Sorry to say I would never advise laying a tray directly onto floor boards, they never last, can just about guarantee that the boards will move over time and cause problems with the tray in a couple of years, if not sooner. If you are going to lay a tray @ floor level then I'd receommend you lift the floorboards to the footprint of the tray. Crossbrace the joists (dwangs) and ensure there is a joist or a support all the way around the perimeter of the tray, then lay at least 18mm/22mm WBP ply/USB3 board into the space to the level of the existing fllor.
Screw @ 100mm spacing, ensuring the floor is dead level. If this is an upper floor then I'd also recommend you tank the space up to at least knee height, wall covering dependent.
You can then cover the whole floor in 3mm WBP ply if needed or just up to the tray, either or. Don't ever use softwood ply. What floor covering is goign down? If it's tile then 3mm really isn't thick enough. Don't foget a separation/decouple layer if it's tile too.
Oh ... how deep are your joists and is that a 11/2" waste pipe notched into it?