Ammonium dihydrogen 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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please see the read-across document as attached in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Specific details on test material used for the study:

The target substance contains between 45 and 86% water.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on pure target substance

Remarks:

As the target substance is an aqueous solution with 45 to 86% water, these values need to be multiplied by at least a factor of 1.8.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on pure target substance

Remarks:

As the target substance is an aqueous solution with 45 to 86% water, these values need to be multiplied by at least a factor of 1.8.

Details on results:

Based on the results with the source test substance and on the supporting results from the SIDS of citric acid and ammonium sulfate, the pure target substance ammonium dihydrogen citrate can be considered to be acutely toxic to aquatic algae at concentrations of 56 mg/L and higher. As the target substance is an aqueous solution with 45 to 86% water, these values need to be multiplied by at least a factor of 1.8. These results are acceptable to be used for the aquatic freshwater algae toxicity endpoint as the source substance was tested in marine species and according to the SIDS, is was shown that marine species tended to be more sentitive to ammonium sulfate than freshwater species.

Validity criteria fulfilled:

not applicable

Conclusions:

Based on the results with the source test substance and on the supporting results from the SIDS of citric acid and ammonium sulfate, the pure target substance ammonium dihydrogen citrate can be considered to be acutely toxic to aquatic algae at concentrations of 56 mg/L and higher. The 72h-ErC50 = 74.1 mg/L and the 72h-NOECr = 32 mg/L. As the target substance is an aqueous solution with 45 to 86% water, these values need to be multiplied by at least a factor of 1.8. These results are acceptable to be used for the aquatic freshwater algae toxicity endpoints as the source substance was tested in marine species and according to the SIDS, it was shown that marine species tended to be more sensitive to ammonium sulfate than freshwater species.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-19 May 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical support was included in the test. For validity justification, please refer to "overall remarks"

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

soluble at 1000 mg/L in seawater after 20 hours stirring

Analytical monitoring:

no

Remarks:

see "overall remarks"

Vehicle:

no

Details on test solutions:

As the substance was classified as soluble, a dilution series was prepared, where an appropriate weight of test material was added to prepare to initial stock. Appropriate volumes were taken from this stock to prepare subsequent test concentrations which were brought to volume with ISO culture medium (see attached background material)

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source: Pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating nutrient medium (ISO culture medium) to a cell density of approximately 10^4 cells per millilitre. The pre-cultures were incubated at approximately 20±2 °C under constant illumination for 3d ±1d, and were used as the inoculum source for subsequent toxicity tests.
Culture Number: C5B1 C

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

19.4 - 20.6 ºC

pH:

in the ISO culture medium and test material stocks at t=0: 7.99 - 8.07
at t=72h: 8.24 - 8.92

Dissolved oxygen:

No information

Salinity:

Not indicated

Conductivity:

50.7 - 53.6 mS/cm

Nominal and measured concentrations:

10, 18, 32, 56 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:100 ml borosilicate glass conical flasks, to which 80 ml of test medium seawater was added. Following inoculation, all flasks were loosely covered with aluminium foil caps, and mounted on an orbital shaker (approximately 18 mm orbit, 150 rpm).
- Aeration: no information
- Initial cells density: approximately 10,000 cells per ml
- Control end cells density: no reported (only growth rates were reported)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: ISO culture medium (no further information available)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 μm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, pH of stock solution of 1000 mg/L was adjusted from 5.47 to 7.98 at test commencement in the range-finding test
- Photoperiod: constant
- Light intensity and quality: 8300 - 9640 lux; the flasks were placed under cool white illumination as specified in the ISO guidelines. The illumination consists of four 40W tubes mounted at a distance of approximately 40 cm directly above the test area. Light intensity values were measured daily during the test.
- Salinity (for marine algae): not indicated

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: The initial inoculum was checked microscopically using a haemocytometer. In the definitive test, counts of algal cell numbers were carried out daily by microscope using either haemocytometer or Sedgewick-Rafter chamber, depending upon cell numbers present or by Fluorometer measurements. Three readings were performed on each test vessel (SOP 106).

