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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 JULY 2020 - 2 DEC 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD No. 23 (Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals)
Version / remarks:
2nd Ed., February 08, 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At 20 ± 5 °C, in the dark
Analytical monitoring:
yes
Details on sampling:
The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): The algae were originally supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae are cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
other: Reconstituted water (OECD medium)
Remarks:
The culture medium was prepared 13 days before test start to allow pH to stabilise.
Limit test:
no
Total exposure duration:
72 h
Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
Test temperature:
The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 21.3 to 23.0 °C
pH:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
- pH value in the control at test start: 8.2,
- pH value in the control at test end: 9.9
- pH values in the test item treatments at test start: 7.9 to 8.4,
- pH values in the test item treatments at test end: 8.6 to 10.0.
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
700, 221.5, 70.1, 22.2 and 7.0 mg test item/L.
The test medium of the highest test concentration of nominal 700 mg test item/L was prepared by dissolving 691.5 mg test item into 987.9 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of at least 50 mL volume
- Type: closed
- Material, size, headspace, fill volume: as much test medium as possible so the remaining head space will be reduced to a technical possible minimum of some mL
- Initial cells density: initial nominal algal cell density of 5000 cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): one replicate of each test concentration and of the controls will be prepared without algae.

The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, were placed in a random order and were repositioned each day to minimize differences in test conditions.
The vessels were kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.

GROWTH MEDIUM
- Standard medium used: yes

Macro-nutrients:
NaHCO3: 50.0 mg/L
CaCl2 x 2 H2O: 18.0 mg/L
NH4Cl: 15.0 mg/L
MgSO4 x 7 H2O: 15.0 mg/L
MgCl2 x 6 H2O: 12.0 mg/L
KH2PO4: 1.6 mg/L

Trace Elements:
Na2EDTA x 2 H2O: 100.0 µg/L
FeCl3 x 6 H2O: 64.0 µg/L
MnCl2 x 4 H2O: 415.0 µg/L
H3BO3: 185.0 µg/L
Na2MoO4 x 2 H2O: 7.0 µg/L
ZnCl2: 3.0 µg/L
CoCl2 x 6 H2O: 1.5 µg/L
CuCl2 x 2 H2O: 0.01 µg/L

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity:4860 lux (range: 4590 to 5210 lux)
- The test water will be enriched with 300 mg NaHCO3/L to enable algae growth in closed vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Pre-experiments (non-GLP) were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
116 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
50.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
20.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
29.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
70.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
28.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 700 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 281.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
151 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
60.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
51 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
20.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
70.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
28.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Growth inhibition:
The 72-hour EyC50 was calculated to be 116 mg test item/L and the ErC50 > 700 mg test item/L. The 72-hour EyC10 was calculated to be 29.5 mg test item/L and the ErC10 51.0 mg test item/L. The 72-hour NOEyC was determined to be 22.2 mg test item/L and the associated 72-hour LOEyC is 70.1 mg test item/L. The 72-hour NOErC was determined to be 22.2 mg test item/L and the associated 72-hour LOErC is 70.1 mg test item/L.
Reported statistics and error estimates:
Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Weibull analysis.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat(R) Solutions GmbH.

Signs of Phytotoxicity evaluated by Microscopic Observations:

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to the highest nominal test concentration of 700 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested

Validity criteria fulfilled:
yes
Conclusions:
The influence of Methylamine (aqueous solution 40 %) on the growth of the freshwater green algae Desmodesmus subspicatus was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 116 mg test item/L (46.7 mg a.i./L) and the 72-hour ErC50 value was calculated to be > 700 mg test item/L (> 281 mg a.i./L). The 72-hour NOEC for yield and growth rate was determined to be 22.2 mg test item/L (8.9 mg a.i./L) and the associated 72-hour LOEC for yield and growth rate was determined to be 70.1 mg test item/L (28.2 mg a.i./L).
Executive summary:

The influence of Methylamine (aqueous solution 40 %) on the growth of the freshwater green algae Desmodesmus subspicatus was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 116 mg test item/L (46.7 mg a.i./L) and the 72-hour ErC50 value was calculated to be > 700 mg test item/L (> 281 mg a.i./L). The 72-hour NOEC for yield and growth rate was determined to be 22.2 mg test item/L (8.9  mg a.i./L) and the associated 72-hour LOEC for yield and growth rate was determined to be 70.1 mg test item/L (28.2 mg a.i./L).

All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Description of key information

EC50(72h; endpoint: growth rate) > 281.8 mg active ingredient/L for Desmodesmus subspicatus (OECD 201)

EC10(72h; endpoint: growth rate) 20.5 mg active ingredient/L for Desmodesmus subspicatus (OECD 201)

NOEC(72h; endpoint: growth rate) 8.9 mg active ingredient/L for Desmodesmus subspicatus (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
281.8 mg/L
EC10 or NOEC for freshwater algae:
20.5 mg/L

Additional information

The effects of methylamine (40 % aqueous solution) on Desmodesmus subspicatus were investiged by Siche, O. and Wydra, V. (2020). The following nominal values (based on growth rate) were determined: EC50 (72h) > 281.8 mg active ingredient/L; EC20 (72 h) = 60.8 mg active ingredient/L; EC10 (72 h) = 20.5 mg active ingredient/L, a LOEC (72h) = 28.2 mg active ingredient/L and a NOEC (72 h) = 8.9 mg active ingredient/L. Furthermore, the following values based on yield were reported: EC50 (72h) = 46.7 mg active ingredient/L; EC20 (72 h) = 20.5 mg active ingredient/L; EC10 (72 h) = 11.9 mg active ingredient/L, a LOEC (72 h) = 28.2 mg active ingredient/L and a NOEC (72 h) = 8.9 mg active ingredient/L. All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

In addition, there is a supporting QSAR calculation available. The result is an estimation by Ecosar v1.11 using an extensive set of structure-activity relationships (SARs). The substance methylamine is classified as 'Aliphatic Amine'. The EC50 estimated for green algae is 43 mg/L. This value is significantly lower than the experimentally determined value in a test according to OECD 201 and 23.

Furthermore, there is supporting information available. The information is, however, derived of older non guideline publications. In one investigation, the test organism was Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). 21% growth inhibition at 1mM (31 mg/L) was investigated. This experimental data was published by Andreozzi et.al.(2000). This value is in line with the reported values of the new key study (EC20 = 20.5 mg/L versus EC21 = 31 mg/L).

Last but not least, 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.