DOI: 10.1594/pangaea.956789
title: Hydrolysis rates of biodegradable plastics by the gastric fluid and enzymatic activities of the gastric fluid of the edible crab Cancer pagurus

description: The hydrolytic degradation of biodegradable and conventional plastics by the gastric fluid of the edible crab Cancer pagurus were assayed in-vitro with pH Stat titration. Suspensions of different microplastics were incubated with gastric fluid at 15 °C in artificial seawater. Rates of hydrolysis, as determined by counter-titration with a diluted base (NaOH), was recorded for two hours. The gastric fluid was separated by anion exchange chromatography into 65 fractions. Again, three pools of fractions were tested for their hydrolytic potential with a plastic based on polylactic acid at 30°C. Enzyme assays with fluorogenic substrates were performed with each of the 65 fractions of the gastric fluid. Methylumbelliferone derivatives of fatty acid esters (MUF-butyrate, MUF-heptanoate and MUF-oleate) were used as substrates in buffer at different pH ranging from 5 to 9 and at a temperature of 25°C. The increase in fluorescence was measured with a microplate reader. Analogously, the effect of a detergent (sodium dodecyl sulfate) on the enzymatic activity was measured. All measurements were conducted under controlled laboratory conditions.
description: Adult Cancer pagurus were collected by beam trawling with the research vessel FK Uthörn in the North Sea near Helgoland, Germany (54°09'54.7N 7°52'06.2E), between April 2020 and September 2022. After collecting the crabs, they were kept in the laboratory at 15 ± 1 °C. The crabs weighed 300 to 1100 grams. Gastric fluid of up to 1 mL was extracted from the crabs every two weeks.The excel sheets labeled as "HydrolysisRates" contain hydrolysis rates of different biodegradable polymers, measured with an automatic pH Stat titration device. The hydrolysis rates of the reaction with gastric fluid from the edible crab Cancer pagurus were determined by addition of a diluted base over time to keep a constant pH. Biodegradable polymer particles (30 mg) were suspended in 10 ml artificial seawater and incubated with gastric fluid or pooled fractions thereof at 15 °C and 30 °C. Data were recorded for two hours, one hour before enzyme addition and one hour after enzyme addition. The columns headings are abbreviated and mean the following. Min: Duration of the titration in minutes; ml: Volume of added NaOH; pH: pH value of the suspension, set to 8.2; °C: Temperature of the suspension. Unless otherwise specified, the concentration of the added NaOH was 10 mM.The designations of the biodegradable plastic materials are composed of the abbreviations of the project in which framework the data were collected, the intended use of application and the predominant base polymer.BPE: BioPlastics EuropeAMF: Agricultural mulch filmsSP: Soft packagingRP: Rigid packagingC: CutleryT: ToysPLA: Polylactic acidPBS: Polybutylene succinatePHBV: Poly(hydroxybutyrate-co-valerate)The pdf-file ChromatographyReport includes information about the separation of the gastric fluid of C. pagurus into 65 different fractions via anion exchange chromatography. This file provides information about the system used, a detailed report on different method steps and an event log of the run.The excel sheets labeled as "EnzymeActivity" contain measurements of enzymatic activity assayed with different 4-methylumbelliferone (MUF) derivatives. The gastric fluid of C. pagurus was separated via anion exchange chromatography into 65 different fractions. Each fraction was incubated in a 96-well microplate with MUF-derivatives of fatty acid esters of different chain length and the resulting increase of fluorescence was recorded every 15 seconds for 5 minutes at 25 °C. The fluorescence was measured at 355 nm excitation and 460 nm emission with a microplate reader. The substrates used were MUF-butyrate (C4, short chain length), MUF-heptanoate (C7, medium chain length) and MUF-oleate (C18, long chain length). Enzyme activities of the different fractions with each substrate were measured with Britton-Robinson buffer at pH 5, pH 6, pH 7, pH 8 and pH 9. Furthermore, enzyme activities were measured with the addition of 0.1% sodium dodecyl sulfate (SDS) in Tris-HCl buffer at pH 7 to determine the effect of detergents on enzymatic activity.The files with EnzymeActivity in the titles contain several worksheets, of which the first one includes the measurement results. Column A in the first sheet includes the number of readings, column B the average time of the measurement after start and the columns C – BO the measured fluorescence of the fractions at a given time. Column BP includes the blank measurement of the substrate without a fraction. The samples were applied in ascending order of their number, with fraction 1 in column C (designated A01) and fraction 65 in column BO (designated F05). The other worksheets include general information about the instrument and details about protocol parameters.The excel sheets labeled as "StandardCurve" contain fluorescence measurements of 4-methylumbelliferone standards to compare the fluorescence signals from the fractions with known concentrations of 4-methylumbelliferone. Standard curves were prepared in Britton-Robinson buffer at pH 5, pH 6, pH 7, pH 8 and pH 9, and Tris-HCl buffer at pH 7.The files with StandardCurve in the titles contain several worksheets, of which the first one includes the measurement results. The other worksheets include general information about the instrument and details about protocol parameters. Standards were applied in triplicate onto the well-plate. In the first sheet, Column B corresponds to the highest amount of MUF, while column M corresponds to the lowest amount of MUF used for the standards. The concentrations used were:Column B: 35 µmol·L-1 (Std0001 – Std0003)Column C: 30 µmol·L-1 (Std0004 – Std0006)Column D: 25 µmol·L-1 (Std0007 – Std0009)Column E: 20 µmol·L-1 (Std0010 – Std00012)Column F: 15 µmol·L-1 (Std0013 – Std0015)Column G: 10 µmol·L-1 (Std0016 – Std0018)Column H: 7.5 µmol·L-1 (Std0019 – Std0021)Column I: 5 µmol·L-1 (Std0022 – Std0024)Column J: 3.75 µmol·L-1 (Std0025 – Std0027)Column K: 2.5 µmol·L-1 (Std00028 – Std0030)Column L: 1.25 µmol·L-1 (Std0031 – Std0033)Column M: 0 µmol·L-1 (Std0034 – Std0036)