[No public or meaningful name is available]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 August - 13 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide

Cas Number:

78687-39-5

Molecular formula:

C11H15BrN2O5

IUPAC Name:

3-(aminocarbonyl)-1-ß-D-ribofuranosyl-pyridinium bromide

impurity 1

Reference substance name:

Nicotinamide

EC Number:

202-713-4

EC Name:

Nicotinamide

Cas Number:

98-92-0

Molecular formula:

C6H6N2O

IUPAC Name:

nicotinamide

impurity 2

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 3

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

impurity 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 5

Reference substance name:

inorganic salts by sulphated ash

IUPAC Name:

inorganic salts by sulphated ash

impurity 6

Reference substance name:

Bromic acid

EC Number:

232-158-3

EC Name:

Bromic acid

Cas Number:

7789-31-3

Molecular formula:

BrHO3

IUPAC Name:

bromic acid

impurity 7

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid

Details on test material:

Batch / Lot No. 1000146/01

Specific details on test material used for the study:

Lot no.: 1000146/01
Storage conditions: At approximately -15 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test media including the control at the start of the test (without algae) and after 24, 48 and 72 hours of exposure (containing algae). For sampling at the start of the test the samples were taken from the freshly prepared test solutions as well as from the pure test water serving as control. For all subsequent sampling time points, aliquots from the respective replicates were pooled per treatment before sampling.
Immediately after sampling, acetonitrile containing 0.2% acetic acid was added to each sample to obtain a composition of sample:acetonitrile (1:1; v:v) containing 0.1% acetic acid. Thereafter, all samples were stored deep-frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution: containing test water, test item and algae (six replicates)
- Control: containing test water and algae (six replicates)
Since the test item is soluble, the test solution was prepared by adding 100 mg/l of test item to test water in sterile glassware and stirring it for five minutes. 100 ml of the resulting test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Pure test water handled as the test solutions and inoculated with the exponentially growing preculture of algae mentioned above served as control.
For the incubation the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per minute. The test flasks were repositioned daily during the exposure.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Test organism: Raphidocelis subcapitata (SAG Nr. 61.81), supplied by the SAG Culture Collection of Algae at Göttingen University, 37073 Göttingen, Germany.
- Culture: 250 ml flask containing 100 ml of OECD medium inoculated with an exponentially growing preculture of Raphidocelis subcapitata.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation, source: Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).
- Temperature: 21–24 °C maintained at ±2 °C in a thermo-controlled room.
- Control of sensitivity: For evaluation of the algal quality and the test procedure, potassium dichromate is tested as a positive control twice a year.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

The calculated water hardness of the test water is 24.2 mg/l as CaCO3.

Test temperature:

21–24 °C maintained at ±2 °C in a thermo-controlled room.

pH:

7.8 - 8.1

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

NOMINAL CONCENTRATIONS
The definitive test was performed as a limit test at a nominal concentration of 100 mg/l.

MEASURED CONCENTRATIONS
The concentrations of Nicotinamide-beta-D-riboside bromide (CAS no. 78687-39-5) in the test media from 0, 24, 48 and 72 hours of exposure were determined by HPLC with UV detection. These analyses showed that the test item was correctly dosed. However, the test item concentration decreased and was not within 80–120% of the nominal value at the end of the test. Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentration of the test item over the entire 72 hours test period, which was 81.3 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flask, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers
- Test medium: Reconstituted water (OECD medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Source: Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).
- Light intensity and homogeneity: Continuous, 4440 – 8880 lux ± max. 15% variation. The test flasks were repositioned daily during the exposure.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: Approximately 5’000 cells/ml; all test media contained the same initial density of algal cells.

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test (three replicates for the control and two replicates per test concentration) with nominal concentrations of 1, 10 and 100 mg/l of Nicotinamide-beta-D-riboside bromide was performed. No relevant inhibitory effects were observed in the non-GLP range-finding test.

EFFECT PARAMETERS MEASURED
- Biomass: Algal biomass is determined at 24, 48 and 72 hours of exposure using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). To this end, aliquots of 5 ml are removed from each test flask. At test start (0 hours) the nominal value is applied.

