(2-hydroxyethyl)ammonium mercaptoacetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH
This scenario covers the category approach for which the hypothesis is based on transformation to a common compound. For the REACH information requirement under consideration, the effects obtained in studies conducted with different source substances are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect is observed for the different source substances; this may include absence of effects for every member of the category. No relevant differences in strengths of effect are observed for several source substances.
There are no relevant variations in properties among source substances and the same potency is predicted for all target substances. This corresponds to Scenario 5 of the RAAF (ECHA, 2017). The substances ATG, MEATG, KTG, CaTG, and NaTG are different inorganic salts of a common acid, thioglycolic acid (TGA; synonym: 2-mercaptoacetic acid). They dissociate rapidly in aqueous media to the common thioglycolate anion and to their different counter ions. The water solubility of all category members is high.
This approach serves to use existing data on aquatic toxicity endpoints for substances in this category.
It can be predicted with high confidence that the target substances will display the same mode of action and lead to the same type and strength of effects as observed with the source substances.


2. CATEGORY APPROACH JUSTIFICATION
For details, refer to Justification for read-across attached to Iuclid section 13

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: read-across, molecular weight correction

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: read-across, molecular weight correction

Basis for effect:

growth rate

Conclusions:

The results of studies conducted with TGA, MeaTG, and KTG were combined and the geometric mean was calculated. After correction for molecular weight this resulted in a 72 h ErC50= 6.8 mg/L and 72 h NOEC = 0.73 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 29/06/03 to 09/02/05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dept. of Health, UK

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not relevant

Analytical monitoring:

yes

Details on sampling:

The test samples were analysed following filtration through glass wool to remove algal cells.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test, the test material was dissolved directly in culture medium.
An amount of test material (195.5 mg) was dissolved in culture medium and the volume adjusted to 0,5 L to give a 200 mg ai/L stock solution from which serial delutions were made to give 100, 50, 25, 12.5 mg ai/L stock solutions. An aliquot (250 mL) of each stock solution was mixed with algal suspension (250 mL) to give the required test concentrations of 6.25, 12,5, 25, 50 and 100 mg ai/L.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Strain: CCAP 276/20
- Source: Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria.
- The culture was maintained in the laboratory at a temperature of 21°C (+ or - 1°C), under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

21 °C (+ or - 1°C)

pH:

From pH 7.3 at 0 hours to pH 8.3 at 72 hours

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks.
- Flasks plugged with polyurethane foam bungs
- 3 flasks each containing 100 ml of test preparation were used for the control and each treatment group.
- Initial nominal cell density: 10^4 cells per mL
- control end cell density: 4.45E05 cells per ml

OTHER TEST CONDITIONS
- Illumination: continuous (intensity approximately 7000 lux)
- Shaking: constant, at 150 rpm for 72 hours.

TEST CONCENTRATIONS
- Range finding study:
- Test concentrations: nominal test concentrations of 0.10, 1.0, 10 and 100 mg ai/l.
- Results used to determine the conditions for the definitive study: After 72 hours no effect on algal growth was observed.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 34 - 45 mg ai/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 58 - 74 mg ai/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: There were no statistically significant differences between the control, the 6.25 and the 12.5 mg ai/L test concentrations (P≥0.05).

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 2.4 – 2.8 mg ai/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 3.0 – 3.5 mg ai/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The test material was unstable in the test medium for the duration of the test.

Results with reference substance (positive control):

no data

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

 

Observations on test material solubility

At 0 hours all control and test cultures were clear colourless solutions. After the 72-Hour test period the control, 6.25, 12.5 and 25 mg ai/l test cultures were green  dispersions and the 50 mg ai/l test cultures were pale green dispersions. The 100 mg ai/l test cultures remained as clear colourless solutions throughout the test period.

 

Verification of test concentrations
Data supplied by the Sponsor indicated that the test material was prone to oxidation to give Di(monoethanolamine) dithiodiglycolate (DiMEADTDG). Analysis of the test preparations therefore included quantitation of both the test material Monoethanolamine thioglycolate (MEATG) and DiMEADTDG.
Recovery and stability analyses confirmed that the test material oxidised in aqueous media forming DiMEADTDG, however the oxidation rate was concentration dependent with greater losses of parent test material being observed at the lower test concentrations. As a difference in stability at different concentrations was observed it was clear that degradation of MEATG was not first order and hence a true half-life for the test material could not be calculated.

