Setup

## [1] "All packages loaded successfully"

SEABIRD TISSUES

Read in data and derive semi-quant values

Raw Data files were processed by Anna Robuck and peak area counts shared in file 20230626_PrelimDataCleanUp4Jackie.csv

Deriving quant and semi-quant values using Shirley Pu’s expertise and code in the next Markdown chunk.

Read in processed data

Now that we have the quant and semi-quanted values created for the seabird tissue data, we read in data.

## New names:
## • `` -> `...1`

QC data

Now that the data is all read into R, we can start looking at the data.

% Detects by compound

Most basic summary of detects above the method reporting limit (MRL) for Seabird data.

Bird detects summary
Compound n >MRL Min 25th Median 75th Max
Nafion_byproduct_6 81 14.81 0 0.00 0.00 0.00 0.01
PFO5DA 93 96.77 0 0.06 0.10 0.20 1.02
PFO6 93 96.77 0 0.03 0.05 0.11 0.65
PFO7 89 55.06 0 0.00 0.01 0.02 0.14
PFO8 81 3.70 0 0.00 0.00 0.00 0.01

Boxplot

Looking at PFAS levels across seabird tissues. Values are graphed on a log scale. Values under MRL are not included in this boxplot.

#Tissue Blood Ratios (TBRs)

PFO6 TBRs

Looking at tissue to blood ratios (TBR) to understand how PFO6 distributes across seabird organs. The dotted line is one to one line.

One way t-test for TBR6

Want to know if TBRs for PFO6 are statistically different than 1.

## 
##  One Sample t-test
## 
## data:  tbr6_hb$Restimate
## t = 2.3663, df = 8, p-value = 0.04551
## alternative hypothesis: true mean is not equal to 1
## 95 percent confidence interval:
##  1.036836 3.856726
## sample estimates:
## mean of x 
##  2.446781
## 
##  One Sample t-test
## 
## data:  tbr6_kb$Restimate
## t = 2.0703, df = 6, p-value = 0.08385
## alternative hypothesis: true mean is not equal to 1
## 95 percent confidence interval:
##  0.7598959 3.8798047
## sample estimates:
## mean of x 
##   2.31985
## 
##  One Sample t-test
## 
## data:  tbr6_lb$Restimate
## t = 1.4436, df = 8, p-value = 0.1868
## alternative hypothesis: true mean is not equal to 1
## 95 percent confidence interval:
##  0.6140726 2.6779167
## sample estimates:
## mean of x 
##  1.645995
## 
##  One Sample t-test
## 
## data:  tbr6_lub$Restimate
## t = 0.59941, df = 6, p-value = 0.5708
## alternative hypothesis: true mean is not equal to 1
## 95 percent confidence interval:
##  0.4877713 1.8446040
## sample estimates:
## mean of x 
##  1.166188
## 
##  One Sample t-test
## 
## data:  tbr6_bb$Restimate
## t = -5.5144, df = 7, p-value = 0.0008928
## alternative hypothesis: true mean is not equal to 1
## 95 percent confidence interval:
##  0.1836605 0.6736552
## sample estimates:
## mean of x 
## 0.4286579

PFO7 TBRs

Test to see if TBRs for PFO7 statistically are similar to PFO6 TBRs.

Kolmogorov–Smirnov test

Run K-S test to see if PFO7 TBRs are statistically similar to PFO6 TBRs. Look within each ratio category. Only 5 TBR groupings have both PFO7 and PFO6 points to tests.

## 
##  Exact two-sample Kolmogorov-Smirnov test
## 
## data:  Restimate by Compound
## D = 0.77778, p-value = 0.2182
## alternative hypothesis: two-sided
## 
##  Exact two-sample Kolmogorov-Smirnov test
## 
## data:  Restimate by Compound
## D = 0.41667, p-value = 0.6182
## alternative hypothesis: two-sided
## 
##  Exact two-sample Kolmogorov-Smirnov test
## 
## data:  Restimate by Compound
## D = 1, p-value = 0.25
## alternative hypothesis: two-sided
## 
##  Exact two-sample Kolmogorov-Smirnov test
## 
## data:  Restimate by Compound
## D = 0.85714, p-value = 0.5
## alternative hypothesis: two-sided
## 
##  Exact two-sample Kolmogorov-Smirnov test
## 
## data:  Restimate by Compound
## D = 0.44444, p-value = 0.7091
## alternative hypothesis: two-sided

Summary tables

Summary of the data for seabird data for inclusion in the manuscript.

