser. It is soluble in ether but insoluble in water).

SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3. As described below, the selected insoluble substance, such as BaP, can be added to the feed supplement before adding agar. 
SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3.1. a.	Sum up the weights of the agar, cellulose, and feed supplement to know the total weight and determine the concentration of the insoluble substance that can be added per gram of diet.

SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3.2. b.	Prepare the BaP-supplemented diet in a 300-ml glass beaker by combining the 2.72 g of granulated feed supplements and 10 mL acetone with or without BaP (NB: high volume of carrier solvent was used to facilitate even distribution of toxicant).

SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3.3. c.	Prepare diets with 0 (control), acetone-treated (control), and 50, 250, or 1250 μg BaP (nominal)/g diet and mix for approximately 2 min (NB: concentrations can be changed based on the insoluble substance used and the determined effective concentration).

SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3.4. d.	Keep the mixture under a fume cupboard to evaporate acetone for 3 min.

 

SECTION: Step II – Supplementing Insoluble Substance (BaP) in Feed Tabs
3.5. e.	Add 1.08 g cellulose and complete the feed preparation as in Step I above.

(NB: Acetone was chosen as the carrier solvent because it can quickly evaporate under a stream of air and has been shown to cause minimal biological effects in previous studies (Hallare et al., 2006; Shatilina et al., 2020).
SECTION: Step III – Drying Feed Tabs
4. a.	Pour the mixture into 36 cylindrical moulds of 1.1 cm diameter and 0.6 cm height (custom‐built aluminium device).
SECTION: Step III – Drying Feed Tabs
4.1. b.	The tablets may initially have a convex surface that quickly flattens during agar solidification at 6 °C in refrigerator. 

SECTION: Step III – Drying Feed Tabs
4.2. c.	Scrape the surfaces using an even scraper before removing them from the moulds.

 

SECTION: Step III – Drying Feed Tabs
4.3. d.	Remove the feed tabs from the moulds after approximately 15 min in a refrigerator at 6 °C.
SECTION: Step III – Drying Feed Tabs
4.4. e.	Then, dry them in an incubator at 45 °C for 1440 min.

 

SECTION: Step III – Drying Feed Tabs
4.5. f.	After this, place the feed tabs in a desiccator or fume cupboard for 30 min and measure the initial dry weight (dw1) with an Analytical Balance.

 

 

SECTION: Step III – Drying Feed Tabs
4.6. g.	Feed tabs can be stored at room temperature for use within months or at -20 °C for long-term use.

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5. In this step, the effects of drying and prewetting on the weight stability of the feed tabs at different handling steps were evaluated using a fine analytical balance.
Water Stability – The stability of feed tabs in water was evaluated by mimicking their replacement cycles during exposure to P. hawaiensis (NB: this section evaluates the expected weight loss of the feed tabs without amphipods feeding on them).
SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.1. Measure the initial dry weight (dwI) of at least four (4) feed tabs, transfer to seawater and prewet them for 1440 min before the experiment.

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.2. After 1440 min of prewetting, transfer them into a mock exposure chamber (a 1 L glass beaker filled with 500 mL seawater with continuous aeration but no test organisms).

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.3. Add three (3) glass marbles in the beaker as hiding materials without amphipods and keep in a temperature-controlled room with a 12:12-h light:dark cycle at approximately 30 °C for 2880 min (NB: this is to mimic the amphipod exposure scenarios).

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.4. After the 2880 min, remove the feed tabs, gently blot on tissue papers, and dry them for 1440 min in the incubator at 45 °C.

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.5. Place them in a desiccator or fume cupboard for 30 min and weigh them again using an analytical balance to obtain the final dry weight (dwF).

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.6. The difference between dwI and dwF (mean weight loss [MWL]) per day provides stability of the feed tabs and allows for comparisons between them.
SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.7. 
 

SECTION: Step IV – Evaluating Feed Tabs Water Stability and Adjustment Factor
5.8. Use the dry weight difference in milligrams per day as an adjustment factor for the feeding rate calculation of the amphipod. This served as the expected weight loss of a particular feed tabs formulation without any organism feeding on them.
SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6. a.	Experiment with a 1 L glass beaker with an appropriate volume (500 mL) of aerated artificial seawater.

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.1. b.	Add three (3) glass marbles as hiding places in each beaker to reduce the stress on the organisms.
 

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.2. c.	Place three pairs of matured male and female P. hawaiensis in each prepared beaker and feed them ad libitum with weighed and 24h-prewetted feed tabs.
 

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.3. d.	Weight the feed tab before feeding the organisms to obtain initial dry weight (dwI).
SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.4. e.	Prewet the feed tabs in seawater for 1440 min in a temperature-controlled room (exposure environment) before transferring them to the experimental beakers (NB: prewetting facilitates organisms' easy feeding of the feed tabs).

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.5. f.	Replace the feed tabs with new ones in an alternating cycle of three days (4320 min) throughout the exposure duration.

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.6. g.	After every removal, dry the used feed tabs for 1440 min in an incubator at 45 °C, weigh and record the final dry weight (dwF)

SECTION: Step V – Feeding Experimental Organisms Feed Tabs and Feeding Rate Calculation
6.7. h.	The feeding rate (FR) per day of Parhyale with dwI and dwF of the specific feed tabs was adjusted by the number of feeding days and the mean weight loss (MWL) of the feed tabs per day calculated from the stability measurement.
SECTION: Determination of the Feeding Rate
6.8. a.	As explained above, after feeding the organisms with weighed (dwI) and 24h-prewetted feed tabs, remove the leftover feed tabs with a spoon every 4320 min from all the experimental containers and transfer to a small pre-dried glass Petri dishes on a metal tray.

SECTION: Determination of the Feeding Rate
6.9. b.	Dry the leftover feed tabs for 1440 min in an incubator at 45 °C and then place in a desiccator or fume cupboard for 30 min.

SECTION: Determination of the Feeding Rate
6.10. c.	After 30 min, weigh the feed tabs with an analytical balance to obtain the feed leftover final dry weight (dwF).

SECTION: Determination of the Feeding Rate
6.11. d.	Calculate the feeding rate by subtracting leftover final dry weight (dwF) from the initial dry weight of feed tabs (dwI)
SECTION: Determination of the Feeding Rate
6.12. e.	Therefore, divide the estimated weight loss by the number of living P. hawaiensis at the end of 4320 min and calculate per day. Use the formula below:


 
Where:
FR – Feeding rate.
dwI: initial dry weight of the feed tab before 24 h pre-wetting and 48 h feeding to the organisms.
dwF – final dry weight of the feed leftover after the organisms feed on it
n = number of living organisms at the end of a particular feeding cycle 
MWL: adjustment factor as mean weight loss of the feed tab without organisms feeding on it.