Sampling procedure and measurements of life-history traits
Immediately prior to dopamine exposure on day 0 of the experiment, a
random sample of thirty F0 neonates were photographed
for body length measurements (mm, measured from the upper margin of the
eye to the junction of the carapace and spine, using ImageJ v1.52a,
National Institutes of Health, Bethesda, MD). Individual measurements of
body length at first reproduction for the F0 generation
(after releasing their first clutch of eggs) and at birth for the
F1 generation was measured in the same way. All body
length measurements (mm) were converted to dry mass (mg) using the
equation by Yashchenko et al., (2016): Dry mass = 0.00535 × Body
length2.72. The somatic growth rate in the
F0 generation was calculated for each individual as:\(Somatic\ growth\ rate=\frac{\ln\left(\text{dry\ mass}_{\text{end}}\right)-\ln\left(\text{dry\ mass}_{\text{start}}\right)}{\text{duration}}\),
where dry mass start is the average dry mass (in mg) at
the neonatal stage (day 0), dry mass end is the
individual dry mass (in mg) at maturation, and duration is the number of
days between the two stages.
As in Issa et al. (2020), conductivity (WTW LF 330 conductivity meter),
pH (WTW pH 340i) and dissolved oxygen (WTW Multi 3410 multiprobe meter)
were measured. This was done on two separate occasions throughout the
experiment (Table 1), after medium renewal, in the new exposure
solutions and ADaM medium used for the controls (N = 4; 2 samples
collected in total from each of the
dopamine and control treatments). Simultaneously, the new exposure
solutions and ADaM medium were sampled for dopamine analysis (N = 4; 2
samples collected in total from each of the dopamine and control
treatments).
The samples were stored at -80 °C for a maximum of five months after
collection (during closure of the labs due to the Covid-19 outbreak),
prior to dopamine analysis. Two complementary sample preparation
protocols were used: (1) dilute-and-shoot; and (2) liquid-liquid
extraction. Subsequent analysis was performed by ultra-performance
liquid chromatography coupled to a triple quadrupole mass analyser
(UPLC-MS/MS). This analysis was done as described in Issa et al. (2020),
where single reaction monitoring (SRM) chromatograms were monitored for
dopamine. Typical SRM chromatograms for dopamine in the analysed samples
are depicted in Appendix 1. Over the course of the experiment, pH,
conductivity and dissolved oxygen were within the recommended range for
testing of chemicals in D. magna , according to OECD guidelines
(OECD, 2012). The conductivity remained at 1.6 mS/cm, mean dissolved
oxygen at 8.9 mg/L and pH at 8.0 across treatments, whereas measured
average concentrations of dopamine were ca. 2% of their nominal
concentrations (Table 1). Much lower than expected concentrations of
this compound were likely caused by degradation during unforeseen
long-term storage due to the Covid-19 outbreak.