This summary serves as a broad overview and notes of the acute (48hour) single stressor polyvinyl chloride (PVC) and polypropylene (PP) leachate exposure to Anthopleura elegantissima.

The following is a timeline overview of the experiment.

Note

Sample processing for Mitotic index, chlorophyll content, and DNA & RNA sequencing still have to be done!

This timeline was made using the vistime¹ package

¹ Checkout the vistime package vignette here

1 Specimen collection & acclimation

We collected anemones under Washington Dept. of Fish & Wildlife scientific collection permit Tanja-010 at Owens Beach Park in Tacoma on Monday March 18th and at Constellation Park in West Seattle on Saturday March 30th. After collection, each anemone was placed on a acid-washed 60mm glass petri dish to adhere and heal their pedal disc during an acclimation phase. All anemones were acclimated for at least 24 days in a recirculating seawater table at 10C prior to the start of the treatment exposure.

2 Leachate preparation

We prepared leachate for PVC (<500um) and for PP (aged) according to the leachate preparation protocol.

In short, we created 1L artificial seawater using salt and deionized water, and adjusted the pH down from ~8.5 to 8.2 by slowly, iteratively, adding a very small amount of 0.1M hydrochloric acid (HCl) via a burette.

Weighed out 41.953 grams of ASTM D1141-98 sea salt

We then carefully measured out 250mg of each microplastic type, and added it to a dry flask.

Initial pH was ~8.4, we lowered it to ~8.2 using 0.1M HCl

After, we transferred 250mL of the pH-adjusted artifical seawater to each flask, set them in a shaker plate rotating at 90 rotations per minute, and left them to ‘shake and soak’ for 7 days.

250mL of artificial seawater and 250mg of aged polypropylene microplastic. Note that this microplastic is buoyant.

Flasks of seawater and microplastics were covered with foil to ensure no further photodegradation, and left to shake at 90rpm for 7 days

After the 7 days were up, we filtered the microplastics from the seawater using 45micron filter paper.

Here we can see the microplastics accumulating on the filter as the leachate passes through the funnel.

Leachate filtration was a simple gravity filtration process and didn’t require the use of vacuum filtration.

After the leachate was filtered, we put it on ice and transported it to a 4C fridge where it was stored for 48 hours prior to dilution and application to the anemones.

3 Leachate dilution

We performed a serial dilution on each prepared leachate to create leachate treatments that represent a mass of microplastic type to volume of water:

100 mg/L
10 mg/L
1 mg/L
0.1 mg/L
0.01 mg/L

For this we took 20mL of the 1000 mg/L leachate, and added it to 180mL of filtered seawater to make the 100mg/L concentration.

We then serially diluted the remaining concentrations in a similar fashion.

4 Exposure

We exposed individual anemones to the leachate treatments for 48 hours in closed-systems. For this, we used 0.5 pint mason jars set in a recirculating seawater bath temperature-controlled by a heat exchanger and the Neptune Apex aquarium controller system.

The water bath was kept at 10C throughout the exposure.

5 Response measurements

After 48 hours we measured:

Rapid Light Curves using a Walz Diving II PAM

30 minutes of respirometry in the dark-adapted state (oxygen consumption, cellular respiration)
30 minutes of respirometry in the light-saturated state (oxygen production, photosynthesis)
wet weight

And then placed anemones in labelled 5mL centrifuge tubes and flash froze them in liquid-nitrogen

6 Initial results

# Install packages
if ("tidyverse" %in% rownames(installed.packages()) == 'FALSE') install.packages('tidyverse')
if ("dplyr" %in% rownames(installed.packages()) == 'FALSE') install.packages('dplyr')
if ("car" %in% rownames(installed.packages()) == 'FALSE') install.packages('car')

# Load packages
library(dplyr)
library(tidyverse)
library(car)

Warning: package 'car' was built under R version 4.2.3

Loading required package: carData

Warning: package 'carData' was built under R version 4.2.3


Attaching package: 'car'

The following object is masked from 'package:dplyr':

    recode

The following object is masked from 'package:purrr':

    some

rates <- read_csv('rates.csv')

Rows: 55 Columns: 17
── Column specification ────────────────────────────────────────────────────────
Delimiter: ","
chr  (2): id, treatment
dbl (15): run, channel, auto_resp_rate, auto_resp_r2, resp_bg_adj.rate, resp...

ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

6.1 Respiration

6.1.1 First glimpse of data

ggplot(rates) +
  aes(x = treatment, y = massnorm_resp_rate, color = treatment) +
  geom_jitter() +
  theme(legend.position = "none")

6.1.2 Data normality

resp_aov <-  aov(massnorm_resp_rate ~ treatment, data = rates)

par(mfrow = c(1, 2)) # combine plots

# histogram
hist(resp_aov$residuals)

# QQ-plot
qqPlot(resp_aov$residuals,
  id = FALSE # id = FALSE to remove point identification
)

6.1.3 Boxplot

ggplot(rates) +
  aes(x = treatment, y = massnorm_resp_rate) +
  geom_boxplot() +
  theme(axis.text.x = element_text(angle = 45, hjust = 1))

6.1.4 ANOVA

resp_aov <- aov(massnorm_resp_rate ~ treatment,
  data = rates
)

summary(resp_aov)

            Df    Sum Sq   Mean Sq F value Pr(>F)
treatment   10 5.268e-10 5.268e-11   1.302  0.259
Residuals   44 1.780e-09 4.045e-11

6.2 Photosynthesis

6.2.1 First glimpse of data

ggplot(rates) +
  aes(x = treatment, y = massnorm_phot_rate, color = treatment) +
  geom_jitter() +
  theme(legend.position = "none")

6.2.2 Data normality

res_aov <-  aov(massnorm_phot_rate ~ treatment, data = rates)

par(mfrow = c(1, 2)) # combine plots

# histogram
hist(res_aov$residuals)

# QQ-plot
qqPlot(res_aov$residuals,
  id = FALSE # id = FALSE to remove point identification
)

6.2.3 Boxplot

ggplot(rates) +
  aes(x = treatment, y = massnorm_phot_rate) +
  geom_boxplot() +
  theme(axis.text.x = element_text(angle = 45, hjust = 1))

6.2.4 ANOVA

phot_aov <- aov(massnorm_phot_rate ~ treatment,
  data = rates
)

summary(phot_aov)

            Df    Sum Sq   Mean Sq F value Pr(>F)
treatment   10 1.433e-10 1.433e-11   1.355  0.233
Residuals   44 4.652e-10 1.057e-11

7 Coming up next

Start the PAM Rapid Light Curve analysis
Extract RNA for gene expression of host anemone and symbiont dinoflagellate

8 Gaps & improvements

Important

We still need to be able to take the background signature of our filtered seawater using C18 SPME fiber extractions, preserve the fibers, and send them to the Saliu Lab for analysis

Note

We are limited to short exposures because of the 48 hour window we have to work with fresh leachate. We can get around this by making leachate in batches and doing water changes every 48 hours, but we will need larger stocks of microplastic with which to make the leachate if we want to pursue this

