Triammonium citrate

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:

(Q)SAR

Adequacy of study:

key study

Study period:

2019-09-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE

ECOSAR v 1.11
2. MODEL (incl. version number)

ECOSAR v 1.11; The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

CAS-No.: 3458-72-8
SMILES: O=C(CC(CC(=O)ON(H)(H)(H)H)(O)C(=O)ON(H)(H)(H)H)ON(H)(H)(H)H
Log Kow (estimated) = -1.43 at 25°C
Water solubility (estimated) = 4.255E+005 mg/L at 25°C
Melting point (measured) = 160°C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For more detailed information please refer to the 'Attached justification' section
5. APPLICABILITY DOMAIN
For more detailed information please refer to the 'Attached justification' section
6. ADEQUACY OF THE RESULT
For more detailed information please refer to the 'Attached justification' section

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version:
ECOSAR v 1.11
- Model(s) used:
ECOSAR v 1.11; The ECOSAR (ECOlogical Structure Activity Relationship) Class Programm for Microsoft Windows; U.S. Environmental Protection Agency; Office of Chemical Safety and Pollution Prevention (Kelly Mayo-Bean, June 19, 2012)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

19 868.549 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: QSAR results for the Neutral organics class

Validity criteria fulfilled:

not applicable

Conclusions:

According to a QSAR estimation performed with ECOSAR v1.11 the toxicity of Triammonium citrate to aquatic algae and cyanobacteria is low. An EC50 value of 19868.549 mg/L at 25 °C was estimated.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Turbidimetrically determination of algal growth
- Short description of test conditions: The test item is dissolved in double distilled water and diluted with a spacing factor of 2. To each dilution (40 mL) 5 mL Stock solution I and 5 mL algal suspension of a distinct extinction value is added. These test vessels are incubated for 7 days and shaken once a day. At termination of the incubation period, the extinction of each vessel is photometrically determined: The concentration at which the inhibitory action of a" pollutant begins is present in that dilution from a series of dilutions of a pollutant having, at the end of the test period, a mean extinction value that is ≥ 3% below the mean value of the extinction value for non-toxic dilutions of the test cultures.
- Parameters analysed / observed: algal growth

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Scenedesmus quadricauda

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Scenedesmus quadricauda
- Source (laboratory, culture collection): not reported
- Age of inoculum (at test initiation):
- Method of cultivation: Store stock cultures of the test strain Scenedesmus quadricauda in 20mL nutrient solution I in 100-mL Erlenmeyer flasks stoppered with metal caps, on a white surface protected against daylight and exposed to constant lighting by. luminescent worm white tubes at 60 cm distance from each other, at 27°C and a relative humidity of 50%. For maintenance of the test strain, prepare fresh stock cultures continuously at 10days' intervals.

ACCLIMATION
- Acclimation period:10 days
- Culturing media and conditions (same as test or not):Nutrient solution I is changed to Stock solution I during test.

Test type:

static

Water media type:

freshwater

Total exposure duration:

8 d

Details on test conditions:

TEST SYSTEM
- Test vessel:Kapsenberg tubes
- Material, size, headspace, fill volume: glass, fill volume: 10 mL
- Aeration: no
- Initial cells density: not reported
- No. of vessels per concentration (replicates):triplicate


GROWTH MEDIUM
- Detailed composition if non-standard medium was used:
Nutrient solution I (for stock and preliminary cultures)
Dissolve in double-distilled water:
496 mg sodium nitrate, NaNO3, A.R.;
39 mg dipotassium hydrogen phosphate, K2HPO, anhydrous, high purity;
75 mg magnesium sulphate, MgSO4x 7H2O, A.R.;
36 mg calcium chloride, CaCl2 x 2 H2O, A.R.;
40 mg sodium metasilicate, Na2SiO3;
58 mg sodium carbonate, Na2CO 3, anhydrous, A.R.;
3 mg citric acid, C6H8O7 x H2O, A.R.;
3 mg iron (III) citrate C6H5FeO7 x 5 H2O;
10mg disodium salt of ethylene diamine tetracetic acid, C10H14N2Na2O8 x 2 H2O.