Growth rate was measured in terms of increase in cell number or in biomass.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Test concentrations: 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: the range-finding test exhibited a 72h EC50 of 59.50 mg/L (dilution series).

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol at 5.6, 3.2, 1.8, 1.0 and 0.56 mg/L which were prepared from a main stock of 100 mg/L. The 100 mg/L stock was stirred for a minimum of one hour, or until completely dissolved.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

74.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 72.2 - 75.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

see attached background information

Results with reference substance (positive control):

the 72h EC50 of the reference material was 3.84 mg/L (95%CI 3.01 - 4.5 mg/L). This was within the ranges as specified in the guideline 72h EC50 2.0 - 4.0 mg/L.

Reported statistics and error estimates:

The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of cell volume. Growth rate was calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period, using an exponential model:
Nt = No .ekt
where
No = volume or number at beginning of test
Nt = volume or number at time t
t = time in days
k = growth rate (.d-1)

The average value of k for each time interval was calculated for each concentration. Since the criterion of effect was the concentration causing 50% reduction in growth rate with respect to the controls, the response for each concentration was estimated from;
effect = 1- (control k/treatment k)
The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculated using an appropriate statistical method from the ToxCalc Version 5 software.

Validity of the test (ISO Guideline)

- the control cell density shall have increased by a factor of more than 16 in 72 h. This corresponds to a growth rate of 0.96/day. The report indicated a growth rate increase of 1.5 (the cell densities of the controls were not reported at the end of the test, so it cannot be verified).

- the pH in the controls shall not have varied by more than +/- 1.0 unit during the test. The pH ranged from 8.18 to 8.81 during the test.

Validity criteria fulfilled:

yes

Remarks:

see "any other information on results"

Conclusions:

Ammonium citrate dibasic exhibited a 72h ErC50 of 74.1 mg/L (based on nominal concentrations) to the marine phytoplankton Skeletonema costatum in the aqueous phase. The NOErC was 32 mg/L.

Executive summary:

In a 72 -h acute toxicity study conducted according to ISO 10253I guidelines and GLP principles, marine phytoplankton Skeletonema costatum was exposed to the test substance under static conditions at the nominal concentrations of 10, 18, 32, 56 and 100 mg/L and a blank seawater control (2 replicates per concentration, 4 replicates for control).

Exposure concentrations were not analytically verified but this was considered not to invalidate the study. The 72h-ErC50 was 74.1 mg/L and the NOErC was 32 mg/L based on nominal concentrations. The study is considered to be reliable with restrictions.

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:

data from handbook or collection of data

Test organisms (species):

Chlorella vulgaris

Water media type:

freshwater

Total exposure duration:

18 d

pH:

7.0

Duration:

18 d

Dose descriptor:

EC50

Effect conc.:

ca. 2 700 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Remarks:

expressed as (NH4)2SO4 concentrations

Basis for effect:

cell number

Remarks on result:

other: Tam and Wong (1996)

Details on results:

In long term test the green algae Chlorella vulgaris were exposed to ammonium sulfate for 21 days (Tam and Wong, 1996). The cell number at start time was 1*10^6 /mL compared to about 1*10^5 as recommended in standard guidelines like OECD TG 201. In control medium, the N-source was KNO3, which was replaced by different concentrations of ammonium sulfate in the test media (47 - 4710 mg/L ammonium sulfate, calculated from 10 – 1000 mg/L NH3-N). The pH value was adjusted to 7 +/- 0.2 before algae inoculation and was maintained at neutral pH during the study. In the first about 10 days after start, in all test concentration and in the control a lag phase was observed. Thereafter in the control and some test concentrations an exponential increase in cell number was observed up to day 17 or 18. No data are available to calculate the 72-h-EC50. Based on data presented in Tam and Wong (1996) an EC50 (18 d) for the endpoint cell count of about 2700 mg/L ammonium sulfate, but no EC10 can be calculated for Chlorella vulgaris. At concentrations of 20 - 500 mg/L N, no significant differences of growth compared to control were observed.

