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EC number: 204-317-7
CAS number: 119-36-8
One reliable key study showed that methyl salicylate nominal EC50 72h is 27 mg/L and NOEC 72h is 6.25 mg/L. Use of nominal concentration is supported by the well documented ability of algae to metabolize a number of chemicals through diverse metabolization enzymes, among them carboxyesterases. So nominal EC50 and NOEC represent methyl salicylate and produced metabolites (methanol and salicylic acid) mixed toxicity in 72 hours.
This is supported by a screnning (reliability 3) study that shows no effect in water extracted fraction from a 100 mg/L substance solution.
One reliable key study is available for this endpoint (Vryenhoef H. and
Mullee D.M. , 2010).
effect of the test item Methyl Salicylate on the growth of the
freshwater green algal speciesDesmodesmus subspicatuswas
investigated in a72‑hour static test according to OECD Guideline 201
(2006), and the method C.3. of Commission Regulation (EC) No
440/2008, C.3. The study was compliant with the GLP.
preliminary range-finding test,Desmodesmus subspicatuswas exposed
to an aqueous solution of the test item at concentrations of 6.25,
12.5, 25, 50 and 100 mg/l(three replicate flasks per concentration)
and a control (six replicate flasks) for 72 hours, under constant
illumination and shaking at a temperature of 24 ± 1°C.
the algal populations were removed daily and cell concentrations
determined for each control and treatment group, using a Coulter®Multisizer
the test preparations at 0 hours showed measured test concentrations to
range from 97% to 106% of nominal.Analysis of the test preparations
at 72 hours showed a concentration dependant decline in measured
concentrations in the range of less than the limit of quantitation (LOQ)
of the analytical method employed to 24% of nominal. This decline
was inline with the preliminary stability analyses conducted which
indicated slight instability over the test period. The further decline
in measured test concentrations was considered by the author to be due
to adsorption of the test item to the algal cells present. Additional
stability analyses conducted under identical test conditions confirmed
the unstable nature of the test item over the 72-Hour exposure period
and the losses of the test item when the algal cells are present.
to current regulatory advice that in cases where a decline in measured
concentrations is observed, geometric mean measured concentrations
should be used for calculating EC50 values, results were not only based
on nominal concentrations but also on the geometric mean measured test
concentrations in order to give a “worst case” analysis of the data. In
cases where the measured concentration was less than the LOQ of the
analytical method following current regulatory advice a value of half
the LOQ (i.e. 0.095 mg/l) was used to enable calculation of the
geometric mean measured concentration.
results obtained withnominal concentrationswere as follows:
= 27 mg/L (growth rate)
= 13 mg/L (biomass)
= 6.25 mg/L (growth rate and biomass)
results obtained with thegeometric mean of the measured concentrationswere
= 1.6 mg/L (growth rate)
= 1.1 mg/L (biomass)
= 0.79 mg/L (growth rate and biomass)
Thehigh level of methyl salicylate decrease
observed in this study when algae are present in the assay medium has
been attributed by the author, to adsorption of the substance on algal
cells.This was only an unverified hypothesis that is contradicted by
the substance water solubility and log Kow that do not let predict such
a strong adsorption.The moderate volatility of methyl salicylate has
been taken into account in the experiment by using flasks plugged with
polyurethane foam bungs. This leads to investigate whether methyl
salicylate metabolization could take place in algae.
At first, an absorption rather than an
adsorption of the substance could be expected, as suggested by results
from Wang and Lay (1989): the ionizable salicylic acid showed
unexpectedly high bioconcentration (log BCF = 3) in green algae, which
may be related to its effect on algal growth. Methyl salicylate could
then be expected to accumulate similarly in algae. However, absorption
is sometimes not necessary when the enzymes are released onto the
medium, as shown in results hereafter.
