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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 1. Acceptable, well-documented publication which meets basic scientific principles; however, test method is not comparable to current guidelines. 2. Only one test concentration used.
Principles of method if other than guideline:
No details about the experimental conditions are given.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Ankistrodesmus braunii, strain CCAP 202-7a
- Cultured in Bold's Basal Medium in Erlenmeyer flasks at 23 +/- 1 deg. C with irradiance of 100 uE/s/m2 provided by daylight fluorescent lamps, photoperiod 16 h light: 8 h dark.
- Placed on shaking apparatus.
- Inoculum of 1.5 E05 cells/mL from axenic cultures in exponential phase of growth
Test type:
not specified
Water media type:
not specified
Total exposure duration:
60 min
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
- Algae were grown in methylamine-containing medium (1 mM) at 23 °C and 16 h light-8 h dark photoperiod
- Each test tube contained the algal inoculum and 6 mL of test solution, incubated on shaking apparatus
- Growth of algae was monitored daily either by measuring the absorbance increase at 550 nm with a colorimeter or by counting the cell number with a Thoma blood-counting chamber when the turbidity of the solution made it difficult to estimate the colorimeter.
- Controls contained only distilled water diluted three times with Bold's Basal Medium at pH = 7.0
- Growth experiments were carried out five times and the results based upon the average of the five tests.
Reference substance (positive control):
not specified
Dose descriptor:
other: growth inhibtion
Effect conc.:
21 other: %
Nominal / measured:
meas. (initial)
Conc. based on:
other: at 1mM (31mg/L)
Basis for effect:
growth rate
Remarks on result:
other: no duration time given
Details on results:
21% growth inhibition at 1mM (31 mg/L)
Validity criteria fulfilled:
yes
Executive summary:

The test organism was Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). 21% growth inhibition at 1mM (31 mg/L) was investigated.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data. Please refer also to the detailed read-across justification attached in section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar environmental fate and behaviour and ecotoxicological properties due to their structural similarity, resemblance to their chemical reactivity, and biotransformation products in environmental compartments. In other words, there is a clear chemical analogy (“biotransformation to common compound", scenario 1 of the Read Across Assessment Framework (ECHA 2017)).
The solvation of both methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (“common breakdown product", scenario 1 of the Read Across Assessment Framework (ECHA 2017)). The toxicity of the respective counterion (“non-common compound” – Cl-) is in this case negligible, as Cl does not drive toxicity effects neither in mammalian species nor in aquatic organisms because it is one of the main electrolytes and is required in large amounts in living organisms. So any toxicity of the amounts originated from the methylammonium chloride at maximum dose levels that would be used in ecotoxicity studies is not expected.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance:
methylamine hydrochloride (methylammonium chloride)
structural formula: CH5N.ClH
SMILES: [Cl-].C[NH3+]
CAS 593-51-1
purity: not specified

target substance:
methylamine
structural formula: CH5N
SMILES: CN
CAS 74-89-5
purity: ≥ 80 – 100 %