In the OECD SIDS of Ammonium sulfate, it was shown that marine phytoplankton is more sensitive to ammonium sulfate than freshwater species. The marine dinoflagellates Gymnodinium splendens and Gonyaulax polyedra showed growth inhibition in a 17d-test at concentrations of 0.706 mg/L ammonium sulfate (calculated from 150 μg/L NH4-N). A reduction in photosynthesis within 3 hours is also shown for Gymnodinium splendens at 0.471 mg/L ammonium sulfate (calculated from 100 μg/L NH4-N). No significance-levels are reported. A LOEC of 0.706 mg/L and a NOEC of 0.471 mg/L (calculated from 150 and 100 μg/L NH4-N) can be estimated for the endpoint growth reduction for Gymnodinium splendens and Gonyaulax polyedra. Also, a LOEC of 0.471 mg/L and a NOEC of 0.235 mg/L (calculated from 100 and 50 μg/L NH4-N) can be estimated for the endpoint reduction in photosynthesis for Gymnodinium splendens. No EC50 can be derived from the study. No information on pH in the test media is given (Thomas, Hastings and Fujita, 1980).

Validity criteria fulfilled:

not specified

Conclusions:

Based on data of a long term test with green algae Chlorella vulgaris presented in Tam and Wong (1996), the 18d- EC50 for the endpoint cell count was about 2700 mg/L ammonium sulfate, but no EC10 could be calculated. At concentrations of 20 - 500 mg/L N, no significant differences of growth compared to control were observed.
Results on marine species indicate a higher sensitivity than freshwater species.

Reference 4

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:

data from handbook or collection of data

Test organisms (species):

Scenedesmus quadricauda

Details on test organisms:

freshwater green algae

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Duration:

7 d

Dose descriptor:

other: TLC

Remarks:

toxic limit concentration

Effect conc.:

640 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

The results refer to toxic limit concentrations from which no dependable EC50 can be derived.

The SIDS also documents toxicity TLC for saltwater species:
Pavlova lutheri (saltwater chrysophytes): 7-day TLC = 1–300 mg/L, method not stated
Chaetoceros gracilis, Navicula ramosissima (saltwater diatoms): 7-day TLC = 1–300 mg/L, method not stated

Validity criteria fulfilled:

not specified

Conclusions:

In freshwater, citric acid appears to be of low toxicity to algae, with TLC value of several hundred milligrams per litre.
The few marine species for which data are available seem to be somewhat more sensitive to citric acid although for two algae and diatoms, the subacute toxic limit concentration is only given as a wide range between 1 and 300 mg/L. Still, at least for the few tested organisms citric acid does not seem to be highly or acutely toxic.

Description of key information

Based on the results with the source test substance and on the supporting results from the SIDS of citric acid and ammonium sulfate, the pure target substance ammonium dihydrogen citrate can be considered to be acutely toxic to aquatic algae at concentrations of 56 mg/L and higher. The 72h-ErC50 = 74.1 mg/L and the 72h-NOErC = 32 mg/L. As the target substance is an aqueous solution with 45 to 86% water, these values need to be multiplied by at least a factor of 1.8.

As the source substance was tested in marine species and according to the SIDS, it was shown that marine species tended to be more sensitive to ammonium sulfate than freshwater species, the results from the saltwater species are considered as worst-case values for the aquatic freshwater algae toxicity endpoints.

Key value for chemical safety assessment

EC50 for freshwater algae:

74.1 mg/L

EC50 for marine water algae:

74.1 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L

EC10 or NOEC for marine water algae:

32 mg/L