Summarize the birds
Compound Tissue n %>MRL Min 25th Median 75th Max
Nafion_byproduct_6 Blood 10 10.00 0.00 0.00 0.00 0.00 0.01
Nafion_byproduct_6 Brain 12 0.00 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Fat 6 0.00 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Heart 12 25.00 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Kidney 8 12.50 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Liver 13 30.77 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Lungs 7 14.29 0.00 0.00 0.00 0.00 0.00
Nafion_byproduct_6 Muscle 13 15.38 0.00 0.00 0.00 0.00 0.00
PFO5DA Blood 14 100.00 0.06 0.09 0.11 0.20 1.02
PFO5DA Brain 12 100.00 0.01 0.03 0.05 0.10 0.33
PFO5DA Fat 6 50.00 0.00 0.00 0.02 0.07 0.17
PFO5DA Heart 12 100.00 0.05 0.10 0.22 0.27 0.48
PFO5DA Kidney 8 100.00 0.04 0.13 0.19 0.28 0.71
PFO5DA Liver 21 100.00 0.06 0.09 0.09 0.19 0.35
PFO5DA Lungs 7 100.00 0.07 0.10 0.14 0.30 0.35
PFO5DA Muscle 13 100.00 0.03 0.05 0.08 0.15 0.30
PFO6 Blood 14 100.00 0.02 0.03 0.04 0.06 0.65
PFO6 Brain 12 100.00 0.01 0.01 0.02 0.03 0.12
PFO6 Fat 6 50.00 0.00 0.00 0.01 0.02 0.09
PFO6 Heart 12 100.00 0.02 0.07 0.14 0.20 0.28
PFO6 Kidney 8 100.00 0.01 0.05 0.07 0.13 0.31
PFO6 Liver 21 100.00 0.04 0.05 0.06 0.11 0.21
PFO6 Lungs 7 100.00 0.03 0.05 0.07 0.13 0.16
PFO6 Muscle 13 100.00 0.02 0.02 0.03 0.06 0.20
PFO7 Blood 10 40.00 0.00 0.00 0.00 0.01 0.10
PFO7 Brain 12 16.67 0.00 0.00 0.00 0.00 0.00
PFO7 Fat 6 16.67 0.00 0.00 0.00 0.00 0.14
PFO7 Heart 12 83.33 0.00 0.01 0.02 0.03 0.05
PFO7 Kidney 8 75.00 0.00 0.00 0.01 0.01 0.04
PFO7 Liver 21 100.00 0.01 0.01 0.01 0.02 0.04
PFO7 Lungs 7 28.57 0.00 0.00 0.00 0.01 0.02
PFO7 Muscle 13 23.08 0.00 0.00 0.00 0.00 0.02
PFO8 Blood 10 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Brain 12 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Fat 6 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Heart 12 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Kidney 8 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Liver 13 23.08 0.00 0.00 0.00 0.00 0.01
PFO8 Lungs 7 0.00 0.00 0.00 0.00 0.00 0.00
PFO8 Muscle 13 0.00 0.00 0.00 0.00 0.00 0.00

Statistics

Data normality

Looking at seabird data normality for both non-logged data and log transformed data.

Neither are normal so will use parametric tests.

## 
##  Shapiro-Wilk normality test
## 
## data:  data$estimate
## W = 0.54218, p-value < 2.2e-16
## 
##  Shapiro-Wilk normality test
## 
## data:  data1$newlogspace
## W = 0.71345, p-value < 2.2e-16

Dunn tests & CLD

Run Kruskal Wallis and Dunn tests to get compact letter display (grouping). Currently have results hidden since it prints out a very long table.

Only running stats on PFO6 in Seabird data since no other PFAS was detected above 75% of tissue samples.

## 
##  Kruskal-Wallis rank sum test
## 
## data:  newestimate by TxC
## Kruskal-Wallis chi-squared = 275.71, df = 31, p-value < 2.2e-16

Plot Dunn & CLD

Now plot the CLD results over top of the PFO6 data to visualize.

Correlations

Running correlations on PFO5 to PFO6 in seabird data. Other compounds not detected often enough for Corr analysis.

