The 32-year-old is likely to miss his side's opening two World Cup group games after picking up a knee injury in their 3-1 loss to Mexico in a friendly on Saturday
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Castillo requires 15 to 21 days to recover from the strained ligament, ruling him out of their Group E opener against Switzerland.
He is likely to miss their second match against Honduras but could be available for the June 25 meeting with France.
Stuttgart midfielder Carlos Gruezo, 19, has been included in the squad as additional cover in the centre of the park.
Manchester United winger Antonio Valencia is the star of Reinaldo Rueda's squad, who face England in a friendly on Wednesday.
Ecuador squad in full:
Goalkeepers: Maximo Banguera (Barcelona), Adrian Bone (El Nacional), Alexander Dominguez (LDU)
Defenders: Gabriel Achilier (Emelec), Walter Ayovi (Pachuca), Oscar Bagui (Emelec), Frickson Erazo (Flamengo), Jorge Guagua (Emelec), Juan Carlos Paredes (Barcelona)
Midfielders: Michael Arroyo (Atlante), Segundo Castillo (Al Hilal), Carlos Gruezo (Stuttgart), Renato Ibarra (Vitesse), Fidel Martinez (Tijuana), Edison Mendez (Independiente Santa Fe), Christian Noboa (Dynamo Moscow), Luis Fernando Saritama (Barcelona), Antonio Valencia (Manchester United)
Forwards: Jaime Ayovi (Tijuana), Felipe Caicedo (Al Jazira), Jefferson Montero (Morelia), Joao Rojas (Cruz Azul), Enner Valencia (Pachuca)