N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

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:

2018-06-06 to 2018-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

EC Number:

205-181-1

EC Name:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Cas Number:

135-16-0

Molecular formula:

C19H23N7O6

IUPAC Name:

(2S)-2-[(4-{[(2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.00 - 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Sampling method: All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 h) and at the end of the exposure (72 h) with algae. The test item and the main degradation product were analysed. The samples were analysed with an LC-MS method. The method was implemented under non-GLP but documented in the raw data and validated according to Annex I. The method validation is not part of this GLP study.
- Sample storage conditions before analysis: Due to the rapid degradation of the test item, the concentrations of the main metabolite of the test item were analytically verified via LC-Q-TOF at the start (0 h) and at the end of the exposure (72 h) in all concentration levels and in the control.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg test item/L were freshly prepared with dilution water. The dispersion was treated with ultrasound for 5 min at room temperature. Thereafter, the dispersion was stirred for 1 h at room temperature at 1100 rpm.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green fresh water alga
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG),  Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

The medium has a nominal hardness of 0.24 mmol Ca+Mg/L

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

pH:

Between 7.27 at start of exposure to 9.50 at the end of exposure

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
Measured: 0.804 - 2.52 - 8.18 - 23.8 - 79.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:Erlenmeyer flasks
- Type (delete if not applicable): closed, with cotton wool plugs
- Material, size, headspace, fill volume: Sterile Erlenmeyer flasks with a fill volume of 250 mL, volume of test medium 100 mL
- Aeration: No
- Initial cells density: Nominal: approximately 5 x E+03 - E+04 cells/mL , Current: 5523 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4- 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 E-03
Na2MoO4 x 2 H2O 7 E-03
COCI2 x 6 H2O 1.5E-03
CuCl2 x 2 H2O 1E-05
NaHCO3 50
pH 8.1 ± 0.2
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Chlorophyll-a-fuorescence and microscopic evaluation
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the range finding test. Therefore, a separate replicate of each test item concentration was prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Five concentrations were tested in a geometrical series with a (nominal) dilution factor of √10
- Range finding study
- Test concentrations: 12.0 and 120 mg/L
- Results used to determine the conditions for the definitive study: Due to the marked inhibition of growth and yield the test concentrations were chosen below 120 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.6 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:

EC50

Effect conc.:

21.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

14.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

28.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

8.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 11

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

14.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 12

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

- Results with reference substance valid? Yes, validity criteria: growth: (Valid Range (average ± 3 x SD)) 0.752 ± 0.554; yield: (Valid Range (average ± 3 x SD)) 0.399 ± 0.294.
- EC50: growth: 0.574 (CI: 0.549 - 0.596), yield: 0.337 (CI: 0.313 - 0.345)

Reported statistics and error estimates:

EC/EL-values and statistical analyses: EC/EL10-, EC/EL20- and EC/EL,so-values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response.
NOEC/NOEL, LOEC/NOEL and statistical analyses: The NOEC/NOEL and LOEC/NOEL were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted: Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01. Levene’s test on variance homogeneity was calculated with a significance level of 0.01. Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done for growth rate with a significance level of 0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Tetrahydrofolic acid was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values based on calculated concentrations of the test item Tetrahydrofolic acid, measured as the degradation product: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 21.5 (21.3 - 21.7) mg/L and 11.3 (10.4 - 13.1) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 h were 2.52 mg/L.
Based on the nominal loadings of the test item Tetrahydrofolic acid, the EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 h were 28.3 (28.1 – 28.6) mg/L and 14.2 (12.9 – 16.6) mg/L, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 h were 3.16 mg/L.