--- title: "Single stressor leachate exposure summary" subtitle: "A 48 hour exposure to polyvinyl chloride (PVC) leachate and to aged polypropylene (PP) leachate" author: "Sarah Tanja" date: '05/03/2024' image: 'images/PXL_20240424_190134501.RAW-02.ORIGINAL.jpg' categories: [lab records] 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 link-external-icon: true link-external-newwindow: true --- This summary serves as a broad overview and notes of the acute (48hour) single stressor polyvinyl chloride (PVC) and polypropylene (PP) leachate exposure to *Anthopleura elegantissima*. ```{r include=FALSE} library(tidyverse) library(vistime) ``` ![](../field-collection/field-pics/12/PXL_20240319_022405096.jpg) ```{r include=FALSE} gantt <- read.csv('gantt.csv') # format start and end as POSIXct date objects gantt <- gantt %>% mutate(start = as.POSIXct(as.Date(start,format = "%m/%d/%Y"))) %>% mutate(end = as.POSIXct(as.Date(end,format = "%m/%d/%Y"))) head(gantt) ``` The following is a timeline overview of the experiment. ::: callout-note Sample processing for Mitotic index, chlorophyll content, and DNA & RNA sequencing still have to be done! ::: ```{r echo=FALSE} vistime(gantt, col.event = "task", col.group = "phase", col.start = "start", col.end = "end", optimize_y = FALSE, linewidth = 15, title = "Timeline of single-stressor leachate experiment") ``` This timeline was made using the `vistime`[^1] package [^1]: Checkout the `vistime` package vignette [here](https://cran.rstudio.com/web/packages/vistime/vignettes/vistime-vignette.html) # Specimen collection & acclimation We collected anemones under Washington Dept. of Fish & Wildlife scientific collection permit `Tanja-010` at [Owens Beach Park](https://sarahtanja.github.io/quarto-blog/posts/projects/anemone/field-collection/18MAR24-owens-beach.html) in Tacoma on Monday March 18th and at [Constellation Park](https://sarahtanja.github.io/quarto-blog/posts/projects/anemone/field-collection/30MAR24-alki.html) in West Seattle on Saturday March 30th. After collection, each anemone was placed on a acid-washed 60mm glass petri dish to adhere and heal their pedal disc during an acclimation phase. All anemones were acclimated for at least 24 days in a recirculating seawater table at 10C prior to the start of the treatment exposure. # Leachate preparation We prepared leachate for PVC (\<500um) and for PP (aged) according to the [leachate preparation protocol](https://sarahtanja.github.io/quarto-blog/posts/projects/anemone/leachate-protocol.html). In short, we created 1L artificial seawater using salt and deionized water, and adjusted the pH down from \~8.5 to 8.2 by slowly, iteratively, adding a very small amount of 0.1M hydrochloric acid (HCl) via a burette. ::: column-margin ![Weighed out 41.953 grams of ASTM D1141-98 sea salt](images/PXL_20240412_230820563.jpg) ::: We then carefully measured out 250mg of each microplastic type, and added it to a dry flask. ::: column-margin ![Initial pH was \~8.4, we lowered it to \~8.2 using 0.1M HCl](images/PXL_20240412_232609016.jpg) ::: After, we transferred 250mL of the pH-adjusted artifical seawater to each flask, set them in a shaker plate rotating at 90 rotations per minute, and left them to 'shake and soak' for 7 days. ::: column-margin ![250mL of artificial seawater and 250mg of aged polypropylene microplastic. Note that this microplastic is buoyant.](images/PXL_20240416_001441079.jpg) ::: ::: column-margin ![Flasks of seawater and microplastics were covered with foil to ensure no further photodegradation, and left to shake at 90rpm for 7 days](images/PXL_20240422_230547996.jpg) ::: ::: column-margin ![After the 7 days were up, we filtered the microplastics from the seawater using 45micron filter paper.](images/PXL_20240423_005801129.jpg) ::: ::: column-margin ![Here we can see the microplastics accumulating on the filter as the leachate passes through the funnel.](images/PXL_20240423_005933927.jpg) ::: ![Leachate filtration was a simple gravity filtration process and didn't require the use of vacuum filtration.](images/PXL_20240423_011215280.jpg) ::: column-margin ![After