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Los Leones booked a place in the competition for the first time in 24 years in March through the Rugby Europe Championship.
However, World Rugby announced in April that Spain had been booted out of the competition after being found to have fielded an ineligible player in two preliminary matches.
South African Gavin Van den Berg played twice for Spain, but his eligibility was questioned by rivals Romania, with World Rugby deciding he did not fit the criteria.
To qualify on residency rules, players involved in qualifying needed to have lived in their chosen country for 36 consecutive months.
The Spanish Rugby Federation (FER) pinned the blame on an alleged forgery of Van den Berg's passport, and they submitted new evidence to help support their appeal.
However, it was ruled inadmissible because the FER "had not adequately explained" why the evidence had not been made available prior to the first hearing.
World Rugby therefore upheld the previous decision and added that Spain – who also suffered a points deduction for fielding an ineligible player in their failed 2019 World Cup qualifying campaign – had no further right to appeal.
Romania will replace Spain in Pool B, which also contains Ireland, Scotland, reigning world champions South Africa and either an Asian or Pacific qualifier.
Portugal will take Romania's place in the Final Qualification Tournament in November 2022, with the competition itself scheduled to begin in France on September 8 next year.