A ministerial inquiry into the decision to site the EU's Food and Veterinary Office in Grange, Co Meath, has been sought by Friends of the Irish Environment.
Green MEP Ms Patricia McKenna and Fine Gael MEP Mr John Cushnahan also said serious questions must be asked about the development.
Ms McKenna has lodged two complaints with the EU Court of Auditors and the Ombudsman while Mr Cushnahan has submitted a dossier on the issue to the EU's environmental directorate. Ms McKenna said the text of the EU decision ordered that the office be located in a town, but Grange was not a town.
Friends of the Irish Environment also filed a petition with the president of the European Parliament over the choice of location. The site was involved in further controversy this week when it was confirmed that the area's group water scheme had shown E.coli contamination when tested on two occasions last September.
However, the OPW pointed out that the water supplying the EU centre would go through a very sophisticated treatment process.
Documents released under the Freedom of Information Act show that the North Eastern Health Board found evidence of contamination of the Kiltale Water Group Scheme (KWGS) supply with one coliform and one E.coli on September 12th and 19th. Its environmental health officer judged the water samples as "unfit for human consumption without sterilisation (e.g. boiling)".
Coliforms indicate recent faecal contamination of water. E.coli contamination can cause severe gastroenteritis and other complications.
A spokesman for Meath County Council said the presence of just one E.coli or coliform in a sample would not have been a major cause for concern.
"It would be seen as a very, very minor contamination and could have happened at the sampling point," he said.
Follow-up tests by the NEHB in November showed the water was clear and the health board indicated there was no cause for concern. "The OPW carried out six samples over that period of time, all of which were clear," the spokesman said.
Mr Cushnahan said the evidence confirmed his original statement that the water had been contaminated.
He called for an apology from the KWGS and the OPW who, he said, had dismissed his claims.
Ms McKenna said: "The conflicting data are very unsettling and worrying." She has sent copies of this evidence to the European Commission as part of her complaint.
An OPW spokesman emphasised these results could not be applied to the quality of water which would supply the EU office as the water would go through a thorough treatment process now being constructed.
The KWGS had dug a second well and would have a new water treatment plant in operation by late summer. The OPW also had the option of using its own well to supply the new office, he said. The water would also pass through the OPW's own water treatment plant.
The Food and Veterinary Office is being constructed about five miles from Trim. About 160 officials are expected to move to the new site from the temporary office in Dublin early next year.
The development has been dogged by controversy since it began. European staff complained about the move to a rural area and queried the quality of transport and education in the area.
Mr Eric Mamer, EU spokesman on administration and personnel issues, said some staff had left Ireland but he could not say if this was related to the move to Grange.
He said the EU had received guarantees from the OPW on the quality of water which would supply the new centre. "They have guaranteed that there will also be extra precautions regarding water treatment and we have accepted these guarantees," he said.