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Read across from the structural analogue to the target substance is based on the high structural similarity of the analogue with the substance of interest and the similarity of their environmental fate and behaviour and (eco-) toxicological characteristics.
The target substance MMA (CAS 74-59-5), as well as the source substance MMA-HCl (CAS 593-51-1) belong to the category of the “Aliphatic amines” according to the profiler “US EPA New Chemical Categories” in the OECD QSAR Toolbox v4.1. According to the inclusion rules of this category, the environmental toxicity of amines is related to the length of the hydrophobic carbon chains: the longer (or greater the number of carbons) the chain the more toxic to aquatic organisms (when the number of amines is constant); and the greater the number of amines, the greater the toxicity given a constant carbon chain length (Explanation tool of profiler “US EPA New Chemical Categories” in the OECD QSAR Toolbox v4.1). In this regard, the target and the source substance – bearing identical hydrophobic carbon chains – are expected to have a similar/equal environmental toxicity.
Moreover, the basicity of amines increases with the length of the aliphatic rest due to electron releasing properties of alkyl groups: the higher the pKa value, the weaker the acid, so the stronger the base. Monomethylamine (the primary amine with a central nitrogen atom and 1 methyl group and 2 hydrogen-atoms) is the least basic of the aliphatic (primary) amines. Monomethylammonium chloride (composed also of one methyl group attached to the nitrogen atom, which is regarded as the common structure / functional group) is no longer basic, but neutralised (please also refer to the following paragraphs). Therefore, it has to be kept in mind that MMA-HCL is an example of a worst case read-across according to RAAF, as MMA tested as salt may achieve very high doses because it is not corrosive.
Furthermore, the chemicals are characterized by a common Mode Of Action (MOA) in detail as “narcotic amines” according to Acute aquatic toxicity MOA by OASIS in the OECD QSAR Toolbox v4.1.
Methylamine and methylammonium chloride - as primary aliphatic amines - undergo similar reactions and resemble each other in their physico-chemical properties. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987).
Typical reactions of amines are salt formation, alkylation, and conversion into amides and Hofmann elimination from quaternary ammonium salts (Morrison and Boyd, 1987).
As already mentioned above, they are linked by the common functionality of one central nitrogen atom which bears an unshared pair of electrons and tends to share these electrons determining a similar chemical behaviour. This unshared electron pair can accept a proton - forming a substituted ammonium ion. The tendency to share this electron pair underlies the entire chemical behaviour of amines as a group and this was considered as main / basic parameter, which is suitable for read-across within an analogue approach within an/a analogue/category approach.
The dissociation constant of MMA allows the conclusion that virtually all molecules of methylamine - when dissolved in an excess of water - are present as the methylammonium cation. Moreover, the available data of MMA with hydrochloric acid shows clearly that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids. Only the ionic form is the relevant species present. So, in consequence, the solvation of both methylamine and methylammonium chloride in water would result in solutions of the methylammonium cation (“common breakdown product"). Therefore, one must only regard the physico-chemical properties of the respective counterion. Methylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the methylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)toxicological effects. Both anions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions.
Besides the influence of HCl on the pH value of an aqueous solution, it does not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. Generally, it should be denoted that very commonly in literature there is no differentiation made between MMA and MMA-HCl.
The source substance MMA HCL and the target substance MMA have a certain toxicity potential towards fish, aquatic invertebrates or aquatic algae and cyanobacteria, but both do not have to be classified (no classification and labelling for aquatic acute nor chronic toxicity) according to Regulation (EC) No 1272/2008.
Primary amines are similar in their environmental fate endpoints (hydrolytically stable, readily biodegradable, negative log Koc values, no evaporation into the atmosphere expected). Furthermore, the results show that the substances do not have a significant potential for persistence (not P not vP) or bioaccumulation in organisms (not B not VB).
Concerning the toxicity potential towards aquatic organisms, MMA has been found to be harmful towards fish (LC50 = 16 mg/L). Moreover, MMA and MMA-HCl have been shown not to be toxic/harmful towards aquatic invertebrates or aquatic algae/cyanobacteria. However, these results are not severe enough to trigger classification or labelling (no C&L) for MMA and/or MMA-HCl.
The similar findings (refer also to the data matrix outlined below) for both substances support the conclusion that the identical molecule will be formed from both substances, and this molecule, i.e. the methylammonium cation, is responsible for their behaviour in the environmental compartment and the observed effects on aquatic organisms including aquatic algae and cyanobacteria. In consequence, the methylammonium cation is indeed what is left to be considered in both cases and similar effects can reasonably be expected when using data from MMA-HCl for the lacking endpoints, compared to the data obtained with MMA.
Hence, MMA-HCl may perfectly serve as a worst case read-across substance for MMA. So, the available data on MMA-HCl can be used to cover all systemic endpoints currently lacking from MMA, making further testing obsolete.

