Urea

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all
- Sampling method: Samples of the test solution were taken from the bulk solution at the test start or fresh prepared test solutions and spent test solutions were sampled as composite specimens per treatment group. 50 mL specimen were stored deep frozen (≤ - 18 °C) until analysis. Concentrations of urea were analysed in the test solutions at the start and at the end of the test and once a week during the test in the aged and fresh prepared test solutions.
- Sample storage conditions before analysis: Freezer

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item Urea was soluble in the test medium. An aqueous stock solution was prepared by adding the test item to test medium, followed by stirring for 1 minute on a magnetic stirrer. The concentrations were prepared by serial dilutions of the stock solution A. The stock solution was clear and visible without particles.
- Controls: blank controls (dilution medium only)
- Test concentration separation factor: 2.5 to 2.9
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: from Landesanstalt für Umweltschutz Baden-Württemberg, Griesbachstr. 1, 76185 Karlsruhe, Germany
- Age at study initiation (mean and range, SD): < 24 h

- Method of breeding: breeding was performed in glass beakers (50 mL) under the same conditions as in the test. Test animals were separated from the adults by means of a pipette. Breeding medium: Elendt M4 medium (according to the OECD Guideline 211, Annex 2)

- Source: Landesanstalt für Umweltschutz Baden-Württemberg, Griesbachstr. 1, 76185 Karlsruhe, Germany
- Feeding during test :Yes
- Food type: Alga Desmodesmus subspicatus purchased from MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany,
- Amount: Daily, depending on age the Daphnia were fed with a defined suspension of algae:
<= 1 day old: 0.5 mL/50 mL M 4
3 to 10 days old: 1.0 mL/50 mL M 4
> 10 days old: 1.5 mL/50 mL M 4
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

between 166 and 169 mg/L CaCO2.

Test temperature:

20.4 – 20.8 °C (continuously measured)

pH:

6.94 to 7.90

Dissolved oxygen:

6.26 to 9.03 mg/L

Nominal and measured concentrations:

Nominal Control, 39.5, 106.6, 288.1, 777.8, 2100.1 mg urea/L
Measured: within 80 to 120% of nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (approximately 60 mL)
- Material, size, headspace, fill volume: 50 mL test solution
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10 + 3 additional vessels without animals (1 for oxygen and pH measurements, 1 for analysis and 1 for retained specimens)
- No. of vessels per control (replicates): 10 + 3 additional vessels without animals (1 for oxygen and pH measurements, 1 for analysis and 1 for retained specimens)
- Biomass loading rate: 50 mL test solution per daphnid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium (according to the OECD Guideline 211, Annex 2) Prepared at least one day in advance
- Culture medium different from test medium: no
- Intervals of water quality measurement: 3 to 4 days

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h light : 8 h dark, daily
light intensity: approximately 20 µE m-2s-1,
equivalent to 1000 lux

- Light intensity: light intensity: approximately 20 µE m-2s-1, equivalent to 1000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
EC10, EC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5 to 2.9
- Test concentrations: nomial 39.5, 106.6, 288.1, 777.8, 2100.1 mg/L test item

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

140.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities: No visible signs of abnormalities of surviving animals were observed in any assessment.
- Observations on body length and weight: The NOEC for the body length was determined to be higher or equal than 2100.1 mg/L test item, nominal. Hence, the LOEC was higher than 2100.1 mg/L test item. EC10, EC20 and EC50 values could not be determiend.
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Immobilisation of control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The total number of living offspring per introduced parent and per survived parent animal alive at test end was determined. Furthermore, the age of first reproduction, body length and the percent mortality of parent animals at the test end was assessed.

The NOEC/LOEC values and effect concentrations ECx values at 21 days for the reproductive output of the parent animals (number of offspring per introduced parent and number of offspring per survived parent). Additionally, LOEC/NOEC values for the mortality and age of first reproduction of the parent animals were determined.
The Welch-t-test after Bonferroni-Holm was used in relation to the cumulative number of offspring per introduced (= survived parent). The multiple sequentially-rejective U-test after Bonferroni-Holm was used for the age of first reproduction. The Dunnett`s Multiple t-test Procedure was used for the endpoint body length.
For the calculation of the ECx values (including 95 % confidence limits), Probit analysis using linear max. likelihood regression as linear regression method was used.

Data were analysed using appropriate statistical methods, using ToxRat Professional 3.3 (2018).

Validity criteria fulfilled:

yes

Conclusions:

21-d EC10 reproduction 140.7 mg/L Daphnia magna

Executive summary:

A Klimisch 1 long-term Daphnia GLP reproduction toxicity test according to OECD 211 was performed by Juckeland (2021) to assess the effects of the test item Urea on the reproductive output of Daphnia magna during 21 days of exposure. Nominal concentrations were Control, 39.5, 106.6, 288.1, 777.8 and 2100.1 mg urea/L.

 

The calculated study endpoints are based on nominal concentrations for test item because the recoveries of the active substance were in the range between 80 - 120% of nominal concentrations.

These concentrations caused no mortality of the paretal daphnids in any test group. The first offspring in control replicates was observed at day 10 and 11 after test start. No aborted brood were observed in all test concentrations.

Reproduction, however, was affected.

 

The lowest EC₁₀ value was 140.7 mg/L test item for offspring per introduced (=survived) parent.

This result is considered relevant and reliable for the risk assessment.

Description of key information

21-d EC10 reproduction 140.7 mg/L Daphnia magna

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

140.7 mg/L

Additional information

A Klimisch 1 long-term Daphnia GLP reproduction toxicity test according to OECD 211 was performed by Juckeland (2021) to assess the effects of the test item Urea on the reproductive output ofDaphnia magnaduring 21 days of exposure. Nominal concentrations were Control, 39.5, 106.6, 288.1, 777.8 and 2100.1 mg urea/L.

 

The calculated study endpoints are based on nominal concentrations for test item because the recoveries of the active substance were in the range between 80 - 120% of nominal concentrations.

These concentrations caused no mortality of the paretal daphnids in any test group. The first offspring in control replicates was observed at day 10 and 11 after test start. No aborted brood were observed in all test concentrations.

Reproduction, however, was affected.

 

The lowest EC₁₀value was 140.7 mg/L test item for offspring per introduced (=survived) parent.

This result is considered relevant and reliable for the risk assessment.