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EFFECTS ASSESSMENT
biodegradability tests (Annex V C.4C,F,E,B; OECD 301B,C,D,E, 1992f). No-effect or EC0
values were 1.5 to 10 times lower than the corresponding EC50 values. The authors recommend
as a provisional rule that biodegradation testing should therefore be conducted at one-tenth of the
EC50 concentration to ensure that a “probable non-inhibitory level” is employed in
biodegradation testing. It would, therefore, seem appropriate to consider the test concentration
from a positive ready biodegradability test to be an acceptable alternative to a NOEC obtained
from a microbial toxicity test for the purposes of determining a PNECmicroorganisms. This is
particularly the case if domestic sludge is used as the source of microorganisms and if there is no
indication of toxicity for the test concentration, e.g. due to other available test results. Similarly,
data from inherent biodegradability testing may also prove useful. However, some additional
issues have to be considered:
Only Ready Biodegradability Tests (RBT) relying on continuous monitoring, i.e. the MITI I test
(EU Annex V C.4F; OECD 301C, 1992f) and the Manometric Respirometry test (EU Annex V
C.4D; OECD 301F, 1992f), are considered reliable for observing the effects of a chemical on the
inoculum, i.e. activated sludge diluted by factors ranging from ca. 100 to 1000. In parallel to the
test itself, a toxicity control is run in extra bottles containing both the test chemical and a
reference chemical that is easily degraded in the system. If for that purpose sodium acetate is
used, the toxic effect is most often manifest as a delayed mineralisation of the substance.
However, even if the vast majority of microorganisms are initially killed in the test system, such
a delay may only be in the order of a few hours or days before rapid mineralisation of sodium
acetate takes place. If measurements are carried out only weekly, which is the case in most
RBT's, a delay in mineralisation of sodium acetate of only a few days may not be detected,
leading erroneously to the conclusion that the test chemical is not inhibitory. Sodium benzoate
may provide an acceptable alternative to sodium acetate when an inhibitory control test (i.e. the
official term, not 'toxicity test') is performed with an RBT method that is not based on
continuous monitoring, because mineralisation of benzoate occurs at a much slower rate.
Subject to expert judgement, consideration of data from biodegradation/removal studies using
the laboratory/pilot scale Activated Sludge Simulation, Continuous Activated Sludge or Aerobic
Sewage Treatment Coupled-Units tests (OECD 303A, 2001b; ISO-11733) may also prove useful
in any consideration of PNECmicroorganisms. These tests are laboratory scale models for simulation
of activated sludge, representing realistic approximation to actual conditions within full scale
STPs. A NOEC from well-conducted simulation studies using domestic activated sludge would
correspond to the concentration of the chemical substance that does not perturb the proper
functioning of the Continuous Activated Sludge unit with regard to performance parameters such
as:
• test substance elimination;
• COD removal;
• nitrification;
• denitrification;
• phosphorus removal;
• effluent quality etc.
when compared to a parallel non-dosed control.
Additionally, the results from tests with ciliated protozoa can be used for deriving a
PNECmicroorganisms. In this case protozoa have to be regarded as additional species, not as an
additional trophic layer. Ciliated protozoa, constituting the most important class of protozoa in
STPs, are, except for certain industrial plants, important for their functioning. The toxicity data
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