Reference substance (positive control):

yes

Remarks:

Control of sensitivity: Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 81.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

81.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 81.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

81.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

AVERAGE SPECIFIC GROWTH RATE
With respect to the endpoint average specific growth rate no significant effects (Student t-test, one-sided smaller, a = 0.05) were observed at 81.3 mg/l mean measured concentration compared to the control.
The 72-hour EC50 based on average specific growth rate of Nicotinamide-beta-D-riboside bromide to Raphidocelis subcapitata was therefore estimated to be >81.3 mg/l, while the NOEC was determined to be 81.3 mg/l.

YIELD
With respect to the endpoint yield no significant effects (Student t-test, one-sided smaller, a = 0.05) were observed at 81.3 mg/l mean measured concentration compared to the control.
The 72-hour EC50 based on yield of Nicotinamide-beta-D-riboside bromide to Raphidocelis subcapitata was therefore estimated to be >81.3 mg/l, while the NOEC was determined to be 81.3 mg/l.

OBSERVATIONS AND ENVIRONMENTAL CONDITIONS
The microscopic examination of the algal cells at the end of the test showed no abnormal appearance of the algae growing at the single test concentration of nominal 100 mg/l. The appearance of the algal cells was not affected by the test item up to at least this concentration.
No remarkable observations were made concerning the appearance of the test media.
The pH value in the control replicates changed by 0.3 units. The light intensity was maintained at 4883 lux ± 8.2%. The temperature ranged from 22 to 24 °C. All values were within the guideline ranges.

Results with reference substance (positive control):

The result of the positive control test performed in February 2019 showed that the sensitivity of the test system based on average specific growth rate (72 h EC50 = 1.16 mg/l) was in line with a ring test yielding 1.19 mg/l, SD = 0.27 (ISO 8692:2012: Water quality - Fresh water algal growth inhibition test with unicellular green algae).

Reported statistics and error estimates:

Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Pro.

Any other information on results incl. tables

Validity of the test:

Validity criterion (applies to control cultures)	Required value	Achieved value
Average specific growth rate [d^-1]	>=0.92	1.89
CV of average specific growth rate over 3 days (%)	<=7	0.6
CV of section-by-section specific growth rates (%)	<=35	4.2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See table in section "any other information on results incl. tables".

Conclusions:

Nicotinamide-beta-D-riboside bromide (CAS no. 78687-39-5) had no significant inhibitory effects on the green algae Raphidocelis subcapitata up to the mean measured concentration of 81.3 mg/l.

Executive summary:

The growth inhibitory effects of Nicotinamide-beta-D-riboside bromide (CAS no. 78687-39-5) to the green alga Raphidocelis subcapitata were investigated according to OECD guideline 201 over a period of 72 hours.

The test item Nicotinamide-beta-D-riboside bromide is solid, 96.2% pure and soluble in test water up to at least 100 mg/l. Consequently, the single test solution was prepared by adding 100 mg/l of the test item to test water and stirring it until dissolution. The single nominal concentration tested was 100 mg/l. Six parallel test vessels were used for the test item and six for the control.

The test item concentrations in the test media from 0, 24, 48 and 72 hours of exposure were determined by HPLC with UV detection. These analyses showed that the test item was correctly dosed. However, the test item concentration decreased and was not within 80–120% of the nominal value at the end of the test. Therefore, the effective concentrations ECx were assessed based on the geometric mean of the measured concentration of the test item over the entire 72 hours test period, which was 81.3 mg/l.

With respect to the endpoint average specific growth rate no significant effects were observed at 81.3 mg/l mean measured concentration compared to the control.

With respect to the endpoint yield no significant effects were observed at 81.3 mg/l mean measured concentration compared to the control.

The results of the average specific growth rate and yield inhibition of Nicotinamide-beta-D-riboside bromide to the green alga Raphidocelis subcapitata are summarized in the following table based on the geometric mean of the measured concentrations:

Parameter (0 -72 h)	Growth rate (mg/L)	Yield (mg/L)
EC50	>81.3	>81.3
NOEC	81.3	81.3

In conclusion, Nicotinamide-beta-D-riboside bromide (CAS no. 78687-39-5) had no significant inhibitory effects on the green algae Raphidocelis subcapitata up to the mean measured concentration of 81.3 mg/l.

All validity criteria were fulfilled.