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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-04-23 - 2001-05-02 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute Immobilization Test and Reproduction Test, Part 1,1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992
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: At room temperature at about 20 °C, away from direct sunlight
- Stability under test conditions: Stability in water: Unlimited
- Solubility and stability of the test substance in the solvent/vehicle: Solubility in water: 5.5 g/l

OTHER SPECIFICS:
pH: 7 (aqueous extract
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/l
- Sampling method: aliquots were taken
- Sample storage conditions before analysis: The samples were stored deep frozen and protected from light until analysis was performed.

Analysis of the Test Item Concentrations
For the analysis of the actual test item concentrations the following samples were taken:
Just before test start:
- duplicate samples from the test medium (without daphnids)
- duplicate samples from the control (without daphnids)
After 48 hours (stability samples):
- duplicate samples from the test medium
- duplicate samples from the control
For the 48-hour stability samples, the contents of the two respective test beakers were combined.
All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP), the test item is sufficiently stable in the test water under these storage conditions.
The concentrations of the test item 1,3-Dimethyl-4-aminouraciI were analyzed in the duplicate test medium samples from the single test concentration of both sampling times (0 and 48 hours).
From the control, only one of the duplicate samples was analyzed from each of both sampling times. The analytical procedure and the results are described in the attached analytical report.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the only test concentration of nominal 100 mg/l was prepared by dissolving 31 mg test item completely in 300 ml test water by ultrasonic treatment for 15 minutes and intense stirring for 10 minutes at room temperature.
The test medium was prepared just before introduction of the daphnids (= start of the test).
- Controls: A control was tested in parallel (test water without addition of the test item).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: daphnids
- Strain/clone: Daphnia magna Straus
- Justification for species other than prescribed by test guideline: The test species Daphnia magna are recommended by the international testing guidelines.
- Source: A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5. Since this date, the clone is bred in the laboratories in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Feeding during test: no

ACCLIMATION
n/a
At the start of the test, the used test organisms were 6-24 hours old and were not first brood progeny.
Test type:
static
Water media type:
freshwater
Remarks:
reconstituted water
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
2.5 mmol/l (= 250.0 mg/l) as CaCO3
Test temperature:
21 °C during the test period
pH:
the pH values ranged from 7.7 to 7.9
Dissolved oxygen:
At the beginning and the end of the test period, the dissolved oxygen concentrations in the test medium and the control were at least 8.2 mg/l.
Salinity:
Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the following nominal concentrations:
CaCI2 x 2H2O: 2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O: 0.5 mmol/L (= 123 mg/L)
NaHCO3: 0.75 mmol/L (= 65 mg/L)
KCI: 0.075 mmol/L (= 5.8 mg/L)
Nominal and measured concentrations:
0, 100 (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): The beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: 100 ml glass beakers, filled with 50 ml test medium
- Aeration: The test water was aerated until oxygen saturation was reached. During the test period the test water was not aerated.
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: The loading rate was lower than one daphnia per 2 ml test solution

TEST MEDIUM / WATER PARAMETERS
- Chlorine: 0.075 mmol/l KCl, 2.0 mmol/l CaCl2 x 2H2O
- Alkalinity: 0.8 mmol/l
- Ca/mg ratio: Ca : Mg = 4:1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH: none stated
- Photoperiod: A 16-hour light to 8-hour darkness photoperiod (with a 30 minute transition period).
- Light intensity: Light intensity at light period between 200 and 1200 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The daphnids were observed for immobility or mortality after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile).

VEHICLE CONTROL PERFORMED: not applicable
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none stated
- Mortality of control: none
- Other adverse effects control: none stated
- Abnormal responses: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no
Validity criteria fulfilled:
yes
Conclusions:
The test was performed scientifically reasonable according to OECD 202 under GLP, the report is well documented and the validity criteria are met. Hence, the results can be considered as sufficiently reliable to assess the toxicity of 1,3-Dimethyl-4-aminouracil to daphnia magna.
The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours) and the 48-hour EC0 of 1,3-Dimethyl-4-aminouracil to Daphnia magna were determined to be at least 100 mg/l. The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations in excess of 100 mg/l have not been tested according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/l. With an EC50 > 100 mg/l, 1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.
Executive summary:

The acute toxicity of the test item 1,3-Dimethyl-4-aminouracil to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202, Part I (1984), under GLP.

