This method would avoid the previous definition of reference conditions and is not fully in agreement with the WFD. (2) The second approach considered up-dated calculations based on pristine conditions (several 1000 years ago). The lack of knowledge and data for this situation as well as high uncertainties with respect to the model application prohibited this method. (3) The third approach assumed Torin 1 cell line a realistic historic reference situation and calculated targets based on that. (4) The fourth approach considered a transfer of historic reference conditions to the presence. The models would have calculated potential reference conditions based on recent basic data (e.g. land-use cover, population density). In a
second step the effects of future climate change would have taken into account. This was the most innovative and scientifically challenging approach, but included assumptions which by some authorities were considered as too subjective. Therefore, the national working group favoured approach 3. During the second meeting possible reference conditions were discussed. The WFD common implementation strategy (CIS) provides additional explanations (REFCOND, 2003): ‘Reference conditions do not equate necessarily to totally undisturbed, pristine conditions.’ They ‘…shall MK-2206 chemical structure be established for each water body type.’ CIS-COAST
 further states: ‘…it is unrealistic to base reference conditions upon historic Ribociclib nmr landscapes that no longer exist in modern Europe.’ ‘The description of the biological reference conditions must permit the comparison
of monitoring results with the reference conditions…’. ‘A hierarchical approach for defining reference conditions is suggested using the various methods in the following order: An existing undisturbed site or a site with only very minor disturbance; or historical data and information; or models…’. Existing literature shows the complexity of finding and defining a high ecological status for WFD biological elements (benthic invertebrates, macroalgae and angiosperms, phytoplankton) especially for German lagoons, fjords and bays. However, compiled historic data, maps and evaluations indicate that at least water transparency and macrophyte coverage in the sea and in all coastal waters were high before the year 1900 [6,32,49,51,57]. Danish and Swedish data support the need to define a very early ‘pre-industrial’ state, like 1880, as reference condition ,  and . However, other authors refer to the minor changes that took place between 1880 and the 1950s, indicate a high ecological status still for the 1950s and early 1960s and suggest this period as a possible reference state  and . Phytoplankton biomass in Kiel Bight, for example, did not increase during the first half of the 20th century but may have doubled during the 1960s . These results are supported by model applications .