## 
##  Kendall's rank correlation tau
## 
## data:  Cor_dataPF_graph$PFO5DoDA and Cor_dataPF_graph$PFO6
## z = 9.7635, p-value < 2.2e-16
## alternative hypothesis: true tau is not equal to 0
## sample estimates:
##       tau 
## 0.7915493

SPECIES SERUM

Now switching gears from Seabird data to the species serum comparison data

Read in raw data files

Derive quant & Semi-quant Species Serum data

## 
##    0    2    3    4 
## 1848   92  500   24

Read in processed data

QC data

% Detects & Summary Tables

Lots of summary tables. Looking at each level of data organization. trying to get an idea of the data and what test might be appropriate based on what we see above MRL.
Summarize all serum
Cmp n %>MRL Min 25th Median 75th Max
PFO6 154 28.57 0 0 0 2.27 34.57
PFO7 154 24.68 0 0 0 0.00 32.78
PFO8 154 7.14 0 0 0 0.00 35.94
Summarize the species serum
Animal Cmp n %>MRL Min 25th Median 75th Max
Alligator PFO6 65 46.15 0 0 0.00 4.39 34.57
Alligator PFO7 65 35.38 0 0 0.00 1.40 7.59
Alligator PFO8 65 1.54 0 0 0.00 0.00 1.59
Bass PFO6 29 48.28 0 0 0.00 9.28 20.67
Bass PFO7 29 51.72 0 0 0.56 10.47 32.78
Bass PFO8 29 34.48 0 0 0.00 7.57 35.94
Dog PFO6 28 0.00 0 0 0.00 0.00 0.00
Dog PFO7 28 0.00 0 0 0.00 0.00 0.00
Dog PFO8 28 0.00 0 0 0.00 0.00 0.00
Horse PFO6 32 0.00 0 0 0.00 0.00 0.00
Horse PFO7 32 0.00 0 0 0.00 0.00 0.00
Horse PFO8 32 0.00 0 0 0.00 0.00 0.00
Summarize the gator and fish by site
Animal Site Cmp n %>MRL Min 25th Median 75th Max
Alligator CFR PFO6 41 65.85 0.00 0.00 2.54 8.29 34.57
Alligator CFR PFO7 41 51.22 0.00 0.00 0.56 2.23 7.59
Alligator CFR PFO8 41 2.44 0.00 0.00 0.00 0.00 1.59
Alligator LW PFO6 24 12.50 0.00 0.00 0.00 0.00 6.98
Alligator LW PFO7 24 8.33 0.00 0.00 0.00 0.00 2.00
Alligator LW PFO8 24 0.00 0.00 0.00 0.00 0.00 0.00
Bass CFR PFO6 11 90.91 0.00 8.96 12.36 16.88 20.67
Bass CFR PFO7 11 100.00 0.62 8.97 18.72 25.65 32.78
Bass CFR PFO8 11 90.91 0.00 7.24 9.97 20.85 35.94
Bass ROANOKE PFO6 18 22.22 0.00 0.00 0.00 0.00 3.05
Bass ROANOKE PFO7 18 22.22 0.00 0.00 0.00 0.00 0.95
Bass ROANOKE PFO8 18 0.00 0.00 0.00 0.00 0.00 0.00
Summarize Gator CFR
Animal Site Cmp Sex n %>MRL Min 25th Median 75th Max
Alligator CFR PFO6 F 17 70.59 0 0 2.54 4.99 20.05
Alligator CFR PFO6 M 24 62.50 0 0 2.71 9.05 34.57
Alligator CFR PFO7 F 17 52.94 0 0 0.56 1.65 5.87
Alligator CFR PFO7 M 24 50.00 0 0 0.45 2.38 7.59
Alligator CFR PFO8 F 17 0.00 0 0 0.00 0.00 0.00
Alligator CFR PFO8 M 24 4.17 0 0 0.00 0.00 1.59
Summarize Fish CFR by sex
Animal Site Cmp SEX n %>MRL Min 25th Median 75th Max
Bass CFR PFO6 F 1 100 16.29 16.29 16.29 16.29 16.29
Bass CFR PFO6 M 10 90 0.00 8.80 11.39 17.06 20.67
Bass CFR PFO7 F 1 100 13.61 13.61 13.61 13.61 13.61
Bass CFR PFO7 M 10 100 0.62 8.22 21.28 25.98 32.78
Bass CFR PFO8 F 1 100 9.97 9.97 9.97 9.97 9.97
Bass CFR PFO8 M 10 90 0.00 7.08 10.59 20.86 35.94
Summarize all Fish CFR
Animal Site Cmp n %>MRL Min 25th Median 75th Max
Bass CFR PFO6 11 90.91 0.00 8.96 12.36 16.88 20.67
Bass CFR PFO7 11 100.00 0.62 8.97 18.72 25.65 32.78
Bass CFR PFO8 11 90.91 0.00 7.24 9.97 20.85 35.94

RSDs

Looks at RSDs across the non-censored PFAS data.

