DOI: 10.5061/dryad.6djh9w16c
title: No evidence that the widespread environmental contaminant caffeine alters energy balance or stress responses in fish

description: Anthropogenic sources of environmental pollution are ever-increasing as
 urban areas expand and more chemical compounds are used in daily life. The
 stimulant caffeine is one of the most consumed chemical compounds
 worldwide, and as a result, has been detected as an environmental
 contaminant in all types of major water sources on all continents.
 Exposure of wildlife to environmental pollutants can disrupt the energy
 balance of these organisms, as restoration of homeostasis is prioritised.
 In turn, energy allocated to other key biological processes such as growth
 or reproduction may be affected, consequently reducing the overall fitness
 of an individual. Therefore, we aimed to investigate if long-term exposure
 to environmentally relevant concentrations of caffeine had any energetic
 consequences on wildlife. Specifically, we exposed wild eastern
 mosquitofish (Gambusia holbrooki) to one of three nominal concentrations
 of caffeine (0, 100, and 10,000 ng/L) and assayed individuals for
 metabolic rate, general activity, antipredator and foraging behaviour, and
 body size as measures of energy expenditure or energy intake. We found no
 differences in any measured traits between any of the given exposure
 treatments, indicating that exposure to caffeine at current environmental
 levels may not adversely affect the energy balance and fitness of
 vulnerable freshwater fish.
description: R and RStudio
description: # Title of Dataset: No evidence that the widespread environmental
 contaminant caffeine alters energy balance or stress responses in fish ***
 Dataset is comprised of R scripts and .csv files that contain data used in
 the data analysis for the manuscript accepted at Ethology, doi:
 10.1111/eth.13403, with the same title as the dataset. Briefly, the
 activity, antipredator behaviour, foraging behaviour, metabolic rate, and
 morphological traits of eastern mosquitofish (Gambusia holbrooki) exposed
 to the environmental contaminant caffeine were analysed. ## Description of
 the data and file structure Contents of this dataset include 3 seperate R
 markdown (RMD) files for data analysis, and 8 .csv files containing data.
 Titles of each R markdown file indicates which results section has been
 analysed. Data from .csv files pertaining to each RMD file is included
 within the R script in each RMD. The RMD file "Tan 2023 foraging and
 morph data" details the R scripts used to analyse the foraging
 behaviour and morphological traits of eastern mosquitofish exposed to
 caffeine. Specifically, comparing caffeine exposure treatments (exposure),
 we analyse the latency to first consume a food item (time.to.first.eat)
 using a cox proportional hazards survival model, and the number of food
 items eaten (worms.eaten) using a zero-inflated poisson distributed
 generalised linear model. Body mass (weight.g) and sex (sex.corr) are
 included as covariates in these models. For morphological traits, body
 mass (weight.g) and total lengths (Total.Length) were analysed with
 analysis of variance models to determine differences between caffeine
 exposure treatment groups. The RMD file "Tan 2023 activity
 antipredator behaviour" details the R scripts used to analyse the
 baseline activity and antipredator responses of mosquitofish exposed to
 caffeine. Specifically, using linear mixed effect models, the distance
 travelled (distance.moved.cm) was compared between caffeine exposure
 treatment groups before mosquitofish were presented with a predatory
 stimulus as a measure of baseline activity, and after being presented with
 a predatory stimulus as a measure of antipredator response (time.bin).
 Body mass (weight.g) and sex (sex.corr) are included as covariates in
 these models. The RMD file "Tan 2023 Mosquitofish metabolic
 rate" details the R scripts used to analyse the oxygen
 uptake/metabolic rate of mosquitofish exposed to caffeine. Specifically,
 the formula to calculate VO2 (i.e., non-mass specific rate of oxygen
 uptake) is presented in this file. VO2 (vo2.mcg.h) was then analysed with
 linear mixed effect models to compare rates of oxygen uptake between
 caffeine exposure treatment groups (TREATMENT). Body mass (weight.mg), sex
 (sex.correct), daily round of trial (ROUND), and activity (activity.cm)
 were included as covariates. A random intercept for the 8 respirometry
 channels available \(1\|TANK\.ID\) was also included\. ## Code/Software
 The open-source free software R (version used was 4.3.0) and RStudio is
 required to open RMD files and run through R scripts included in this
 dataset. R packages required to run analyses are detailed within each RMD
 file. Each RMD file also details which .csv files are required to be
 loaded. ## Description of variables in CSV files #### CSV File Name:
 fish.measurements.foraging.csv **Description of file:** Wet body weight
 data used as a covariate for models analysing mosquitofish foraging
 behaviour, see details in Tan et al. 2023 (doi: 10.1111/eth.13403).