the leachate was filtered, we put it on ice and transported it to a 4C fridge where it was stored for 48 hours prior to dilution and application to the anemones.](images/PXL_20240423_012640301.jpg) ::: # Leachate dilution We performed a serial dilution on each prepared leachate to create leachate treatments that represent a mass of microplastic type to volume of water: - 100 mg/L - 10 mg/L - 1 mg/L - 0.1 mg/L - 0.01 mg/L For this we took 20mL of the 1000 mg/L leachate, and added it to 180mL of filtered seawater to make the 100mg/L concentration. We then serially diluted the remaining concentrations in a similar fashion. # Exposure We exposed individual anemones to the leachate treatments for 48 hours in closed-systems. For this, we used 0.5 pint mason jars set in a recirculating seawater bath temperature-controlled by a heat exchanger and the Neptune Apex aquarium controller system. ![](images/PXL_20240424_190134501.RAW-02.ORIGINAL.jpg) The water bath was kept at 10C throughout the exposure. # Response measurements After 48 hours we measured: - Rapid Light Curves using a Walz Diving II PAM ![](PXL_20240426_162505396.PORTRAIT.jpg) ![](images/PXL_20240426_224927703.jpg) - 30 minutes of respirometry in the dark-adapted state (oxygen consumption, cellular respiration) - 30 minutes of respirometry in the light-saturated state (oxygen production, photosynthesis) - wet weight ![](images/PXL_20240428_004714187.jpg) And then placed anemones in labelled 5mL centrifuge tubes and flash froze them in liquid-nitrogen # Initial results ```{r} # Install packages if ("tidyverse" %in% rownames(installed.packages()) == 'FALSE') install.packages('tidyverse') if ("dplyr" %in% rownames(installed.packages()) == 'FALSE') install.packages('dplyr') if ("car" %in% rownames(installed.packages()) == 'FALSE') install.packages('car') # Load packages library(dplyr) library(tidyverse) library(car) ``` ```{r} rates <- read_csv('rates.csv') ``` ## Respiration ![](images/PXL_20240426_191325446.jpg) ![](images/PXL_20240426_191336067.jpg) ### First glimpse of data ```{r} ggplot(rates) + aes(x = treatment, y = massnorm_resp_rate, color = treatment) + geom_jitter() + theme(legend.position = "none") ``` ### Data normality ```{r} resp_aov <- aov(massnorm_resp_rate ~ treatment, data = rates) ``` ```{r} par(mfrow = c(1, 2)) # combine plots # histogram hist(resp_aov$residuals) # QQ-plot qqPlot(resp_aov$residuals, id = FALSE # id = FALSE to remove point identification ) ``` ### Boxplot ```{r} ggplot(rates) + aes(x = treatment, y = massnorm_resp_rate) + geom_boxplot() + theme(axis.text.x = element_text(angle = 45, hjust = 1)) ``` ### ANOVA ```{r} resp_aov <- aov(massnorm_resp_rate ~ treatment, data = rates ) summary(resp_aov) ``` ## Photosynthesis ### First glimpse of data ```{r} ggplot(rates) + aes(x = treatment, y = massnorm_phot_rate, color = treatment) + geom_jitter() + theme(legend.position = "none") ``` ### Data normality ```{r} res_aov <- aov(massnorm_phot_rate ~ treatment, data = rates) ``` ```{r} par(mfrow = c(1, 2)) # combine plots # histogram hist(res_aov$residuals) # QQ-plot qqPlot(res_aov$residuals, id = FALSE # id = FALSE to remove point identification ) ``` ### Boxplot ```{r} ggplot(rates) + aes(x = treatment, y = massnorm_phot_rate) + geom_boxplot() + theme(axis.text.x = element_text(angle = 45, hjust = 1)) ``` ### ANOVA ```{r} phot_aov <- aov(massnorm_phot_rate ~ treatment, data = rates ) summary(phot_aov) ``` # Coming up next - Start the PAM Rapid Light Curve analysis - Extract RNA for gene expression of host anemone and symbiont dinoflagellate # Gaps & improvements ::: callout-important We still need to be able to take the background `signature` of our filtered seawater using C18 SPME fiber extractions, preserve the fibers, and send them to the Saliu Lab for analysis ::: ::: callout-note We are limited to short exposures because of the 48 hour window we have to work with fresh leachate. We can get around this by making leachate in batches and doing water changes every 48 hours, but we will need larger stocks of microplastic with which to make the leachate if we want to pursue this :::