4. DATA MATRIX
There is data available on the environmental fate and behaviour, ecotoxicological and toxicological properties of MMA. Data on MMA-HCl covers data on Biodegradation, Toxicity to aquatic algae and cyanobacteria, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vivo and Toxicity to reproduction/Developmental toxicity. Hence, the identification and discussion of common properties of MMA and MMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (MMA is - as a pure substance - gaseous at room temperature, MMA-HCl is a solid primary ammonium salt) triggers some differences in the physico-chemical properties like Melting point, Boiling point, Decomposition temperature and Vapour pressure. Nevertheless, regarding the application of both substances, i.e. their distributed form, the gaseous character of MMA becomes less relevant as the substances are usually not applied in their pure forms but rather as aqueous solutions.
The available data for the following physico-chemical properties, which are among others relevant for absorption into living organisms, are very similar. Both substances are small molecules with a low molecular weight of 31.042 (MMA) resp. 67.019 (MMA-HCl), they are both very soluble in water (completely miscible in water (MMA) and at least 1080 mg/L at 20°C (MMA-HCl)), have a negative logPow (-0.713 (aqueous solution, 25°C, pH 11.1 - 11.4) (MMA) and -3.82 (MMA-HCl)), and both are readily biodegradable. Although being expected to be hydrolytically stable in the natural environment, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, MMA has a pKa of 10.79 at 20°C (≙ pKb = 3.21), which indicates that methylamine exists almost entirely in the cationic form as methylammonium cation at pH values of 5 to 9.
MMA (not neutralised) has a toxicity potential towards fish and aquatic invertebrates, but does not have to be classified as hazardous. MMA (neutralised) and MMA-HCl have a lower toxicity potential to aquatic organisms and do not have to be considered to be acutely harmful to fish or aquatic invertebrates, nor to microorganisms (no C & L). MMA and MMA-HCL have been shown to be toxic to aquatic algae and cyanobacteria (shown in Pseudokirchnerella subcapitata, Scenedesmus obliquus and Microcystis aeruginosa). Their aquatic toxicity potential under real environmental conditions has to be judged carefully, since, methylamine and methylamine hydrochloride are readily biodegradable in nature. As such, both can be considered as non-toxic to aquatic organisms and thus do not have to be classified as hazardous as per the CLP classification criteria.
Regarding their toxicity towards mammals, both substances exert their acute oral toxicity in the same range and both are classified as Acute tox 4 and MMA also as STOT SE 3 (C≥5 %). Moreover, MMA is corrosive / irritative to the skin and the eyes (MMA (gas) = Skin Irrit. 2 and Eye Dam 1; MMA (aqueous solution) = Skin Irrit 1B); in the case of MMA –HCl no classification is warranted.
Regarding their repeated dose toxicity, the No-observed-adverse-effect-levels of both substances to differ to a certain extent (NOAEL ≥ 10 versus NOAEL = 500 mg/kg bw/day). For MMA HCL there is also data for toxicity to reproduction and developmental toxicity available (NOEL (reproduction/systemic tox) = 500 mg/kg bw/day and NOAEL (maternal/developmental tox.) = 155 mg/kg bw/day).
No data on long-term toxicity to fish and data on long-term toxicity to aquatic invertebrates was available.
Reason / purpose:
read-across source
Duration:
96 h
Dose descriptor:
other: TTC
Effect conc.:
4 mg/L
Executive summary:

Scenedesmus quadricauda was investigated.

A TTC of 4 mg/L within 96 hours.

The target substance methylamine and the source substance methylammonium chloride used in this study belong to the group of primary aliphatic amines. The solvation of both, methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. The methylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.

Therefore both substances are expected to follow the same toxicokinetic pattern.

Description of key information

Key value for chemical safety assessment

Additional information

The test organism was Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). 21% growth inhibition at 1mM (31 mg/L) was investigated. This experimental data is published by Andreozzi et.al.(2000).

Furthermore, the effects of methylamine hydrochloride on Scenedesmus quadricauda were investiged by Bringmann et al. (1959). A TTC (96 h) of 4 mg/L was found.

Scenedesmus obliquus was investigated to evaluate the toxicity of monomethylamine by Abeliovich, A. and Azov, A. in 1974.

The scientists investigated the effects on the assimilation rate compared to controls at different pH values. The test concentration was chosen to be 160 mg/L. At a pH of 7.9 an 35 % increase can be investigated, whereas inhibition of 20 % and 97 % are given at pH values of 8.3 and 9.0, respectively.