62 Alternative Methods
biomarkers, whole-organism tests and biological early warning systems for bio-
logical monitoring (Allan et al., 2006). These tools, many being under validation,
even if they are commercially available, are actually designed for water bodies mon-
itoring and very few for wastewater. However, considering their complementary
nature with reference and other alternative methods, there are several new methods
for biological monitoring. Further developments will be devoted to direct applica-
tion to wastewater quality. Meanwhile, a lot of emerging tools can already be used
for discharge toxicity monitoring, such as bioassays and biological early warning
systems (see Chapter 5.1). Other emerging tools designed for chemical monitoring
are passive samplers, immersed in a stream, for the selective adsorption and con-
centration of micropollutants. A recent review (Vrana et al., 2005) has pointed out
the huge development of this approach for water quality monitoring. Even if only
a few applications exist for wastewater quality monitoring with analysis of polar
organic compounds (Alvarez et al., 2005) or trace metals and organic micropollu-
tants (Petty et al., 2004), the use of passive samplers appears to be a very promising
technique, even if the calibration is difficult as it is strongly dependent on the com-
position of water. This is the reason why applications deal with wastewater discharge
impact.
1.4.4 COMPARABILITY OF RESULTS
The purpose of this section is not to give an exhaustive overview of the tools for qual-
ity control and assurance for water quality (the reader will find complete information
in Quevauviller, 2002), but rather to stress a simple procedure to check the compa-
rability of results between a reference method and an alternative one (candidate for
being recognised as an equivalent method).
There exist very few standards for the purpose. The French experimental stan-
dard (AFNOR XP T90-210, 1999) on the evaluation protocol of a physico-chemical
quantitative analysis (for water analysis) regarding a reference method, defines some
principles and tools for the comparability of methods. Considering the complexity
of the problem, this standard is still experimental, and discussions still exist. How-
ever, the principles of this standard have been chosen for the evaluation procedure
for comparing two methods intended for the detection or quantification of the same
target group or species of microorganisms (ISO 17994, 2004). ISO 17994 provides
the mathematical basis for the evaluation of the average relative performance of two
(quantitative) methods against chosen criteria of equivalence. Another international
standard (ISO 11726, 2004) describes procedures for validating alternative (quan-
titative) methods of analysis for coal and coke either directly by comparison with
the relevant international standard method or indirectly by comparison with refer-
ence materials that have been exhaustively analysed using the relevant international
standard method.
Comparability of Results 63
Reference method (x)
Alternative
method (y)Theoretical line
y = x
Experimental line
y = a.x + b
0
Figure 1.4.2 Comparison between reference and alternative methods
The principles of comparison are simple and schematically based on two steps:
rThe first step aims to calculate the analytical characteristics of the two methods
(reference and alternative), including the reproducibility for a given value (from a
standard solution). A first comparison is carried out on the average values, from a
Fisher–Snedecor test. If the test is conclusive (if the two values are not statistically
different), the second step can be performed.
rThen, the equivalence between methods must be statistically verified by plotting the
results (Figure 1.4.2) and checking the coordinates of the experimental regression
line [comparison of the slope and intercept values which must be not statistically
different from, respectively, 1 and 0, values of the theoretical line (y=x)]. For
the purpose a Student test is carried out.
An example is given in Table 1.4.1, showing the results of the Student test of a compar-
ison from real urban and industrial wastewater (grab samples) for the measurement of
total Kjeldahl nitrogen (TKN). Reference and alternative methods are, respectively,
standard NF EN 25663 and UV/UV procedure (Roig et al., 1999) for TKN. The re-
gression line between the estimated (by the alternative method) and measured values
(by the reference method) is: TKNest =0.96 TKNref +0.86 (R2=0.98). The re-
sults obtained from the comparison of the slope and intercept values to, respectively,
1 and 0, show that the alternative method can be considered as equivalent.
In fact, the scientific decision must be determined by other considerations, such as
the improvement of the alternative method if it brings some consistency advantages
regarding the reference methods (very cheap, rapid, etc.), and the acceptability of
the procedure (Figure 1.4.3).
Once the equivalence between methods is confirmed, the validation procedure
results given for on-/off-line instruments (permanent measurement) must be com-
pleted, taking into account the sampling procedure is different for a laboratory
method and a permanent measurement. For example, considering that regulation
64 Alternative Methods
Table 1.4.1 Results of Student test (95 % confidence interval) for TKN
measurement by UV (method described in Roig et al., 1999)
Student test Values
Slope δ0.9643
Y intercept γ0.8609
Sδ0.02
Sγ0.63
Degree of freedom 55
t0.975 2.01
δ−t0.975*Sδ0.92
δ+t0.975*Sδ1.0045
δ−t0.975*Sδ<1<δ+t0.975*Sδ0.92 <1<1.0045
γ−t0.975*Sγ−0.405
γ+t0.975*Sγ2.127
γ−t0.975*Sγ<0<γ +t0.975*Sγ−0.405 <0<2.127
constraints require 24 h composite sampling before laboratory analysis, the challenge
is to obtain equivalent results with this procedure and with permanent measurement.
In this case, the results to be compared are the mean values for each measurement
during the permanent acquisition, with the reference value of the corresponding
composite sample (Thomas and Pouet, 2005).
Proposal for
alternative method
Characterisation of standard
and alternative methods
Test of comparability
(reliability)
Proposal for
alternative method
Comparable?
YN
Optimisation
of method
Validation (method)
Abandon
Improvement?
Y
N
Use method
Seek acceptance
Relevance?
Y
N
Validation
Figure 1.4.3 Validation procedure of a candidate alternative (equivalent) method (adapted from
Bruner et al., 1997)
References 65
Finally, the international standards already cited (ISO 17381, 2003; ISO 15839,
2003) should be considered for the general evaluation of ready-to-use test kits
methods and on-line systems. Other procedures can also be cited (Battelle, 2002,
2004), including works in progress in the frame of the European project Swift-WFD
(www.swift-wfd.com).
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