 **Variables:** date: date of measurement sex: sex of individual; JF and JM
 = juveniles (eventually consolidated into one group within analyses), M =
 males, F = females weight.g: wet body weight of individuals in grams
 exp.tank.id: experimental tank ID used for behavioural trial
 exp.tank.temp: experimental tank temperature during behavioural trial in
 degrees celcius Observation.id: unique observation ID assigned to each
 focal experimental individual (these are not tracked across different
 behavioural trials as animals are grouped housed, i.e., individuals with
 the same unique identifier are only specific to each type of behavioural
 trial). exposure: caffeine exposure treatment group; c = control, l = low
 nominal treatment, h = high nominal treatment *** #### CSV File Name:
 count.worms.eaten.csv **Description of file:** Data of number of food
 items consumed as one of the foraging behaviour endpoints measured in the
 foraging behaviour trial, see details in Tan et al. 2023 (doi:
 10.1111/eth.13403). **Variables:** Observation.id: unique observation ID
 assigned to each focal experimental individual (these are not tracked
 across different behavioural trials as animals are grouped housed, i.e.,
 individuals with the same unique identifier are only specific to each type
 of behavioural trial). worms.eaten: number of food items (bloodworms)
 eaten across the length of the trial period *** #### CSV File Name:
 caftimebin.csv **Description of file:** Activity data used within models
 analysing the general activity and antipredator behaviour of
 caffeine-exposed mosquitofish, see details in Tan et al. 2023 (doi:
 10.1111/eth.13403). **Variables:** date: data of measurement fish.id:
 unique observation ID assigned to each focal experimental individual
 (these are not tracked across different behavioural trials as animals are
 grouped housed, i.e., individuals with the same unique identifier are only
 specific to each type of behavioural trial). exposure.tank.id: caffeine
 exposure treatment tank ID that focal individuals were housed in prior to
 experimentation exposure: caffeine exposure treatment group; c = control,
 l = low nominal treatment, h = high nominal treatment trial: trial ID used
 for identification purposes in ethovision tracking software arena: arena
 ID used for identification purposes in ethovision tracking software (four
 arenas were present in each trial) time.bin: time intervals trials were
 split into for analysis; Start-0:10:00 = initial 10 min acclimation period
 not included in analyses, 0:10:00-0:20:00 = 10 min baseline general
 activity period, 0:20:00-0:30:00 = 10 min post-predator strike activity
 distance.moved.cm: distance travelled in centimeters (tracking software
 output) mean.velocity.cm/s: mean velocity in centimeters per second
 (tracking software output) cum.dur.in.arena.s: duration fish was tracked
 in seconds (tracking software output) latency.to.arena.s: latency for fish
 to first enter arena (tracking software output) sex: sex of individual; JF
 and JM = juveniles (eventually consolidated into one group within
 analyses), M = males, F = females weight.g: wet body weight of individuals
 in grams experimental.tank.id: experimental tank ID used for behavioural
 trial experimental.tank.temp: experimental tank temperature during
 behavioural trial in degrees celcius experimental.round: round of trial
 per day *** #### CSV File Name: foragingdata.csv **Description of file:**
 Foraging zone latency data measured as one of the foraging behaviour
 endpoints in the foraging behaviour trial, see details in Tan et al. 2023
 (doi: 10.1111/eth.13403). **Variables:** Observation id: unique
 observation ID assigned to each focal experimental individual (these are
 not tracked across different behavioural trials as animals are grouped
 housed, i.e., individuals with the same unique identifier are only
 specific to each type of behavioural trial). Observation date: date of
 measurement Total length: total length of trial video in seconds
 acclimation period: trial video timestamp where acclimation period ends in
 seconds first entry including acclimation period: first entry into
 foraging zone recorded in trial video first entry: true first entry
 latency (first entry in trial video minus acclimation period timestamp)
 *** #### CSV File Name: eat.csv **Description of file:** Foraging
 behaviour latency data measured as one of the foraging behaviour endpoints
 in the foraging behaviour trial, see details in Tan et al. 2023 (doi:
 10.1111/eth.13403). **Variables:** Observation id: unique observation ID
 assigned to each focal experimental individual (these are not tracked
 across different behavioural trials as animals are grouped housed, i.e.,
 individuals with the same unique identifier are only specific to each type
 of behavioural trial). Observation date: date of measurement Total length:
 total length of trial video in seconds time to first eat including
 acclimation period: time to first consume a food item recorded in trial
 video (this includes the acclimation portion of the video and is not the
 true latency data) *** #### CSV File Name: o2.slope.csv **Description of
 file:** Oxygen consumption data (% oxygen saturation over time) used to
 analyse metabolic rates of caffeine-exposed mosquitofish, see details in
 Tan et al. 2023 (doi: 10.1111/eth.13403). **Variables:** label: Arbitrary
 consecutive identifier TRIAL ID: unique observation ID assigned to each
 focal experimental individual (these are not tracked across different
 behavioural trials as animals are grouped housed, i.e., individuals with
