Cytidine 3'-(dihydrogen phosphate)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 11, 2017 - September 14, 2017

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

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: room temperature
- Stability under test conditions: yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 100 mg/L (nominal)
- Sampling method: Analytical measurement was performed at the applied test concentration level and from the control at the beginning and at the end of the experiment.
- Sample storage conditions before analysis: No storage was necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath for approximately 5 minutes. As a Limit Test was carried out, further dilution of stock solution was not be performed.
- Controls: Algal growth medium (OECD Medium) was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

ACCLIMATION
- Acclimation period: no.
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

2.5 - 22.8

pH:

6.56 - 8.83

Nominal and measured concentrations:

- Nominal: 0 (control), 100 mg/L.
- Measured: 0 (control), and 103 mg/L at the start and 98.1 mg/L at the end of the experiment. As the analytically measured concentration deviated not more than 20 percent from the nominal, the biological results are based on the nominal test item concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmayer flasks
- Type: covered by air-permeable stoppers.
- Material, size, headspace, fill volume: glass; size: 250 mL; fill volume: 100 mL test solution.
- Initial cells density: 1 E04
- Control end cells density: 70.17 E04
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water.
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous ilumination
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8373 lux (equivalent to ~113 E/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:Because no toxic effect was observed during the preliminary range-finding test, only one test concentration of the test item in the test medium (100 mg/L) and one control group were tested in a Limit Test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes.
- Observation of abnormalities (for algal test): no.
- Unusual cell shape:no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

TThe results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate, areas and yield were not statistically significantly different from the untreated control value.

Table 2:  Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

Test group	Growth ratemand % inhibition ofm
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0589	0.0	0.0614	0.0	0.0590	0.0
100 mg/L (nominal)	0.0553	6.0	0.0605	1.4	0.0590	0.1

Table 3:  Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	38.0	0.0	294.0	0.0	1342.0	0.0
100 mg/L (nominal)	34.0	10.5	276.0	6.1	1310.0	2.4

 

Table 4:Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	69.2	0.0
100 mg/L (nominal)	68.8	0.5

 

Table 5:pH-Values in the Test Media at the Start and End of the Test

Test group	pH-values
	Start	End
Control	7.35	8.80
		8.72
		8.83
		8.76
		8.43
		8.68
100 mg/L (nominal)	6.56	6.80
		6.65
		6.64
		6.67
		6.71
		6.84

 

 Table 6:Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.8	22.7	22.5	22.8
temperature (ºC) of the climate chamber	max	23.1	22.9	22.8	23.2
	min	22.2	22.4	22.2	22.4

 

Table 7:Cell Number (× 10⁴cell/mL) determined in the Main Experiment

 

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	19	72
		1	5	22	73
		1	3	17	70
		1	4	19	71
		1	4	17	68
		1	5	21	67
	Mean	1.00	4.17	19.17	70.17
	SD	0.0	0.8	2.0	2.3
100 mg/L (nominal)		1	4	18	69
		1	5	21	72
		1	4	17	70
		1	4	17	68
		1	3	19	71
		1	3	18	69
	Mean	1.00	3.83	18.33	69.83
	SD	0.0	0.8	1.5	1.5

 

Validity criteria fulfilled:

yes

Remarks:

All validty criteria were met (see "Overall remarks"), therefore the study can be considered as valid.

Conclusions:

The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L and the NOEC was determined to be 100 mg/mL, both based on growth rate.

Executive summary:

A freshwater algae growth inhibition test was performed on the test item in accordance with the OECD Guideline 201 and EU Method C.3, following GLP. Based on the results of a range-finding study performed at concentrations of 0.1, 1, 10 and 100 mg/L of the test item, where no toxic response was observed, a Limit Test was performed using only one test concentration of 100 mg/L (nominal) and one control group. Pseudokirchineriella subcapitata was exposed to 100 mg/L test item (nominal) or medium (negative control) for 72 h under static conditions. Six replicates were run for both the test and control items. Analytical monitoring was performed using a validated method, measurements were taken at the start and at the end of the test. As the analytically measured concentration deviated not more than 20 percent from the nominal, the biological results are based on the nominal test item concentration. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met. The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate, yield, and biomass). The overall NOEC was determined to be 100 mg/L (nominal) and the overall LOEC was determined to be higher than 100 mg/L (nominal).

Description of key information

Key study: OECD 201 and EU method C.3. GLP study. The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L and the NOEC was determined to be 100 mg/mL, both based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Key study: A freshwater algae growth inhibition test was performed on the test item in accordance with the OECD Guideline 201 and EU Method C.3, following GLP. Based on the results of a range-finding study performed at concentrations of 0.1, 1, 10 and 100 mg/L of the test item, where no toxic response was observed, a Limit Test was performed using only one test concentration of 100 mg/L (nominal) and one control group. Pseudokirchineriella subcapitatawas exposed to 100 mg/L test item (nominal) or medium (negative control) for 72 h under static conditions. Six replicates were run for both the test and control items. Analytical monitoring was performed using a validated method, measurements were taken at the start and at the end of the test. As the analytically measured concentration deviated not more than 20 percent from the nominal, the biological results are based on the nominal test item concentration. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met. The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate, yield, and biomass). The overall NOEC was determined to be 100 mg/L (nominal) and the overall LOEC was determined to be higher than 100 mg/L (nominal).