As such, following current regulatory advice, it was considered appropriate to expose test organisms to parent test material and allow degradation to DiMEADTDG to occur over the study period rather than expose test organisms to the degradation product alone.
Preliminary work showed that the addition of ascorbic acid to test samples (at a rate of 1 ml of 1 M ascorbic acid/10 ml test sample) stabilised MEATG and prevented any further degradation occurring. Analytical standards confirmed that this method of stabilising the samples was sufficient to maintain MEATG at nominal concentrations for at least 8 hours. It was therefore considered appropriate to acidify the test samples immediately upon sampling in order that oxidation of MEATG to DiMEADTDG did not occur during the time between sampling and analysis.
Whilst it was known that the test material was unstable in culture medium, following current regulatory advice samples were taken for chemical analysis at 0 and 72 hours only. Taking of samples for analysis at 24 and 48 hours was not possible due to the sacrificial nature of sampling. Analysis of the test preparations at 0 hours (see Appendix 2) showed measured concentrations of MEATG to range from 83% to 100% of nominal values with the exception of the 6.25mg ai/l test concentration which was 71% of nominal. Analysis of the duplicate frozen sample for this test group gave a measured concentration of 68% of nominal. Critical examination of the data could
not determine the reason for this low measured concentration, however as the NOEC was shown to be 12.5 mg ai/l, this low value was considered to have no adverse impact on the study.
Analysis of the 0-Hour test samples showed measured concentrations of DiMEADTDG to range from less than the limit of quantitation (LOQ) of the analytical method (which was assessed down to 0.30 mg ai/l) to 1.49 mg ai/l. These measured concentrations were in line with those observed in analytical standards and were considered to be due to the presence of small quantities of DiMEADTDG in the sample of test material supplied by the Sponsor rather than due to oxidation of MEATG during preparation of the 0-Hour test samples.
At 72 hours the concentration of MEATG in all test samples was less than the LOQ of the analytical method which was assessed down to 0.37 mg ai/l. Measured concentrations of DiMEADTDG were shown to have increased to 5.02 to 87.8 mg ai/l in the 72-Hour test samples. The decline in MEATG concentrations and corresponding increase in DiMEADTDG was greater than that observed in the stability analyses. This effect was considered to be due to differences in the storage/incubation of the stability and test samples.
The samples analysed for chemical stability were prepared in sample bottles and allowed to stand in the laboratory for the 72-Hour stability period prior to analysis. The test samples were incubated in 250 ml glass conical flasks under constant shaking and with the addition of algal cells. It was therefore considered that the constant shaking and subsequent re-oxygenation of the test media in the test samples may have resulted in greater oxidation in these when compared to the ‘static’ storage of the stability samples.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations of MEATG 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. 0.185 mg ai/l) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were
determined to be:

Nominal Test Concentration (mg ai/l)	Geometric Mean Measured Test Concentration (mg ai/l)	Expressed as a % of the Nominal Test Concentration
6.25	0.907	15
12.5	1.45	12
25	1.96	8
50	3.04	6
100	4.24	4

 

 

Validity criteria fulfilled:

yes

Remarks:

The cell concentration of the control cultures increased by a factor of 49 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Conclusions:

The 72h-EC50 (biomass) based on the nominal test concentrations was 39 mg ai/L (nominal) and the 72h- EC50 (growth rate) was 65 mg ai/L (nominal). The 72h NOEC was 12.5 mg ai/L (nominal).
The
EbC50 (72 h) based on the geometric mean measured test concentrations was 2.6 mg ai/l; 95% confidence limits 2.4 – 2.8 mg ai/l and the ErC50 (0 - 72 h) was 3.2 mg ai/l; 95% confidence limits 3.0 – 3.5 mg ai/l. The No Observed Effect Concentration was 1.45 mg ai/l.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).
Following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg active ingredient (ai)/l (three replicate flasks per concentration) 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.

