Making PAE treatments and controls – Sarah Tanja’s Open Lab Notebook

1 Phthalates

I used the EPA 506 phthalate esters mix 1, which comes as a 1mL ampule vial with a glass break-off cap. It includes 6 common phthalates each at a concentration of $500\mu g/mL$, aka $(500,000 \mu g/L)$ in methanol as a carrier solvent to stabilize the phthalate esters mix.

The mix contained the following 6 phthalates:

Benzyl butyl phthalate (BBP)
Bis(2-ethylhexyl) adipate (DEHA)
Bis(2-ethylhexyl) phthalate (DEHP)

Caution

DEHP is listed on EPA’s Toxics Release Inventory (TRI)

Caution

DEHP is regulated under the Safe Drinking Water Act. The highest concentration allowed, the maximum contaminant level (MCL), is $0.006 mg/L$ , or ($6\mu g/L$)

Dibutyl pthalate (DBP)

Caution

DBP is listed on EPA’s Toxics Release Inventory (TRI)

Diethyl phthalate (DEP)
Dimethyl phthalate (DMP)

1.0.1 Safety Considerations

Please review the SDS for EPA 506 phthalate mix, 500$\mu$g/mL in methanol before working with this chemical!

Hazard statements:

H225 Highly flammable liquid and vapor
Toxic if swallowed, in contact with skin or if inhaled
Causes damage to organs (Eyes, Central nervous system)

Caution

This chemical mix is toxic and can absorb through skin, and vaporize and be inhaled. WEAR GLOVES, SAFETY GLASSES, & MASK. WORK UNDER A HOOD! If you are pregnant, consider alternatives like preparing leachate similar to (Tetu et al. 2019)

Tetu, Sasha G., Indrani Sarker, Verena Schrameyer, Russell Pickford, Liam D. H. Elbourne, Lisa R. Moore, and Ian T. Paulsen. 2019. “Plastic Leachates Impair Growth and Oxygen Production in Prochlorococcus, the Ocean’s Most Abundant Photosynthetic Bacteria.” Communications Biology 2 (May): 184. https://doi.org/10.1038/s42003-019-0410-x.

2 Environmentally Relevant Doses

Concentrations of phthalates in environmental sea water samples reported in the literature:

Reference	Study Area	DEHA	DEHP	DBP	DEP	DMP	$\sum$PAE
(Lynch, Knauer, and Shaw 2022)	Review						0.5 - 10 $\mu$g/L
(Jebara et al. 2021)	Tunisia		<LOD-168$\mu$g/L	<LOD-30.5$\mu$g/L	<LOD-17.0$\mu$g/L
(Sánchez-Avila, Tauler, and Lacorte 2012)	Mediterranean Coastal Spain	0.0021-0.304$\mu$g/L	0.031-0.617$\mu$g/L		0.024-0.483$\mu$g/L	0.0028 - 0.142 $\mu$g/L

Lynch, Jennifer M., Katrina Knauer, and Katherine R. Shaw. 2022. “Plastic Additives in the Ocean.” In Plastics and the Ocean, edited by Anthony L. Andrady, 1st ed., 43–76. Wiley. https://doi.org/10.1002/9781119768432.ch2.

Jebara, Amel, Ambrogina Albergamo, Rossana Rando, Angela Potortì, Vincenzo Turco, Hedi Ben Mansour, and Giuseppa Di Bella. 2021. “Phthalates and Non-Phthalate Plasticizers in Tunisian Marine Samples: Occurrence, Spatial Distribution and Seasonal Variation.” Marine Pollution Bulletin 163 (January). https://doi.org/10.1016/j.marpolbul.2021.111967.

Sánchez-Avila, Juan, Romà Tauler, and Silvia Lacorte. 2012. “Organic Micropollutants in Coastal Waters from NW Mediterranean Sea: Sources Distribution and Potential Risk.” Environment International 46 (October): 50–62. https://doi.org/10.1016/j.envint.2012.04.013.

Jebara et al. (2021) detected DEHP in 92.7% of 165 water samples. The mean concentration of DEHP in those samples were: $71.1\mu g/L$, and the median was: $45.7 \mu g/L$.

