Triammonium citrate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

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:

48 h

Dose descriptor:

LC50

Effect conc.:

98 638.648 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: QSAR result for the Neutral organics class

Details on results:

Estimates provided below use the Neutral Organics QSAR equations which represent baseline toxicity potential (minimum toxicity) assuming a simple non-polar narcosis model. Without empirical data on structurally similar chemicals, it is uncertain if this substance will present significantly higher toxicity above baseline estimates. Based on the acid moeity found the predicted values were multiplied by 10.

Validity criteria fulfilled:

not applicable

Conclusions:

According to a QSAR prediction performed with ECOSAR v1.11, the toxicity of Triammonium citrate to aquatic invertebrates is low. A LC50 value of 98638.648 mg/L at 25°C was estimated.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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:

equivalent or similar to guideline

Guideline:

other: Meth- ods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians.

Version / remarks:

1975

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

other: Standard Methods for the Examination of Water and Wastewater, 16th ed.

Version / remarks:

1985

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. ASTM Standard E 729-80.

Version / remarks:

1980

Deviations:

not specified

Principles of method if other than guideline:

- Principle of test: The principle is based on the guidelines mentioned above, but uses seven species from five phyla simultaneously.
- Short description of test conditions: The static multispecies bioassays were performed in seamless glass, 30.5-cm cuboidal, Pyrex chromatography jars to which 20 liters of test solution was added. Activated carbon-filtered, de-chlorinated and tempered industrial service water from Lake Ontario was used in all tests. Based on a recommended expected environmental concentration cutoff level, the maximum concentration used in these bioassays was 100 mg/L. Chemicals known to be readily soluble at 100 mg/L were added directly to the diluent water in each aquarium in the appropriate amounts to give nominal concentrations of 100, 10, 1 and 0.1 mg/L. Test chemical concentrations were not analyzed. All test organisms were acclimated to the control diluent water in the breedinghearing tanks. Food was withheld for the 24 h preceding start of the test. Juveniles of each species, as uniform in size as possible, were collected from the colonies. Ten juvenile organisms of each species were routinely exposed to the test chemical in each treatment. Stratified randomization was accomplished by proportioning out no more than 20% of any one species into an aquarium at any one time. Biological loading was kept below 0.5 gram wet weight per liter of test solution.The remaining five species were segregated in welded stainless steel, 55-mesh wirecloth baskets (5.5 cm in diameter x 7.5 cm in depth). Each basket was suspended from a 1-rpm motor-driven mechanism (Fig. 1) that raised and lowered the baskets in the water column. A stainless steel band, slotted every 0.5 cm, facilitated the positioning of the baskets so that the submerged volumes changed from one-third to two-thirds during each cycle. The baskets were spaced around the test vessel rim so that they did not interfere with each other. One-half of the volume of the submerged basket was exchanged with the main tank volume every minute.
- Parameters analysed / observed: Determinations of the temperature, dissolved oxygen and pH of each test solution were made in conjunction with the daily biological observations.
The test temperature target was 20 + 1°C. If the dissolved oxygen concentration in a test chamber fell below 40% of the starting level in a test, the test was repeated with 0.05 L/min glass-sparger aeration. All tests were conducted within the extremes of 6.5 to 8.5 pH units. The photoperiod duration was 16 h of light. The air-water interface of each tank received approximately 50 ft-c of cool-white fluorescent light. Biological observations were made daily. Survival, condition and behavorial information were recorded. Dead organisms were removed when observed. A test organism was considered dead if it appeared motionless and exhibited no response to gentle prodding. If more than one-half of the population of a species exposed in any treatment was determined to be dead, additional aquaria containing lower concentrations of test solution were set up. All seven species were exposed to each dose level. At any time during the test when all 10 organisms of a species were considered dead, these biological parameters were determined and recorded. All species in these tests were exposed for the same time period, 96 h. As in any bioassay that determines a dose response, the LCSO value can be achieved at any time during the exposure. It is recognized by the authors that some invertebrates usually are exposed for only 48 h.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Chemicals known to be readily soluble at 100 mg/L were added directly to the diluent water in each aquarium in the appropriate amounts to give nominal concentrations of 100, 10, 1 and 0.1 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna
- Age at study initiation (mean and range, SD): juvenile
- Weight at study initiation (mean and range, SD): 0.5g wet weight per L
- Feeding during test
No
ACCLIMATION
- Acclimation period: 24h
- Acclimation conditions (same as test or not): yes

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

CaCO3 130 mg/L

Test temperature:

20 ± 1°C

pH:

6.5 to 8.5

Conductivity:

260 µmhos/cm

Nominal and measured concentrations:

100, 10, 1, 0.1 mg/L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: seamless glass, 30.5-cm cuboidal, Pyrex chromatography jars
- Volume of solution: 20L
- Aeration: yes
- Type of flow-through: The remaining five species were segregated in welded stainless steel, 55-mesh wirecloth baskets (5.5 cm in diameter x 7.5 cm in depth). Each basket was suspended from a 1-rpm motor-driven mechanism that raised and lowered the baskets in the water column. A stainless steel band, slotted every 0.5 cm, facilitated the positioning of the baskets so that the submerged volumes changed from one-third to two-thirds during each cycle. The baskets were spaced around the test vessel rim so that they did not interfere with each other. One-half of the volume of the submerged basket was exchanged with the main tank volume every minute.
- No. of organisms per vessel: 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: 1.8 mg/L
- Chlorine: 6.6 µg/L
- Alkalinity: 93 mg/L CaCO3
- Conductivity: 260 µmhos/cm
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: yes by the addition of 10% (v/v) NaOH or 10% (v/v) H2SO4.

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Validity criteria fulfilled:

not applicable

Conclusions:

In a multispecies test which was conducted according to Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians (US EPA, 1975), Standard Methods for the Examination of Water and Wastewater, 16th ed. (American Public Health Association, American Water Works Association and Water Pollution Control Federation,1985) and Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. ASTM Standard E 729-80. (American Society for Testing and Materials) water fleae from the Daphnia magna strain were exposed to 100, 10, 1, 0.1 mg/L Ammonium sulphate (nominal) for 24h. No immobility was detected at the highest tested concentration, thus, Ammonium sulphate does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Executive summary:

In a multispecies test which was conducted according to Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians (US EPA, 1975), Standard  Methods for  the Examination of  Water and  Wastewater,  16th  ed. (American  Public  Health  Association,  American Water Works Association and Water Pollution Control  Federation,1985) and Standard practice for conducting acute toxicity tests with  fishes,  macroinvertebrates,  and  amphibians. ASTM  Standard E  729-80. (American Society for Testing and Materials) water fleae from the Daphnia magna strain were exposed to 100, 10, 1, 0.1 mg/L Ammonium sulphate (nominal) for 24h. No immobility was detected at the highest tested concentration, thus, Ammonium sulphate does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

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:

according to guideline

Guideline:

other: Meyer et al. 1982

Deviations:

not specified

Principles of method if other than guideline:

- Short description of test conditions:
This assay uses brine shrimp, Artemia franciscana (Prime Artemia Incorporation, Salt Lake City, UT), a tiny salt water crustacean, to determine the toxicity of plant secondary metabolites. In summary, the brine shrimp eggs (cysts) are allowed to hatch for 48 hours. The brine shrimp are then put in wells of a 96 flat-bottom well plate (Nunc Microwell plate). Each column (12 per plate) contains a two-or four-fold dilution series representing 8 doses. Each chemical, or combination of chemicals, was repeated five to six times (columns) in each experiment. The percent of brine shrimp dead after 24 hours represents the response per dose used in isobolographic analysis. We used 4% DMSO as a negative control and CuSO 4 as a positive, toxic control, repeated two to three times (columns) in each experiment. Each experiment was replicated three to four times.
Hatching brine shrimp eggs
The sea water (SW) was prepared as 70.3 g sea salt (Instant Ocean by Aquarium Systems, Mentor, OH) dissolved in 2 liters of bottled spring water (Mount Olympus Water, Inc., Salt Lake City, UT). The specific gravity of the SW ranged from 1.020–1.025 g/mL. The starting pH of the SW was 8–8.5. After 48 hours of hatching, the pH dropped to 7.5–8. We used an Imhoff settling cone (VWR ScientificProducts, Salt Lake City, UT) for hatching. To 500 ml SW in the cone, we added 0.5 g brine shrimp eggs and 3.0 mg yeast. We placed the cone in a water bath at 28°C, allowed the eggs to hydrate for one hour and then turned on a 60 watt light, 20 cm above the cone. We aerated the SW in the cone and simultaneously aerated the excess
SW. The eggs were allowed to hatch for 48 hours.
Brine shrimp assay
Following hydration and 48 hours of light and aeration, the assay was initiated. To prepare the brine shrimp for the wells, we turned off the aeration and redirected the light to the bottom of the Imhoff cone because the brine shrimp nauplii are phototactic and will aggregate near the light, allowing more efficient removal. To prepare the SW with yeast (SWY) solutions, we added 1.2 mg yeast, Candida utilis (Bioserve, Frenchtown, NJ) to 200 ml SW. For rows B-H, we
prepared the stock SWY containing 6.67% DMSO for two-fold dilutions or 6.0% DMSO for the four-fold dilutions (DMSO-SWY).
Brine shrimp assay count
After 24 hours of exposure to the toxins, the number of living and total (determined after killing by freezing) brine shrimp for each well were determined and the percent mortality was used to calculate the LD50s. All combinations were analyzed using two-fold dilutions.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Citric acid 123 mg/mL DMSO (stocksolution) : For two-fold dilutions, 280 mL of SWY were added to each well in row A of the 96 well microtitre plates. Then 20 mL of the stock solution of sample were added. For a four-fold dilution, 251
mL of SWY were added to each well in row A and 16 mL of sample. For the two-fold dilution, we added 150 mL of 6.67% DMSO-SWY to rows B–H. After mixing the compounds thoroughly in row A, 150 mL were removed from row A and serially diluted this in rows B–H. The last 150 mL from row H was discarded. Similarly, for the four-fold dilution, 200 mL of 6.0% DMSO-SWY were added to rows B–H and transferred 67 mL from row to row. After the serial dilution process,
100mL brine shrimp (approximately 10–20 brine shrimp) were added to each well. Only wells with five or more brine shrimp were included in the analyses. In the two-fold dilution, each well had a total volume of 250 mL. In the four-fold dilution, each well had a total of 300 mL. The final DMSO content was 4% for all wells in which DMSO was used as a solvent
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO

Test organisms (species):

Artemia sp.

Details on test organisms:

TEST ORGANISM
- Common name: brine shrimp
- Strain/clone: Artemia franciscana
- Justification for species other than prescribed by test guideline: The brine shrimp assay is used widely in natural products chemistry and is presented here as an illustration.
- Age at study initiation (mean and range, SD): brine shrimp eggs (cysts)
- Source: Prime Artemia Incorporation, Salt Lake City, UT
- Feeding during test
- Food type: Yeast
- Amount: 0.6 mg/100mL
- Frequency: complete experimental time 24h

ACCLIMATION
- Acclimation period: hatching for 48h
- Acclimation conditions (same as test or not): yes
- Type and amount of food: yeast

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

24 h

Hardness:

not reported

Details on test conditions:

TEST SYSTEM
- Test vessel: 96 flat-bottom well plate (Nunc Microwell plate)
- Volume of solution: 300 µL
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): Each column (12 per plate) contains a two-or four-fold dilution series representing 8 doses. Each chemical, or combination of chemicals, was repeated five to six times (columns) in each experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The sea water (SW) was prepared as 70.3 g sea salt (Instant Ocean by Aquarium Systems, Mentor, OH) dissolved in 2 liters of bottled spring water (Mount Olympus Water, Inc., Salt Lake City, UT).
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: The starting pH of the SW was 8–8.5. After 48 hours of hatching, the pH dropped to 7.5–8.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality after 24h

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
Not reported

Reference substance (positive control):

yes

Remarks:

CuCO4

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

270 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Reported statistics and error estimates:

The data were analyzed by nonlinear estimation (Statistica, 2nd edition, Statsoft, Inc., Tulsa, OK) with a user-specified, half-maximal response regression. and a least squares loss function. Default starting values and initial step sizes with the Quasi-Newton or Hooke-Jeeves pattern move algorithm to estimate the parameters, b 0 , b 1 , and b 2 . In this model, b 2 is the LD50 or median effect, b 1 is the slope of the function, and b 0 is the expected response at saturation, in this case set at 1, or 100% mortality. In addition to parameters, the standard errors were given when the matrix was well-defined, i.e., when there was sufficient
variance. In a number of replicates (for chlorogenic acid, citric acid, and PPMS), there was so little variance that the standard errors could not be estimated. In such cases, no confidence intervals werecalculated.

Validity criteria fulfilled:

not applicable

Conclusions:

In the present study the LD50 of citric acid for birne shrimps were determined according to the method of Meyer et al. (1982). The median 24h LD50 for birne shrimps was determined to be 226.67 mg/L.

Executive summary:

In the present study the LD50 of citric acid for birne shrimps were determined according to the method of Meyer et al. (1982). The median 24h LD50 for birne shrimps was determined to be 226.67 mg/L.

Description of key information

QSAR prediction of toxicity to aquatic invertebrates using ECOSAR v1.11, result LC50 = 98638.648 mg/L at 25°C.

The mean LD50 value for citric acid in birne shrimps was determined to be 226.67 mg/L. Additionally, a 24h LD50 value for Ammonium sulphate could not be established because no immobility occurred when juvenile Daphnia magna were exposed to 100 mg/L Ammonium sulphate. Thus, for precautionary reasons the LD50 for birne shrimps is used for further determination of ecotoxicity.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

226.67 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).

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 predicted values.

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.