 the same unique identifier are only specific to each type of behavioural
 trial). TREATMENT: caffeine exposure treatment group; C = control, L = low
 nominal treatment, H = high nominal treatment, B = blanks used to measure
 oxygen depletion in chambers not including a focal individual DATE: date
 of measurement TANK ID: experimental tank ID used in trial ROUND: round of
 trial per day Lbound: summary statistic provided by summary of function
 rankLocReg in R package LoLinR; left bound of time series local regression
 Rbound: summary statistic provided by summary of function rankLocReg in R
 package LoLinR; right bound of time series local regression alph: summary
 statistic provided by summary of function rankLocReg in R package LoLinR;
 window size used to fit local regression as proportion of data b0: summary
 statistic provided by summary of function rankLocReg in R package LoLinR;
 y-axis intercept (% oxygen saturation) of local regression b1: summary
 statistic provided by summary of function rankLocReg in R package LoLinR;
 slope of local regression (% oxygen saturation over time/every 30 seconds)
 b1LoCI: summary statistic provided by summary of function rankLocReg in R
 package LoLinR; lower bound of 95% confidence interval for the slope (b1)
 of the local regression b1UpCI: summary statistic provided by summary of
 function rankLocReg in R package LoLinR; upper bound of 95% confidence
 interval for the slope (b1) of the local regression ciRange: summary
 statistic provided by summary of function rankLocReg in R package LoLinR;
 range of 95% confidence interval skew: summary statistic provided by
 summary of function rankLocReg in R package LoLinR; skewness of
 standardized residuals for the local regression estimated as the
 Fisher-Pearson standardised third moment coefficient bgN: summary
 statistic provided by summary of function rankLocReg in R package LoLinR;
 modified Breusch-Godfrey statistic used to estimate autocorrelation of
 standardised residuals Lz: summary statistic provided by summary of
 function rankLocReg in R package LoLinR; combined linearity metric used to
 rank local regressions with implicitly weighting of each component metric
 by their empirical variances Leq: summary statistic provided by summary of
 function rankLocReg in R package LoLinR; combined linearity metric used to
 rank local regressions with component metrics weighted equally Lpc:
 summary statistic provided by summary of function rankLocReg in R package
 LoLinR; combined linearity metric used to rank local regressions with
 summed percentile-ranks of Zmin scores for each component metric. This
 ranking method was used in data analyses. regressions.n: number of fitted
 local regressions per focal individual time series SEX: sex of individual;
 JF and JM = juveniles (eventually consolidated into one group within
 analyses), M = males, F = females sex.correct: sex of individuals with
 juveniles consolidated into one group; J = juveniles, M = males, F =
 females WEIGHT: wet body weight of individuals in grams chamber.vol:
 volume of respirometry chambers in milliliters blanko2: slope of oxygen
 depletion of blank chambers used to account and control for background
 respiration, matched by experimental tank ID and day of trial activity.cm:
 activity of individual within chamber measured as a subset of trial period
 to account for movement during oxygen consumption *** #### CSV File Name:
 Fish.Lengths.Cleaned.csv **Description of file:** Standard and total
 lengths of mosquitofish used across all experimental trials, extracted
 from images **Variables:** Aim: Timepoint of measurement and corresponding
 experimental trial; Pre = pre-exposure prior to caffeine treatment, 1 =
 fish used in foraging and metabolic rate trials 21-days since start of
 caffeine exposure, 2 = fish used in activity and antipredator trials
 42-days since start of caffeine exposure Treatment: caffeine exposure
 treatment group; PE = pre-exposure, C = control, L = low nominal
 treatment, H = high nominal treatment Treatment #: exposure tank ID Fish
 Number: unique ID arbitrarily assigned to each fish within each exposure
 tank, treatment group, and timepoint Date Taken: data photograph of fish
 was taken (following completion of respective experimental trial, or prior
 to exposure for pre-exposure fish) Measurement: type of measurement taken;
 Standard.Length = standard length of fish in millimeters, Total.Length =
 total length of fish in millimeters Order: arbitrary order of measurement
 taken Length: length of corresponding measurement in millimeters *** ####
 CSV File Name: fish.measurements.full.csv **Description of file:** Wet
 body weight of mosquitofish used across all experimental trials, with
 measurements taken following completion of respective experimental trial
 **Variables:** date: data of measurement sex: sex of individual; JF and JM
 = juveniles (eventually consolidated into one group within analyses), M =
 males, F = females weight.g: wet body weight in grams exp.tank.id: focal
 individual's experimental tank ID within each experimental trial
 Observation.id: unique ID assigned to each fish within each experimental
 trial exposure: caffeine exposure treatment group; c = control, l = low
 nominal treatment, h = high nominal treatment, fwh and fwl = fish returned
 to freshwater from their respective low and high caffeine exposure
 treatments. However, note that these fish were not eventually used in
 analyses. trial: type of experimental trial respective to each focal
 individual timepoint: timepoint of measurement following start of caffeine
 exposure, 1 = 21 days since start, 2 = 42 days since start ***