The goal of this ILC is to demonstrate the performance of the (reference) methods using reference materials (RMs) developed within the project.

Both MS-based method (chemical) and Effect-Based Methods (EBMs bioassays) will be tested on the same test materials

Chemical measurement
For chemical measurement are to be considered as mandatory:
– Measurements of the three WFD priority substances: 17-beta-estradiol (17βE2), 17-alpha-ethinylestradiol (17EE2), and estrone (E1)
Optional, but strongly recommended:
– Measurement of Estrogens 17-alpha-estradiol (17E2) and estriol (E3).
The method validated will particularly aim to measure selected endocrine estrogens by GC-MS-MS, LC-MS-MS, GC-HRMS and LC-HRMS. The laboratories, if possible, may apply more than one instrumental detection system. For the laboratories the following pre-treatment steps are mandatory:
i) A stable isotope dilution method (i.e. isotopically labeled internal standard for each estrogen)
ii) Sample preparation technique based on SPE or SPE-DISK (C18, polymeric phase (HLB, DVB, polymeric reversed phase))
iii) Purification step (LC-NH2, SiOH or selective SPE cartridges for estrogens)

EBMs measurement
EBMs particularly aim to measure the overall effect of the selected endocrine estrogens. The application of ERα-Calux and/or A-YES is preferred, while the use of other bioassays is optional. For the laboratories the following pre-treatment steps are mandatory:
i) Sample preparation technique based on SPE or SPE-DISK (C18, polymeric phase (HLB, DVB, polymeric reversed phase)
ii) Purification step (LC-NH2, SiOH or selective SPE cartridges for estrogens)

Materials
In order to define precision data characterizing the performance of the method, the interlaboratory comparison organizer will provide two (2) Reference materials as kit.
Each kit is composed by:
– 2 units of natural water (1L each)
– Ampoules of SPMs (suspended particulate matters) and DOC (Dissolved Organic Compounds)
– Vials containing the standard of each targeted 5 substances
The two kits will have different level of concentration.
Each test sample reconstituted by the laboratory participating in the ILC, following a specific protocol provided by the organizers (including also a video), will be analyzed under repeatability conditions in duplicate (independent measurements). The overall number of samples to be analyzed will be four (4).

Regarding internal quality control the laboratory has to measure 1 extra unit of water for quality control (blank) provided by the organizers together with the kits.
The laboratories interested in the ILC will receive detailed instructions for the participation.

We ask your interest in participating in this interlaboratory comparison.
Participation will be free of charge, but you will be required to strictly follow the protocol and report the measurement results to the organizer.
In order to best anticipate the results that will be returned during this ILC and to be able to adapt (as much as possible) the number of samples/kits to be provided, participating laboratories are also asked, in the pre-registration form the techniques (both chemical methods and/or bioassays) that they will be able to perform during the ILC and for each of them to give an indication of the compounds that will be analyzed (both mandatory and recommended).

Timing
– The provisional schedule for this ILC includes the sending of the samples in the first week of November 2022. The precise dates will be communicated later in due time, according to the progress of the project and to the registrations of the participants.
– The results must be delivered within three weeks of sending the test material to the laboratories

– The intermediate report will be returned to the laboratories by four weeks from receiving the measurement results from the laboratories.
In order to formalize your pre-registration for this ILC, we kindly ask you to fill the following pre-registration form (google survey ) by the 23rd September 2022.

Thank you in advance for your collaboration

EDC-project Coordination team

ILC contact persons:
Paolo de Zorzi (ISPRA): paolo.dezorzi@isprambiente.it

Teemu Näykki (SYKE): teemu.naykki@syke.fi

Sabrina Barbizzi (ISPRA): sabrina.barbizzi@isprambiente.it