Stock solution I (for test cultures)
Dissolve in double-distilled water:
248 mg sodium nitrate, NaNO3, A.R.;
19.5 mg disodium hydrogen phosphate, K2HPO 4, anhydrous, high purity;
750 mg magnesium sulphate, MgSO4 x 7 H,O, A.R.;
360 mg calcium chloride, CaCI2 x 2 H2O, A.R.:
30 mg iron (III) citrate, C6H5FeO7 x 5 H2O.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

8 d

Dose descriptor:

other: toxicity threshold

Effect conc.:

640 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: inhibition of growth was determined by photometric measurement

Validity criteria fulfilled:

not applicable

Conclusions:

In the present publication of Brinkmann and Kühne 156 pollutants were tested to evaluate the toxicity threshold for green algae. The toxicity threshold (TT) should be regarded to as LOEC. The TT for citric acid was determined to be 640 mg/L. Considering the spacing factor of 2 in dosing the NOEC is expected to be 640mg/L /1.5 = 427 mg/L. Based on this concentration which is above the limit concentration of 100 mg/L, Citric acid is not classified according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals with respect to algal toxicity.

Executive summary:

In the present publication of Brinkmann and Kühne 156 pollutants were tested to evaluate the toxicity threshold for green algae. The toxicity threshold (TT) should be regarded to as LOEC. The TT for citric acid was determined to be 640 mg/L. Considering the spacing factor of 2 in dosing the NOEC is expected to be 640mg/L /1.5 = 427 mg/L. Based on this concentration which is above the limit concentration of 100 mg/L, Citric acid is not classified according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals with respect to algal toxicity.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: stock algal suspension was transferred to 250 ml conical flasks containing 150 ml sterilized culture media of different ammonia concentrations. The culture media were prepared in the same method as that of the Bristol medium except the nitrate component was replaced by different amounts of ammonium sulphate. A total of 12 ammonia concentrations: 0, 10, 20, 40, 50, 60, 80, 125, 250, 500, 750 and 1000 mg N 1-1, were prepared. The algae were axenically grown at 20 + 2°C, with light-dark cycles of 16-8 h for 21 days. The commercial Bristol medium was used as the control. The initial cell density was 1 x E+06 cells mL-1. The pH values of all culture media were adjusted to 7.0 ± 0.2 before algal inoculation. Each flask was aerated with filtered air which provided atmospheric CO2 and a mixing process. All treatments were in duplicate.
At 3 or 4 day intervals, algal cell number was determined by using the improved Neubauer haemacytometer and two counts were performed for each flask.

- Parameters analysed / observed: The specific growth rate constant (k, day -1) of C. vulgaris in each culture was determined by a simple linear regression analysis on ln(Nt/No) and t, where Nt, No denoted the final and initial cell
numbers, respectively. The k value was the slope of the regression line. The pH values of the cultures were measured and maintained at neutral pH by the addition of either sterilized and diluted NaOH or HCI. The chlorophyll content was determined at 7-day intervals by methanol-chloroform extraction. At the same time intervals, algal suspensions were centrifuged at 3000 g for 10 min and the amounts of ammonia and nitrate ions remaining in the culture medium were respectively examined by Nesslerization method and ultraviolet spectrophotometry (APHA, 1989). All samplings and measurements were carried out at the same time of the day and during the light period. At the end of the cultivation period, the percentages of NHa-N removal and the specific NHa-N uptake rates were calculated. The algal proteins were extracted in 0.5 N NaOH and assayed by modified Folin-Lowry method. The algal growth (cell number) and chlorophyll content were treated by two-way analysis of variance to determine any significant difference between ammonia concentration and incubation time.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A total of 12 ammonia concentrations: 0, 10, 20, 40, 50, 60, 80, 125, 250, 500, 750 and 1000 mg N 1-1, were prepared.
- Controls: The commercial Bristol medium was used as the control.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: fresh water green alga
- Strain: Chlorella vulgaris
- Source (laboratory, culture collection): Carolina Biological Supply
- Age of inoculum (at test initiation): At least 14 days
- Method of cultivation: Stock culture of Chlorella vulgaris was kept aseptically in a commercial Bristol medium (also called Bold Basal medium containing 40mgN L-1 in the form of KNO3 and 53 mg P L-1 in the form of KH2PO4).