## `geom_smooth()` using formula = 'y ~ x'

Boxplots

Gator & fish by site

We have one gator who is missing metadata and is labeled as site = NA. this will get fixed when I am able to get the information from Scott Belcher.

Statistics

PFO6 sex-based diff in CFR gators

## 
##  Wilcoxon rank sum test with continuity correction
## 
## data:  mean_estimate_censored by Sex
## W = 197.5, p-value = 0.8713
## alternative hypothesis: true location shift is not equal to 0

Gator vs bass PFAS at CFR site

Want to test if the levels of the PFAS are different between fish and gator at the CFR site.

First need to look at data normality then run KW and Dunn test. 

## 
##  Shapiro-Wilk normality test
## 
## data:  fgdata$mean_estimate_censored
## W = 0.68483, p-value < 2.2e-16
## 
##  Kruskal-Wallis rank sum test
## 
## data:  mean_estimate_censored by SxC
## Kruskal-Wallis chi-squared = 83.783, df = 5, p-value < 2.2e-16
## Dunn (1964) Kruskal-Wallis multiple comparison
##   p-values adjusted with the Bonferroni method.
##                       Comparison           Z      P.unadj        P.adj
## 1  AlligatorPFO6 - AlligatorPFO7  2.28215483 2.248020e-02 3.372030e-01
## 2  AlligatorPFO6 - AlligatorPFO8  5.25311494 1.495481e-07 2.243222e-06
## 3  AlligatorPFO7 - AlligatorPFO8  2.97096011 2.968703e-03 4.453055e-02
## 4       AlligatorPFO6 - BassPFO6 -2.48475504 1.296406e-02 1.944609e-01
## 5       AlligatorPFO7 - BassPFO6 -3.96916813 7.212398e-05 1.081860e-03
## 6       AlligatorPFO8 - BassPFO6 -5.90161022 3.599708e-09 5.399562e-08
## 7       AlligatorPFO6 - BassPFO7 -3.12436832 1.781873e-03 2.672809e-02
## 8       AlligatorPFO7 - BassPFO7 -4.60878141 4.050359e-06 6.075538e-05
## 9       AlligatorPFO8 - BassPFO7 -6.54122350 6.101756e-11 9.152634e-10
## 10           BassPFO6 - BassPFO7 -0.50934520 6.105103e-01 1.000000e+00
## 11      AlligatorPFO6 - BassPFO8 -2.40913573 1.599035e-02 2.398552e-01
## 12      AlligatorPFO7 - BassPFO8 -3.89354882 9.878831e-05 1.481825e-03
## 13      AlligatorPFO8 - BassPFO8 -5.82599092 5.677470e-09 8.516206e-08
## 14           BassPFO6 - BassPFO8  0.06021815 9.519819e-01 1.000000e+00
## 15           BassPFO7 - BassPFO8  0.56956335 5.689739e-01 1.000000e+00

Gator and bass diff PFO7 and PFO8

using wilcox rank sum test

Correlations

Going to look at correlations for PFO6/7/8 in fish since there is a high detection frequency. Correlations between PFO5DA and PFO6 is not possible for the species serum comparison.

## 
##  Kendall's rank correlation tau
## 
## data:  cor_fdataPF_graph$PFO6 and cor_fdataPF_graph$PFO7
## T = 39, p-value = 0.002213
## alternative hypothesis: true tau is not equal to 0
## sample estimates:
##       tau 
## 0.7333333
## 
##  Kendall's rank correlation tau
## 
## data:  cor_fdataPF_graph$PFO7 and cor_fdataPF_graph$PFO8
## T = 38, p-value = 0.004687
## alternative hypothesis: true tau is not equal to 0
## sample estimates:
##       tau 
## 0.6888889
## 
##  Kendall's rank correlation tau
## 
## data:  cor_fdataPF_graph$PFO6 and cor_fdataPF_graph$PFO8
## T = 34, p-value = 0.04662
## alternative hypothesis: true tau is not equal to 0
## sample estimates:
##       tau 
## 0.5111111