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Nuno Rocha joined Tosno from Romanian club Universitatea Craiova last year
The incident comes less than two months before Russia hosts the World Cup and is sure to fuel fears of racism marring the tournament.
Wednesday’s game in Moscow culminated with a penalty shootout between hosts Spartak and the small Russian Premier League side Tosno.
Rocha scored the winning spot-kick for Tosno before running past Spartak supporters while celebrating the upset.
Footage of the episode posted on the Championat.ru sports site showed the 26-year-old cupping his hand to his ear as what sounded like a chorus of ape noises rang down from the crowd.
The Russian Football Union’s (RFU) anti-discrimination department chief Alexei Smertin said the chants came from Spartak fans.
“We are currently studying game material, including the chanting that was heard from the Spartak supporters’ stand after Nuno Rocha’s penalty,” he told Russian media.
Football officials are expected to issue their ruling during a disciplinary committee meeting next week.
The investigation comes with concern about racial abuse in Russia already running high after a spate of recent incidents.
The world football governing body FIFA has opened disciplinary proceedings against Russia over monkey chants heard at a World Cup warmup against France last month.
Manchester United midfielder Paul Pogba and Barcelona winger Ousmane Dembele were allegedly targeted during the match in Saint Petersburg.
Racism has been plaguing Russian football since clubs began purchasing foreign players after the Soviet Union collapsed in 1991.
The Football Against Racism in Europe (FARE) anti-discrimination network reported 89 racist and far-right incidents at Russian games in the 2016/17 season.
The number was about the same as that recorded in the preceding three seasons — a sign that Russia’s campaign to clean up its game has not had the desired effect.
The World Cup final will be played on July 15 in Moscow while Saint Petersburg will host one of the semi-finals.
Both cities are home to large groups of hardcore nationalist supporters and have witnessed numerous ugly episodes during games.
Spartak have already been fined for monkey chants and other racial abuse heard after its July 2017 Super Cup victory against city rivals Lokomotiv.
They were penalised a second time for what RFU disciplinary committee chief Artur Grigoryants had said were “50 seconds of chanting” hurled at Lokomotiv’s Brazilian goalkeeper Guilherme Marinato during a league game in March.
Spartak were hit by more controversy when centre-back Georgi Dzhikiya used the team’s official Twitter account in January to send out a message likening its three Brazilian players to “chocolates”.
Rocha played in Portugal and Romania before joining Tosno last year.
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