ACCLIMATION
- Acclimation period: 14 days
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: conical flasks 250 mL fill volume and 150 mL test volume
- Aeration: yes, each flask was aerated with filtered air which provided atmospheric CO2 and a mixing process.
- Initial cells density: ´1 x E+06 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, the pH values of the cultures were measured and maintained at neutral pH by the addition of either sterilized and diluted NaOH or HCI.
- Photoperiod: 16-8 h
- Light intensity and quality: 4300 ± 300 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 3 or 4 day intervals, algal cell number was determined by using the improved Neubauer haemacytometer and two counts were performed for each flask.
- Chlorophyll measurement: The chlorophyll content was determined at 7-day intervals by methanol-chloroform extraction.
- Other: At the same time intervals, algal suspensions were centrifuged at 3000 g for 10 min and the amounts of ammonia and nitrate ions remaining in the culture medium were respectively examined by Nesslerization method and ultraviolet spectrophotometry (APHA, 1989). All samplings and measurements were carried out at the same time of the day and during the light period. At the end of the cultivation period, the percentages of NHa-N removal and the specific NHa-N uptake rates were calculated. The algal proteins were extracted in 0.5 N NaOH and assayed by modified Folin-Lowry method.

Key result

Duration:

18 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Total ammonia content

Basis for effect:

growth rate

Key result

Duration:

18 d

Dose descriptor:

EC50

Effect conc.:

2 700 mg/L

Nominal / measured:

nominal

Conc. based on:

other: (NH4)2SO4

Remarks:

calculated from total ammonia content

Basis for effect:

growth rate

Validity criteria fulfilled:

not applicable

Conclusions:

In the present study the effect of ammonia concentration on growth and physiology of a unicellular green alga, Chlorella vulgaris, was investigated. Green alga were exposed to 0, 10, 20, 40, 50, 60, 80, 125, 250, 500, 750 and 1000 mg N L-1, where NL-1 means the total ammonia content of the growth medium. The EC50 was determined to be > 1000 mg N x L-1 corresponding to 2700 mg (NH4)2SO4/L. There was no significant reduction in algal growth up to the highest tested concentration, thus, Ammonium sulphate is not classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In the present study the effect  of ammonia  concentration  on  growth  and physiology  of  a  unicellular  green  alga,  Chlorella vulgaris,  was  investigated. Green alga were exposed to 0,  10,  20,  40,  50,  60,  80, 125,  250,  500,  750  and  1000 mg N  L-1, where NL-1 means the total ammonia content of the growth medium. The EC50 was determined to be > 1000 mg N x L-1 corresponding to 2700 mg (NH4)2SO4/L. There was no significant reduction in algal growth up to the highest tested concentration, thus, Ammonium sulphate is not classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Description of key information

QSAR prediction of toxicity to aquatic algae and cyanobacteria using ECOSAR v1.11, result EC50 = 19868.549 mg/L at 25°C for Triammonium citrate.

There is no data available regarding the toxicity to algar and cyanobacteria for Triammonium citrate.

Published results are available for the constituents of Triammonium citrate, namely citric acid and ammonium ions.

Determination growth rate inhibition by ammonia was conducted with Chlorella vulgaris, measurement of growth rate via determination of chlorophyll content during a 18 days period. EC50 = 2700 mg (NH4)2SO4/L or 1000 mg N x L-1.

For citric acid the toxicity threshold was determined, Scenedesmus quadricauda were incubated with up to 14 concentrations of the test item with a spacing factor of 2, the toxicity threshold was determined at 640 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

2 700 mg/L

Additional information

Triammonium citrate is the ammonia salt of citric acid in which all carboxy groups are deprotonated and associated with ammonium ions as counter-cations. Its physical appearance are white crystals at 1013 Pa and ambient temperature. Furthermore, Triammonium citrate is very water soluble (>1000 mg/L, Merck Index, 2001). It is used as food additive mainly due to its buffer capacity, an acceptable daily intake (ADI) was not derived, the daily intake was not limited (JEFCA report, 1979), thus it is expected to be of low acute toxicity.

Once, dissolved in water the ions will dissociate thereby generating free citric acid and ammonium ions. Both molecules are ubiquitously present and also integral parts of the eukaryotic intermediary metabolism.

Based on this reaction behavior it can be assumed that during testing of toxicity to aquatic invertebrates, algae and cyanobacteria, these organisms are mainly exposed to the free ions, namely ammonium-ions and citrate.