A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of nominal 100 mg/l. Thus, the only concentration tested was nominal 100 mg/l and a control.

During the test period, the test item concentration in the duplicate samples from the start and the end of the test was measured to be 93 and 91% of the nominal value. Under the test conditions, the test item 1,3-Dimethyl-4-amhouracil was sufficiently stable during the test period of 48 hours. Therefore, all reported results are related to the nominal concentration of the test item.

In the control and at the test item concentration of 100 mg/l, no immobilized or dead test organisms or other signs of intoxication were determined during the test period of 48 hours.

The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours) and the 48-hour EC0 of 1,3-Dimethyl-4-aminouracil to Daphnia magna were determined to be at least 100 mg/l. These values might even be higher, but concentrations in excess of 100 mg/l have not been tested according to the EU Commission Directive. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/l. These values could not be quantified due to the absence of toxicity of 1,3-Dimethyl-4-aminouracil at the test concentration of 100 mg/l.

1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study. Scientifically acceptable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
The test concentration (100 mg/l, nominal) was prepared in the M4 medium and treated ultrasonically for 3 minutes. Following this, the test solution was stirred for about 20 hours at 20 +/- 2 °C.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: Daphnia magna Straus, originally obtained from the Institute National de Recherche Chimique Appliquee, France and cultured in the laboratories of BASF since 1978.
- Age at study initiation (mean and range, SD): 2 - 24 hours
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
2.20 - 3.20 mmol/l
Test temperature:
19.6 - 20.3 °C
pH:
8.0 - 8.7
Dissolved oxygen:
8.7 - 8.7 mg/l
Nominal and measured concentrations:
Nominal test concentrations: 100 mg/l Additionally, a control was tested in parallel.

Analytically determined concentrations:
after 0 h = 99.7 % after 48 h = 102 % of nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: flat bottom glass tubes
- Fill volume: 10 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.5 animal/ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium, according to ISO 10706
- Alkalinity: up to pH 4.3: 0.80 - 1.00 mmol/l
- Ca/mg ratio: approx. 4 : 1
- Conductivity: 550 - 650 microS/cm


OTHER TEST CONDITIONS
- Photoperiod: 16 : 8 hours day-night regime
- Light intensity: diffuse light, 1 - 8 microE/(m2*s) at a wave length of 400 - 700 nm


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility after 0, 24, 48 h
Key result
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Reported statistics and error estimates:
The EC50 was determined directly from the raw data
Immobility of Daphnia after x hours [%]: 
Nominal test concentration (mg/l)
/ 0 h / 24 h / 48 h /
Control / 0 / 0 / 0 /
100.0 / 0 / 0 / 0 /

Description of key information

Short-term toxicity to aquatic invertebrates

- EC50 > 100 mg/l, EC0 ≥ 100 mg/l for Daphnia magna Straus over 48h (OECD 202, static, GLP)

- EC50 > 100 mg/l, EC0 ≥ 100 mg/l, NOEC ≥ 100 mg/l for Daphnia magna Straus over 48h (OECD 202, static, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

According to Regulation 1272/2008, Table 4.1.0, Classification categories for hazardous to the aquatic environment, a substance must be classified as Acute (short-term) aquatic hazard, Acute Category 1, if i.a. the 48 hr EC50 (for crustacea) is ≤ 1 mg/l. It must be classified as Chronic Category 3 if the 48 hr LC50 (for crustacea) is > 10 to ≤ 100 mg/l, and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log Kow ≥ 4).

In both available studies, the following values were determined: EC50 > 100 mg/l, EC0 ≥ 100 mg/l, for daphnia magna. Hence, the substance does not need to be classified as hazardous to the aquatic environment, neither acute nor chronic.