Algae are known to have various enzymes
able to metabolize a variety of chemicals(Tagaki, 2010):
- oxidases (P450 enzymes in marine algae
(Pflugmacher and Sandermann 1998b))
- reductases (most relevant information on
reductive metabolism mechanism and the enzymes involved is limited to
algae and aquatic macrophytes),
-glucose transferases (salicylic acid has
been shown to be metabolized by Lemna gibba by glucosidation(Ben-Tal
and Cleland (1982)),
- sulfotransferases (Enzyme activity has
been reported in various aquatic organisms such as bivalva, crustacea,
- glutathione-S-transferases (In emergent
and submergent aquatic macrophytes and various algae, GST activity
toward typical substrates were detected in both microsomal and cytosolic
factions (Pflugmacher and Steinberg 1997, Pflugmacher et al. 1999, 2000),
- acyltransferases (Acetylation and
formylation reactions are also known to occur mostly in algae,
macrophytes, and bivalves).
-esterases, among them carboxyesterases(for
example, Baeza-Squiban et al. (1988, 1990) have shown that the green algaDunaliellasp.
can hydrolyze deltamethrin to the corresponding acid and alcohol by
carboxyesterase released into medium. Propanil was hydrolyzed by various
green and blue-green algae to 3,4-dichloroaniline (Wright and Maule
Moreover,esterase activity is used as a
basis for algal bioassays, as shown for example in Regel et al.
(2002), a study that investigated the potential for using algal esterase
activity of Microcystis aeruginosa and Selenastrum capricornutum as a
rapid measure of biological effects of some pollutants, or a more recent
report published on the Royal Society of Chemistry website (Shi and Tai,
2010) using algal esterase as indicator for environmental toxicity.This
shows that esterase activity is a very common feature in algae.
In the light of results above, the methyl
salicylate decay observed in 72h in presence of algae could be explained
by (absorption and) metabolization of the substance rather than to
adsorption that is not supported by physico-chemical properties. In such
a situation, the nominal EC50 and NOEC found reflects the combination of
a toxic effect of methyl salicylate, and its hydrolysis catalyzed by the
algal carboxyesterases, giving methanol and salicylic acid that are not
harmful for algal growth (ECHA, 2010), (Henschel et al., 1997). When a
substance hydrolyses fast (at least within the tests time span), it is
relevant to assess the hydrolysis products hazard for assessing the
In these conditions,nominal
concentrations more appropriately reflect overall methyl salicylate
toxicity (substance itself and hydrolysis products released within the
test time span), rather than the concentrations measured after 72h
contact with algae that reflects substance residues remaining in the
Therefore a chronic toxicity
classification is not appropriate, as reflected by the NOEC expressed as
nominal concentration: 6.25 mg/L.
This is supported as weight of evidence by a
screening algal test considered with reliability 3 because of lack of
substance concentration control and expression of results in biomass
inhibition only, that concluded that no effect was observed in algae
Baeza-Squiban et al. (1988), cited in Tagaki
Baeza-Squiban et al. (1990), cited in Tagaki
Ben-Tal and Cleland (1982), cited in Tagaki
ECHA (2010) Methanol REACH dissemination
Henschel KP, Wenzel A, Dietrich M and
Fliedner A (1997), Environmental hazard assessment of pharmaceuticals,
Regulatory Toxicology and Pharmacology, 25, 220-225.
Pflugmacher et al. (1999), cited in Tagaki
Pflugmacher et al. (2000), cited in Tagaki
Pflugmacher and Steinberg 1997, cited in
Regel RH, Ferris JM, Ganf GG and Brookes JD
(2002), Algal esterase activity as a biomeasure of environmental
degradation in a freshwater creek, Aquat. Toxicol. 59 (3-4), 209-223.
Shi W and Tai Y-C (2010), Algal biotoxicity
assay using µflow cytometer for environmental monitoring,
Takagi T (2010) Bioconcentration,
bioaccumulation and metabolism of pesticides in aquatic organisms, D.M.
Whitacre (ed.), Reviews of Environmental Contamination and Toxicology
Wang and Lay (1989), cited in Tagaki (2010)
Wright and Maule (1982), cited in Tagaki (
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