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Toxicity to aquatic algae and cyanobacteria

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Description of key information

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.

Key value for chemical safety assessment

EC50 for freshwater algae:
27 mg/L
EC10 or NOEC for freshwater algae:
6.25 mg/L

Additional information

One reliable key study is available for this endpoint (Vryenhoef H. and Mullee D.M. , 2010).

In this study, the 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.

 

Following a 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.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

 

Analysis of 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.

 

According 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.

 

The results obtained withnominal concentrationswere as follows:

72h-ErC50 = 27 mg/L (growth rate)

72h-EbC50 = 13 mg/L (biomass)

72h-NOEC = 6.25 mg/L (growth rate and biomass)

The results obtained with thegeometric mean of the measured concentrationswere as follows:

72h-ErC50 = 1.6 mg/L (growth rate)

72h-EbC50 = 1.1 mg/L (biomass)

72h-NOEC = 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, green algae,),

- 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 1982)).

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 substance itself.

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 assay solution.

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 after 72h.

REFERENCES

Baeza-Squiban et al. (1988), cited in Tagaki (2010)

Baeza-Squiban et al. (1990), cited in Tagaki (2010)

Ben-Tal and Cleland (1982), cited in Tagaki (2010)

ECHA (2010) Methanol REACH dissemination dossier.

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 (2010)

Pflugmacher et al. (2000), cited in Tagaki (2010)

Pflugmacher and Steinberg 1997, cited in Tagaki (2010)

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, http://www.rsc.org/binaries/LOC/2010/PDFs/Papers/117_0056.pdf

Takagi T (2010) Bioconcentration, bioaccumulation and metabolism of pesticides in aquatic organisms, D.M. Whitacre (ed.), Reviews of Environmental Contamination and Toxicology 204, 1-132.

Wang and Lay (1989), cited in Tagaki (2010)

Wright and Maule (1982), cited in Tagaki (