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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-07 to 2018-09-26

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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

Remarks:

LC-Q-TOF

Details on sampling:

- Concentrations: 3.88 - 8.54 - 18.8 - 41.3 - 90.9 - 200 mg/L
- Sampling method and Sample storage conditions before analysis: At the start of the exposure and at renewal (0 and 24 h), the fresh media samples were taken after preparation of the test item loadings, diluted with methanol (dilution factor 2) and analyzed. At the renewal and at the end of the exposure (24 and 48 h), samples of the 24-h old media were taken directly from the test vessels, diluted with methanol (dilution factor 2) and analyzed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (200 mg/L of the test item was weighed out) was freshly prepared with dilution water prior to the start of the exposure (day 0) and prior to the renewal of the test solutions (day 1). An appropriate amount of the test item was transferred with an appropriate amount of the dilution water into a glass flask. The stock solution was treated with ultrasound for 5 min at room temperature and mixed by constant stirring with a magnetic stirrer at approximately 1100 rpm for 1 h at room temperature.
- Controls: Dilution water without test item incubated under the same conditions as the test groups
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the highest loading level of 200 mg/L, a fine sediment was observed after 24 h and the color of the test solution changed from yellow to a yellowish to brownish color.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna STRAUS
- Source: from continuous laboratory cultures; Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): Less than 24 h old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.
- Type and amount of food, frequency: The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

211 to 217 mg CaCO3/L

Test temperature:

20°C ± 2°C

pH:

7.11 to 7.70

Dissolved oxygen:

7.98 to 8.49 mg/L

Conductivity:

501 to 509 µS/cm

Nominal and measured concentrations:

Nominal: 3.88 - 8.54 - 18.8 - 41.3 - 90.9 - 200 mg/L
geometric mean measured: 3.42, 7.20, 14.9, 35.0, 78.4 and 173 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): loosely covered with watch glasses
- Material, size, headspace, fill volume: glass, 50 mL fill volume, test volume: 20 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): renewal of the test medium after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same composition as the culture medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Prior to the start of the exposure (0 h) and at the renewal of the test solutions (24 h), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured. At the start of the exposure and at the renewal (0 and 24 h), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) per loading level and control. At the renewal and at the end of the exposure (24 and 48 h), the water quality parameters of the old media were measured in one appropriate replicate (containing daphnids) per loading level and control. The replicate with the highest immobilization rate per loading level and control was measured.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h
- Light intensity: max.1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization was determined in all groups after 24 and 48 h. An animal was considered immobile, if it was not able to swim in thewater phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.


RANGE-FINDING STUDY
- Test concentrations: 12.0 and 120 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

117 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

107 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

173 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

173 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: not reported
- Other biological observations: not reported
- Effect concentrations exceeding solubility of substance in test medium: In the preliminary test all tested concentration levels were visually clear throughout the exposure. The stock solution of 120 mg/L was yellowish colored throughout the test period. In the main test, a fine sediment was observed after 24 h and the color of the test solution changed from yellow to a yellowish to brownish color at the highest loading level of 200 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, validity criteria: EC50 (24 h): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5), EC50 (24 h): 0.6 - 2.1 mg/L, according to OECD 202 (clone A
Measured value: 2.03 mg/L (CI:1.00 - 4.00 mg/L)

Reported statistics and error estimates:

The EC/EL10- and EC/EL50-values (after 24 h of exposure) were calculated by straight line regression with the software GraphPad Prism. Only the two highest concentration/loading levels were taken into account, representing the EC/EL0- and the EC/EL100-level, since only one partial effect occurred. The respective 95% confidence limits were empirically derived from the observation data as follows: The highest concentration/loading level without any effect (EC/EL0) and the lowest concentration/loading level causing 100% immobilization (EC/EL100) were used as 95% confidence limits. The EL-values were calculated accordingly. The EC/EL10- and EC/EL50-values (after 48 h) were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution.
An EC50 value was calculated for the reference item by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The respective 95% confidence limits were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as 95% confidence limits.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the calculated concentrations (geometric mean) of the test item Tetrahydrofolic acid, measured as the degradation product, the 48 h EC50 for Daphnia magna was 107 mg/L (95 % confidence limits: 80.3 - 170 mg/L).
Based on the nominal loadings of the test item Tetrahydrofolic acid, the 48 h EL50 for Daphnia magna was 123 mg/L (95 % confidence limits: 93.0 - 196 mg/L).