Based on these sources reporting environmentally relevant concentrations of phthalates in surface waters, I chose to dose the coral gametes and larvae at the following concentrations:

$0\mu g/L$ : control
$0.5\mu g/L$ ($0.0005 \mu g/mL$): environmentally relevant value
$5 \mu g/L$ ($0.005 \mu g/mL$): would be considered just under acceptable drinking water
$50 \mu g/L$ ($0.05 \mu g/mL$) : increasing by factor of 10
$150 \mu g/L$ ($0.150 \mu g/mL$): max environmental relevance $168 \mu g/L$)

Tip

Doses will be graphed on a log scale x axis

3 Methanol ‘Solvent’ Control

c1(ug/mL)	c2(ug/mL)	v2(mL)	v1(mL)
200	0.15	800	0.6
0.15	0.05	600	200
0.05	0.005	500	50
0.005	0.0005	500	50

--- title: "Making PAE treatments and controls" author: "Sarah Tanja" date: '11/14/2023' categories: [protocols] draft: false toc: true toc-title: Contents <i class="bi bi-bookmark-heart"></i> toc-depth: 5 toc-location: left reference-location: margin citation-location: margin bibliography: "../library.bib" --- ## Phthalates I used the [EPA 506 phthalate esters mix 1](https://www.sigmaaldrich.com/US/en/product/supelco/48223?gclid=CjwKCAjw-8qVBhANEiwAfjXLrp8fD-G8yiB_NyrA3OLy3r2jdylgBmHVPUfGFnMp4CMfuVwePGi1dxoCBQIQAvD_BwE), which comes as a 1mL ampule vial with a glass break-off cap. It includes 6 common phthalates each at a concentration of $500\mu g/mL$, aka $(500,000 \mu g/L)$ in methanol as a carrier solvent to stabilize the phthalate esters mix. The mix contained the following 6 phthalates: 1. Benzyl butyl phthalate **(BBP)** 2. Bis(2-ethylhexyl) adipate **(DEHA)** 3. Bis(2-ethylhexyl) phthalate **(DEHP)** ::: callout-caution **DEHP** is listed on EPA's Toxics Release Inventory (TRI) ::: ::: callout-caution **DEHP** is regulated under the Safe Drinking Water Act. The highest concentration allowed, the maximum contaminant level (MCL), is $0.006 mg/L$ , or ($6\mu g/L$) ::: 4. Dibutyl pthalate **(DBP)** ::: callout-caution **DBP** is listed on EPA's Toxics Release Inventory (TRI) ::: 5. Diethyl phthalate **(DEP)** 6. Dimethyl phthalate **(DMP)** #### Safety Considerations Please review the [SDS](https://www.sigmaaldrich.com/US/en/sds/supelco/48223) for EPA 506 phthalate mix, 500$\mu$g/mL in methanol before working with this chemical! Hazard statements: - H225 Highly flammable liquid and vapor - Toxic if swallowed, in contact with skin or if inhaled - Causes damage to organs (Eyes, Central nervous system) ::: callout-caution This chemical mix is toxic and can absorb through skin, and vaporize and be inhaled. **WEAR GLOVES, SAFETY GLASSES, & MASK. WORK UNDER A HOOD!** *If you are pregnant, consider alternatives like preparing leachate similar to* [@tetu2019] ::: ## Environmentally Relevant Doses Concentrations of phthalates in environmental sea water samples reported in the literature: | Reference | Study Area | BBP | DEHA | DEHP | DBP | DEP | DMP | $\sum$PAE | |----------------------|-----------------------------|-----|----------------------|---------------------|--------------------|---------------------|-------------------------|-------------------| | [@lynch2022] | Review | | | | | | | 0.5 - 10 $\mu$g/L | | [@jebara2021] | Tunisia | | | \<LOD-168$\mu$g/L | \<LOD-30.5$\mu$g/L | \<LOD-17.0$\mu$g/L | | | | [@sanchez-avila2012] | Mediterranean Coastal Spain | | 0.0021-0.304$\mu$g/L | 0.031-0.617$\mu$g/L | | 0.024-0.483$\mu$g/L | 0.0028 - 0.142 $\mu$g/L | | @jebara2021 detected **DEHP** in 92.7% of 165 water samples. The mean concentration of **DEHP** in those samples were: $71.1\mu g/L$, and the median was: $45.7 \mu g/L$. Based on these sources reporting environmentally relevant concentrations of phthalates in surface waters, I chose to dose the coral gametes and larvae at the following concentrations: - $0\mu g/L$ : control - $0.5\mu g/L$ ($0.0005 \mu g/mL$): environmentally relevant value - $5 \mu g/L$ ($0.005 \mu g/mL$): would be considered just under acceptable drinking water - $50 \mu g/L$ ($0.05 \mu g/mL$) : increasing by factor of 10 - $150 \mu g/L$ ($0.150 \mu g/mL$): max environmental relevance $168 \mu g/L$) ::: callout-tip Doses will be graphed on a log scale x axis ::: ## Methanol 'Solvent' Control | c1(ug/mL) | c2(ug/mL) | v2(mL) | v1(mL) | |-----------|-----------|--------|--------| | 200 | 0.15 | 800 | 0.6 | | 0.15 | 0.05 | 600 | 200 | | 0.05 | 0.005 | 500 | 50 | | 0.005 | 0.0005 | 500 | 50 | | | | | |