The senior New York police officer at the centre of the Occupy Wall Street pepper spray controversy fired the gas at protesters a second time just moments later.

After new video emerged on Wednesday showing the second incident, New York police commissioner Ray Kelly told reporters that the Civilian Complaint Review Board would investigate the officer, deputy inspector Anthony Bologna.

The New York Police Department's own internal affairs bureau also plans to open an investigation, the New York Times reports.

The investigations were announced after bloggers and activists drew attention to video posted online which showed that Bologna fired pepper spray on two occasions last Saturday as officers broke up a protest march through Greenwich Village.

The first footage shows him targeting a group of female protesters who were being penned in by officers on East 12th Street. The latest video shows another incident on the same street, shortly after the first, when he fired more pepper spray towards at least one of the same women, after they were recovering from the first incident.

On both occasions, the officer appears to have violated New York Police Department guidance on how the gas should be used.

In response to the Guardian's appeal to readers to help us reconstruct Saturday's events on East 12th Street, one protester wrote to say that she was sprayed with gas by the officer both times.

The protester, Ashley Drzymala, also sent us a link to this raw footage, which shows - at about the 3:56 mark - the officer spraying protesters as they retreated from the area of West 12th Street where he had used the gas on another group about a minute earlier.

Early on Wednesday, Charlie Grapski, in a diary for the Daily Kos, posted a slow-motion edit of just the portion of the video that showed Inspector Bologna spraying the retreating protesters.

Drzymala, 21, a student at a state university outside New York, told the Guardian that she had also been sprayed in the first incident.

We saw police throw a guy who had a video camera into a car. I remembered a police officer pushed the kid and he was trying to get away he was just videotaping, he was not inciting anything. I was saying "What are you doing? Stop it, we're peaceful" I kept saying "what are you doing" They shoved him into a car, they just attacked him, it was uncalled for." Then the girl next to me was pulled through the net by police. She had a black-t-short and black curly hair. Then she was dragged across the ground.

Drzymala, who was also shooting video, said she captured an image the woman in the black t-shirt before the attack, when she was calling police fascists and she was uninjured, and afterwards, when they were trying to leave the pavement, her mouth was bloody.

"I was watching her and then I looked up and I saw the police officer, the one in the white shirt coming up with his hand up and he had a vial in his hand."

Her own video shows the first use of pepper spray quite clearly, although her camera was pointing away from inspector Bologna when he sprayed her the second time.

Drzymala added that since she was in Cairo as an exchange student in January and took part in the Tahrir Square protests, she managed to escape the worst of the pepper spray. Being teargassed in Egypt had taught her to turn her head away, she said.

Asked about the first, more highly-publicized use of pepper spray by Bologna, the NYPD commissioner said on Wednesday, "I don't know what precipitated that specific incident." Chelsea Elliott, who was among the first group to be sprayed, drew our attention to this raw footage, which shows more of what the women were doing moments before the gas was fired at them - arguing with police officers.

The section of the NYPD patrol guide that outlines how pepper spray should, and should not, be used, was posted online in a 2000 report on the matter by the Civilian Complaint Review Board, an independent agency that examines allegations of police misconduct.

According to the guidance, officers are permitted to use pepper spray when "necessary to effect an arrest of a resisting suspect, for self-defense or defense of another from unlawful force, or to take a resisting emotionally disturbed person into custody." The patrol guide also specifies that officers should "not use pepper spray on subjects who passively resist." Officers with special training, however, do have latitude "in the use of pepper spray for disorder control."

Donna Lieberman, director of New York Civil Liberties Union, said: "There's no excuse for using pepper spray in the faces of peaceful demonstrators whether or not they are engaging in minor disorderly conduct. The use of pepper spray appears to be gratuitous and in violation of police department rules. What the video demonstrates how harmful it is for the police to engage in excessive force against protesters because it causes fear and how harmful it is for the police department itself."

The NYPD were forced to change some of their policing practices during demonstrations, specifically their use of pens, after a lawsuit brought by NYCLU which accused them of excessive restrictions in movements of protesters during the February 2003 anti-war protests in New York.

According to recent statistics, 1722 people complained of being wrongfully pepper-sprayed by New York police officers between 2006 and 2010. Of that number, the civilian review board substantiated just 22 complaints.