Exposure of Scenedesmus subspicatus to the test material based on nominal test concentrations gave an EbC50 (72 h) value of 39 mg ai/l; 95% confidence limits 34 - 45 mg ai/l and an ErC50 (0 - 72 h) value of 65 mg ai/l; 95% confidence limits 58 - 74 mg ai/l. The NOEC was 12.5 mg ai/l.  The EbC50 (72 h) based on the geometric mean measured test concentrations was 2.6 mg ai/l; 95% confidence limits 2.4 – 2.8 mg ai/l and the ErC50 (0 - 72 h) was 3.2 mg ai/l; 95% confidence limits 3.0 – 3.5 mg ai/l. The NOEC was 1.45 mg ai/l.

Data supplied by the Sponsor indicated that the test material was prone to oxidation to give Di(monoethanolamine) dithiodiglycolate (DiMEADTDG). Analysis of the test preparations therefore included quantitation of both the test material Monoethanolamine thioglycolate (MEATG) and DiMEADTDG.
Recovery and stability analyses confirmed that the test material oxidised in aqueous media forming DiMEADTDG, however the oxidation rate was concentration dependent with greater losses of parent test material being observed at the lower test concentrations. As a difference in stability at different concentrations was observed it was clear that degradation of MEATG was not first order and hence a true half-life for the test material could not be calculated. As such, following current regulatory advice, it was considered appropriate to expose test organisms to parent test material and allow degradation to DiMEADTDG to occur over the study period rather than expose test organisms to the degradation product alone.
Analysis of the test preparations at 0 hours showed measured concentrations of MEATG to range from 83% to 100% of nominal values with the exception of the 6.25 mg ai/l test concentration which was 71% of nominal. Analysis of the duplicate frozen sample for this test group gave a measured concentration of 68% of nominal. Critical examination of the data could not determine the reason for this low measured concentration, however as the NOEC was shown to be 12.5 mg ai/l, this low value was considered to have no adverse impact on the study.
Analysis of the 0-Hour test samples showed measured concentrations of DiMEADTDG to range from less than the limit of quantitation (LOQ) of the analytical method (which was assessed down to 0.30 mg ai/l to 1.49 mg ai/l). These measured concentrations were in line with those observed in analytical standards and were considered to be due to the presence of small quantities of DiMEADTDG in the sample of test material supplied by the Sponsor rather than due to oxidation of MEATG during preparation of the 0-Hour test samples.
At 72 hours the concentration of MEATG in all test samples was less than the LOQ of the analytical method which was assessed down to 0.37 mg ai/l. Measured concentrations of DiMEADTDG were shown to have increased to 5.02 to 87.8 mg ai/l in the 72-Hour test samples.
The decline in MEATG concentrations and corresponding increase in DiMEADTDG was greater than that observed in the stability analyses. This effect was considered to be due to differences in the storage/incubation of the stability and test samples.
The samples analysed for chemical stability were prepared in sample bottles and allowed to stand in the laboratory for the 72-Hour stability period prior to analysis. The test samples were incubated in 250 ml glass conical flasks under constant shaking and with the addition of algal cells. It was therefore considered that the constant shaking and subsequent re-oxygenation of the test media in the test samples may have resulted in greater oxidation in these when compared to the ‘static’ storage of the stability samples.
Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations of Monoethanolamine thioglycolate. The EbC50 (72 h) based on the geometric mean measured test concentrations was 2.6 mg ai/l; 95% confidence limits 2.4 – 2.8 mg ai/l and the ErC50 (0 - 72 h) was 3.2 mg ai/l; 95% confidence limits 3.0 – 3.5 mg ai/l. The No Observed Effect Concentration was 1.45 mg ai/l.

Description of key information

The overall EC50 and NOEC for MeaTG are 6.8 mg/L and 0.73 mg/L, respectively. (OECD TG 201, Pseudokirchneriella subcapitata, RL1, GLP; read-across from TGA, MeaTG, and KTG; molecular weight correction)

Key value for chemical safety assessment

EC50 for freshwater algae:

6.8 mg/L

EC10 or NOEC for freshwater algae:

0.73 mg/L

Additional information

Experimental data on toxicity to algae are available for TGA, KTG and MeaTG.

 

TGA

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata exposed to the test item Thioglycolic acid for a duration of 72 hours was performed according to OECD Guideline 201.

Algae were exposed to the test item at nominal concentrations of 0 (control), 4.0, 7.7, 14.6, 27.2, 52.6 and 100 mg/L over a time period of 72 hours.

