Urea

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal: Control, 1560, 3120, 6250, 12500, 25000, 50000 mg urea/L,
- Sampling method: direct sampling from test solutions
- Sample storage conditions before analysis: Specimens were stored deep frozen (≤ -18°C) until they were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by adding the test item to test medium.
The stock solution was stirred for 2 minutes on a magnetic stirrer.
Stock solution and test vessels were prepared in the following way:
- a stock solution (stock A) was prepared by weighing 50.0006 g test item into a graduated flask and volume to 250 mL (= 200.0024 g/L test item)
- the test solutions were prepared by direct dilution of the stock solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green alga – Pseudokirchneriella subcapitata KORSHIKOV
- Strain: strain: 61.81 SAG
(further cultivation in the test facility)
purchased from MBM ScienceBridge GmbH,
Hans-Adolf-Krebs-Weg 1, 37077 Göttingen
Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.8 – 23.9 °C

pH:

8.10 – 8.57

Nominal and measured concentrations:

Nominal: Control, 1560, 3120, 6250, 12500, 25000, 50000 mg urea/L,
Mean measured: 0 (control), 1521.7, 2936.9, 4724.3, 11570.7, 24538.2, 50362.6 mg a.s./L. The results were based on mean measured concnetrations and active substace.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flask
- Type:with air-permeable stoppers
- Material, size, headspace, fill volume: 100 mL test volume
- Initial cells density:
- Control end cells density: 280000 cells/mL
- No. of organisms per vessel: 5000 cells/mL at start
- 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: OECD medium (according to OECD 201)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: cold white fluorescent light; light intensity was measured once before test start: light intensity: on average 77 µE•m-2•s-1 measured at 400-700 nm differences from the selected light intensity over the test area did not exceed the range ± 15%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:[Neubauer counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study : Yes

CULTURING APPARATUS
-Details on culturing apparatus used:
axenic stock cultures grew in culturing vessels for 4 days prior to test initiation; cultivation was performed in glass flasks with the medium described above; algae were kept at the same temperature and light conditions as in the test itself

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6 895.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 541.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No differences such as sedimentation of test solution, cell aggregation or colour differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test.
- Effect concentrations exceeding solubility of substance in test medium:no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.41 mg/L
- Other:
The EC50 values for average specific growth rate and yield determined in the most recent study (BioChem project No.: 20 48 AAL 0022, performed from 13.10. – 16.10.2020, Pseudokirchneriella subcapitata) were ErC50 = 1.41 mg/L test item for growth rate and EyC50 = 0.57 mg/L for yield.
The reference toxicity test was valid because the cell density increased by a factor of more than 16 within the test period of 72 hours (actually 66.8). This increase corresponds to a growth rate of 1.336 d-1. The variation coefficient of the growth rate was < 7 % (actually 1.0 %). The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35% (actually 22.9 %).

Reported statistics and error estimates:

Regression analysis was performed using individual replicate responses, not treatment group means.
From average specific growth rates and yield, recorded in a series of test solutions, effect concentrations of ErC10, ErC20 and ErC50, (average specific growth rate) and EyC10, EyC20 and EyC50 (yield) was determined using concentrations-response modelling (non-linear regression, 3 parameters Normal). To determine a LOEC and to derive a NOEC for effects on growth rate, it was necessary to compare treatment means using analysis of variance (ANOVA) techniques. Shapiro-Wilk´s Test on Normal Distribution was performed. The mean for each concentration was compared with control means using an appropriate multiple comparison test method. Williams’s t-test was used if variance-homogeneity requirements are fulfilled. The Welch-t-test After Bonferroni-Holm for non-homogeneous variances with Bonferroni-Holm-adjustment was used. As a test for homogeneity of variances, Levene’s test was used.

Statistical analysis was performed using the software ToxRat Professional (Version 3.3, 20.10.2018).

Validity criteria fulfilled:

yes

Remarks:

Biomass in control cultures increased exponentially by a factor of 53.6. Mean coefficient of variation for section-by-section specific growth rates: 27.4%. CV average specific growth rates 0-72h in replicate control cultures was 1.3%.

Conclusions:

72-h ErC10: 6895.8 mg a.s./L
72-h ErC50: 24541.9 mg a.s./L

Executive summary:

A Klimisch 1 GLP growth inhibition test was performed to assess the effects of the test item Urea to green algae (Pseudokirchneriella subcapitata)during 72 hours of exposure according to OECD 201 (Juckeland, 2021). The test fulfilled all validity criteria outlined in the guideline.

 

Measured concentrations of the test item in test solutions were within a range of 86 to 101% of nominal values at test start and after 72 hours the concentrations ranged from 68 to 106% in spent test solutions. Thus, all endpoints are based on mean measured concentrations.

The72-h ErC10 and 72-h ErC50 were 6895.8 and 24541.9 mg a.s./L.

No differences such as sedimentation of test solution, cell aggregation or colour differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test.

The results of the test are considered relevant and reliable for the risk assessment.

 

Description of key information

72-h ErC10: 6895.8 mg a.s./L

72-h ErC50: 24541.9 mg a.s./L

Key value for chemical safety assessment

EC50 for freshwater algae:

24 541.9 mg/L

EC10 or NOEC for freshwater algae:

6 895.8 mg/L

Additional information

Multiple studies are available for the effect of urea on the growth of algae and cyanobacteria.

Various studies including a guideline conform study with freshwater algae and further studies with limited reliability demonstrate that green-algae are or low sensitivity to urea.

A Klimisch 1 GLP growth inhibition test was performed to assess the effects of the test item Urea to green algae (Pseudokirchneriella subcapitata)during 72 hours of exposure according to OECD 201 (Juckeland, 2021). The test fulfilled all validity criteria outlined in the guideline. Measured concentrations of the test item in test solutions were within a range of 86 to 101% of nominal values at test start and after 72 hours the concentrations ranged from 68 to 106% in spent test solutions. Thus, all endpoints are based on mean measured concentrations. The72-h ErC10 and 72-h ErC50 were6895.8and 24541.9 mg a.s./L. No differences such as sedimentation of test solution, cell aggregation or colour differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test. The results of the test are considered relevant and reliable for the risk assessment.

In the study from Bringmann and Kuhn (1978) it was proposed that the 192 hour toxicity threshold for Microcystis aeruginosa was 47 mg urea/l. This is considered to be not realistic since information from Huang et al. (2014) indicate that urea is used by Microcystis aeruginosa as N-source and that the EC50 is > 2500 mg/L. Therefore, it can be concluded that Microcystis aeruginosa is not specifically sensitive to urea and that the result from Bringmann and Kuhn (1978) was therefore considered as not relevant for the risk assessment.