It is well known that citric acid due to its pivotal role in intermediary metabolism has a low toxicity. The acute oral toxicity was evaluated in several rodent and non-rodent species revealing LD50 values between 3000 mg/kg bw and 5500 mg/kg bw and a LDLo of 7000 mg/kg bw in rabbits, respectively[1][2][3]. Low toxicity was also reported for aquatic species like Carcinus maenas, 48h LC50 = 160 mg/L[4].Nelson & Kursar determined the median 24h LD50 for brine shrimp to be 226.7 mg/L[5].In the green alga Scenedesmus quadricauda the toxicity threshold was determined to be 640 mg/L which corresponds to an estimated NOEC of 427 mg/L[6]. However, possible adverse effects may result from the irritation potential of citric acid based on changes in pH. However, the simultaneous presence of ammonium ions in the substance of interest will prevent changes in pH, thus, no toxicity up to the limit concentration is expected. Additionally, in an OECD assessment report test results from several aquatic toxicity test with citric acid in several aquatic species were summarized (OECD SIDS Initial Assessment Report for 11th SIAM, 2001). In none of the tests citric acid exhibit a LC50 or EC50 value beneath the limit concentration although there were EC0 values reported for Daphnia magna of 73 and 85 mg/L, respectively. A considerable change in pH in these tests cannot be excluded, hence these effect values are assumed to be related to an increased acidity.

Similar information are available for ammonium. The ionized form of ammonia is well tolerated in green algae for ammonium assimilation and subsequent metabolism (Turpin et a.., 1990[7];[8][9]). In an OECD report (SIDS Initial Assessment Report For SIAM 19, 2004) for Ammonium sulphate it was described that the ration of unionized ammonia to ionized ammonium is increasing in some of the conducted tests, thus, leading to signs of toxicity.

NH⁴⁺and NH₃coexist in aqueous solution in adynamic pH-dependent equilibrium. Under basic conditions (pH >10), ammonia (NH₃) redominates whereas the ammoniumion (NH⁴⁺) is the dominant species in weakly basic to neutral (environmental) conditions. With decreasing pH, the ammonium cation becomes the only species.

This increasing ratio is known to be correlated with pH- and temperature changes. Since the substance of interest exhibits a buffer capacity due to the contained citric acid it can be assumed that an increased generation of unionized ammonia is negligible for the substance of interest. However, again neither in Daphnia magna nor in green freshwater algae the LC/EC50 values were below the limit concentration, thus, low toxicity is expected. These results are in line with the results of QSAR predictions that were performed with either Triammonium citrate or citric acid. Although the results are afflicted with a high uncertainty based on the ionic structure which is not applicable with the simple non-polar narcosis model used and therefore possibly underestimates the toxicity, due to the aforementioned test results with the constituents of the salt underpin the determined values.

Due to the stoichiometric distribution of ammonium in Triammonium citrate, the low toxicity of citric acid and the sensitive balance between ammonia and ammonium depending on the pH, the ammonium ion is considered to be the toxicity-determining component and the EC50 for ammonium sulfate is used as the value for the chemical safety assessment

Taken together, both constituents (ions) of the substance exhibit a low toxicity and based on the ionic structure and its dissociation into free ions, aquatic organisms are expected to be exposed only to these ions and thus, the substance itself is also considered to be of very low toxicity and does not need to be classified as hazardous.

 

 

 

[1]Oyo Yakuri. Pharmacometrics., 43(561), 1992

[2]Takeda Kenkyusho Ho. Journal of the Takeda Research Laboratories., 30(25), 1971

[3]Industrial and Engineering Chemistry., 15(628), 1923

[4]Portmann JE, Wilson KW; Shellfish Information Leaflet No.22 (2nd ed) Ministry of Agric Fish Food:12 (1971)

[5]Nelson, Ann C., and Thomas A. Kursar. "Interactions among plant defense compounds: a method for analysis."Chemoecology 9.2 (1999): 81-92.

[6]Bringmann, G., and R. Kühn."Comparison of the toxicity thresholds of water pollutants to bacteria, algae, and protozoa in the cell multiplication inhibition test."Water Research 14.3 (1980): 231-241.

[7]Turpin, David H., et al. "Regulation of carbon partitioning to respiration during dark ammonium assimilation by the green alga Selenastrum minutum."Plant Physiology 93.1 (1990): 166-175.

[8]Vanlerberghe, Greg C., Kenneth W. Joy, and David H. Turpin. "Anaerobic metabolism in the N-limited green alga Selenastrum minutum: III. Alanine is the product of anaerobic ammonium assimilation." Plant physiology 95.2 (1991): 655-658.

[9]Weger, Harold G., et al."Ammonium assimilation requires mitochondrial respiration in the light: a study with the green alga Selenastrum minutum."Plant Physiology 86.3 (1988): 688-692.