The study was performed using 100 ml glass erlenmeyer flasks stoppered with cotton wool wrapped in sterilised, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1E04 cells/ml. Tests flasks were incubated at 23 ± 1 °C continuously shaken and constantly illuminated. The algal concentration was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test.

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.  Microscopic observation confirmed that the algae appeared normal at the end of the test excepted at the highest concentration where algae appeared a little smaller in comparison to the control.

During the test, the control pH varied by 0.23 units.

The quality criterion related to the algal growth was respected since the increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R = 88). The validity criterion specific to C3 92/69EEC method and related to the test item stability during the test was not respected:

the final concentrations of the test were not maintained within the designated limit of 80 % of the initial concentrations. Therefore, effect levels are given as geom. mean measured concentrations.

The 72 h ErC50 was 27 mg/L, the 72 h ErC10 was 12 mg/L, the 72 h NOECr was 15.2 mg/L.

The 72 h EbC50 was 13 mg/L, the 72 h EbC10 was 4.8 mg/L, the 72 h NOECb was 7.6 mg/L.

 

Supporting data are available for the oxidation product Diammonium dithioglycolate. Diammonium dithioglycolate is the main degradation product of thioglycolic acid when released in the environment occurring few hours after the release (Devaux, 2003 - Smolin and Fölsing, 2009).

 

Diammonium dithioglycolate toxicity to Scenedesmus subspicatus has been investigated through a test in accordance with OECD Guideline 201 and GLP requirements.

Scenedesmus subspicatus were exposed for 72 hours to an aqueous solution of Diammonium Dithiodiglycolate at a nominal concentration of 100 mg/L.

EC 50 - 72h determined was higher than 100 mg/L according to either growth rate and biomass and NOEC was of 100 mg/L.

 

KTG

The purpose of this test was to determine the inhibitory effect of KTG on the growth of the freshwater green algae Pseudokirchneriella subcapitata. Exponentially growing cultures of this unicellular green algal species were exposed to nominal concentrations of 10, 32, 1.0, 0.3 and 0.1 mg test item/L (spacing factor 3.16) and a control (corresponding to following geometric mean measured concentrations of the test item: 8540, 2480, 81.5, 7.70 and 0.0143 µg test item/L and a control).  The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Th study was conducted in accordance with OECD Guideline 201.

Cell density in the control: Achieved: 48.3-fold increase (criterion: ≥ 16) within 72 hours;

Coefficient of Variation (CV) of sectional (daily) growth rate of the control: Achieved: 11.5 % (criterion: ≤ 35%);

CV of average growth of control replicates: Achieved: 6.4 % (criterion: ≤ 7%);

and thus, the validity criteria were met.

The 72-hour EyC50 was calculated to be 26.4 µg test item/L, and the 72-hour ErC50 value was calculated to be 1582 µg test item/L. The 72-hour NOEyC was determined to be 0.0143 µg test item/L and the associated 72-hour LOEyC was 7.70 µg test item/L. The 72-hour NOErC was determined to be 7.70 µg test item/L and the associated 72-hour LOErC was 81.5 µg test item/L. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal initial concentrations during the test.

 

MeaTG

A study according to OECD TG 201 was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. Following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg active ingredient (ai)/l (three replicate flasks per concentration) 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.

Exposure of Scenedesmus subspicatus to the test material based on nominal test concentrations gave an EbC50 (72 h) value of 39 mg ai/l; 95% confidence limits 34 - 45 mg ai/l and an ErC50 (0 - 72 h) value of 65 mg ai/l; 95% confidence limits 58 - 74 mg ai/l. The NOEC was 12.5 mg ai/l.  The EbC50 (72 h) based on the geometric mean measured test concentrations was 2.6 mg ai/l; 95% confidence limits 2.4 – 2.8 mg ai/l and the ErC50 (0 - 72 h) was 3.2 mg ai/l; 95% confidence limits 3.0 – 3.5 mg ai/l. The NOEC was 1.45 mg ai/l.

 

Conclusion

There were differences in effect levels in the studies conducted with TGA, MeaTG and KTG. Since no obvious cause could be identified, all three results are combined in a geometric mean value after adjustment to thioglycolate. The overall EC50 and NOEC for MeaTG are 6.8 mg/L and 0.73 mg/L, respectively.